A comparative study between a commercial mixture compound and its individual active ingredients on the cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) on tomatoes under semi-field conditions

Tomato, Solanum lycopersicum L. (Solanales: Solanaceae) is the second most important vegetable crop in Egypt and is infested with many insect pests. The cotton leafworm, Spodoptera littoralis (Boisd.) causes severe economic losses in tomatoes and many other crops. Many management strategies were developed in order to manage the economic losses obtained. In this context, the present study was conducted to evaluate the effectiveness using a mixture of Emamectin benzoate and lufenuron or using them solely against the 2nd and 4th instar larvae of S. littoralis under semi-field conditions. The obtained results showed that the mixture compound show high initial killing effect against 2nd and 4th instar larvae in both growing seasons. Furthermore, the residual effect of the tested compounds also showed the efficiency of the mixture over the solitary active ingredients. In addition, the treatment of the 4th instar larvae with the LC50 of the tested compounds showed significant impacts against the soluble protein, carbohydrate, lipid contents, and the detoxification enzymes. In conclusion, the results showed that the emamectin benzoate and lufenuron could be safe and effective substitute for conventional insecticides either applied solely or in combination.


2021) and genetically modified crops
were developed in order to lessen the economic losses obtained. The management strategy of cotton leafworm in Egypt has depended on preserving and extending the insecticidal efficacy based on rotating various insecticides including organophosphates, carbamates, insect growth regulators, and pyrethroids every year. The extensive use of conventional insecticidal compounds caused many serious problems such as high resistance to many chemical pesticides, resurgence, and residues of chemical pesticides in the environment (Forgash, 1984;Hawkins et al., 2019). Consequently, considerable effort should be performed to develop alternative or additional techniques, which would allow a rational use of pesticides and provides adequate crop protection for sustainable food, feed, and fiber production. Among the most promising and excellent alternatives are avermectin insecticide group and insect growth regulators (IGRs) (Abdel-Baky et al., 2019; Barrania & Selim, 2020;El-Sheikh, 2015;Metayi et al., 2015). The major advantage of using IGRs is that they have impacts on insect growth regulator hormones that are specific for insects and not for animals or humans. In addition, IGRs have great selectivity to the target insect species, so they are likely less harmful to natural enemies when compared with the broader spectrum insecticides (El-Sheikh, 2015;Grafton-Cardwell et al., 2005). Lufenuron, a chitin synthesis inhibitor, influences the development of lepidopteran larvae and causes the production of infertile eggs. Treated insects develop normally until molting then fail to complete the molt due to the inhibition of the synthesis of new cuticle (Tunaz & Uygun, 2004). Emamectin benzoate is a second-generation avermectin analog with exceptional activity against lepidopterans (Terán-Vargas et al., 1997). Emamectin benzoate acts as a chloride channel activator, which decreases the excitability of neurons. Shortly after exposure, the insect larvae stop feeding, become irreversibly paralyzed, and die in 3-4 days (Grafton-Cardwell et al., 2005). Recently, many agricultural services companies offer commercial mixture compounds. Using such compounds can grant a noteworthy progress for Insect Pest Management programs (IPMs), including the potential impact for lowering the quantities of each agent used. Such reduction would mean supposedly lowering costs, lowering environmental pollution, lessening damage to beneficial organisms and reducing selection pressure leading to the development of resistance to each agent (El-Sheikh, 2015;Kandil et al., 2020;Khatun et al., 2015;Korrat et al., 2012). Accordingly, the current study was conducted to detect the efficiency of Lufenuron and Emamectin benzoate either alone or in combination against the 2nd and 4th instar larvae of S. littoralis under semi-field conditions. In addition, the biochemical effect of the tested compounds alone and in combination on the soluble vital contents like proteins, carbohydrates and lipids was investigated. Furthermore, the effect of the tested compounds on some enzyme activity was also determined.

Tested insects
A laboratory strain of S. littoralis was obtained as egg masses from the Research Division of the cotton leaf worm, Plant Protection Research Institute, Agricultural Research Center, Dokki, Giza, Egypt. These eggs were kept in plastic cups covered with gauze under laboratory condition of 27 ± 2 °C and 65 ± 5% R.H. until hatching. The newly hatched larvae were offered fresh and clean castor bean leaves, Ricinus communis L., and were checked on daily basis for adding more leaves if needed (Eldefrawi et al., 1964;El-Guindy et al., 1979). The 2nd and 4th instar larvae were employed for further investigations.

Tested compounds
Three commercial insecticidal compounds were tested against the 2nd and 4th instar larvae of S. littoralis. An emamectin benzoate compound under the trade name Pasha ® (EC 1.9%) with a recommended application rate is 250 ml/feddan. It was obtained from ElHelb Pesticides and Chemicals-Egypt. A chitin synthesis inhibitor compound (Lufenuron) under the trade name Cymex ® (EC 5%) was obtained from Shoura Chemicals-Egypt and has a recommended application rate of 160 ml/feddan. The third compound was a commercial mixture of both Lufenuron and emamectin benzoate with the trade name Heater ® (Lufenuron 2% + Emamectin benzoate 1%) (SC 3%). It was supplied from Starchem Industrial Chemicals-Egypt at the application rate of 100 ml/100L (Table 1).

Semi-field application
In order to evaluate the efficacy of the tested compounds against S. littoralis larvae, a semi-field experiment was executed. The study was carried out throughout 2019 and 2020 late winter season at El-Dakhaly village (30°42′38.3"N 30°45′52.9"E), the western side of Rashid branch, Kom Hamada Center, Beheira Governorate, Egypt. The field area was cultivated with Alisa tomato variety on February the 9th, 2019 and February the 8th, 2020, respectively. The standard agricultural practices were applied. The experimental area was divided into plates of 1/16 feddan (Feddan = 4168.27m 2 ; 1/16 feddan = 262.5 m 2 ). The treatment was arranged in randomized complete blocks design (RCBD) with four replicates each. Application of the tested compounds was on March 11 in both growing seasons. Temperature in the experiment area were 23-27 ± 2 °C and the relative humidity was 65-75 ± 10%. The tomato leaves were sprayed using a backpack sprayer. To determine the initial (24 h. post spraying) and residual (7-and 10-days post spraying) effects of the tested compounds, treated tomato leaves were collected after 24 h, 7-days, and 10-days post spraying. Collected leaves were then transferred directly to the laboratory and offered to separate sets of the 2nd and 4th instar larvae of the cotton leaf worm. For the control group, 2nd and 4th instar larvae were offered untreated tomato leaves. Larvae were left to feed on treated leaves for 48 h and larval mortalities were recorded. Mortality percentage was corrected according to Abbott's formula (Abbott, 1925).

Determination of LC values of the tested compounds
In order to determine the LC 50 and LC 90 values of the tested compounds for the 2nd and 4th instar larvae, a toxicity test was carried out using leaf-dipping technique (Abo El-Ghar et al., 1994). Dry and clean castor bean leaves were dipped for 10 s in six different concentrations of the tested compounds, then left to air dry at room temperature and then offered to 2nd and 4th instar larvae in clean jars, each jar containing 20 larvae. Four replicates were used for each concentration of each treatment. Leaves dipped in water served as untreated group.

Biochemical assay Preparation of insect samples
The insects were prepared as previously described by (Amin, 1998). The 4th instar larvae were treated with the LC 50 of tested compounds for 24 h. One gram of the larvae that survived treatment was weighed and were homogenized in distilled water (50 mg/1 ml). Homogenates were centrifuged at 8000 rpm for 15 min. at 4 °C in a cooling-centrifuge. The deposits were discarded and the supernatant, which is referred as enzyme extract, can be stored for at least one week without significant loss of activity when stored at 50 °C.

Determination of total proteins, total carbohydrates, and total lipids
The impact of the LC 50 of tested compounds on the total proteins, total carbohydrate, and total lipids of the 4th instar larvae was assayed according to (Bradford, 1976), (DuBois et al., 1956), and (Knight et al., 1972), respectively.

Determination of enzyme activities
The activity of α-and β-esterases were determined according to (van Asperen, 1962). The activity of chitinase was assayed according to (Bade & Stinson, 1981). The Glutathione S-transferase (GST) activity was determined according to (Habig et al., 1974).

Statistical data analysis
All evaluated toxicity and physiological parameters were analyzed based on three replicates and the values are expressed as mean ± standard error. The data were statistically analyzed separately for each experiment and were subjected to analysis of variance (ANOVA) using SPSS 17.0 release 17.0.0 software (Statistical Package for Social Sciences, USA). Means were compared according to (Snedecor & Cochran, 1980) and they were considered significant at P ≤ 0.05. Differences between the treatments were determined by Tukey's multiple range test (P ≤ 0.05) (Snedecor and Cochran, 1989). The LC 50 values that obtained by regression lines according to (Finney, 1971) using "LdPLine ® " software. The reduction percentage for each treatment was calculated by Henderson and Tilton's formula (Henderson & Tilton, 1955).

Semi-field application
The initial (24 h. post spraying) and residual (7-and 10-days post spraying) effects of Heater ® , Pasha ® , and Cymex ® against the 2nd and 4th instar larvae were evaluated during 2019 and 2020 growing seasons under semifield conditions. With regards to the initial effect of the tested compounds after one-day post treatment, the Pasha ® and Cymex ® exhibited more toxic effect than heater ® against both the 2nd and 4th instar larvae during both growing seasons. Moreover, the residual effect of the tested compounds against the 2nd and 4th instar larvae showed that Heater had the highest residual effect through both growing seasons (Tables 2 and 3). On the other hand, it was noted that the larval mortality caused by tested compounds was decreased in the 2nd growing season compared to the 1st one.

Toxicity of the tested compounds
Results in Table 4 shows the LC 50 and LC 90 values of the tested compounds against the 2nd and 4th instar larvae of S. littoralis. Results showed that heater ® exhibited the highest toxic effect according to the obtained LC 50 values. In addition, the 2nd instar larvae were more susceptible than the 4th instar larvae. This was observed through the low LC 50 values. Moreover, Cymax ® was toxic than Pasha ® . Furthermore, results showed that the 2nd instar larvae were more susceptible than the 4th instar larvae. This was also observed through the low LC 50 values determined for 2nd instar larvae compared to the 4th instar.

Effect of the tested compounds on total protein, total carbohydrates, and total lipids
The latent effect of treatment of the 4th instar larvae with the LC 50 of the tested compounds on total proteins, total carbohydrates, and total lipids is presented in Table 5. Treatment with the tested compounds decreased the total proteins, total carbohydrates, and total lipids compared to the control. In addition, Heater ® was the most efficacious among the tested compounds as the reduction in the total proteins, total carbohydrates, and total lipids was more obvious.

Effect of the tested compounds on the detoxifying enzyme activities:
Results presented in Tables 6 and 7 show the effect of treatment of the 4th instar larvae with the LC 50 of the tested compounds on some detoxifying enzymes; nonspecific esterases (α-and β-esterase), chitinase, and glutathione s-transferase (GST) activity. Results showed increased α-esterase activity which was significant in case of Heater ® and cymax ® but not significant in Pasha ® treatment compared to control. Furthermore, results also revealed increased β-esterase activity which was insignificant compared to control. Results also manifested that nevertheless the tested compound α-esterase activity exhibited higher activity for detoxification than β-esterase. Moreover, the activity of both chitinase and GST increased due to treatment with the tested compounds. However, a significant increase in chitinase activity was detected in both Heater ® and Cymax ® but insignificant increase was observed in Pasha ® treatment. In addition, a significant increase was observed in GST activity in both Heater ® and Pasha ® treatment and no significant difference was detected in case of Cymax ® treatment.

Discussion
The present study was conducted in order to evaluate the efficacy of lufenuron and emamectin benzoate applied alone and as a mixture. Three commercial compounds were selected; Heater ® , Pasha ® , and Cymax ® , which were a commercial mixture of emamectin benzoate and lufenuron, emamectin benzoate, and lufenuron, respectively. The selected compounds were tested against the 2nd and 4th instar larvae under semi-field conditions and in vitro. The results obtained showed that the tested compounds displayed high initial kill against both 2nd and 4th instar larvae. Further, the mixture compound (Heater ® ) showed higher toxicity against both instar larvae more than the sole compounds. These results were in the same trend (Abdu-Allah, 2011; El-Sheikh, 2015) when treating S. littoralis larvae with emamectin benzoate and IGR compounds under semi-field conditions. Moreover, results showed that Heater ® exhibited the highest toxic effect according to obtained LC 50 values. These results agreed with (Abdel-Baky et al., 2019;El-Sheikh, 2015;Khatun et al., 2015). In addition, the 2nd instar larvae  were more susceptible than the 4th instar larvae. This may be due to differences in size and defense mechanisms between instars. This was well documented previously (Abdu-Allah, 2011; Bengochea et al., 2014;Qayyum et al., 2020). Furthermore, the obtained results showed that the total proteins, total carbohydrates, and total lipids were decreased due to treatment with the LC 50 of tested compounds. The changes in energy reserves such as carbohydrates, lipids, proteins, and glycogen indicate the susceptibility of the insect to insecticide and its function alterations (Piri et al., 2014). Proteins are important for individual-level fitness associated traits such as body size, growth rate, and fecundity, and at higher levels of organization they have been linked to population dynamics, life histories, and even biological diversification (Fagan et al., 2002). Reduction in total proteins may be attributes to the high toxicity of tested compounds either in mixture (Heater) or solitary (Pasha ® and Cymex ® ). In addition, the decreased content of proteins could be due to the breakdown of protein into amino acids, so with the entrance of these amino acids to tricarboxylic acid cycle (TCA) as a keto acid, they will help supply energy for the insect. So, protein depletion in tissues may constitute a physiological mechanism and might play a role in compensatory mechanisms under insecticidal stress to provide intermediates to the TCA cycle by retaining free amino acid content in hemolymph (Nath et al., 1997). These results agreed with (Abdel-Hafez & Osman, 2013;Assar et al., 2016;Kola et al., 2015;Saleh & Abdel-Gawad, 2018;Talleh et al., 2020) who detected reduction in soluble protein contents when treated different insects with emamectin and IGRs insecticides. Carbohydrates are an important source of energy for insects. Carbohydrates may be converted to lipids and may contribute to the production of amino acids. Many carbohydrates such as sugars are powerful feeding stimulants (Genç, 2006). The carbohydrate reduction may be because the increased metabolism under toxicant stress. The carbohydrate reduction suggests the possibility of active glycogenolysis and glycolytic pathway to provide excess energy in stress condition (Abdel-Hafez & Osman, 2013;Balan et al., 2008;Franeta et al., 2018;Vojoudi et al., 2017). Our results were concurrent with (Abdel-Mageed et al., 2018;Assar et al., 2016;El-Sobki & Ali, 2020;Hamadah et al., 2015;Kola et al., 2015;Osman et al., 2015;Saleh & Abdel-Gawad, 2018) who found reduction in total carbohydrate contents in different insects after treatment with sublethal concentrations of different insecticides. Lipids in living organisms consist of free and bound fatty acids, short and long chain alcohols, steroids and their esters, phospholipids, and other groups of compounds. Insects are able to convert carbohydrates into lipids, and many insects can synthesize lipids and accumulate them in fat body tissue. Fatty acids, phospholipids, and sterols are components of cell walls in addition to having other specific functions (Piri et al., 2014). Similar reduction in total lipid contents were also reported when treated S. littoralis larvae with emamectin and IGRs (Assar et al., 2016;Awadalla et al., 2017). The reduction in total lipid contents could be due to that the detoxification process in larvae, demands the transformation of large quantity of consumed food into energy after treatment with insecticides (Xu et al., 2016). In addition, treatment with the LC 50 of the tested compounds increased the activity of the non-specific esterases compared to control. The esterase enzymes belong to the detoxifying enzymes which are responsible for the detoxification of any foreign substance in insect's body. Moreover, esterase is an important detoxifying enzyme which hydrolyzes the esteric bond in any toxicant. Also, esterase is one of the enzymes showing the strongest reaction to environmental stimulation (Hemingway & Karunaratne, 1998). Their high activity may be an indication of the insect's response to body intoxication and may be consider as a remark of resistance development (Ahmed & Freed, 2021;Chen et al., 2017;Serebrov et al., 2006). Furthermore, it is well known that any infectious disease for insect regardless of the infection-causing factor, leads to increased activity of detoxifying enzymes in general, and the esterases in particular (Zibaee, Bandani, et al., 2009;Zibaee, Bandani, et al., 2009;Zibaee, Sendi, et al., 2009;Zibaee, Sendi, et al., 2009). The obtained results agreed with (Abdel-Baky et al., 2019;El-Helaly et al., 2020;Korrat et al., 2012;Pineda et al., 2007) as treating S. littoralis larvae with sublethal concentration of emamectin benzoate and IGRs. Over and above, results revealed significant increase in GST activity due to treatment. The exposure to sublethal doses of insecticides will activate immune response and induce detoxifying enzymes such as glutathione S-transferases that are responsible for insecticide tolerance or resistance (Vojoudi et al., 2017). These enzymes degrade the toxic chemicals in insects before reaching the target sites (Bogwitz et al., 2005). GST gained its importance from its role in the degradation of insecticides and toxic substances. In addition, GST takes part in metabolite removal, protection of tissues from damage by free radicals, and may play a role in protecting insects from pathogen infection and toxicants (Hayes et al., 2005;Papadopoulos et al., 2000). The increased GST may be due to overproduction induced by the treatment with the tested compounds as a protective mechanism against those compounds (Ismail, 2020;Kristensen, 2005). Our results were correspondence to (Ahmed & Freed, 2021;Franeta et al., 2018;Vojoudi et al., 2017) when treating different insects with insecticides.

Conclusion
In conclusion, the results showed that the emamectin benzoate and lufenuron could be safe and effective substitute for conventional insecticides either applied solely or in combination. In addition, the mixture of both active ingredient has a high initial kill against the 2nd and 4th instar larvae which can help lowering the quantity of the applied compounds.