Chemical composition and bioactivities of Melaleuca alternufolia essential oil and its main constituents against Spodoptera littoralis (Boisaduval, 1833)

Spodoptera littoralis is mostly controlled by the use of synthetic insecticides. Nonetheless, the use of these insecticides causes a slew of issues. On this pest, the antifeedant activity of Melaleuca alternifolia essential oil (EO) and its two principal components was investigated. The gas chromatography–mass spectrometry (GC/MS) analysis revealed that the M. alternifolia EO was composed of eleven compounds. Terpinen-4-ol (40.1%) and γ-terpinene (21.9%) were chosen as the major constituents. In terms of antifeedant efficacy, treatment with M. alternifolia EO and these components reduced leaf consumption and the efficiency of food conversion in larvae in a concentration-dependent manner. When compared to untreated larvae, weight, growth, and pupation percentage were all significantly lower. The findings show that M. alternifolia EO and its components, terpinen-4-ol and γ-terpinene can be effectively combined for cotton leafworm management. The chemical composition of M. alternifolia EO was identified by GC–MS. The EO of M. alternifolia and its components, terpinen-4-ol and γ-terpinene, had potent bioactivity against S. littoralis. M. alternifolia EO and their components, terpinen-4-ol and γ-terpinene have great potential as a biopesticide in integrated pest management programs. The chemical composition of M. alternifolia EO was identified by GC–MS. The EO of M. alternifolia and its components, terpinen-4-ol and γ-terpinene, had potent bioactivity against S. littoralis. M. alternifolia EO and their components, terpinen-4-ol and γ-terpinene have great potential as a biopesticide in integrated pest management programs.


Background
The cotton leafworm is a destructive pest that causes significant damage and economic losses in Egypt and other nations. Larval instars are the insect's most damaging stage, capable of entirely destroying or severely reducing crop yields in over 100 economically significant species, including cereal crops, vegetables, and ornamental plants (Ismail et al. 2020). For decades, chemical synthetic insecticides have been used to control this pest, with injudicious use contributing to an increase in the likelihood of resistance evolution (Whalon et al. 2006; Ismail Page 2 of 6 Ismail et al. Bulletin of the National Research Centre (2022) 46:157 2019), as well as risks to human health, untargeted organisms, the environment, and residue issues (Sharma et al. 2019). As a result, successful pest control necessitates the development of new compounds with unique modes of action that are also low-risk. EOs have recently gained popularity as an alternative pest management method, due to its quick biodegradability, economic application, minimal toxicity to mammals, and environmental safety (Gerwick and Sparks 2014; Nollet and Rathore 2017). Furthermore, EOs are made up of a variety of compounds whose mode of action inhibits pest resistance from evolving (Nollet and Rathore 2017). On the other hand, EOs have mostly been studied as natural fumigants for the control of stored-product insects and are rarely used in lepidoptera prevention and control. The nutritional indicators and digestion were the important characteristics examined in this study to evaluate the M. alternifolia EO and its two main components, terpinen-4-ol and γ-terpinene, for cotton leafworm larvae.

Insect rearing
The second-instar larvae of Spodoptera littoralis used in bioassays were obtained from a laboratory susceptible culture in the Department of Insect Population Toxicology, Central Agricultural Pesticides Laboratory, Agriculture Research Center, Giza, Egypt. For several years, the culture was maintained at 25 ± 1 °C, 65 ± 5% relative humidity and a photoperiod of 16:8 (L:D) in the rearing chamber without being exposed to any pesticides. Larvae were fed soft, fresh castor bean leaves (Ricinus communis L.) in sterilized glass jars with muslin coverings. The growing larvae were transferred daily to other clean, sterile glass jars to avoid infection from feces and provided with fresh leaves for feeding. All of the experiments in this study were carried out under the same controlled conditions mentioned above.

Melaleuca alternifolia EO analyzed by gas chromatography-mass spectrometry
The composition of Melaleuca alternifolia essential oil was measured with a Trace GC Ultra-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG-5MS (30 m × 0.25 mm × 0.25 µm film thickness). M. alternifolia EO was diluted in diethyl ether before being injected to the GC/MS. The used carrier gas was helium (flow rate of 1 mL/min). The solvent delay was 3 min, and diluted sample (1 µL) was injected automatically in splitless mode with Autosampler AS1300 coupled with GC. The column oven temperature program and the separation conditions were as follows: At the temperature of 50 °C, the column oven was initially held, then by 5 °C/min, the temperature was increased to 250 °C and held for 2 min. By 30 °C/min, the final temperature was increased to 300 °C and held for 2 min. The temperatures of the injector and MS transfer line were kept at 270 and 260 °C, respectively. At 70 eV ionization voltages, the EI mass spectra were collected at the m/z range of 50-650 in full-scan mode. The temperature of ion source was set at 200 °C. Chemical constituents were identified based on their retention time (RT), with the mass spectra with those of Wiley 09 and NIST 14 mass spectral database the percentage of components was calculated by the GC peak area.

Efficiency measures of nutritional indices
To evaluate the effect of M. alternifolia EO (purchased from local market, Egyptian Natural Co.), terpinen-4-ol, and γ-terpinene were purchased from Sigma-Aldrich Chemical Ltd. (St. Louis, MO, USA) on the nutritional physiology in larvae of S. littoralis. Fresh castor bean leaves were used to prepare the leaf discs (8 cm diameter). A monopan balance was used to measure all of the weights, which were accurate to 0.1 mg (Sartorius GMBH, Type: A 120 S). For 20 s, leaf discs were dipped in five concentrations of each M. alternifolia EO, terpinen-4-ol and γ-terpinene (250, 500, 1000, 2000, and 4000 mg/L). Control leaves were submerged in diluted water containing Triton X-100. Newly molted secondinstar larvae (< 24 h) were chosen and weighed after being denied nourishment for 4 h. This experiment was conducted in 4 replications, each with 10 larvae/concentration. Larvae were allowed to feed on treated leaves for 72 h, which were changed every 24 h and replaced with fresh treated leaves, after which they were allowed to feed on untreated leaves. Each replicate's feces and unconsumed treated leaves were weighed every 24 h during the 72-h feeding period. Farrar et al. 's formula (1989) was used to calculate nutritional indicators as follows: Approximate digestibility: Efficiency of conversion of ingested food: Efficiency of conversion of digested food: Consumption rate: Relative growth rate: CR (mg/mg/day) = C/TA, where A weight of larvae during the feeding period (mg), C weight of food consumed (mg), F weight of feces produced (mg), G weight gain of larvae (mg), T the duration of feeding period (day). The larvae that survived were then transferred to sterilized glass jars, fed fresh untreated leaves, and monitored daily until pupation. When larvae were probed with fine brush and did not move, they were assumed to be dead. The larval growth index was calculated as follows (Itoyama et al. 1999): Larval growth rate: where P pupation (%), T the duration of the larval period (day).

Statistical analysis
The data were calculated as mean ± SE and analyzed statistically. One-way analysis of variance (ANOVA) was used to evaluate statistically significant differences between individual means using SAS software. Mean values were analyzed with Tukey's test at the 0.05 level of probability or less.

Results
The chemical components of the M. alternifolia EO GC/MS revealed 11 aromatic components, accounting for 92.6 percent of the total oil (Table 1). The main components of M. alternifolia EO were terpinen-4-ol (40.1%), γ-terpinene (21.9%), and other minor components. The oxygenated monoterpenes were the most common chemical group in the oil's chemical composition.

Nutritional indices
All nutritional indicators of different concentrations of M. alternifolia EO, terpinen-4-ol, and γ-terpinene on the feeding efficiency of S. littoralis larvae were significantly reduced when compared to the control ( Table 2). The weight gain was the lowest in larvae treated with M. alternifolia EO. All treatments led to a significant decrease in weight gain (P ≤ 0.05) than the control group due to lower consumption rate (CR) and relative growth rate (RGR). In treated larvae, there was a significant decrease in digestibility (AD), with significant differences from untreated larvae. The efficiency of converting ingested food (ECI) and conversion of digested food (ECD) values decreased with increasing concentration. M. alternifolia EO was the most effective at a concentration of 4000 mg/L, showing the lowest values of ECI (4.29%) and ECD (4.51%).

Antifeedant activity
The results in Table 3 show that the antifeedant activity on second instar larvae of S. littoralis reveal different values according to treatments and concentrations. M. alternifolia EO treatment at all concentrations (250, 500, 1000, 2000, and 4000 mg/L) was higher than terpinen-4-ol, and γ-terpinen treatments as compared to the untreated control. The most significant increase has been found with 4000 mg/ L of M. alternifolia EO. Table 4 shows that the treatments with M. alternifolia EO, terpinen-4-ol, and γ-terpinene greatly slowed larval growth after 72 h of treatment even at the lowest concentration (250 mg/L). Larval growth index (LG) values steadily decreased with increasing concentrations tested for all treatments. The most effective was M. alternifolia EO, followed by terpinen-4-ol, and the least effective was γ-terpinene, with LG values of 1.08, 1.86, and 3.51%, respectively, at 4000 mg/L.

Discussion
The quality and quantity of food consumed by insects can influence their growth, development, and reproduction (Dmitriew 2011). S. littoralis larvae fed on treated leaves showed a lower consumption rate (CI) than larvae fed on untreated leaves, according to the feeding indices studied at varied concentrations of M. alternifolia EO and two of its main components. According to the findings, reduced CI is associated with slower larval growth, which is most likely owing to longer food retention in the gut in order to maximize approximate digestibility (AD) to satisfy increasing nutritional requirements (Akhtar and Isman 2004). In treated larvae, the levels of converting ingested food (ECI) and converting digested food (ECD) also reduced significantly, suggesting that plant allelochemicals are toxic to the peritrophic membrane and that damage to the midgut's cellular surfaces has occurred (Mukherjee 2002; Sun et al. 2019;Braga et al. 2020). As a result, the EO of M. alternifolia is high in bioactive compounds (terpinen-4-ol and γ-terpinene) that have an antifeedant impact on insects and can be employed as natural insecticides (El-Wakeil 2013; Thomsen et al. 2013;Liao et al. 2017;Dehsheikh et al. 2020;Manfron et al. 2021).

Conclusions
Finally, the M. alternifolia EO and its components, terpinen-4-ol, and γ-terpinene had potent antifeedant activity on S. littoralis via effects on important metabolic processes. As a result, M. alternifolia EO and its compounds should be used as natural insecticides in IPM to combat the cotton leafworm.
Abbreviations EO: Essential oil; AD: Approximate digestibility; ECI: Efficiency of conversion of ingested food; ECD: Efficiency of conversion of digested food; CR: Consumption rate; RGR : Relative growth rate; FD: Feeding deterrent index; LG: Larval growth index.