To examine the insecticidal potential of phytochemicals under investigation against M. persicae, active constituents α-pinene and β-caryophyllene with 97% and 98% purity respectively were purchased from Innochem Science and Technology Co., Ltd. (Beijing, China).

Stock culture of M. persicae was sustained under an innocuous and insect-free environment at the insectarium of Anhui Agriculture University (China) for over four years. Cabbage plants were used to nurture the insects and then placed in an incubator at ± 26°C and 78% relative humidity in a photoperiod of 16:8 h (L:D) [32].

To assess the fumigant toxicity of compounds α-pinene and β-caryophyllene caryophyllene extracted from Rosemary (Rosemainus officinalis) and Piper cubeba essential oil. For the fumigation bioassay, a 500 ml glass container was used. In each treatment, three replication and 30 adult M. persicae were placed. Five different doses were chosen (3.5, 4.0, 4.5, 5.0, and 6.0 μL L⁻¹). Using a microinjector, drops of each dose of α-pinene and β-caryophyllene were functional to a part of filter paper (3 × 3 cm) attached to the bottom side of the jar lid. Cabbage leaf was used to provide a suitable environment for M. persicae. Then glass containers were preserved in the culturing environments stated overhead. Those insects which were deprived of the constituent’s handling were taken as a control. All dealings and panels were executed thrice autonomously. Insect death was noted at 24, 48, and 72 h post-treatment. Abbott’s formula was applied to calculate the mortality of M. persicae [33]. Furthermore, the lethal concentrations (LC₃₀ and LC₅₀) were calculated using probit analysis.

qRT-PCR was carried out to probe the countenance levels of five selected genes under LC₃₀ strength of both compounds α-pinene and β-caryophyllene. After 24 h of post-treatment, at least 50 M. persicae were composed and immediately deposited in liquid nitrogen, where they were preserved at −80°C until further analysis. The constructor’s commands extracted the entire RNA from each treatment using TRIZOL reagent (Invitrogen, Carlsbad, MX, USA). The Biophotometer Plus (Eppendorf, Hamburg, Germany) was used to assess and quantify RNA integrity. To improve RNA stability, cDNA was synthesized using the Prime Script^™ reagent kit (Takara, Dalian, China) according to the manufacturer’s instructions. For the quantification of OSD and gene, qRT-PCR analysis was performed using β-actin as internal control, whereas the ACE gene was used as an inner switch for quantification of ANT, HSP 60, and FPPS I. All primers for qRT-PCR analysis are enlisted in Table 1. Bio-Rad iCycler iQ Real-Time Detection System (Bio-Rad, Hercules, CA, USA) which contained 2x UltraSYBR Mixture (Progema Corporation, Beijing, China) 7.5 μL, cDNA 2 μL, each primer (10 μM) 1 μL, and of RNase-free water 3.5 μL in a final volume of 15 μL, was used for the execution of qRT-PCR. Every treatment was repeated three times for all qRT-PCR. The 2^−ΔΔCT technique was used to quantify the gene expression (mean ± SD) as a relative fold change [34]. The comparative quantifiable fold appearance change was assessed using one-way ANOVA [35].

The Log-Probit model analysis was applied to mortality rate of M. persicae to determine the 30% and 50% lethal concentrations (LC₃₀ and LC₅₀). The data for mortality rate of M. persicae was statistically analyzed by three-way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences (SPSS) version 16.0. The Tukey HSD analysis was applied at 5% probability of Type I error (α) to separate the means of the obtained data using ANOVA. The mean values were compared through alphabetical letters using Statistix 8.1 and standard error were calculated through software MS Excel version 16.0.

We performed a fumigant bioassay to investigate the toxicity of α-pinene and β-caryophyllene against the horticultural pest M. persicae. Five different doses (3.5, 4, 4.5, 5, and 6 μL L⁻¹) were finalized for both constituents. The LC₃₀ and LC₅₀ values of mortality rate after 24, 48, and 72 h of α-pinene and β-caryophyllene application were calculated through probit analysis (Table 2). After 24 h LC₃₀ and LC₅₀ α-pinene and β-caryophyllene were 4.2, 5.2, and 5.5, 6.8 μL L⁻¹ of air. The constituent’s fumigant control proved effective against M. persicae. Both constituents performed well; however, the response of α-pinene was superior to that of β-caryophyllene. The highest mortality rates of 98% and 74.80% after 72 h were observed by exposing to the highest concentration of α-pinene and β-caryophyllene (6 μL L⁻¹ of air). At the same time, at the lowest concentration of constituents α-pinene and β-caryophyllene (3.5 μL L⁻¹ of air) mortality rate was also low up to 15.6% and 4.3% after 24 h of exposure, respectively (Fig. 1 and Table S1).

Three-way analysis of variance (ANOVA) revealed significant mortality rates individually in treatments, doses, time, and the interactions between treatments × doses and doses × time (Table 3). However, the interactions between treatments × time and treatments × doses × time were non-significant (p < 0.05). The interactions between treatments × doses and doses × time were analyzed through two-way ANOVA, and results are given in Tables 4 and 5. The treatment × doses interactions revealed the highest mortality in the sequence of 6.0 µL L⁻¹ > 5.5 µL L⁻¹ > 5.0 µL L⁻¹ > 4.0 µL L⁻¹ > 3.5 µL L⁻¹, while application of α-pinene reported more mortality than β-caryophyllene (Table 4). These doses cause a higher mortality rate as time duration increases (Table 5). Applying α-pinene and β-caryophyllene up to 72 h time reported maximum mortality followed by 48 and 24 h of incubation.

The interaction between treatment and doses was statistically significant at p < 0.05; the values that share different letters in the lower case along the column and row were statistically significant at p < 0.05 and vice versa; The treatments and doses values that share different letters in the upper case along last column and last row were statistically significant at p < 0.05 and vice versa; SE stands for standard error.

The interaction between doses and time was statistically significant at p < 0.05; the values that share different letters in the lower case along the column and row were statistically significant at p < 0.05 and vice versa; The doses and time values that share different letters in the upper case along last column and last row were statistically significant at p < 0.05 and vice versa; SE stands for standard error.

To find out the difference in gene expression patterns, gene expression profiling was performed on M. persicae (adult) at sub-lethal concentrations (LC₃₀). Various researchers have examined gene expression responses to several treatments in relative quantity (RQ) to that in controlled M. persicae. A two-fold increase or decrease in expression was considered biologically significant [38]. The variation in the expression of five genes (HSP60, FPPS I, OSD, TOL, and ANT) was studied [39] in adult M. persicae exposed to α-pinene and β-caryophyllene for 24 h. OSD, TOL, and ANT (dispersal-related genes) have previously been shown to be overexpressed in M. persicae in response to stress. The results of this study indicate all three genes showed varying levels of overexpression, except for OSD genes that showed several times greater upregulation in M. persicae, at an LC₃₀ dose of α-pinene and β-caryophyllene.

In adult M. persicae, a 5-fold increase in OSD gene expression was observed after exposure to the lethal dose (LC₃₀) of α-pinene (Fig. 2A). The LC₃₀ dose of β-caryophyllene has been shown to have a maximum expression of the OSD gene. At the same lethal dose (LC₃₀) of β-caryophyllene, α-pinene causes a 2-fold less augmentation in the expression of OSD gene in M. persicae after the same period. However, essential oil constituents (α-pinene and β-caryophyllene) boosted strongly upregulated gene expression in comparison to the control (Fig. 2A). Initial studies reveal that fecundity can be suppers at enhanced levels of OSD in M. persicae, which correlates with reduced reproduction [36].

The constituents α-pinene and β-caryophyllene incite markedly increased TOL gene expression at LC₃₀ doses. On exposure to α-pinene and β-caryophyllene, an unexpectedly different TOL gene expression pattern was recorded compared to the OSD gene. Compared to α-pinene and β-caryophyllene, the highest expression of the TOL gene was recorded at the LC₃₀ dose of α-pinene (Fig. 2B). Previous results demonstrate that TOL can be overexpressed in response to starvation [40,41] and fluctuation in JH (Juvenile hormones) titers [42] during courtship and mating [43]. At an LC₃₀ dose of α-pinene and β-caryophyllene, significant upregulation of the TOL gene was recorded. It has been previously reported that [41–43] upregulation of; the TOL gene causes starvation due to juvenile hormones binding proteins. It is carried out by fluctuation in JH titers, affecting antennal responses to food.

In contrast to OSD gene expression under treatment of α-pinene LC₃₀ dose, ANT gene expression is high. However, its expression is lower than TOL gene. A comparison between α-pinene and β-caryophyllene also shows the variation at the same dose. At the LC₃₀ dose of α-pinene, ANT gene expression is 2 folds high in M. persicae as compared to β-caryophyllene LC₃₀ dose (Fig. 2C). ANT gene specifically regulates mitochondrial proteins. These proteins are responsible for vital functions such as carriers of essential metabolites, which facilitate several mitochondrial roles, catalyzing trans-membranous (mitochondrial) transport of ADP to synthesize ATP [44]. With the modest comeback of the ANT gene to an at LC₃₀ dosage of β-caryophyllene, ANTgene expresses moderate response, which shows negligible energy expenditures for M. persicae.

One of the stress response genes is HSP 60, and its expression varies according to each stressor type. Multiple studies have proven the downregulation of the HSP gene family (HSP 60, HSP 70, and HSP 90) associated with a recovery response after prolonged exposure to even mild stress. On the other hand, the upregulation of HSP 60 demonstrates the buildup of impaired proteins after stress or injury to an organism [45]. In addition, HSP accumulation has been reported to decrease fecundity [46]. In this study, after 24 h exposure to topical treatment of adult M. persicae with LC₃₀ dose of α-pinene and β-caryophyllene, an almost 6 and 9-fold upregulation in the expression of the HSP 60 gene was obtained (Fig. 2D). Meanwhile, three folds reduced expression was noticed in response to LC₃₀ dose of α-pinene. Compared to previous studies [46], it can be concluded that α-pinene and β-caryophyllene at higher doses negatively affect M. persicae reproduction by reducing fecundity. Results show that α-pinene has a low effect on HSP 60 gene compared to β-caryophyllene.

FPPS I gene expression was enhanced post-treatment with LC₃₀ of α-pinene and β-caryophyllene (Fig. 2E). At LC₃₀ dose of α-pinene, FPPS Igene showed the highest (4 folds) upregulation. β-caryophyllene while 2-fold downregulation in FPPS Igene expression was noted in β-caryophyllene compared to α-pinene (Fig. 2E). Previously, it came to know that JH biosynthesis is affected by [47] FPPS I by catalyzing the synthesis of farnesyl diphosphate (FPP). The production of sexual pheromones in M. persicae is stimulated by JH. At the same time, increased JH titers in female insects promote the growth of apterous forms via inhibition of wing growth. FPPS I downregulation is related to reduced production of β-farnesene (EBF) [47,48], which may raise egg-laying capacity in M. persicae.

In short, it was concluded that an enhancement in the expression of FPPS I gene could negatively affect the reproduction and fecundity in M. persicae. Recent studies show significantly upregulated FPPS I gene expression after 24 h treatment with (LC₃₀) of α-pinene and β-caryophyllene in adult M. persicae. Hence, the phytochemical constituents (α-pinene and β-caryophyllene) were found to be inversely related to the fecundity and reproduction of M. persicae.

In this study, α-pinene and β-caryophyllene of Rosemary and Piper cubeba oils have demonstrated potent fumigant toxic effects for controlling adults M. persicae. A comparative gene expression analysis has further supported that α-pinene upregulates the expression of TOL, ANT, and FPPS I genes more drastically than β-caryophyllene at the same dose (LC₃₀) and time intervals (24 h).