Monitoring Resistance and Biochemical Studies of Three Egyptian Field Strains of Spodoptera littoralis (Lepidoptera: Noctuidae) to Six Insecticides

Background: Spodoptera littoralis (Boisd.) is a prominent agricultural insect pest that has developed resistance to a variety of insecticide classes. In this study, the resistance of three field strains of S. littoralis, collected over three consecutive seasons (2018 to 2020) from three Egyptian Governorates (El-Fayoum, Behera and Kafr El-Shiekh), to six insecticides was monitored. Methods: Laboratory bioassays were carried out using the leaf-dipping method to examine the susceptibility of the laboratory and field strains to the tested insecticides. Activities of detoxification enzymes were determined in an attempt to identify resistance mechanisms. Results: The results showed that LC50 values of the field strains ranged from 0.0089 to 132.24 mg/L, and the corresponding resistance ratio (RR) ranged from 0.17 to 4.13-fold compared with the susceptible strain. Notably, low resistance developed to spinosad in all field strains, and very low resistance developed to alpha-cypermethrin and chlorpyrifos. On the other hand, no resistance developed to methomyl, hexaflumeron or Bacillus thuringiensis. The determination of detoxification enzymes, including carboxylesterases (α- and β-esterase), mixed function oxidase (MFO) and glutathione-S-transferase (GST), or the target site of acetylcholinesterase (AChE), revealed that the three field strains had significantly different activity levels compared with the susceptible strain. Conclusion: Our findings, along with other tactics, are expected to help with the resistance management of S. littoralis in Egypt.


Introduction
The cotton leaf worm Spodoptera littoralis (Boisd.) is an insect, which causes serious losses in over 80 economically significant crop species [1]. It is spread in Africa and in the Middle Eastern nations [2]. Excessive dependence on chemical control has led to the development of resistance to numerous classes of insecticides, and insect resistance to insecticides has been documented in more than 600 arthropod species [3].
Resistance was first reported in S. littoralis in 1968 to methyl-parathion, which belongs to organophosphates [1]. In recent years, S. littoralis has evolved high levels of resistance to different groups of insecticides, such as organophosphates, pyrethroids, carbamates and insect growth regulators (IGRs) [4,5], as well as to several newer insecticides, including indoxacarb and chlorantraniliprole [6,7]. In addition, S. littoralis is resistant to bioinsecticides, including Bacillus thuringiensis [5], spinosad [7,8] and spinotram [7], due to the acceleration caused by the absence of a hibernation period in this pest [9]. Currently, S. littoralis is in the top 30 most resistant species in the world, according to the Arthropod A susceptible laboratory strain of S. littoralis was continuously inbred on castor bean (Ricinus communis) leaves under laboratory conditions [7,26] for more than ten generations with no exposure to any pesticides. The adult moths fed on sugar solution (10%) as a dietary supplement [27]. Three field strains of S. littoralis were collected from three Egyptian governorates (Fayoum; 29 • 18 30 N and 30 • 50 39 E, Beheira; 30 • 59 00 N and 30 • 12 00 E and Kafr-El-Shiekh; 31 • 06 42 N and 30 • 56 45 E) over three consecutive seasons (2018 to 2020). Egg masses of S. littoralis were collected from cotton and vegetable fields in May-July, before pesticide application. These masses were maintained in a rearing room at 60-70% relative humidity, at a temperature of 25 ± 1 • C and a 16 h:8 h light/dark regimen, to obtain the 4th instar larvae for bioassays and biochemical studies.

Insecticides and Reagents Used
The insecticides used in bioassays are presented in Table 1. The substrates and reagents used for biochemical studies were purchased from Sigma Aldrich, Germany.

Bioassay
The leaf-dipping method was conducted as described by Moustafa et al. [29] and Awad et al. [30]. Fourth-instar larvae of S. littoralis were used for the bioassays. As shown in Table S1, 6 concentrations, ranging from 0.002 to 16 mg/L, of each tested insecticide were used. Castor bean leaves were immersed in each concentration for 20 s; untreated leaves were immersed in water for the control group then t allowed to air-dry. Leaves with ten larvae were then transferred into a glass container (0.25 L), and five replicates were performed for each concentration [7,31]. For conventional insecticides, i.e., chlorpyrifos, methomyl and alpha-cypermethrin, lethal concentrations (LCs) were calculated after 24 h of treatment. Meanwhile, for bioinsecticides, i.e., hexaflumeron, B. thuringiensis and spinosad, the larvae fed on insecticide-treated leaves for one day, then on untreated leaves for two days, and mortality was recorded by the end of the third day to calculate LC 50 and LC 90 . For enzyme activity assays, 50 mg of untreated fourth instar larvae from susceptible or field strains were homogenized in 1 mL distilled water in a chilled glass Teflon tissue homogenizer. Homogenates were centrifuged at 8000 rpm for 15 min at 4 • C. The supernatants were kept at −20 • C prior to biochemical assays. Three replicates were used for each strain. Protein content was determined as described by Bradford [32].

Detoxification Enzyme Assays Carboxylesterase (CarE)
Determination of αand β-esterases' activity was performed as described by Van Asperen [33]. The hydrolysis of αor β-naphthyl acetate was spectrophotometrically measured at 600 and 550 nm, respectively. The total CarE activity was calculated using the standard curves of αand β-naphthol and protein content.

Mixed Function Oxidase (MFO)
The activity of MFO was measured as described by Hansen and Hodgson [34]. The reaction mixture of enzyme solution, NADPH, glucose-6-phosphate and Glucose-6-phosphate dehydrogenase was initiated by adding p-nitroanisole and incubated at 37 • C for 30 min. The reaction was then terminated by adding HCl. The optical density was measured spectrophotometrically for 10 min at 405 nm.

Glutathione S-Transferase (GST)
The activity of GST was determined according to Habig et al. [35]. 1-chloro 2,4dinitrobenzene (CDNB) was used as a substrate. The mixture of potassium phosphate buffer, GSH, enzyme solution and the substrate was incubated for 5 min at 30 • C. The absorbance increase at 340 nm was then recorded versus a blank mixture. The nanomoleconjugated substrate/min/mg protein was then determined.

Acetylcholine Esterase (AChE)
The activity of AChE was determined as indicated by Simpson et al. [36]. Acetylcholine bromide (AChBr) was used as a substrate. The reaction mixture of the enzyme solution, phosphate buffer and substrate was incubated for 30 min at 37 • C. The AChBr decrease was read at 515 nm.

Statistical Analysis
Probit analysis was used to calculate median lethal concentrations (LC 50 ) and their 95% confidence limits (CLs) [37]. To calculate the resistance ratio (RR), the LC 50 of a field strain was divided by the LC 50 of the laboratory strain. In addition, one-way ANOVA with GraphPad-Prism v. 9.3 statistical analysis software were used to analyze the enzymes' activity data. Differences among the means of strains were assessed using Tukey's HSD test at a significance level of p ≤ 0.05.

Susceptibility of Laboratory Strain of S. littoralis to the Tested Insecticides
The toxicity levels of the tested insecticides (chlorpyrifos, methomyl, alphacypermethrin, hexaflumeron, B. thuringiensis and spinosad) to the susceptible strain of S. littoralis are shown in Table 2. Overall, based on LC 50 values, insecticide toxicity to S. littoralis was as follows, in descending order: Spinosad > B. thuringiensis > hexaflumeron > alpha-cypermethrin > chlorpyrifos > methomyl. It is obvious that the bioinsecticides (spinosad, B. thuringiensis and hexaflumeron) were more toxic than the conventional ones, with LC 50 of 0.0089, 6.11 and 14.57 mg/L, respectively. Table 2. Susceptibility of the 4th instar larvae of a laboratory strain of S. littoralis to the tested insecticides. Castor leaves were dipped in six concentrations of each tested insecticide. Five replicates, ten larvae each, were used for each concentration. (i.e., 50 larvae/concentration). The larvae were allowed to feed for 24 h on leaves treated with conventional insecticides, and for 72 h post-treatment with bioinsecticides. LC 50

Susceptibility of Field Strains of S. littoralis to Conventional Insecticides
As shown in Tables 3-5, in 2018, the Fayoum and Kafr El-Shiekh strains of S. littoralis showed very low resistance levels to chlorpyrifos (2.37-and 2.08-fold, respectively). In 2019, the resistance ratios were 1.19, 1.38 and 1.18-fold for the Fayom, Beheira and Kafr El-Shiekh strains, respectively. In 2020, resistance to chlorpyrifos decreased to 1.10-and 0.65-fold in Beheira and Kafr El-Shiekh strains, respectively). As to methomyl, very low levels of resistance to it were observed in the Fayoum strain (3.11-fold) in 2020 (Table 3) and in Beheira strain (2.31-fold) in 2018 (Table 4). In contrast, a resistance ratio of <2-fold was found in the Kafr El-Sheikh strain (Table 5). Regarding alpha-cypermethrin, a very low level of resistance to it was found in the Fayoum strain (2.02-fold) in 2018 (Table 3); in the Beheira strain (2.65-and 2.08-fold) in 2018 and 2019, respectively (Table 4); and in the Kafr El-Sheikh strain (2.79-fold) in 2019 (Table 5). Table 3. Susceptibility of fourth instar larvae of Fayoum field strain of Spodoptera littoralis to six insecticides over three consecutive seasons (2018 to 2020). Egg masses of S. littoralis, collected over three consecutive seasons (2018 to 2020), were kept in the rearing room until the fourth instar larvae. Six concentrations of each tested insecticide were prepared, then castor bean leaves were immersed in each concentration for 20 s. For each concentration, 50 larvae were used to calculate the LC 50 , LC 90 and the resistance ratio.

Insecticides
Season a LC 50

Susceptibility of Field Strains of S. littoralis to Bioinsecticides
The data showed no resistance to hexaflumeron in any of the field strains (Tables 3-5) during the three seasons (2018-2020), and the resistance ratios ranged from 0.07-to 0.31-fold. Regarding B. thuringiensis, no resistance was detected in the Fayoum and Bereira strains (Tables 3 and 4), while a moderate level of resistance (2.19-fold) was recorded in the Kafr El-Shiekh strain in 2020 (Table 5). Nevertheless, a low level of resistance to spinosad was observed in all field strains (Tables 3-5), and the resistance ratios ranged from 1.79-to 4.31-fold. Table 4. Susceptibility of the fourth instar larvae of Beheira field strain of Spodoptera littoralis to six insecticides over three consecutive seasons (2018 to 2020). Egg masses of S. littoralis, collected over three consecutive seasons (2018 to 2020), were kept in the rearing room until the fourth instar larvae. Six concentrations of each tested insecticide were prepared, then castor bean leaves were immersed in each concentration for 20 s. For each concentration, 50 larvae were used to calculate the LC 50 , LC 90 and the resistance ratio.

Activity of Detoxification Enzymes
To examine the prospective role of detoxification enzymes in the susceptibility of S. littoralis to the tested insecticides, enzymes assays were performed to determine the levels of carboxylesterases (α and βesterases), acetylcholine esterase (AChE), glutathione-S-transferase (GST) and mixed-function oxidase (MFO) in the tested field strains in comparison with the laboratory one. Data are shown in Tables 6 and 7. Table 5. Susceptibility of the fourth instar larvae of Kafr El-Shiekh field strain of Spodoptera littoralis to six insecticides over three consecutive seasons (2018 to 2020). Egg masses of S. littoralis, collected over three consecutive seasons (2018 to 2020), were kept in the rearing room until the fourth instar larvae. Six concentrations of each tested insecticide were prepared, then castor bean leaves were immersed in each concentration for 20 s. For each concentration, 50 larvae were used to calculate the LC 50 , LC 90 and the resistance ratio. As shown in Table 6, the level of α-esterase, expressed as folds of that of the susceptible strain, was reduced to 0.96-, 0.92-and 0.79-fold in Fayoum strain; to 0.89-, 0.85-and 0.88-fold in Beheira strain; and to 0.81-, 0.92-and 0.93-fold in the Kafr El-Sheikh strain over the three seasons, respectively. A similar reduction was also recorded for the level of β-esterases enzyme. In addition, AchE significantly decreased in the Beheira strain in all seasons, but only in 2020 for the Fayoum strain, and in 2018 for the Kafr El-Sheikh strain.  As shown in Table 7, the levels of MFO and GST significantly decreased in the Beheira strain in all seasons. MFO levels ranged from 1.26 to 1.32 mg/mg of protein, while GST ranged from 1.92 to 2.01 mmol/min/mg of protein. Similarly, the level of both enzymes significantly decreased, but only in 2020 for the Fayoum strain and in 2018 for the Kafr El-Sheikh strain.

Discussion
The indiscriminate use of conventional and newer insecticides has caused the development of resistance to almost all kinds of insecticides in the Noctuidae species [7,12,13,[38][39][40][41]. According to the Egyptian Agricultural Pesticides Committee, EAPC (2022), several groups of biochemical or chemical insecticides, such as organophosphorus, pyrethroids, insect growth regulators (IGRs), diamides, oxadiazin, spinosyns, emamectin benzoate and B. thuringiensis, are used for S. littoralis management. Hence, insecticide resistance has developed in this insect pest [7,42,43]. Consequently, the history of insect resistance to various insecticides, including S. littoralis, should be studied to monitor the tolerance changes and detect any problems that may occur. In addition, continuous monitoring of the insect strains for changes in resistance frequencies is needed for the development of effective management strategies [44]. Therefore, monitoring insecticides is considered a pre-requisite in IPM programs [45], and becomes a remarkable aspect of resistance management [3]. The present study investigated the susceptibility of the fourth instar larvae of three field strains of S. littoralis to six insecticides with different modes of action over three consecutive seasons (2018-2020). To explore the mechanism of resistance, if it existed, the activities of the relevant enzymes were also studied.
The results showed that bioinsecticides were more toxic to S. littoralis than conventional ones. Spinosad had the highest toxicity, with an LC 50 value of 0.0089 mg/L. These results are congruent with previous studies by Ahmed et al. [8] and Tamilselvan et al. [46], who found that spinosad and other bioinsecticides, such as emamectin benzoate and spinotram, were more toxic to S. littoralis and Plutella xylostella (L.) than conventional insecticides. Based on the resistance ratio, all field strains developed resistance to spinosad compared with other insecticides. In line with this finding, resistance to spinosad has been reported in several insect pests, including S. littoralis [7,8], S. litura [19], S. exigua [14] and P. xylostella [46]. On the contrary, a slight level of resistance was developed to chlorpyrifos (organophosphorus) and methomyl (carbamates) in some cases, but in others, no resistance was found. These findings can be attributed to the fact that in Egypt, bioinsecticides, including spinosad and newer chemical insecticides, have been extensively used due to the health and environmental issues associated with conventional ones. The fluctuation of resistance levels recorded in this study can be connected with the type of insecticides used and the sequence of usage in each governorate.
Metabolic resistance relies on enzymatic systems that can detoxify and/or sequester toxic molecules, interrupting or decreasing its harmful effect [47]. Mostly, metabolic resistance, which involves three major families of enzymes (CarE, MFO and GST), is one of the most common defense mechanisms in insects [7,25,48,49]. The point mutation at the target site is involved in the insect resistance mechanism, like that of AChE, which is involved in organophosphates and carbamates insecticides' resistance [50]. Therefore, the activity of these enzymes together with AChE was determined in the three field strains of S. littoralis to assess their roles in resistance and to identify resistance mechanisms for the sake of enlightened pest management. In this regard, our results revealed, unexpectedly, a reduction in the activities of all enzymes in field strains. This might suggest that the resistance of these strains to the tested insecticides is not always associated with higher detoxification activity, but is, rather, related to a different mechanism. These results are not consistent with those of Hu et al. [51] and Zhang et al. [52], who found that the overexpression of CarE and GST was related to resistance to insecticides. In fact, most of the resistance to insecticides is associated with an increase in the activity of detoxification enzymes [7,25,53,54]. However, modification of the target site could lead to insensitivity of insect pests to insecticides [55]. In addition, factors such as UV light, sunlight, photolysis in water and shelf life could affect the insecticide efficiency and delay the development of the resistance/prevent it from occurring [56]. Thus, we speculate that the inconsistency with other studies might be due to the different species of insect, type of insecticide, time of sampling or method of treatment.
Moreover, studies have confirmed cross-resistance between spinosad and newer chemical insecticides [46,52,57]. Cross-resistance between different groups of insecticides might be due to metabolic detoxification mechanisms [58]. Therefore, it is necessary to develop effective management plans to delay any resistance development.

Conclusions
In summary, our study provides evidence of very low to non-resistance development by S. littoralis to some commonly used insecticides in some Egyptian governorates due to the successful resistance management strategies used in Egypt. However, monitoring resistance to insecticides is an important aspect of insecticide rotation and their mixed application. Thus, regular follow up is needed to specify and confirm the mechanisms by which S. littoralis develops resistance to the tested insecticides, in order to avoid resistance problems and pest control failure.