RESEARCH ARTICLE Bionomic responses of Spodoptera frugiperda (J. E. Smith) to lethal and sublethal concentrations of selected insecticides Kokou Rodrigue FiaboeID 1,2¤*, Ken Okwae Fening1,2, Winfred Seth Kofi Gbewonyo1,3, Sharanabasappa DeshmukhID 4 1 African Regional Postgraduate Programme in Insect Science (ARPPIS), School of Agriculture, College of Basic and Applied Sciences, University of Ghana, Legon, Accra, Ghana, 2 Soil and Irrigation Research Centre (SIREC), School of Agriculture, College of Basic and Applied Sciences, University of Ghana, Legon, Accra, Ghana, 3 Department of Biochemistry, Cell and Molecular Biology, School of Biological Science, College of Basic and Applied Science, University of Ghana, Legon, Accra, Ghana, 4 Department of Entomology, College of Agriculture, Keladi Shivappa Nayak University of Agricultural and Horticultural Sciences (UAHS), Shivamogga, Karnataka, India ¤ Current address: Department of Zoology and Entomology, University of Pretoria, Hatfield, South Africa * rfiaboe@yahoo.com Abstract Since 2016, the invasive insect Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctui- dae) from the Americas has made maize production unattainable without pesticides in parts of Sub-Saharan Africa and Asia. To counteract this pest, farmers often resort to the use haz- ardous pesticides. This study aimed to investigate botanicals, microbials, and semi-syn- thetic insecticides in Ghana for pest control without harming local ecosystems. Under laboratory and on-station conditions, the present study evaluated the acute and sublethal responses of S. frugiperda to: (i) Pieris rapae Granulovirus (PrGV) + Bacillus thuringiensis sub sp. kurstaki (Btk) 5 WP, (ii) Btk + monosultap 55 WP, (iii) ethyl palmitate 5 SC, (iv) aza- dirachtin 0.3 SC, (v) acetamiprid (20 g/l) + λ-cyhalothrin (15 g/l) 35 EC, (vi) acetamiprid (30 g/l) + indoxacarb (16 g/l) 46 EC, and (vii) emamectin benzoate 1.9 EC. The results showed that at 96 hours post-exposure emamectin benzoate-based formulation has the highest acute larvicidal effect with lower LC50 values of 0.019 mL/L. However, the results suggested strong sublethal effects of PrGV + Btk, azadirachtin, and ethyl palmitate on the bionomics of S. frugiperda. Two seasons on-station experiments, showed that the semi-synthetic ema- mectin benzoate and the bioinsecticide PrGV + Btk are good candidates for managing S. frugiperda. The promising efficacy of emamectin benzoate and PrGV + Btk on the bionomics of S. frugiperda in the laboratory and on-station demonstrated that they are viable options for managing this pest. Introduction Agricultural productivity plays a crucial role in determining the economic growth of a country. In sub-Saharan Africa, agriculture is a significant contributor to the GDP of countries and PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 1 / 21 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Fiaboe KR, Fening KO, Gbewonyo WSK, Deshmukh S (2023) Bionomic responses of Spodoptera frugiperda (J. E. Smith) to lethal and sublethal concentrations of selected insecticides. PLoS ONE 18(11): e0290390. https://doi.org/ 10.1371/journal.pone.0290390 Editor: Yonggen Lou, Zhejiang University, CHINA Received: March 12, 2023 Accepted: August 8, 2023 Published: November 15, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0290390 Copyright: © 2023 Fiaboe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: The authors received no specific funding for this work. https://orcid.org/0000-0001-9239-194X https://orcid.org/0000-0001-7447-0126 https://doi.org/10.1371/journal.pone.0290390 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0290390&domain=pdf&date_stamp=2023-11-15 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0290390&domain=pdf&date_stamp=2023-11-15 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0290390&domain=pdf&date_stamp=2023-11-15 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0290390&domain=pdf&date_stamp=2023-11-15 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0290390&domain=pdf&date_stamp=2023-11-15 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0290390&domain=pdf&date_stamp=2023-11-15 https://doi.org/10.1371/journal.pone.0290390 https://doi.org/10.1371/journal.pone.0290390 https://doi.org/10.1371/journal.pone.0290390 http://creativecommons.org/licenses/by/4.0/ employs a large proportion of the population [1,2]. For instance, in Ghana, agriculture accounts for 20% of the GDP of the country and is vital for food security, with maize (Zea mays L., Poales: Poaceae) being a major crop [2,3]. Maize is a staple crop in Ghana that can be cultivated in almost all ecological zones of the country [2,3]. However, agricultural production in Ghana is under threat from various pests, including the fall armyworm (Spodoptera frugi- perda, Lepidoptera: Noctuidae) [4,5]. Spodoptera frugiperda is an invasive pest, native to the Americas, that was first detected in Africa in early 2016 and has since spread rapidly across the continent, posing a serious threat to food security and agricultural livelihoods [6–8]. Infesting other staple crops like sorghum, millet, rice, and wheat, S. frugiperda is a voracious feeder that predominantly targets maize crops [9–11]. Clusters of eggs are laid by female moths, and the resulting larvae are extremely destructive [6,12]. The larvae of the pest feed on the foliage of host plants and possess the ability to tunnel into maize cobs, resulting in significant reductions in both grain quantity and quality [6,13]. The fast reproduction rate, preference for maize, and pesticide tolerance of S. frugiperda make it a challenging pest to manage [14,15]. Farmers have turned to synthetic insecticides for controlling the pest, which are hazardous to the environment and human health [4,16]. Studies have shown that the use of synthetic insecticides can lead to resistance development, environmental pollution, and health hazards [15,17]. As a result, there is a growing interest in alternative pest control methods such as botanicals, microbials, and insect growth regulator pesticides, which are considered safer for the environment and human health, and less likely to induce resistance [5,18–20]. In response to the urgent S. frugiperda infestation in Ghana, farmers were provided with insecticides, encompassing synthetic, botanical, and microbial formulations [21–23]. How- ever, it is important to highlight that these distributions took place without conducting prior efficacy testing or determining suitable application doses. Instead, the application doses were approximated based on those used for similar Lepidopteran pests affecting other crops [4,5]. However, it is important to note that variations in environmental conditions, biological fac- tors, and insecticide resistance can affect the sensitivity of pesticides [24,25]. Furthermore, to ensure the safety of non-target species and the effectiveness of the pesticides, bioassays should be conducted before approving new chemical formulations for use by farmers, as recom- mended by WHO [26]. Meanwhile, traditional approaches of bio-efficacy testing can be chal- lenging due to their narrow focus on individual mortality in a short term and the variation in the mode of action of active ingredients [27]. For instance, an insecticide may not induce acute mortality, but its sublethal toxicity can still significantly impact insect populations, including the development of resistance, disruption of behavior, and reduced reproductive success [27]. Thus, understanding the sublethal effects of insecticides is crucial for developing effective pest management strategies that minimize chronic impacts on non-target organisms and reduce the development of resistance in pest populations. On-station and/or field trials are vital to ensuring that the recommended doses of insecti- cides are the minimum necessary to control the target pests effectively, while minimizing non- targeted effects, such as phytotoxicity [28–30]. Additionally, on-station conditions typically represent more realistic conditions compared to laboratory studies, providing more reliable data for making management decisions [28,30]. Insects in the field are exposed to a range of environmental factors that can affect their behavior and susceptibility to insecticides [30]. In the present study, we hypothesized that not all the insecticide formulation in use or recom- mended to maize farmers for the management of S. frugiperda may demonstrate promising acute efficacy against the pest. Therefore, the primary objective of this study was to assess the efficacy of selected commercial pesticides against S. frugiperda in Ghana. The study was con- ducted in two phases, where lethal concentrations of selected commercial pesticides were determined under laboratory conditions, and the effects of sublethal concentration on the PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 2 / 21 Competing interests: The authors have declared that no competing interests exist. https://doi.org/10.1371/journal.pone.0290390 bionomics, reproduction parameters, and longevity of S. frugiperda were investigated. In the second phase, the effectiveness of selected commercial pesticides in reducing the infestation of S. frugiperda and increasing maize grain yield was evaluated under field conditions. Understanding the sublethal effects of insecticides is crucial for developing effective pest management strategies that minimize the impact on non-target organisms and reduce the development of resistance in pest populations. The findings of this study will provide valuable insights into the efficacy of selected commercial pesticides and their impact on S. frugiperda, contributing to the development of more effective and sustainable pest management strategies for farmers in Ghana. Materials and methods Ethics statement This study was conducted outside of national parks or any protected areas. The crop used in the study, maize (Zea mays L.), and the invasive insect pest, the fall armyworm (Spodoptera frugiperda, Lepidoptera: Noctuidae), are not considered endangered or protected species. Laboratory studies Experimental insect and plant. We obtained test-insects from a colony of S. frugiperda at the Laboratory of Entomology of SIREC. The colony was established in 2017 by collecting egg masses and larvae from insecticide-free maize fields across Ghana during a national inventory survey of the natural enemies of the pest. To rear the insects, we followed the method described by [12] and fed the larvae fresh castor bean leaves and adults a 10% honey solution. We tested the insects under conditions of 27 ± 1˚C temperature, 60 ± 5% RH, and 12 hours of photo- phase. To conduct the larvicidal bioassays, we planted "QPM var. Obatanpa" maize in plastic pots (8 cm diameter × 7.5 cm high) containing 0.5 L planting substrate (manure:soil 1:5). Insecticides. Seven commercial insecticides obtained from different sources were tested in vitro on the larvae of S. frugiperda (Table 1). The insecticide formulations included: Synthet- ics, Strike 1.9 EC™ (19.2 g/L of emamectin benzoate), K-Optimal 35 EC1 (20g/L acetamiprid + 15g/L lambda-cyhalothrin), Viper 46 EC1 (16g/L acetamiprid + 30g/L indoxacarb); Botani- cals, Adepa 5 SC1 (5% ethyl palmitate), Neemgold 0.3 SC1 (3% azadirachtin); and microbials Agoo 55 WP1 (55%Bacillus thuringiensis kurstaki + 45% monosultap), and Bypel 5 WP1 [10000 PIB/mg Peris rapae Granulosis Virus + 16000 IU/mg Bacillus thuringiensis kurstaki). Table 1. Details on insecticides assessed and the concentration ranges used. Trade name Common namea Manufacture Conc. Recommended No. of tested conc.b Conc. Rangesc Strike 1.9 EC™ 19.2 g/L Emamectin benzoate B. Kaakyire Agrochemicals 1 mL/L 5 0.001–10 Viper 46 EC1 16 g/L Acetamiprid + 30 g/L Indoxacarb Arysta Life Science Ltd. 3 mL/L 5 0.03–24 K-Optimal 35 EC1 20 g/L Acetamiprid + 15 g/L ʎ-cyhalothrin Macrofertil Gh. Ltd. 3.3 mL/L 6 1.67–40 Adepa 5 SC1 5% Ethyl palmitate Kwadutsa and Joam Co. Ltd. 6.7 mL/L 7 3.33–1000 NeemGold 0.3 SC1 3% Azadirachtin Foliage Crop Solutions Ltd. 2 mL/L 6 0.50–54 Bypel 1 WP1 10,000 PIB/mg PrGV + 16,000 IU/mg Btk Wuhan UNIOASIS BioTech Co. Ltd. 1000 mg/L 6 125–4000 Agoo 55WP1 55% Btk + 45% Monosultap Kwadutsa and Joam Co. Ltd. 3333 mg/L 5 863–13333 a Pieris rapae Granulosis Virus (PrGV); PIB: Polyhedra Infective Bodies; Btk: Bacillus thuringiensis subsp. kurstaki; IU: International Units. b Number of concentrations tested (expressed as either w/v or v/v of product per liter of water). c Range of concentrations tested for each formulated insecticide product. Conc.: Concentration of product. No.: Number. https://doi.org/10.1371/journal.pone.0290390.t001 PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 3 / 21 https://doi.org/10.1371/journal.pone.0290390.t001 https://doi.org/10.1371/journal.pone.0290390 Concentrations (w/v or v/v) of the product per liter of water were prepared using distilled water. The baseline concentrations of each insecticide were determined following a prelimi- nary test by exposing batches of 25 third instar larvae per a wide range of concentrations in leaf-dip assay and monitored for 72 h. The minimum lethal concentration (LC5), the maxi- mum lethal concentration (LC95), and the median lethal concentrations (LC30 and LC70) were chosen to establish the number and range of concentrations for each insecticide [26] (Table 1), with the manufacturer-recommended concentrations included in the tests. The concentrations were prepared using serial dilution method [31]. Larvicidal bioassays. The lethal concentrations of each formulation were determined using a leaf-dip method with fresh leaves of potted maize plants aged 14 to 18 days. The foliage of maize plant was briefly dipped into each test dilution, along with a control of distilled water, and air-dried for 40 minutes at room temperature. The poisoned maize leaves were then cut into pieces (2.5 cm) and placed in plastic cups (7.3 × 7.3 × 6.0 cm). Batches of 10 early third instar larvae of S. frugiperda were individually confined with insecticide-treated leaves, and their death time was monitored up to 96 hours. This constituted the biological replicates and replicated with 8–10 batches of larvae per concentration. Experiments were conducted at 27 ± 1˚C, 60 ± 5% RH, and 12:12 h (L:D) photoperiod. When control mortality exceeded 10%, data were corrected using the following Schneider-Orelli’s formula [32]: Corrected mortality pð Þ ¼ %Responded � %Responded in Control 100 � Responded% in Control Sublethal effects on the bionomics of F0 parent generation of S. frugiperda. To evaluate the effects of sublethal insecticide concentrations on S. frugiperda progeny reproduction, maize plants were impregnated with LC25 concentrations in distilled water using the same method as the larvicidal bioassays. A control group was treated with distilled water only. Chopped, treated leaves were placed in plastic cups, and third-instar larvae of S. frugiperda were individually placed in each cup to feed on either untreated or insecticide-treated maize leaves. After 48 hours of feeding, the insecticide-treated leaves were replaced daily with untreated leaves for the surviving larvae until they emerged as adults. The F0 generation moths were then coupled in a 1:1 sex ratio, with five couples being placed in an oviposition cage (40 × 40 × 55 cm) along with individually potted maize plants. The longevity of the pupae and adults (days), as well as fecundity (i.e., number of eggs produced by individual female insect), were meticulously recorded. The experiment was conducted under controlled environmental conditions of 27 ± 1˚C, 60 ± 5% RH, and a 12:12 h (L:D) photoperiod. Sublethal effects on the bionomics of F1 generation of S. frugiperda. Ten individual eggs laid by insecticide-survived parents (F0 generation) from the same treatment plant were gently placed on untreated potted maize plant leaves using a tiny brush pen. Each insecticide treatment was replicated 7–9 times, resulting in 7–9×10 eggs per treatment. Upon hatching, the larvae were fed with untreated maize leaves in separated cups until adult emergence. Ten adult moths (1:1 sex ratio) from the same treatment were independently paired on maize plants (in 20 × 20 × 35 cm Plexiglass cages), and the daily oviposition was monitored and recorded by changing the maize plants until the insects died. The duration of each develop- mental stage was recorded. On-station experiments This research was conducted at the University of Ghana’s Soil and Irrigation Research Centre, (SIREC) at Kpong (00 04’ E, 60 09’ N), in the Lower Manya District of the Eastern Region of PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 4 / 21 https://doi.org/10.1371/journal.pone.0290390 Ghana. SIREC, is located approximately 22 m above sea level and lies within the lower Volta basin of the Coastal Savanna agro-ecological zone of Ghana. It is characterized by an annual rainfall of 700–1100 mm and an average annual temperature of 28˚C. The relative humidity (RH) ranged between 59%-93% throughout the year. The main soil type was the Vertisols (black clay soil). Two consecutive on-station experiments were carried out to assess the effec- tiveness of four insecticides, namely Strike 1.9 EC™ (emamectin benzoate), Bypel 1 WP1 (PrGV + Btk), Agoo 55WP1 (Btk + monosultap), and NeemGold 0.3 SC1 (azadirachtin). The selection of these insecticides was based on their larvicidal potency (see Table 1) and also to represent each of the insecticide classes, including synthetic, microbial, and botanical. The first trial took place from September 3, 2018 to December 26, 2018 during the minor rainy season; the second trial was conducted under surface irrigation and was conducted from January 14, 2019 to April 27, 2019 during the dry season. Plant material. Maize variety “QPM Obatanpa var.” was used for the field trials and arranged in a randomized complete block design (RCBD) with four replicates. Each block had 5-unit plots (10 m × 8 m) representing the single factor, insecticide treatments. Two seeds were sown per hill with an interplant distance of 80 cm between rows and 40 cm between plants. The alley between neighboring plots were 2.5 m to avoid spray drift. A complex fertil- izer (N15P15K15) was applied at 250 kg/ha and supplied with 100 kg N/ha in the form of ammo- nium nitrate. Standard cropping and agronomic practices were carried out, including chemical control of weeds using selective herbicide 2,4-Dichlorophenoxyacetic acid (2,4 D). Handpicking of resistant weed and alternative host of the pest, mainly crabgrass (Digitaria sp.) was performed during both seasons. During the dry season, surface irrigation method was employed to provide 7-day intervals water to the plants. Insecticide preparation, application and data collection. In both trials, recommended doses were used: Strike 1.9 EC™ (emamectin benzoate) at 1 mL per 1 L of water (150 mL / ha), Bypel 1 WP1 (PrGV + Btk) at 1 g per 1 L of water (200g / ha), Agoo 55WP1 (Btk + monosul- tap) at 3.3 g per L of water (400g / ha), and NeemGold 0.3 SC1 (azadirachtin) at 2 mL per 1 L of water (400 mL / ha). Control plots received no insecticide. A 15 L knapsack sprayer with a full-cone nozzle was used for application, calibrated before use to ensure proper flow rate and insecticide dose. Applications were made late afternoon (4–6 pm) beginning 10 days after ger- mination to reduce photodegradation and minimize side effects on pollinators [33]. The num- ber of insecticide applications was decided based on the infestation level determined by visual scouting [12], resulting in two and three treatments during the first and second trials, respectively. Data collected included alive larvae per plant (larval incidence) and maize grain yield. Ten plants per plot were sampled, and larval incidence data were collected 1 day before and 3, and 7 days after treatment. Yield per hectare was estimated by multiplying the yield per 10 plants per plot by theoretical plant density per hectare. Statistical analyses The laboratory data were analyzed using Finney’s Probit Analysis Programme [34] to deter- mine lethal concentrations, fiducial limits (95%), slopes, and Chi-square values. The popula- tion and reproduction parameters were calculated using TWOSEX-MSChart 2023 software [35,36], with the standard errors determined by bootstrapping with 100,000 repetitions. Paired bootstrapping was used to evaluate differences between groups (P< 0.05). The data were checked for normality using Shapiro–Wilk test and equal variances using Levene test before selecting the appropriate statistical analyses. Thus, the analysis of larval mortality induced by the concentrations recommended by the manufacturers was conducted using the Likelihood PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 5 / 21 https://doi.org/10.1371/journal.pone.0290390 ratio test (LR test) applied to a generalized linear model (GLM). Similarly, pupal duration, mortality (between pupa and adult stages), adult longevity, and fecundity were analyzed using the LR test with GLM. The percent population reduction resulting from insecticide application was determined using the following formula: R ¼ ðPreTP � PostTPÞ PreTP � 100 Here, R represents the percent population reduction, while PreTP and PostTP represent the population densities before and after insecticide spray per treatment plot, respectively. The cal- culated R values were subjected to statistical analysis using a one-way analysis of variance (ANOVA). In addition, to analyze the differences between the control treatment (no insecti- cide) and the other treatments (insecticide receiver plots), the Dunnett (two-sided) test was employed [37]. This analysis was conducted with a confidence interval of 95%. Similarly, yield data were analyzed using one-way ANOVA. The means were separated using Tukey’s Range Test (α = 0.05). Except for the population and reproduction characteristics, all data were ana- lyzed in R [38]. Results Larvicidal potency of insecticides on S. frugiperda larvae mortality in laboratory bioassay Table 2 (toxicity bioassay results) showed LC50 values at 96 HAT (hours after treatment) rang- ing from 0.019 mL/L Strike 1.9 EC™ (emamectin benzoate) to 108.5 mL/L Adepa 5 SC1 (ethyl palmitate) with slope values below 1 observed for emamectin benzoate and ethyl palmitate. According to the findings presented in Tables 1 and 2, the LC50 values for emamectin benzo- ate, Viper 46 EC1 (acetamiprid + indoxacarb), Bypel 1 WP1 (PrGV + Btk), and Agoo 55WP1 (Btk + monosultap) were lower than the dosages recommended by the manufacturer. In contrast, the LC50 of K-Optimal 35 EC1 (acetamiprid + γ-cyhalothrin), Adepa 5 SC1 (ethyl palmitate), and NeemGold 0.3 SC1 (azadirachtin) were higher than the recommended dose (Tables 1 and 2). However, it is noteworthy that the calculated LC90 values for all of the insecticides tested were higher than the manufacturer-recommended dosages (Tables 1 and 2). Table 2. Dose-mortality responses of S. frugiperda larvae to different insecticides at 96 HAT. Insecticides N LC10 (95% F.L.) LC50 (95% F.L.) LC90 (95% F.L.) Slope ± SE df χ2 Emamectin benzoate 575 0.0000587 (1.03E-05–3.34E-04) 0.019 (0.003–0.108) 6.09 (1.07–34.69) 0.513±0.385 4 0.8 Acetamiprid + Indoxacarb 585 0.17 (0.08–0.35) 1.78 (0.86–3.65) 18.445 (8.99–37.82) 1.274±0.159 4 0.49 Acetamiprid + ʎ-Cyhalothrin 665 1.41 (0.89–2.23) 7.39 (4.66–11.71) 38.69 (24.40–61.34) 1.786±0.102 5 0.9 Ethyl palmitate 740 2.83 (1.13–7.10) 108.51 (43.28–272.03) 4152.48 (1656.48–10410.36) 0.819±0.204 6 0.42 Azadirachtin 700 0.48 (0.25–0.93) 4.73 (2.48–9.04) 45.92 (24.05–87.68) 1.304±0.143 5 0.89 PrGV + Btk 650 51.06 (31.20–83.57) 257.46 (157.32–421.34) 1298.81 (793.20–2124.33) 1.828±0.109 5 0.97 Btk + Monosultap 650 706.94 (466.93–1070.33) 2766.16 (1827.02–1827.02) 10823.52 (7148.82–16387.13) 2.255±0.092 4 0.32 N: Number of larvae tested. F.L.: Fiducial Limits. LC: Lethal concentration 10% (LC10), lethal concentration 50% (LC50), lethal concentration 90% (LC90). https://doi.org/10.1371/journal.pone.0290390.t002 PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 6 / 21 https://doi.org/10.1371/journal.pone.0290390.t002 https://doi.org/10.1371/journal.pone.0290390 Mortality rates were compared for different insecticide formulations using their recom- mended concentration at various time intervals (Fig 1). Emamectin benzoate had significantly higher mortality rates than other formulations at 24 HAT (LR test with GLM, χ2 = 426.85, P< 0.0001; Fig 1A). Emamectin benzoate and acetamiprid + indoxacarb had significantly higher mortality rates at 48 HAT (LR with GLM, χ2 = 952.05, P< 0.0001; Fig 1B). Emamectin benzoate, acetamiprid + indoxacarb, and PrGV + Btk had the highest mortality rates at 72 HAT (LR with GLM, χ2 = 860.78, P< 0.0001; Fig 1C). PrGV + Btk and emamectin benzoate had the highest mortality rates at 96 HAT (LR with GLM, χ2 = 915.96, P< 0.0001; Fig 1D). Emamectin benzoate and acetamiprid + indoxacarb had the highest mortality rates between 24–72 HAT, while PrGV + Btk and Btk + monosultap had a progressive effect, with a signifi- cantly higher mortality rate at 96 HAT (LR with GLM, χ2 = 915.96, P< 0.0001; Fig 1). Sublethal effects on F0 generation parent bionomics Pupal duration, pupal mortality, adult longevity, and female fecundity of the F0 generation of S. frugiperda were all affected by the sublethal concentration of the tested insecticide formula- tions (LR with GLM, P< 0.0001; Table 3). For instance, pupal duration was significantly Fig 1. Effects of manufacture recommended concentrations on the mortality rate of Spodoptera frugiperda. Bars represent the means ± SE larval mortality at (a) 24 hours after treatment (HAT), (b) 48 HAT, (c) 72 HAT, and (d) 96 HAT. Different letters above bars indicate significant differences between treatments (Likelihood ratio test (LR test) applied to a generalized lineal model (GLM), followed by Tukey’s honest significant difference (HSD) at P< 0.05). https://doi.org/10.1371/journal.pone.0290390.g001 PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 7 / 21 https://doi.org/10.1371/journal.pone.0290390.g001 https://doi.org/10.1371/journal.pone.0290390 lengthened by PrGV + Btk and ethyl palmitate, while shortened by azadirachtin, acetamiprid + ʎ-cyhalothrin, acetamiprid + indoxacarb, and emamectin benzoate, compared to the control treatment (LR with GLM, P< 0.0001; Table 3). The lowest fecundity was recorded with PrGV + Btk, acetamiprid + indoxacarb, and ethyl palmitate treatments (LR with GLM, P< 0.0001; Table 3). Transgenerational sublethal effects on S. frugiperda bionomics Stage duration of F1 generation. Compared to the control treatment, insecticides signifi- cantly reduced the stage length of F1 generation of S. frugiperda (paired bootstrap test, P< 0.05; Table 4; S1–S7 Tables). Azadirachtin (S2 Table) and ethyl palmitate (S3 Table) lengthened the egg stage, but PrGV + Btk (S4 Table) shorten it compared to the control treat- ment (paired bootstrap test, P< 0.05; Table 4; S1–S4 Tables). Azadirachtin (S2 Table) and ethyl palmitate (S3 Table) lengthened the larval stage, but PrGV + Btk (S4 Table), emamectin benzoate (S5 Table), and acetamiprid + indoxacarb (S6 Table) significantly shortened it (paired bootstrap test, P< 0.05; Table 4; S2–S6 Table). Emamectin benzoate (S5 Table) and acetamiprid + indoxacarb (S6 Table) had the shortest preadult stage, whereas azadirachtin Table 3. Effect of sublethal concentrations on parent adults (F0 generation) of Spodoptera frugiperda (Mean ± SE). Insecticide Pupal duration (day) % Mortality (Pupa–Adult) Male longevity (day) Female longevity (day) Fecundity Emamectin benzoate 5.82±0.20c 27.78±6.15ab 8.40±0.47d 8.95±0.51c 238.00±13.65bc Acetamiprid + Indoxacarb 6.02±0.20c 26.79±5.97ab 9.86±0.46bc 11.90±0.50ab 150.50±09.27cd Acetamiprid + ʎ-Cyhalothrin 5.82±0.20c 26.42±6.11ab 9.19±0.46cd 10.00±0.53bc 248.37±32.76b Ethyl palmitate 9.45±0.19a 34.33±5.84b 9.67±0.46bcd 8.87±0.47c 187.12±12.88bcd Azadirachtin 6.37±0.23c 30.00±6.55ab 9.83±0.50bc 10.47±0.54bc 233.75±22.99bc PrGV + Btk 9.36±0.19a 27.87±5.79ab 8.91±0.44cd 8.67±0.49c 115.75±05.32d Btk + Monosultap 7.21±0.21b 32.26±5.99b 10.94±0.50b 10.54±0.46bc 259.25±21.54b Control 7.44±0.18b 8.06±3.49a 12.85±0.41a 13.26±0.41a 580.87±34.38a χ2 89.6 23.37 31.64 42.08 43.55 df 7 7 7 7 7 P< 0.05 < 0.0001 0.0014 < 0.0001 < 0.0001 < 0.0001 Different letters within the same column represent significant differences at P < 0.05 (Likelihood Ratio test (LR) applied to a generalized linear model (GLM), followed by Tukey’s HSD, test). Fecundity: Means number of eggs laid by individual female insect. https://doi.org/10.1371/journal.pone.0290390.t003 Table 4. Response of duration (in day) of developmental stages of Spodoptera frugiperda to sublethal insecticide concentrations (Mean ± SE). Insecticide Egg Larva Pupa Preadult Male adult Female adult Emamectin benzoate 2.83±0.07bcd 10.35±0.22d 5.76±0.11f 19.31±0.25e 9.90±0.26cd 10.57±0.27c Acetamiprid + Indoxacarb 2.80±0.08cd 10.35±0.21d 5.94±0.11f 19.11±0.30e 12.09±0.34b 12.97±6.16a Acetamiprid + ʎ-Cyhalothrin 2.88±0.08bcd 11.10±0.26c 6.32±0.13e 20.45±0.32d 10.62±0.31c 11.00±0.35c Ethyl palmitate 3.28±0.10a 15.31±0.40a 7.45±1.27b 26.41±0.45a 7.00±0.15e 7.15±0.18e Azadirachtin 3.04±0.08a 15.08±0.37a 6.80±0.19c 25.10±0.42b 7.23±0.32e 7.45±0.33e PrGV + Btk 2.67±0.08d 10.41±0.15d 8.77±0.17a 21.97±0.24c 9.97±0.35c 10.33±0.37d Btk + Monosultap 2.74±0.09d 11.19±0.23c 6.62±0.14d 20.57±0.32d 12.48±0.49a 12.68±0.43ab Control 3.00±0.07b 13.96±0.10b 7.22±0.12b 24.24±0.16b 13.31±0.27a 13.49±0.35a The data in the table are mean (days) ± SE. Different superscript letters indicate significant difference P < 0.05 (paired bootstrap test with TWOSEX-MSChart 2023 software [35]). Consult S1–S8 Tables for an in-depth view of the data that informed these analyses. https://doi.org/10.1371/journal.pone.0290390.t004 PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 8 / 21 https://doi.org/10.1371/journal.pone.0290390.t003 https://doi.org/10.1371/journal.pone.0290390.t004 https://doi.org/10.1371/journal.pone.0290390 (S2 Table) and ethyl palmitate (S3 Table) had the longest (paired bootstrap test, P< 0.05; Table 4). Azadirachtin, ethyl palmitate, PrGV + Btk, and emamectin benzoate treatments had the shortest adult stages (paired bootstrap test, P< 0.05; Table 4; S2–S5 Tables). Reproduction parameters of F1 generation. The insecticide treatments did not signifi- cantly affect the fecundity of F1 generation of S. frugiperda (paired bootstrap test, P> 0.05; Table 5; S1–S8 Tables). However, they had a significant impact on the total pre-oviposition period (TPOP), with and azadirachtin (S2 Table) and ethyl palmitate (S3 Table) having the longest TPOP, and emamectin benzoate (S5 Table) and acetamiprid + indoxacarb (S6 Table) having the shortest (paired bootstrap test, P< 0.05; Table 5; S2–S6 Tables). Additionally, the number of oviposition days was significantly reduced by ethyl palmitate (S3 Table), emamectin benzoate (S5 Table), acetamiprid + indoxacarb (S6 Table), acetamiprid + ʎ-cyhalothrin (S7 Table) (paired bootstrap test, P< 0.05; Table 5; S3–S7 Tables). PrGV + Btk (S4 Table), ema- mectin benzoate (S5 Table), acetamiprid + indoxacarb (S6 Table), and acetamiprid + ʎ-cyhalo- thrin (S7 Table) had a significantly negative effect on the longevity of S. frugiperda compared to other treatments (paired bootstrap test, P< 0.05; Table 5; S4–S7 Tables). Population growth parameters of Spodoptera frugiperda at F1 generation. The popula- tion growth parameters of F1 generation were significantly affected by the insecticide treat- ments, with azadirachtin (S2 Table) and ethyl palmitate (S3 Table) having negative effect on the Intrinsic Rate of Increase (r) of S. frugiperda (paired bootstrap test, P< 0.05; Table 6; Table 5. Effect of sublethal insecticide concentrations on reproduction parameters of Spodoptera frugiperda (Mean ± SE). Insecticide Fecundity TPOP (day) Oviposition days Mean Longevity Emamectin benzoate 237.70±09.19a 19.53±0.41e 5.16±0.14d 25.25±0.94g Acetamiprid + Indoxacarb 276.97±10.23a 19.75±0.44e 5.27±0.17d 27.11±1.04de Acetamiprid + ʎ-Cyhalothrin 295.19±10.65a 21.46±0.43cd 5.11±0.14d 26.66±0.96def Ethyl palmitate 232.50±08.29a 26.56±5.06a 5.06±0.11d 30.44±0.91b Azadirachtin 302.55±16.67a 25.21±0.60a 5.86±0.22c 28.75±0.94bc PrGV + Btk 350.43±17.95a 22.37±0.32c 6.67±0.29b 26.37±1.14cd Btk + Monosultap 407.61±14.61a 21.61±0.44c 7.68±0.21a 28.81±1.08b Control 428.82±12.69a 24.69±0.22b 7.05±0.13b 33.36±1.21a TPOP: Total pre-oviposition period. The data in the table are mean values ± SE. Different superscript letters indicate significant difference P < 0.05 (paired bootstrap test with TWOSEX-MSChart 2023 software [35]). Consult S1–S8 Tables for an in-depth view of the data that informed these analyses. https://doi.org/10.1371/journal.pone.0290390.t005 Table 6. Effect of sublethal insecticide concentrations on the population parameters of F1 generation of Spodoptera frugiperda (Mean ± SE). Insecticide Intrinsic Rate of Increase (r in days) Finite Rate of Increase (λ in days) Net Reproductive Rate (R0) Mean Generation Time (T in days) Emamectin benzoate 0.20±0.008abc 1.22±0.010abc 81.03±12.39e 21.88±0.42e Acetamiprid + Indoxacarb 0.21±0.008a 1.24±0.010a 108.80±15.28cde 21.73±0.49e Acetamiprid + ʎ-Cyhalothrin 0.20±0.007ab 1.22±0.009ab 117.44±15.57bcd 23.40±0.45d Ethyl palmitate 0.16±0.006g 1.18±0.008g 101.34±13.54cde 28.14±0.71a Azadirachtin 0.18±0.007def 1.20±0.008def 132.15±17.75b 26.81±0.67ab PrGV + Btk 0.19± 0.006bcde 1.21±0.008bcde 128.20±19.77bc 25.15±0.34c Btk + Monosultap 0.20±0.006a 1.23±0.009a 164.10±23.58a 24.62±0.38c Control 0.19±0.004bcd 1.21±0.005bcd 209.05±24.70a 28.71±0.21a The data in the table are mean values ± SE. Different superscript letters indicate significant difference P< 0.05 (paired bootstrap test with TWOSEX-MSChart 2023 software [35]). Consult S1–S8 Tables for an in-depth view of the data that informed these analyses. https://doi.org/10.1371/journal.pone.0290390.t006 PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 9 / 21 https://doi.org/10.1371/journal.pone.0290390.t005 https://doi.org/10.1371/journal.pone.0290390.t006 https://doi.org/10.1371/journal.pone.0290390 S2 and S3 Tables). These insecticides similarly affected the Finite Rate of Increase (ʎ) of S. fru- giperda, resulting in lowest values (paired bootstrap test, P< 0.05; Table 6; S2 and S3 Tables). The Net Reproductive Rate (Ro) of S. frugiperda was negatively significantly affected by ethyl palmitate (S3 Table), emamectin benzoate (S5 Table), acetamiprid + indoxacarb (S6 Table) (paired bootstrap test, P< 0.05; Table 6; S3–S6 Tables). The Mean Generation Time (T) was significantly reduced by PrGV + Btk (S4 Table), emamectin benzoate (S5 Table), acetamiprid + indoxacarb (S6 Table), acetamiprid + ʎ-cyhalothrin (S7 Table), and Btk + monosultap (S8 Table) (paired bootstrap test, P< 0.05; Table 6; S4–S8 Tables). Age-stage survival of Spodoptera frugiperda at F1 generation. Age-stage specific sur- vival rate Sxj is the expected duration of neonate nymphs that will survive to age x and stage j. Fig 2 displays the effects of sublethal concentrations on Sxj of F1 generation of S. frugiperda, with no significant differences observed between the treatments (paired bootstrap test, P> 0.05; S1–S8 Tables). However, compared to the control treatment (0.85), the probability of neonate larvae reaching the adult stage was lower for all other treatments, with the lowest probability observed for PrGV + Btk (0.73) (paired bootstrap test, P> 0.05; S1–S8 Tables). On-station experiments Minor rainy season Throughout the minor rainy season experiment, the treatments had significant effect on the reduction of S. frugiperda larvae population per maize plant (One-way ANOVA, N = 10, df = 4, P< 0.05; Fig 3A–3D; S9 Table). The initial insecticidal spray administered 15 days after planting demonstrated higher population reduction with emamectin benzoate and PrGV + Btk treatments compared to other treatment groups, observed 3 days after the insecticide treat- ment (DAT) (One-way ANOVA, N = 10, df = 4, F = 8.56, P< 0.001; Fig 3A; S9 Table). Com- pared to the control treatment, emamectin benzoate treatment plot had significantly higher population reduction of 70.04% (Dunnet’s test, P< 0.001; Fig 3A; S9 Table). Similarly, 7 DAT, significant difference was observed between the treatment plots regarding the number of alive S. frugiperda larvae (One-way ANOVA, N = 10, df = 4, F = 19.82, P< 0.0001; Fig 3B; S9 Table). As a result, the population reductions on the emamectin benzoate (72.96%) and PrGV + Btk (71.42%) treatment plots were significantly higher compared to the control plots (Dun- net’s test, P< 0.001; Fig 3B; S9 Table). After the second spray at 3 DAT, none of the insecticide treatments showed a significant difference compared to the control treatment based on Dunnett’s test (P> 0.05; Fig 3C; S9 Table). However, at 7 DAT, significantly higher population reduction rates of 77.29% for the emamectin benzoate treatment and 66.62% for the PrGV + Btk treatment were recorded, com- pared to the control treatment (Dunnett’s test, P< 0.01; Fig 3D; S9 Table). Dry season. During the dry season experiment, a significant difference was observed among the populations of alive larvae of S. frugiperda following insecticide sprays (Fig 4; S10 Table). After the first spray, at 3 DAT, the emamectin benzoate treatment plots demonstrated the highest reduction in the population of S. frugiperda larvae (One-way ANOVA, N = 10, df = 4, F = 14.64, P< 0.0001; Fig 4A; S10 Table), i.e., 58.33% higher than on control plots (Dunnett’s test, P< 0.0001; Fig 4A; S10 Table). Furthermore, at 7 DAT, there were signifi- cantly higher population reductions on the emamectin benzoate (60.54%) and PrGV + Btk (48.35%) treatment plots compared to the control plots., as determined by Dunnett’s test (P< 0.01; Fig 4B; S10 Table). Following the second spray, at 3 DAT, there was no significant difference observed between the treatments in terms of S. frugiperda larvae populations (One-way ANOVA, N = 10, df = 4, F = 0.59, P> 0.05; Fig 4C; S10 Table). However, at 7 DAT, the highest population reduction PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 10 / 21 https://doi.org/10.1371/journal.pone.0290390 rates of 46.10% and 44.49% were recorded in the emamectin benzoate and PrGV + Btk treat- ment plots (One-way ANOVA, N = 10, df = 4, F = 5.84, P< 0.05; Fig 4D; S10 Table), and sig- nificantly different from the control treatment (Dunnett’s test, P< 0.05; Fig 4D; S10 Table). On the third day following the third application of insecticide, significant decrease in the population of S. frugiperda larvae was observed in the plots treated with emamectin benzoate and PrGV + Btk, in comparison to other treatments (One-way ANOVA, N = 10, df = 4, F = 3.30, P< 0.05; Fig 4E; S10 Table). Seventh day following the third insecticide spray, the most significant reduction (77.29%) in population of S. frugiperda larvae was recorded on PrGV + Btk treated plots compared to the other treatments (One-way ANOVA, N = 10, df = 4, F = 6.62, P< 0.01; Fig 4F; S10 Table). In general, only the emamectin benzoate (60.71%) and Fig 2. Age-stage survival rates (Sxj) of Spodoptera frugiperda on maize leaves treated with sublethal concentration LC25 and control (untreated). Sxj is the probability that a newborn egg will survive to age x and stage j. The data evaluation was performed using the paired bootstrap test via the TWOSEX-MSChart 2023 software [35]. Consult S1–S8 Tables for an in-depth view of the data that informed these analyses. https://doi.org/10.1371/journal.pone.0290390.g002 PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 11 / 21 https://doi.org/10.1371/journal.pone.0290390.g002 https://doi.org/10.1371/journal.pone.0290390 Fig 3. Population reduction of Spodoptera frugiperda larvae during the minor rainy season on-station trial. Two insecticide spray events were conducted: The first spray on September 15th, 2018 (A, B), and the second spray on October 4th, 2018 (C, D). The bars in the figure represent the mean population reduction, and the asterisks (*P< 0.05; **P< 0.01 and ***P< 0.001) above the bars indicate the significant difference between the insecticide-treated plots and the control using One-way ANOVA and Dunnett’s Test (α = 0.05). The term "pop." is an abbreviation for the population of live S. frugiperda larvae, while "DAT" indicates the count of days post-treatment. Refer to S9 Table for a detailed breakdown of the data. https://doi.org/10.1371/journal.pone.0290390.g003 PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 12 / 21 https://doi.org/10.1371/journal.pone.0290390.g003 https://doi.org/10.1371/journal.pone.0290390 Fig 4. Population reduction of Spodoptera frugiperda larvae during the dry season on-station trial. Three insecticide spray events were conducted: The first spray on January 29th, 2019 (A, B), the second spray on February 13th, 2019 (C, D), and the third spray on February 27th, 2019 (E, F). The bars in the figure represent the mean population reduction, and the asterisks (*P< 0.05; **P< 0.01 and ***P< 0.001) above the bars indicate the significant difference between the insecticide-treated plots and the control using One-way ANOVA and Dunnett’s Test (α = 0.05). The term "pop." is an abbreviation for the population of live S. frugiperda larvae, while "DAT" indicates the count of days post-treatment. Refer to S10 Table for a detailed breakdown of the data. https://doi.org/10.1371/journal.pone.0290390.g004 PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 13 / 21 https://doi.org/10.1371/journal.pone.0290390.g004 https://doi.org/10.1371/journal.pone.0290390 PrGV + Btk treatments showed significantly higher population reduction compared to the control treatment at both 3 and 7 DAT (Dunnett’s test, P< 0.05; Fig 4E and 4F; S10 Table). Dry grain yield of maize during on-station experiments. The study found that applying insecticide treatments had a positive impact on maize grain yield (Fig 5). During the minor rainy season, there was a significant difference in grain yields among the treatments. Maize plants treated with PrGV + Btk and emamectin benzoate had the highest yields, while the low- est yields were recorded on Btk + monosultap plots (One-way ANOVA, df = 4, F = 70.12, P< 0.0001; Fig 5A; S11 Table). Similarly, during the dry season, PrGV + Btk and emamectin benzoate significantly produced the highest maize grain yields (One-way ANOVA, df = 4, F = 59.97, P< 0.0001; Fig 5B; S11 Table). However, the azadirachtin treatment had the lowest yields among the insecticide-treated maize plants (One-way ANOVA, df = 4, F = 59.97, P< 0.0001; Fig 5B; S11 Table). Discussion The study conducted toxicity bioassays on Spodoptera frugiperda larvae using various insecti- cide classes, including synthetic, botanical, and microbial insecticides. The results showed that emamectin benzoate had the highest larvicidal potency, whereas ethyl palmitate had the least. Previous research found substantial larval mortality in Lepidoptera larvae, especially those fed insecticide-treated diets [39–42]. Emamectin benzoate showed high toxicity to Lepidopterans such as the diamondback moth (Plutella xylostella, Plutellidae), the tomato leafminer (Phthori- maea absoluta, Gelechiidae), and Spodoptera sp. [31,41–44]. For instance, [31] and [41] found 0.0051 mg/L (expressed in ppm) and 0.0023–3.303 mg/L as LC50 for emamectin benzoate on first and second early instar larvae of S. frugiperda in a leaf-dip bioassay, respectively, while [40] found 0.0014 mg/L for neonate larvae of S. littoralis at 48 h post-exposure. Though some- what different, mean LC50 for emamectin benzoate (0.019 mg/L) in our study is within the range established by [31]. These discrepancies in results may be attributed to variations in bio- logical resources, species, age, methods, and exposure time. Nevertheless, our findings, along with other studies, support the notion that emamectin benzoate is a promising candidate for managing Lepidopteran pests, particularly defoliators like S. frugiperda [5,41]. Notably, the LC50 values of emamectin benzoate, acetamiprid + indoxacarb, PrGV + Btk, and Btk + mono- sultap in our study were lower than the manufacturer-recommended dosages, indicating that these insecticides could be effective in controlling S. frugiperda under field conditions using the concentrations suggested by the manufacturers. Comparing the manufacture recommended concentrations of the insecticides, we found that effectiveness of the binary microbial PrGV + Btk was slow in action compared to emamec- tin benzoate, and acetamiprid + indoxacarb but became more effective overtime, mainly at 96 HAT. Yet, little evidences exist from previous studies on the toxicity of PrGV against S. frugi- perda, while, its companion compound Btk toxin has been studied and used against a wide range of Lepidopteran species, including S. frugiperda [45–47]. Studies have, however been reported about the insecticidal activity of PrGV on Pieris rapae Linnaeus (Lepidoptera: Pieri- dae) in vitro [48]. However, since natural entomopathogenic viruses utilized as biological con- trol agents are species-specific, narrow-spectrum insecticides, their toxicity to other arthropod species (pests or beneficials) is essential. Meanwhile, natural entomopathogenic viruses have been shown to be an effective alternative to broad-spectrum insecticides [5,49,50]. Our study suggests that insecticidal efficacy testing should not only focus on determining lethal dosages but also include sublethal effects of formulations, especially with entomopatho- gens and botanicals. Our study found that PrGV + Btk, Btk + monosultap, as well as the botan- icals ethyl palmitate and azadirachtin, had significant sublethal effects on fecundity, reducing PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 14 / 21 https://doi.org/10.1371/journal.pone.0290390 the net reproductive rate of both the S. frugiperda parents (F0 generation) and their offspring (F1 generation). Although these formulations showed weaker potency compared to synthetic insecticides during the toxicity bioassays, their sublethal effects are noteworthy. This finding is consistent with previous research [51,52]. For instance, it is known that azadirachtin com- monly reduces fertility and offspring production in Lepidoptera adults [51]. In contrast, there is limited published research available on the insecticidal potency of ethyl palmitate as a stand- alone insecticide [53,54]. Ethyl palmitate has been used as a solvent or carrier for other insecti- cides and biopesticides, and there are a few studies investigating the efficacy of these formulations. Thus, our study constitutes a baseline for further investigations on the insecti- cidal role of ethyl palmitate and its mode of action. The efficacy of Btk + monosultap and azadirachtin in controlling the incidence of S. frugi- perda larvae showed inconsistency during the on-station experiments, unlike PrGV + Btk and emamectin benzoate treatments. This suggests that the efficacy of Btk + monosultap and aza- dirachtin may be influenced by environmental factors such as temperature, UV radiation, and humidity [55,56]. However, it is worth noting that previous studies have reported the efficacy of Btk against S. frugiperda under field conditions [5,57,58]. It is important to consider that most of these studies utilized genetically engineered Bt-maize, which may explain the discrep- ancy between our findings and the previously reported efficacy of Bt in controlling S. frugi- perda. Moreover, the field-evolved resistance of Lepidopteran pest species to Bt-based formulations [45,59]and the rapid development of resistance to monosultap in Lepidopteran species [60,61] can also explain the limited efficacy of Btk + monosultap against S. frugiperda. Furthermore, considering the LC50 values, it is worth noting that they were higher than the recommended dosages applied for the Btk + monosultap and azadirachtin treatments in field conditions. Therefore, it is advisable to utilize higher dosages of these formulations to achieve Fig 5. Grain yield of maize during minor and dry season on-station experiments. Bars represent the means ± SE of dry grain yields recorded on four plots (replicates). Different letters above bars indicate significant differences between treatments (One-way ANOVA followed by a Tukey’s test, P< 0.05) during (A) the minor rainy and (B) dry season. https://doi.org/10.1371/journal.pone.0290390.g005 PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 15 / 21 https://doi.org/10.1371/journal.pone.0290390.g005 https://doi.org/10.1371/journal.pone.0290390 more effective control of S. frugiperda. On the other hand, the low efficacy in this study of aza- dirachtin-based formulations is due to the effects of manufacturing, storage, and transport conditions that can impact neem-based pesticides [62,63]. Consistently, PrGV + Btk reduced the incidence of S. frugiperda and increased maize grain yield. While the PrGV + Btk-based product has shown promising efficacy in both laboratory and on-station conditions, there is still a need for further investigations to fully understand its potential and serve as a relief for smallholder farmers. One important consideration is the need to confirm the complete profile of the commercial formulation used in our study. Con- ducting microbiological studies will be crucial in assessing the impact of the technical-grade PrGV on target pests, understanding its mode of action, evaluating its persistence, and assess- ing any potential environmental effects [64]. These studies will provide valuable insights into the efficacy and safety of PrGV + Btk and contribute to its appropriate use in pest management strategies. Similar to PrGV + Btk-based formulation, emamectin benzoate has demonstrated effective- ness against S. frugiperda in on-station conditions, and increased maize grain yield, consistent with findings from other studies [5,41,65]. However, it is important to acknowledge the high risk of resistance evolution in S. frugiperda populations exposed to emamectin benzoate [65]. Meanwhile, evidence of field-evolved resistance to emamectin benzoate has been reported in the native range of the pest [66]. Furthermore, it is crucial to investigate the potential impact of these formulations on non-target species, particularly natural enemies of S. frugiperda [5]. Understanding the compatibility of PrGV + Btk and emamectin benzoate with beneficial organisms will help ensure the preservation of natural biological control agents, which are vital for sustainable pest management practices. To support the adoption of these formulations by local farmers, economic analyses are essential. Assessing the cost-effectiveness of PrGV + Btk and emamectin benzoate-based formulations will provide valuable insights into their practical- ity and affordability for farmers, helping them make informed decisions regarding their use. Conclusion Our study evaluated the toxicity of various insecticides against Spodoptera frugiperda larvae, and emamectin benzoate showed the highest larvicidal potency. The binary microbial PrGV + Btk was effective but slow in action, while ethyl palmitate had the lowest potency. The study also found that insecticidal efficacy testing should not only focus on determining lethal dosages but also include sublethal effects of formulations. Although botanical and microbial formula- tions showed weaker potency compared to synthetic insecticides during the toxicity bioassays, their sublethal effects are noteworthy. However, the efficacy of azadirachtin and Btk + mono- sultap was inconsistent in on-station experiments, indicating that their effectiveness depends on environmental factors. Additionally, the study suggests that further investigations are needed to understand the mode of action of ethyl palmitate and PrGV as a standalone insecti- cide. Overall, our study indicated that the semi-synthetic emamectin benzoate and the micro- bial PrGV + Btk are good candidate in managing S. frugiperda. Supporting information S1 Table. Effects of the control treatment on the bionomics of Spodoptera frugiperda. (TXT) S2 Table. Impacts of sublethal doses of azadirachtin on the bionomics of Spodoptera frugi- perda. (TXT) PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 16 / 21 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0290390.s001 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0290390.s002 https://doi.org/10.1371/journal.pone.0290390 S3 Table. Impacts of sublethal doses of ethyl palmitate on the bionomics of Spodoptera fru- giperda. (TXT) S4 Table. Impacts of the combined sublethal doses of Pieris rapae Granulovirus and Bacil- lus thuringiensis subsp. kurstaki on the bionomics of Spodoptera frugiperda. (TXT) S5 Table. Impacts of sublethal doses of emamectin benzoate on the bionomics of Spodop- tera frugiperda. (TXT) S6 Table. Impacts of the combined sublethal doses of acetamiprid and indoxacarb on the bionomics of Spodoptera frugiperda. (TXT) S7 Table. Impacts of the combined sublethal doses of acetamiprid and lambda-cyhalothrin on the bionomics of Spodoptera frugiperda. (TXT) S8 Table. Impacts of the combined sublethal doses of Bacillus thuringiensis subsp. kurstaki and Monosultap on the bionomics of Spodoptera frugiperda. (TXT) S9 Table. Number of alive Spodoptera frugiperda larvae per ten maize plants per plot dur- ing the minor rainy season on-station trial. CT: Control (no insecticide); AZ: NeemGold 0.3 SC1 (azadirachtin); BT: Agoo 55WP1 (Btk + Monosultap); PR: Bypel 1 WP1 (PrGV + Btk); EB: Strike 1.9 EC™ (emamectin benzoate). (DOCX) S10 Table. Number of alive Spodoptera frugiperda larvae per ten maize plant per plot dur- ing the dry season on-station trial. CT: Control (no insecticide); AZ: NeemGold 0.3 SC1 (azadirachtin); BT: Agoo 55WP1 (Btk + Monosultap); PR: Bypel 1 WP1 (PrGV + Btk); EB: Strike 1.9 EC™ (emamectin benzoate). (DOCX) S11 Table. Maize grain yield (t) per hectare (ha) during the minor rainy and dry seasons. CT: Control (no insecticide); AZ: NeemGold 0.3 SC1 (azadirachtin); BT: Agoo 55WP1 (Btk + Monosultap); PR: Bypel 1 WP1 (PrGV + Btk); EB: Strike 1.9 EC™ (emamectin benzoate). (DOCX) Acknowledgments KRF warmly thanks the German Academic Exchange Service (DAAD) for the two-year MPhil Scholarship. We are grateful to the research technicians at the University of Ghana’s SIREC, Mr. Tegbe R and Mr. Awittor RK for their supports during the on-station trials. Author Contributions Conceptualization: Kokou Rodrigue Fiaboe, Ken Okwae Fening, Winfred Seth Kofi Gbewonyo. Data curation: Kokou Rodrigue Fiaboe, Sharanabasappa Deshmukh. Formal analysis: Kokou Rodrigue Fiaboe, Sharanabasappa Deshmukh. PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 17 / 21 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0290390.s003 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0290390.s004 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0290390.s005 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0290390.s006 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0290390.s007 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0290390.s008 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0290390.s009 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0290390.s010 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0290390.s011 https://doi.org/10.1371/journal.pone.0290390 Project administration: Ken Okwae Fening. Supervision: Ken Okwae Fening, Winfred Seth Kofi Gbewonyo. Validation: Ken Okwae Fening. Writing – original draft: Kokou Rodrigue Fiaboe. Writing – review & editing: Kokou Rodrigue Fiaboe, Ken Okwae Fening, Winfred Seth Kofi Gbewonyo, Sharanabasappa Deshmukh. References 1. Ministry of Food and Agriculture (MoFA). Comprehensive Food Security and Vulnerability Analysis (CFSVA) Ghana 2020. Accra, Ghana; 2020. Available: https://ghana.un.org. 2. Wongnaa CA, Awunyo-Vitor D, Mensah A, Adams F. Profit efficiency among maize farmers and impli- cations for poverty alleviation and food security in Ghana. Sci African. 2019; 6. https://doi.org/10.1016/j. sciaf.2019.e00206 3. Badu-Apraku B, Fakorede MAB. Maize in sub-Saharan Africa: importance and production constraints. Advances in genetic enhancement of early and extra-early maize for sub-Saharan Africa. Springer International Publishing AG 2017; 2017. p. 632. https://doi.org/10.1007/978-3-319-64852-1 4. Koffi D, Agboka K, Adenka DK, Osae M, Tounou AK, Anani Adjevi MK, et al. Maize infestation of Fall Armyworm (Lepidoptera: Noctuidae) within agro-ecological zones of Togo and Ghana in West Africa 3 Yr after its invasion. Environ Entomol. 2020; 49: 645–650. https://doi.org/10.1093/ee/nvaa048 PMID: 32346729 5. Agboyi LK, Nboyine JA, Asamani E, Beseh P, Badii BK, Kenis M, et al. Comparative effects of biopesti- cides on fall armyworm management and larval parasitism rates in Northern Ghana. J Pest Sci (2004). 2023. https://doi.org/10.1007/s10340-023-01590-z 6. Goergen G, Kumar PL, Sankung SB, Togola A, TamòM. First report of outbreaks of the fall armyworm Spodoptera frugiperda (J E Smith) (Lepidoptera, Noctuidae), a new alien invasive pest in West and Central Africa. PLoS One. 2016; 11: 1–9. https://doi.org/10.1371/journal.pone.0165632 PMID: 27788251 7. Kuate AF, Hanna R, Doumtsop Fotio ARP, Abang AF, Nanga SN, Ngatat S, et al. Spodoptera frugi- perda Smith (Lepidoptera: Noctuidae) in Cameroon: Case study on its distribution, damage, pesticide use, genetic differentiation and host plants. PLoS One. 2019; 14: 1–18. https://doi.org/10.1371/journal. pone.0217653 PMID: 31163054 8. Abang AF, Fotso Kuate A, Nanga Nanga S, Okomo Esi RM, Ndemah R, Masso C, et al. Spatio-tempo- ral partitioning and sharing of parasitoids by fall armyworm and maize stemborers in Cameroon. J Appl Entomol. 2021; 145: 55–64. https://doi.org/10.1111/JEN.12827 9. Ahissou BR, Sawadogo WM, Sankara F, Brostaux Y, Bokono-Ganta AH, Somda I, et al. Annual dynam- ics of fall armyworm populations in West Africa and biology in different host plants. Sci African. 2022; 16: 0–7. https://doi.org/10.1016/j.sciaf.2022.e01227 10. Montezano DG, Specht A, Sosa-Gómez DR, Roque-Specht VF, Sousa-Silva JC, Paula-Moraes S V., et al. Host plants of Spodoptera frugiperda (Lepidoptera: Noctuidae) in the Americas. African Entomol. 2018; 26: 286–300. https://doi.org/10.4001/003.026.0286 11. Fiteni E, Durand K, Gimenez S, Meagher RL, Legeai F, Kergoat GJ, et al. Host-plant adaptation as a driver of incipient speciation in the fall armyworm (Spodoptera frugiperda). BMC Ecol Evol. 2022; 22: 1– 11. https://doi.org/10.1186/s12862-022-02090-x PMID: 36368917 12. Prasanna BM, Huesing JE, Eddy R, Peschke VM (eds). Fall armyworm in Africa: A guide for intergrated pest management. First Edit. Mexico, CDMX: CIMMYT; 2018. Available: www.maize.org. 13. Nboyine JA, Kusi F, Abudulai M, Badii BK, Zakaria M, Adu GB, et al. A new pest, Spodoptera frugiperda (J.E. Smith), in tropical Africa: Its seasonal dynamics and damage in maize fields in northern Ghana. Crop Prot. 2020; 127. https://doi.org/10.1016/j.cropro.2019.104960 14. Boaventura D, Martin M, Pozzebon A, Mota-Sanchez D, Nauen R. Monitoring of target-site mutations conferring insecticide resistance in Spodoptera frugiperda. Insects. 2020; 11: 1–15. https://doi.org/10. 3390/insects11080545 PMID: 32824659 15. Yu SJ. Insecticide resistance in the fall armyworm, Spodoptera frugiperda (J. E. Smith). Pestic Biochem Physiol. 1991; 39: 84–91. https://doi.org/10.1016/0048-3575(91)90216-9 PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 18 / 21 https://ghana.un.org https://doi.org/10.1016/j.sciaf.2019.e00206 https://doi.org/10.1016/j.sciaf.2019.e00206 https://doi.org/10.1007/978-3-319-64852-1 https://doi.org/10.1093/ee/nvaa048 http://www.ncbi.nlm.nih.gov/pubmed/32346729 https://doi.org/10.1007/s10340-023-01590-z https://doi.org/10.1371/journal.pone.0165632 http://www.ncbi.nlm.nih.gov/pubmed/27788251 https://doi.org/10.1371/journal.pone.0217653 https://doi.org/10.1371/journal.pone.0217653 http://www.ncbi.nlm.nih.gov/pubmed/31163054 https://doi.org/10.1111/JEN.12827 https://doi.org/10.1016/j.sciaf.2022.e01227 https://doi.org/10.4001/003.026.0286 https://doi.org/10.1186/s12862-022-02090-x http://www.ncbi.nlm.nih.gov/pubmed/36368917 http://www.maize.org https://doi.org/10.1016/j.cropro.2019.104960 https://doi.org/10.3390/insects11080545 https://doi.org/10.3390/insects11080545 http://www.ncbi.nlm.nih.gov/pubmed/32824659 https://doi.org/10.1016/0048-3575%2891%2990216-9 https://doi.org/10.1371/journal.pone.0290390 16. Rwomushana I, Bateman M, Beale T, Beseh P, Cameron K, Chiluba M, et al. Fall armyworm: impacts and implications for Africa. Outlooks Pest Manag. 2018; 28: 196–201. https://doi.org/10.1564/v28_oct_ 02 17. Zhang L, Liu B, Zheng W, Liu C, Zhang D, Zhao S, et al. Genetic structure and insecticide resistance characteristics of fall armyworm populations invading China. Mol Ecol Resour. 2020; 20: 1682–1696. https://doi.org/10.1111/1755-0998.13219 PMID: 32619331 18. Akutse KS, Kimemia JW, Ekesi S, Khamis FM, Ombura OL, Subramanian S. Ovicidal effects of ento- mopathogenic fungal isolates on the invasive Fall armyworm Spodoptera frugiperda (Lepidoptera: Noc- tuidae). J Appl Entomol. 2019; 143: 626–634. https://doi.org/10.1111/jen.12634 19. Bateman ML, Day RK, Luke B, Edgington S, Kuhlmann U, Cock MJW. Assessment of potential biopes- ticide options for managing fall armyworm (Spodoptera frugiperda) in Africa. J Appl Entomol. 2018; 142: 805–819. https://doi.org/10.1111/jen.12565 20. Mwamburi LA. Endophytic fungi, Beauveria bassiana and Metarhizium anisopliae, confer control of the fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), in two tomato varieties. Egypt J Biol Pest Control. 2021; 31. https://doi.org/10.1186/s41938-020-00357-3 21. Asare-Nuamah P. Smallholder farmers’ adaptation strategies for the management of fall armyworm (Spodoptera frugiperda) in rural Ghana. Int J Pest Manag. 2021; 68: 8–18. https://doi.org/10.1080/ 09670874.2020.1787552 22. Nboyine JA, Asamani E, Agboyi LK, Yahaya I, Kusi F, Adazebra G, et al. Assessment of the optimal fre- quency of insecticide sprays required to manage fall armyworm (Spodoptera frugiperda J.E Smith) in maize (Zea mays L.) in northern Ghana. CABI Agric Biosci. 2022; 3: 1–11. https://doi.org/10.1186/ s43170-021-00070-7 23. Babendreier D, Koku Agboyi L, Beseh P, Osae M, Nboyine J, Ofori SEK, et al. The efficacy of alterna- tive, environmentally friendly plant protection measures for control of Fall armyworm, Spodoptera frugi- perda, in maize. Insects. 2020; 11. https://doi.org/10.3390/insects11040240 PMID: 32290333 24. Amarasekare KG, Edelson J V. Effect of temperature on efficacy of insecticides to differential grasshop- per (Orthoptera: Acrididae). J Econ Entomol. 2004; 97: 1595–1602. https://doi.org/10.1603/0022-0493- 97.5.1595 PMID: 15568348 25. Matzrafi M. Climate change exacerbates pest damage through reduced pesticide efficacy. Pest Manag Sci. 2019; 75: 9–13. https://doi.org/10.1002/ps.5121 PMID: 29920926 26. WHO. Guidelines for efficacy testing of insecticides for indoor and outdoor ground-applied space spray applications control of neglected tropical diseases who pesticide evaluation scheme. Geneva World Heal Organ. 2009; 2–53. Available: http://www.who.int/iris/handle/10665/70070. 27. Garzón A, Medina P, Amor F, Viñuela E, Budia F. Toxicity and sublethal effects of six insecticides to last instar larvae and adults of the biocontrol agents Chrysoperla carnea (Stephens) (Neuroptera: Chry- sopidae) and Adalia bipunctata (L.) (Coleoptera: Coccinellidae). Chemosphere. 2015; 132: 87–93. https://doi.org/10.1016/j.chemosphere.2015.03.016 PMID: 25828251 28. Hardke JT, Temple JH, Leonard BR, Jackson RE. Laboratory toxicity and field efficacy of selected insecticides against fall armyworm (Lepidoptera: Noctuidae). Florida Entomol. 2011; 94: 272–278. https://doi.org/10.1653/024.094.0221 29. Helps JC, Paveley ND, van den Bosch F. Identifying circumstances under which high insecticide dose increases or decreases resistance selection. J Theor Biol. 2017; 428: 153–167. https://doi.org/10.1016/ j.jtbi.2017.06.007 PMID: 28625474 30. Guedes RNC, Smagghe G, Stark JD, Desneux N. Pesticide-induced stress in arthropod pests for opti- mized integrated pest management programs. Annu Rev Entomol. 2016; 61: 43–62. https://doi.org/10. 1146/annurev-ento-010715-023646 PMID: 26473315 31. Argentine JA, Jansson RK, Halliday WR, Rugg D, Jany CS. Potency, spectrum and residual activity of four new insecticides under glasshouse conditions. Florida Entomol. 2002; 85: 552–562. https://doi.org/ 10.1653/0015-4040(2002)085[0552:PSARAO]2.0.CO;2 32. Schneider-Orelli O. Entomologisches Praktikum Einf. in d. land- u. forstwirtschaftl. Insektenkunde. 2., erw. A. Aarau: Sauerländer; 1947. 33. Katagi T. Photodegradation of pesticides on plant and soil surfaces. Rev Environ Contam Toxicol. 2004; 182: 1–195. https://doi.org/10.1007/978-1-4419-9098-3_1 PMID: 15217019 34. Finney DJ. Probit Analysis. 2nd Ed. Journal of the American Pharmaceutical Association (Scientific ed.). Cambridge University Press., New York; 1952. https://doi.org/10.1002/jps.3030411125 35. Chi H. TWOSEX-MSChart: a computer program for the age-stage, two-sex life table analysis. 2009. Available: http://140.120.197.173/Ecology/. 36. Chi H. Life-table analysis incorporating both sexes and variable development rates among individuals. Environ Entomol. 1988; 17: 26–34. PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 19 / 21 https://doi.org/10.1564/v28%5Foct%5F02 https://doi.org/10.1564/v28%5Foct%5F02 https://doi.org/10.1111/1755-0998.13219 http://www.ncbi.nlm.nih.gov/pubmed/32619331 https://doi.org/10.1111/jen.12634 https://doi.org/10.1111/jen.12565 https://doi.org/10.1186/s41938-020-00357-3 https://doi.org/10.1080/09670874.2020.1787552 https://doi.org/10.1080/09670874.2020.1787552 https://doi.org/10.1186/s43170-021-00070-7 https://doi.org/10.1186/s43170-021-00070-7 https://doi.org/10.3390/insects11040240 http://www.ncbi.nlm.nih.gov/pubmed/32290333 https://doi.org/10.1603/0022-0493-97.5.1595 https://doi.org/10.1603/0022-0493-97.5.1595 http://www.ncbi.nlm.nih.gov/pubmed/15568348 https://doi.org/10.1002/ps.5121 http://www.ncbi.nlm.nih.gov/pubmed/29920926 http://www.who.int/iris/handle/10665/70070 https://doi.org/10.1016/j.chemosphere.2015.03.016 http://www.ncbi.nlm.nih.gov/pubmed/25828251 https://doi.org/10.1653/024.094.0221 https://doi.org/10.1016/j.jtbi.2017.06.007 https://doi.org/10.1016/j.jtbi.2017.06.007 http://www.ncbi.nlm.nih.gov/pubmed/28625474 https://doi.org/10.1146/annurev-ento-010715-023646 https://doi.org/10.1146/annurev-ento-010715-023646 http://www.ncbi.nlm.nih.gov/pubmed/26473315 https://doi.org/10.1653/0015-4040%282002%29085%5B0552%3APSARAO%5D2.0.CO%3B2 https://doi.org/10.1653/0015-4040%282002%29085%5B0552%3APSARAO%5D2.0.CO%3B2 https://doi.org/10.1007/978-1-4419-9098-3%5F1 http://www.ncbi.nlm.nih.gov/pubmed/15217019 https://doi.org/10.1002/jps.3030411125 http://140.120.197.173/Ecology/ https://doi.org/10.1371/journal.pone.0290390 37. Dunnett CW. A multiple comparison procedure for comparing several treatments with a control. J Am Stat Assoc. 1955; 50: 1096–1121. https://doi.org/10.1080/01621459.1955.10501294 38. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2022. Available: https://www.r-project.org/. 39. Cook DR, Leonard BR, Gore J. Field and laboratory performance of novel insecticides against army- worms (Lepidoptera: Noctuidae). Florida Entomol. 2004; 87: 433–439. https://doi.org/10.1653/0015- 4040(2004)087[0433:FALPON]2.0.CO;2 40. Barrania AA. Effects of some insecticides on some biological parameters of cotton leafworm, Spodop- tera littoralis (Lepidoptera: Noctuidae). Alexandria Sci Exch J. 2019; 40: 307–313. https://doi.org/10. 21608/asejaiqjsae.2019.34182 41. Deshmukh S, Pavithra HB, Kalleshwaraswamy CM, Shivanna BK, Maruthi MS, Mota-Sanchez D. Field efficacy of insecticides for management of invasive fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) on maize in India. Florida Entomol. 2020; 103: 221–227. https://doi.org/10. 1653/024.103.0211 42. Taleh M, Rafiee Dastjerdi H, Naseri B, Ebadollahi A, Sheikhi Garjan A, Talebi Jahromi K. Toxicity and biochemical effects of emamectin benzoate against Tuta absoluta (Meyrick) alone and in combination with some conventional insecticides. Physiol Entomol. 2021; 46: 210–217. https://doi.org/10.1111/ phen.12360 43. Wu ZW, Zhang YL, Shang SQ. Effectiveness of 12 insecticides to a laboratory population of Cydia pomonella (Lepidoptera: Tortricidae) newly established in China. J Econ Entomol. 2015; 108: 1271– 1278. https://doi.org/10.1093/jee/tov083 PMID: 26470255 44. Liu KX, Guo Y, Zhang CX, Xue C Bin. Sublethal effects and reproductive hormesis of emamectin benzo- ate on Plutella xylostella. Front Physiol. 2022; 13: 1–8. https://doi.org/10.3389/fphys.2022.1025959 PMID: 36338483 45. Janmaat AF, Myers J. Rapid evolution and the cost of resistance to Bacillus thuringiensis in greenhouse populations of cabbage loopers, Trichoplusia ni. Proc R Soc B Biol Sci. 2003; 270: 2263–2270. https:// doi.org/10.1098/rspb.2003.2497 PMID: 14613613 46. O’Callaghan M, Glare TR, Lacey LA. Bioassay of bacterial entomopathogens against insect larvae. Second Edi. Manual of Techniques in Invertebrate Pathology. Elsevier Ltd; 2012. https://doi.org/10. 1016/B978-0-12-386899-2.00004-X 47. Zheng X, Ren X, Su J. Insecticide susceptibility of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) in China. J Econ Entomol. 2011; 104: 653–658. https://doi.org/10.1603/EC10419 PMID: 21510218 48. Zhang BQ, Cheng R-L, Wang X-F, Zhang C-XZ. The Genome of Pieris rapae Granulovirus. J Virol. 2012; 86: 9544–9544. https://doi.org/10.1128/jvi.01431-12 PMID: 22879615 49. Biondi A, Mommaerts V, Smagghe G, Viñuela E, Zappalà L, Desneux N. The non-target impact of spi- nosyns on beneficial arthropods. Pest Manag Sci. 2012; 68: 1523–1536. https://doi.org/10.1002/ps. 3396 PMID: 23109262 50. Honjo MN, Emura N, Kawagoe T, Sugisaka J, Kamitani M, Nagano AJ, et al. Seasonality of interactions between a plant virus and its host during persistent infection in a natural environment. ISME J. 2020; 14: 506–518. https://doi.org/10.1038/s41396-019-0519-4 PMID: 31664159 51. Koul O. Insect growth regulating and antifeedant effects of neem extracts and azadirachtin on two aphid species of ornamental plants. J Biosci. 1999; 24: 85–90. https://doi.org/10.1007/BF02941111 52. Abedi Z, Saber M, Vojoudi S, Mahdavi V, Parsaeyan E. Acute, sublethal, and combination effects of azadirachtin and Bacillus thuringiensis on the cotton bollworm, Helicoverpa armigera. J Insect Sci. 2014; 14: 1–9. https://doi.org/10.1093/jis/14.1.30 PMID: 25373177 53. Bu CY, Duan DD, Wang YN, Ma LQ, Liu YB, Shi GL. Acaricidal activity of ethyl palmitate against Tetra- nychus cinnabarinus. Adv Intell Soft Comput. 2012; 134 AISC: 703–712. https://doi.org/10.1007/978-3- 642-27537-1_84 54. Zhang H, Chen G, Lü S, Zhang L, Guo M, Beitia J, et al. Insecticidal activities against Odontotermes for- mosanus and Plutella xylostella and corresponding constituents of Tung meal from Vernicia fordii. Insect. 2021; 12. https://doi.org/10.3390/insects12050425 PMID: 34068455 55. Chandler D, Bailey AS, Mark Tatchell G, Davidson G, Greaves J, Grant WP. The development, regula- tion and use of biopesticides for integrated pest management. Philos Trans R Soc B Biol Sci. 2011; 366: 1987–1998. https://doi.org/10.1098/rstb.2010.0390 PMID: 21624919 56. Fenibo EO, Ijoma GN, Matambo T. Biopesticides in sustainable agriculture: A critical sustainable devel- opment driver governed by green chemistry principles. Front Sustain Food Syst. 2021; 5: 1–6. https:// doi.org/10.3389/fsufs.2021.619058 PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 20 / 21 https://doi.org/10.1080/01621459.1955.10501294 https://www.r-project.org/ https://doi.org/10.1653/0015-4040%282004%29087%5B0433%3AFALPON%5D2.0.CO%3B2 https://doi.org/10.1653/0015-4040%282004%29087%5B0433%3AFALPON%5D2.0.CO%3B2 https://doi.org/10.21608/asejaiqjsae.2019.34182 https://doi.org/10.21608/asejaiqjsae.2019.34182 https://doi.org/10.1653/024.103.0211 https://doi.org/10.1653/024.103.0211 https://doi.org/10.1111/phen.12360 https://doi.org/10.1111/phen.12360 https://doi.org/10.1093/jee/tov083 http://www.ncbi.nlm.nih.gov/pubmed/26470255 https://doi.org/10.3389/fphys.2022.1025959 http://www.ncbi.nlm.nih.gov/pubmed/36338483 https://doi.org/10.1098/rspb.2003.2497 https://doi.org/10.1098/rspb.2003.2497 http://www.ncbi.nlm.nih.gov/pubmed/14613613 https://doi.org/10.1016/B978-0-12-386899-2.00004-X https://doi.org/10.1016/B978-0-12-386899-2.00004-X https://doi.org/10.1603/EC10419 http://www.ncbi.nlm.nih.gov/pubmed/21510218 https://doi.org/10.1128/jvi.01431-12 http://www.ncbi.nlm.nih.gov/pubmed/22879615 https://doi.org/10.1002/ps.3396 https://doi.org/10.1002/ps.3396 http://www.ncbi.nlm.nih.gov/pubmed/23109262 https://doi.org/10.1038/s41396-019-0519-4 http://www.ncbi.nlm.nih.gov/pubmed/31664159 https://doi.org/10.1007/BF02941111 https://doi.org/10.1093/jis/14.1.30 http://www.ncbi.nlm.nih.gov/pubmed/25373177 https://doi.org/10.1007/978-3-642-27537-1%5F84 https://doi.org/10.1007/978-3-642-27537-1%5F84 https://doi.org/10.3390/insects12050425 http://www.ncbi.nlm.nih.gov/pubmed/34068455 https://doi.org/10.1098/rstb.2010.0390 http://www.ncbi.nlm.nih.gov/pubmed/21624919 https://doi.org/10.3389/fsufs.2021.619058 https://doi.org/10.3389/fsufs.2021.619058 https://doi.org/10.1371/journal.pone.0290390 57. Horikoshi RJ, Vertuan H, de Castro AA, Morrell K, Griffith C, Evans A, et al. A new generation of Bt maize for control of fall armyworm (Spodoptera frugiperda). Pest Manag Sci. 2021; 77: 3727–3736. https://doi.org/10.1002/ps.6334 PMID: 33624355 58. Sousa FF, Mendes SM, Santos-Amaya OF, Araújo OG, Oliveira EE, Pereira EJG. Life-history traits of Spodoptera frugiperda populations exposed to low-dose Bt maize. PLoS One. 2016; 11: 1–18. https:// doi.org/10.1371/journal.pone.0156608 PMID: 27243977 59. Moar WJ, Pusztai-Carey M, Van Faassen H, Bosch D, Frutos R, Rang C, et al. Development of Bacillus thuringiensis CryIC resistance by Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae). Appl Environ Microbiol. 1995; 61: 2086–2092. https://doi.org/10.1128/aem.61.6.2086–2092.1995 60. Zhou L, Huang J, Xu H. Monitoring resistance of field populations of diamondback moth Plutella xylos- tella L. (Lepidoptera: Yponomeutidae) to five insecticides in South China: A ten-year case study. Crop Prot. 2011; 30: 272–278. https://doi.org/10.1016/j.cropro.2010.10.006 61. Ping He Y, Fen Gao C, Zhang Cao M, Ming Chen W, Qin Huang L, Jun Zhou W, et al. Survey of suscep- tibilities to monosultap, triazophos, fipronil, and abamectin in Chilo suppressalis (Lepidoptera: Crambi- dae). J Econ Entomol. 2007; 100: 1854–1861. https://doi.org/10.1093/jee/100.6.1854 62. Gahukar RT. Factors affecting content and bioefficacy of neem (Azadirachta indica A. Juss.) phyto- chemicals used in agricultural pest control: A review. Crop Prot. 2014; 62: 93–99. https://doi.org/10. 1016/j.cropro.2014.04.014 63. Shaurub EH, El-meguid AA, El-aziz NMA. Effect of some environmental factors on the toxicity of azadir- achtin to the Egyptian cotton leafworm Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae). Ecol Balk. 2014; 6: 113–117. 64. Puglis HJ, Boone MD. Effects of technical-grade active ingredient vs. commercial formulation of seven pesticides in the presence or absence of UV radiation on survival of green frog tadpoles. Arch Environ Contam Toxicol. 2011; 60: 145–155. https://doi.org/10.1007/s00244-010-9528-z PMID: 20422168 65. Muraro DS, de Oliveira Abbade Neto D, Kanno RH, Kaiser IS, Bernardi O, Omoto C. Inheritance pat- terns, cross-resistance and synergism in Spodoptera frugiperda (Lepidoptera: Noctuidae) resistant to emamectin benzoate. Pest Manag Sci. 2021; 77: 5049–5057. https://doi.org/10.1002/ps.6545 PMID: 34216515 66. Muraro DS, Salmeron E, Cruz JVS, Amaral FSA, Guidolin AS, Nascimento ARB, et al. Evidence of field-evolved resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae) to emamectin benzoate in Brazil. Crop Prot. 2022; 162. https://doi.org/10.1016/j.cropro.2022.106071 PLOS ONE Insecticides bio-efficacy on Spodoptera frugiperda PLOS ONE | https://doi.org/10.1371/journal.pone.0290390 November 15, 2023 21 / 21 https://doi.org/10.1002/ps.6334 http://www.ncbi.nlm.nih.gov/pubmed/33624355 https://doi.org/10.1371/journal.pone.0156608 https://doi.org/10.1371/journal.pone.0156608 http://www.ncbi.nlm.nih.gov/pubmed/27243977 https://doi.org/10.1128/aem.61.6.2086%26%23x2013%3B2092.1995 https://doi.org/10.1016/j.cropro.2010.10.006 https://doi.org/10.1093/jee/100.6.1854 https://doi.org/10.1016/j.cropro.2014.04.014 https://doi.org/10.1016/j.cropro.2014.04.014 https://doi.org/10.1007/s00244-010-9528-z http://www.ncbi.nlm.nih.gov/pubmed/20422168 https://doi.org/10.1002/ps.6545 http://www.ncbi.nlm.nih.gov/pubmed/34216515 https://doi.org/10.1016/j.cropro.2022.106071 https://doi.org/10.1371/journal.pone.0290390