Effect of sublethal doses of some insecticides and their role on detoxication enzymes and protein-content of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae)

The indiscriminating and intensive use of insecticides to control the cotton leafworm (CLW), Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), usually induces high levels of resistance. The insecticides are the principal method for controlling this pest because of its critical role in reducing insects when the economic threshold (ETL); therefore, the effectiveness of these insecticides should be maintained. So, the target of the present work was directed to focus on studying the change in the activities of some important enzymes as a result of sublethal treatment concentrations (viz. LC25 values) of tested new insecticides (profenofos, cyfluthrin, emamectin benzoate, lufenuron, and spinetoram). The expected results could offer better understanding and more specific information about the resistance development in field populations of CLW because resistance is a significant challenge to pest control workers and these results may contribute to making the right decision at the right time. Bioassays were performed on fourth instar larvae of S. littoralis field populations compared with the laboratory strain to assess the activity of the emamectin benzoate, lufenuron, and spinetoram by LC25 and study the biochemical activities of some detoxification enzymes, like acetylcholinesterase (AChE), glutathione S-transferases (GST), alkaline phosphatase (ALP), and acid phosphatase (ACP) in fourth larval instar which was treated with LC25; protein content is also determined. The emamectin benzoate was the most toxic compound followed by lufenuron, cyfluthrin, spinetoram, and profenofos with the corresponding LC50 values of 0.05, 49.18, 70.99, 130.26, and 156.78 ppm respectively. The results showed that all the tested insecticides at LC25 value gave a slight inhibition of the acetylcholinesterase (AChE) activity, except profenofos was the most potent one. The activity of glutathione S-transferase (GST) of larvae treated with spinetoram has significantly increased, whereas the enzyme activity was significantly inhibited following cyfluthrin and non-significantly inhibited following profenofos, lufenuron, and emamectin benzoate application. There were no significant differences between treated and untreated larvae in ACP activity. In contrast, the alkaline phosphatase (ALP) activity of larvae treated with tested insecticides significantly increased, while the activity was inhibited following cyfluthrin application. On the other hand, spinetoram, emamectin benzoate, and lufenuron exhibited significant increment in the protein content, whereas there was no significant effect following either cyfluthrin or profenofos treatments. In summary, the present results suggest that not only the lethal effects but also the sublethal effects of newly tested insecticides could have a negative influence on the dynamics of CLW; thus, these sublethal effects can be integrated into pest control to reduce the overuse and misuse of insecticides. This effect appears reduction in the activity of detoxification enzymes, resulting in response to the tested insecticide by the lowest concentration of (sublethal doses). Also, the inhibition of detoxification enzymes, which represents defensive reactions in insects, is playing important role in reducing resistance in S. littoralis, which is one of the most dangerous pests of all agricultural crops in the world. Therefore, these results were valuable for the practical use of these insecticides in IPM programs.


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Results: The emamectin benzoate was the most toxic compound followed by lufenuron, cyfluthrin, spinetoram, and profenofos with the corresponding LC 50 values of 0. 05, 49.18, 70.99, 130.26, and 156.78 ppm respectively. The results showed that all the tested insecticides at LC 25 value gave a slight inhibition of the acetylcholinesterase (AChE) activity, except profenofos was the most potent one. The activity of glutathione S-transferase (GST) of larvae treated with spinetoram has significantly increased, whereas the enzyme activity was significantly inhibited following cyfluthrin and non-significantly inhibited following profenofos, lufenuron, and emamectin benzoate application. There were no significant differences between treated and untreated larvae in ACP activity. In contrast, the alkaline phosphatase (ALP) activity of larvae treated with tested insecticides significantly increased, while the activity was inhibited following cyfluthrin application. On the other hand, spinetoram, emamectin benzoate, and lufenuron exhibited significant increment in the protein content, whereas there was no significant effect following either cyfluthrin or profenofos treatments.
Conclusions: In summary, the present results suggest that not only the lethal effects but also the sublethal effects of newly tested insecticides could have a negative influence on the dynamics of CLW; thus, these sublethal effects can be integrated into pest control to reduce the overuse and misuse of insecticides. This effect appears reduction in the activity of detoxification enzymes, resulting in response to the tested insecticide by the lowest concentration of (sublethal doses). Also, the inhibition of detoxification enzymes, which represents defensive reactions in insects, is playing important role in reducing resistance in S. littoralis, which is one of the most dangerous pests of all agricultural crops in the world. Therefore, these results were valuable for the practical use of these insecticides in IPM programs.

Background
During the last few decades, many species of insects have acquired resistance to insecticides. This considered resistance is proved to be one of the major obstacles in the successful control of insects and the biggest challenge faced by the pesticide research today due to the critical role of insecticide in reducing insect when the economic threshold (ETL) is exceeded, and insecticides are currently the principal method for controlling the Egyptian cotton leafworm (CLW), Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae), in Egypt and will likely continue to be used until more biologically based management systems could be developed (Subramanian and Shankarganesh 2016). Therefore, it was necessary to study the effect of some new insecticides that appeared in the Egyptian market on S. littoralis, in terms of their toxicity and their effect on enzymes that plays a defensive role in changing the response to insecticide in S. littoralis becomes extremely tolerant of the action of insecticides because of the intensive treatments of these insecticides. Therefore, new insecticides with modes of action that differed from conventional insecticide are highly desirable in integrated pest management (IPM) programs among these insecticides are insect growth regulators (IGRs), avermectin, and spinosyn insecticide group (Subramanian and Shankarganesh, 2016;Wei et al., 2018). Different mechanisms of resistance to organophosphate and pyrethroid insecticides have been identified in S. littoralis, including enhanced metabolism, nerve insensitivity, reduced penetration, and target site insensitivity; it is caused by defensive enzymes in the insect (Rong et al., 2017). Among the defensive enzymes are glutathione S-transferases (GST) and phosphatases (APs). GST is an enzyme widespread in both prokaryotic and eukaryotic cells and catalyzes the glutathione conjugation reaction with reduced glutathione (GSH) (Armstrong, 1997;Listowsky et al., 1998).
The present work aims to evaluate the sub-lethal effects of the five compounds (profenofos, cyfluthrin. emamectin benzoate, lufenuron, and spinetoram) on the fourth instar larvae of S. littoralis field populations compared with the laboratory strain. Also, these compounds may manipulate the physical and biochemical processes of the insect by influencing relative enzymes. Therefore, the activities of acetylcholinesterase (AChE), glutathione S-transferases (GST), alkaline phosphatase (ALP), acid phosphatase (ACP), and protein content were determined in surviving larvae after treatment.

Toxicity studies Test insects
Laboratory strain Laboratory strain (L-strain) of S. littoralis was obtained from central laboratory of pesticides, Agricultural Research Center (ARC) Cairo, Egypt, and reared on castor oil leaves under laboratory conditions (27 ± 2°C and 65 ± 5% RH) for several years, according to Eldefrawi et al. (1964).
Field strain Field strain (F-strain) of S. littoralis used in this study was obtained from the cotton field at Vaccus District, El-Sharkia Governorate (Egypt) during the season 2019. Samples were brought to the laboratory as egg masses. The hatched larvae were reared on castor oil leaves under the same conditions.

Bioassays
Bioassays were performed on the 4th instars larvae both L4-and F4-strains to assess the activity of the fivementioned insecticides. A series of aqueous concentrations for each insecticide were prepared using the commercial formulations. The leaf dipping technique was used to determine the median lethal concentration (LC 50 ) values; fresh castor oil leaves were cut into discs (2 cm 2 ). Each disc was dipped for 30 s in one of the prepared concentrations. The treated leaves had discs dried under laboratory conditions before being offered to the larvae. Ten larvae (40-50 mg/larvae), replicated 3× were used for each concentration. Larvae were fed on leaves immersed in only water as a control. Newly moulted 4th larval instars for L-and F-strains were fed on the treated leaves in a glass jar covered with muslin for 24 h with the treated discs insecticides. Another untreated one replaced the treated leaves. Mortality percentages were recorded after 24 h of treatment for profenofos and cyfluthrin and after 72 h for lufenuron, emamectin benzoate, and spinetoram. The corrected mortality was calculated by using Abbott's (1925) formula. Data were subjected to Probit analysis, as described by Finney (1971). Resistance ratio (RR) as field/ laboratory (F/L) was calculated by dividing the LC 50 value of the F-strain over that of the L-strain.

Biochemical studies Preparation of samples
The survived larvae after 24 h of treatment were fed on discs treated with the corresponding LC 25 values concentrations of the tested insecticides. The LC 25 values were 0.332, 4.732, 7.75, 12.09, and 15.35 ppm for emamectin benzoate, lufenuron, cyfluthrin, profenofos, and spinetoram respectively. The collected midguts of the L4 were rinsed and homogenized in distilled water (DW) using a Teflon homogenizer surrounded with a jacket of crushed ice for 3 min. Homogenates were centrifuged at 6000 rpm for 10 min at 5°C; the supernatant was used as an enzyme for AChE, GST, ALP, ACP and the determination of protein content.

Statistical analysis
All the quantitative estimation of toxicity and biochemical parameters were based on three replicated, and the values were expressed as mean ± SE. The data were statistically analyzed separately for each experiment and were subjected to ANOVA using SPSS 12.0 software (Statistical Package for Social Sciences, USA). Mean values were compared using Duncan's multiple range test (1955).  (Table 1). Also, data showed that the F-strain exerts high resistance levels towards profenofos and cyfluthrin resistance ratios were 122.48 and 68.26, respectively. The 4th instar larvae of the F-strain showed no resistance to emamectin benzoate and spinetoram, and resistance ratios 9.42 towards lufenuron.

Effect on AChE
The in vivo effect of tested insecticides on the activity of AChE in larvae of S. littoralis is presented in Table 2. It was found that all the tested insecticides slightly inhibited the activities of AChE except that the profenofos was the only one most inhibitor. Table 2 show that the cyfluthrin inhibited the activity of GST in S. littoralis by 55.47, while spinetoram significantly induced the enzyme activity by 276.14% of control.

Effect on GST Results listed in
Phosphatases ALP activity in larvae treated with profenofos, lufenuron, emamectin benzoate, or spinetoram had significantly increased. However, its activity was significantly dropped following the treatment with cyfluthrin (Table 3). Moreover, Table 3 showed that there were no significant differences in the activity of ACP between treated and untreated larvae.
Effect on total protein Results in Table 4 showed that there was a significant enhancement in the protein content of the larvae treated with spinetoram. However, cyfluthrin and profenofos treatments were similar to those of the control.

Discussion
Insecticide resistance in Lepidopteran is a major concern throughout the world because several species of it have been reported to have developed resistance to several classes of insecticides Wei et al. (2018). The present study revealed high levels of resistance to profenofos and cyfluthrin in the tested field population; at the same time, emamectin benzoate, spinetoram, and lufenuron showed low levels of resistance. The efficiency of different tested compounds in the control of the L4 and F4 of the S. littoralis varied tremendously according to the different modes of action for the tested compounds. These results agreed with those obtained by Ismail (2008), who found that the S. littoralis larvae collected from a cotton field that was heavily sprayed with conventional insecticides showed strong resistance to OPs and pyrethroids. In contrast, the LC 50 values of the F-strains were 120× Activity is expressed as μmole acetylthiocholine hydrolyzed/min. mg protein 2 Activity is expressed as nmole CDNB conjugated formed/min. mg protein Means in the same column followed by the same letters are not significantly at P = 0.05 Table 3 In vivo effect of the tested compounds on the activity of ACP and ALP of S. littoralis larvae and 102× more resistant than the L-strain to the OP chlorpyrifos and the pyrethroid cypermethrin, respectively. Also, our results are similar to El-Sheikh (2015) reported that the F-strain showed low resistance ratios to emamectin benzoate, lufenuron, and spinosad. Meanwhile, fenvalerate-and cypermethrin-resistant larvae of Helicoverpa armigera Hubner (Lepidoptera: Noctuidae) showed higher activities of esterases, APs, and methyl paraoxon hydrolase, compared with susceptible larvae (Oppenoorth, 1985). Since many toxicants show secondary actions at high concentrations, the sublethal dose (LC 25 ) was used in this study to measure the biochemical changes in the L4 larvae of S. littoralis following treatment with five compounds mentioned previously. Current results show that the profenofos recorded a significant decrease in the activity of AChE; this inhibition may be occurred as a result of the blocking of the action potential of the nervous system caused by the toxic effect of larvae treated with profenofos because organophosphorus insecticides are neurotoxic by acting as inhibitors of neuronal cholinesterase activity. Generally, this indicated that the number of active sites on the AChE of the larvae was increased in the F-strain; this explains the low susceptibility of F-strain for profenofos, our results agreement with Richardson et al. (2001); El-Sheikh (2012); Abd El-Aziz and Shaurub (2014). On the contrary, GST activity was increased in treated larvae; this enzyme plays a role in detoxification mechanism in insects; therefore, may be an overproduction led to the toxic effect of treated larvae. This results parallel with Motoyama (1978), Oppenoorth et al. (1979); Kristensen (2005), they reported that GST plays a physiological role in detoxication and elimination of toxic compounds, and therefore, the increment of GST activity may protect against toxic compounds; also, Wang et al. (2010) reported that GST was higher in treated larvae than their control. On the other hand, the present study showed significantly increased ALP activity, and decreased ACP activity; the reaction on ALP and ACP activities might be due to tissue damage, where the enhanced activity could be related to the influence of glucocorticoids or could be released from ruptured cells due to the effect of pesticide (Abou-Donia et al., 1986); also, Trowsdale et al. (1990) reported that the increment of ACP activity seems to result from enhanced enzyme turnover under pesticide stress. In contrast, the reaction of its activity may be related to leakage of the enzyme into the extracellular compartment.
The total protein is a major component necessary for an organism to develop, grow, and perform its vital activities; thus, the effect on protein leads to a cell disorder. From this study, a significant increase in the total protein content was detected in treated larvae with tested compounds; thus, this effect leads to a cell disorder. Our result agreement with Muthusamy and Ramkumar (2011) reported that changing the behavioral patterns associated with enzymes and/or the exchange of chemical information can be due to alteration in total protein reserves of the larvae tissues which could be partly due to the stress of the tested compounds of an imbalance between the rate of protein synthesis and the rate of biodegradation.

Conclusion
The Egyptian cotton leafworm, Spodoptera littoralis, a destructive insect pest for a wide range of host plants. Because of the extensive and continuous use of traditional insecticides, this led to the development of resistance in this insect, so there is a constant demand to use new control agents. Therefore, from this study, it could be recommended to use emamectin benzoate, lufenuron, and spinetoram because these compounds have modes of action that differed from conventional insecticides like profenofos and cyfluthrin. These results are valuable for the practical use of these compounds in Integrated Pest Management (IPM) programs to reduce the phenomenon of resistance in this insect pest.

Author's contributions
The author contributed to the production and writing of the manuscript. The author(s) read and approved the final manuscript.

Funding
There was no funding for this work.

Availability of data and materials
All data generated during this study are included in this published article.

Ethics approval and consent to participate
The manuscript does not contain any studies involving human participants, human data or human tissue.

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Not applicable.

Competing interests
The author declares that there are no competing interests.