Introduction

Tomato, Lycopersicon esculentum Miller, is one of the most important agricultural products in the world and is widely cultivated worldwide due to its high nutritional and economic value1. However, this precious plant is threatened by various insect pests and diseases. Among the pests affecting tomato yield, the tomato leaf miner Phthorimaea absoluta (Meyrick) (Lepidoptera: Gelechiidae) is considered the most important pest2. This invasive pest, native to South America and specifically Peru, was first detected in eastern Spain in late 2006. It rapidly spread across the Mediterranean region, eventually reaching North Africa and the Middle East3. This pest was first collected and identified in July 2010 in a tomato field near Urmia, West Azerbaijan4. It attacks all parts of the tomato plant (including leaves, flowers, stems, and fruits) at any stage of growth, causing devastating crop losses (up to 100% if left uncontrolled)5. Although the primary host of this pest is tomato, potato and wild species of the Solanaceae family are also considered its secondary hosts. This pest can produce 10 to 12 generations per year in South America6 due to the continuous availability of hosts and climate.

Controlling P. absoluta is not easy due to its resistance to many pesticides2. Its mode of feeding (borrowing inside the tomato leaves) is a reason for the difficulty in controlling this pest, and above that, their high reproductive rate and indiscriminate chemical control may lead to genetic mutation and subsequent resistance7. Due to the side effects of chemical pesticides on the environment and humans, and the problem of resistance in many pests including P. absoluta, hence, an integration of several methods must be incorporated in controlling this devastating pest8,9,10.

Vermiwash is a brown extract of organic compost. This bio-fertilizer contains several enzymes, plant growth hormones, vitamins, macro and micronutrients, along with the excreta and mucus secretions of earthworms, which are easily absorbed by plant tissues11,12. The dead earthworm tissues release nitrogen in the form of nitrate, ammonia, and soluble organic matter, which increases the nutrient quality of vermiwash13. Vermiwash is also rich in several enzymes such as proteases, amylases, ureases, and phosphatases. Similarly, several microbial agents including nitrogen-fixing bacteria such as Azotobacter sp., Agrobacterium sp., and Rhizobium sp., and some phosphate-solubilizing bacteria have been reported from vermiwash14. Vermiwash is also composed of urban wastes, organic matter, plant nutrients, and soluble salts, which increases the moisture and nutrient content of the soil15. Aghamohammadi et al. (2016) reported that in vivo and greenhouse studies demonstrated that incorporating vermiwash with azocyclotin in both sterilized and non-sterilized pots led to the highest reduction in Tetranychus urticae populations, achieving 100% mortality, compared to treatments without vermiwash on bean plants16. In a separate study, Nath and Singh (2012) demonstrated the effectiveness of vermiwash spray combined with neem oil and custard apple extract in reducing aphid populations17.

Gibberellins (GAs) are a group of plant growth hormones that play a critical role in plant growth, seed germination, water uptake, and dormancy-inducing substances18. Gibberellins are responsible for the synthesis of hydrolases in plants, which improves seed viability and germination, and on the other hand, play a role in repairing damaged cell membranes19. Gibberellins also act as strong signaling molecules to increase growth and strengthen growth processes to cope with stress conditions and boost their immune system20. Gibberellin treatment not only favors plant growth, but also negatively affects insects21,22,23. There are some reports on the inhibition of food consumption, growth, and reproductive potential of Bactrocera cucurbitae (Coquillett) and Spodoptera litura (Stoll) after gibberellin inclusion in their diets24. Gibberellin also retards the reproduction and fertility in Locusta migratoria L. and the biological parameters in Galleria mellonella L25,26.

Traditional approaches to controlling P. absoluta have fallen short in meeting the standards of environmental sustainability, cost-effectiveness, and health enhancement. Consequently, there is a pressing need to adopt alternative measures, particularly those that can effectively address these concerns. Based on our previous works on gibberellin23 and vermiwash (unpublished data) on the significant effect of these compounds on the life cycle and physiology of P. absoluta, we came to a conclusion on the interaction of these two compounds with P. absoluta performance. Therefore, we applied gibberellin + vermiwash and the tomato leaf miner pest, marking the first examination of such interaction. Additionally, the research delves into assessing the effects of these two compounds on the biology and physiology of the tomato leaf miner, with the aim of identifying potential control measures. This comprehensive investigation aims to pave the way for sustainable and effective pest management practices in tomato cultivation.

Materials and methods

Rearing

The initial population of tomato leaf miner was obtained from the Department of Agricultural Entomology at Tarbiat Modares University in Tehran, Iran. The insects were carefully introduced to recently planted EURO WAAN-F1 tomato plants. Subsequently, the insect colony was maintained in a controlled greenhouse at the Department of Agricultural Entomology, University of Guilan, Rasht, Iran (25 ± 1 °C, 60 ± 5% RH, and 16 L: 8D-h photoperiod). The experimental tomato plants were cultivated in plastic pots, in total 40 pots were allocated for each treatment.

Application to the leaves

For foliar spraying of cultivated tomato plants, pure gibberellin powder (with 99% purity, Merck, Germany) and liquid vermiwash bio-fertilizer (Behkesht farming corporation, Malard, Iran) were purchased. The gibberellin solution at a concentration of 50 µM27 and the vermiwash solution at a concentration of 1:10 (vermiwash: tap water)28 were prepared (GV treatment). These solutions were sprayed three times on the leaves at four-week intervals starting from the four-leaf stage of the plants using a handheld sprayer. The control group received only water. In the 1st stage of spraying, 400 mL of solution were applied to 40 tomato pots. In the 2nd stage, due to the increase in the number of leaves and the size of the plants, the amount of solution was increased to 800 mL. Additionally, in the 3rd stage, this volume was further increased to 1200 mL.

Life table study

To analyze the life table parameters of P. absoluta, we confined 20 pairs of adult insects within a mesh enclosure (70 × 55 × 35 cm) for egg-laying purposes. After 24 h, we collected the deposited eggs for life table experiments. Each egg was individually placed on treated leaves within a plastic container (8.5 × 6.5 × 3 cm), with the petiole of each leaf inserted into moist cotton wool to prevent desiccation. 30 eggs were used to examine the life table in each treatment. We conducted daily observations until egg hatching, replacing leaves every 3 days. The duration of immature stages and survival rates were recorded daily. To determine the larval stage, we used the booklet provided by USDA29. This was accomplished using an ocular micrometer attached to a stereomicroscope, set at ×20 magnification. Upon adult emergence, males and females were transferred to oviposition chambers (18 × 16 × 6 cm) to monitor egg laying. We recorded preoviposition and oviposition periods, daily fecundity, total fecundity, and adult moth longevity until the death of the last adult in the cohort.

Biochemical tests

To assess the activity of detoxifying and antioxidant enzymes, 5 final-instar larvae were homogenized with a hand homogenizer in a 1:1 ratio with phosphate buffer solution (pH 7). Following homogenization, the mixture was centrifuged at 13,000 ×g for 15 min at 4 °C, yielding the supernatant containing the enzyme source. The protein concentration was determined following the protocol described by Lowry et al., using a biochemical kit supplied by Ziest Chem (Ziest Chem. Co., Tehran-Iran). Enzyme extracts were mixed with 100 µl of the reagent and incubated at 25 °C for 30 min, after which the absorbance was measured at 545 nm30.

Catalase (CAT)

Catalase activity was assessed following the method outlined by Wang et al. (2001). Specifically, 50 µL of both the treatment and control samples were mixed with 500 µL of 1% hydrogen peroxide, and the reaction mixture was then incubated at 28 °C for 10 min. Subsequently, the absorbance was measured at 240 nm31.

Peroxidase (POX)

The enzyme peroxidase activity was measured using the method described by Addy and Goodman. The reaction mixture consisted of 50 µL of pyrogallol (0.05 M), phosphate buffer (0.1 M, pH 7), 50 µL of 1% oxygenated water, and 20 µL of enzyme extract. The mixture was incubated for 2 min at room temperature, and absorbance of the reaction mixture was read every 30 s (5 readings) at a wavelength of 431 nm32.

Superoxide dismutase (SOD)

The measurement of superoxide dismutase activity was performed using the method described by McCord and Fridovich. For the measurement of this enzyme, first, 10 mg of bovine serum albumin and 100 µL of xanthine oxidase were added to 2 mL of phosphate buffer (0.1 M, pH 7) (Step 1). Then, 500 µL of phosphate buffer (containing 70 µM of NBT and 125 µM of xanthine) was mixed with 100 µL of the solution prepared in the first step. After adding the enzyme sample, the resulting solution was incubated for 20 min in the dark. Subsequently, the absorbance of the reaction mixture was measured at a wavelength of 560 nm33.

Glucose-6-phosphate dehydrogenase (GPDH)

The activity of this enzyme was measured using the method described by Balinsky and Bernstein. The reaction mixture contained 100 µL of Tris-hydrochloride buffer (100 mM, pH 8.2), 0.2 mM NADP, and 0.1 mM magnesium chloride, along with water and the enzyme sample inside a cuvette. The reaction was initiated by adding 100 µL of glucose-6-phosphate. The increase in absorbance at a wavelength of 341 nm was monitored to assess the enzymatic activity34.

Cytochrome P450

The activity of the enzyme cytochrome monooxygenase was measured using the substrate TMB (3,3’,5,5’-Tetramethylbenzidine dihydrochloride) according to the method of Martin et al. The reaction mixture included 50 µL of sodium phosphate buffer (100 mM, pH 7.2), 50 µL of enzyme extract, and 150 µL of TMB solution (13 mg in 6.5 mL of methanol in 19.5 mL of sodium acetate buffer, 0.25 M, pH 5). Then, 25 µL of hydrogen peroxide (3%) was added, and after 30 min of incubation at room temperature, absorbance was read at 630 nm35.

General esterase (ESTs)

The measurement of the activity of general esterase according to the method of Han et al., was performed using two separate substrates, α-naphthyl acetate and β-naphthyl acetate. 10 µL of each substrate (10 mM) was mixed separately with 5 µL of RR-Salt blue dye and 40 µL of phosphate buffer (20 mM). Then, 5 µL of the enzyme sample was added to them, and after 5 min of incubation at 25 °C, the optical absorption at a wavelength of 450 nm was read36.

Glutathione S-transferase (GSTs)

Measurement of glutathione S-transferase activity was performed according to the method of Oppenorth et al., using two substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB). A volume of 40 µL of phosphate buffer (20 mM) and 20 µL of reduced glutathione were separately mixed with 10 µL of each substrate. Then, 5 µL of enzyme sample was added, and after 5 min of incubation at 25 °C, optical absorption was measured at a wavelength of 340 nm37.

Assessment of primary and secondary metabolites

Total phenol content

The quantification of phenolic compounds followed the Folin-Ciocalteu method, as detailed by Singleton and Rossi. Initially, 0.5 g of leaf sample was meticulously ground with 3 mL of 85% methanol (Merck Co, Germany) in a mortar. The resulting methanol extract underwent filtration, and precisely 300 µL of this extract was combined with 1500 µL of Folin-Ciocalteu reagent, which had been diluted in a 10:1 ratio with distilled water. Following an incubation period of precisely 8 min, 1200 µL of 7% sodium carbonate solution was introduced. The samples were then rigorously agitated on a shaker at a controlled speed of 120 rpm, maintained at room temperature, and shielded from light. Finally, the absorbance of the samples was meticulously measured at a precisely defined wavelength of 765 nm using a spectrophotometer38.

Carbohydrates

The carbohydrate content in the leaf extract was determined following the protocol outlined by Irigoyen et al. Initially, 0.1 mL of an 83% ethanol extract was carefully transferred into a test tube and stored in the refrigerator using a pipettor. Subsequently, 3 mL of freshly prepared anthrone solution (consisting of 150 mg of anthrone dissolved in 100 mL of 72% sulfuric acid) was added to the test tube. The test tubes were then placed in a water bath for 10 min to allow color development. Following cooling, the absorbance of the solution was measured at 625 nm using a spectrophotometer39.

Tannin

The tannin extract (5 mL) underwent dilution with 20 mL of deionized water. Following this, Folin-Ciocalteu reagent was added, succeeded by the introduction of 2.5 mL of saturated sodium carbonate solution after a 5-minute interval. Upon complete development of the blue color within 1–2 h, the solution’s optical absorption was measured at a wavelength of 760 nm using a spectrophotometer40.

Anthocyanin

Fresh plant tissue weighing 0.1 g was thoroughly ground in a mortar and pestle with 10 mL of a methanol solution, composed of pure methanol and hydrochloric acid in a volumetric ratio of 99:1. The resulting extract was transferred to a screw-capped test tube and kept in the dark at 25 °C for 24 h. Afterward, the solution was centrifuged at 4000×g for 10 min, and the absorbance was measured at a wavelength of 550 nm41.

Flavonoid

Fresh plant tissue weighing 0.1 g was crushed in a mortar with 10 mL of an ethanolic acid solution, which consisted of ethyl alcohol and glacial acetic acid in a 1:99 ratio. After centrifuging at 8000×g for 10 min, the extract was heated in a water bath at 80 °C for 10 min. Absorbance readings were then taken at wavelengths of 270 nm, 300 nm, and 330 nm42.

Data analysis

The examination of data was carried out using the TWOSEX-MSChart software43, enabling the computation of various population parameters like the intrinsic rate of increase (r), finite rate of increase (λ), net reproductive rate (R0), and mean generation time (T)44,45. Standard errors for diverse parameters, including fecundity, reproduction period, developmental times, and population parameters, were computed using the bootstrap technique, with 100,000 bootstrap resampling iterations ensuring robust estimates. Paired bootstrap analysis was employed to compare means across different treatments. Both bootstrap resampling and paired bootstrap tests were conducted using the TWOSEX-MSChart computer program43.

Antioxidant and detoxifying enzyme assays followed a completely randomized design with three replications for treatments and controls. Data were normalized using arcsine transformation. Experiments examining tomato plant metabolite levels also adhered to a completely randomized design with five replications. Statistical analyses were performed using SAS 9 software, with mean values compared at a significance level of p < 0.05 using the t-test. Graphs were generated using Excel 2019 software.

Results

Life table

Table 1 illustrates the lengths of various life stages of P. absoluta, including the embryonic period, different larval stages, pre-pupal, pupal, pre-oviposition, and oviposition periods, for the groups treated with GV, and the control group. According to the obtained results, the length of the embryonic period does not show a significant difference among treatments, while each of the four larval instars in the GV treatment exhibits a significant difference compared to the control. However, this significant difference is not observed in the prepupal and pupal periods. Ultimately, the total length of the preadult stage varies significantly among different treatments, with the lengths for GV treatment and control being 32.06 ± 0.39 and 27.38 ± 0.35 days, respectively. The preadult survival rate in the control treatment (0.80 ± 0.07) is higher than that in the GV treatment (0.53 ± 0.09), indicating a statistically significant difference. The total lifespan duration for the GV treatment (44.31 ± 0.51 days) is significantly longer than the control (38.46 ± 0.41 days). Although a significant difference was not observed between the GV treatment and the control in the duration of APOP, the duration of TPOP among different treatments had a significant difference, with 34.44 ± 0.67 days for GV, and 29.50 ± 0.56 days for the control. No significant difference was observed in fertility between the GV treatments and the control.

Table 1 Duration of different life stages, female and male longevity, fecundity and oviposition days (Od) of Phthorimaea absoluta on Gibberellin + Vermiwash (GV) treated plants and control.
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Table 2 displays the population parameters of P. absoluta in different treatments. A comparison of the mean values of various parameters showed that GRR and R0 do not indicate a significant difference between the GV treatment and the control. The parameter r in the GV treatment (0.086 ± 0.009 day− 1) had a lower value compared to the control (0.110 ± 0.008 day− 1), which statistically indicates a significant difference. Additionally, the parameter λ in the GV treatment and the control was 1.090 ± 0.009 day− 1 and 1.116 ± 0.008 day− 1, respectively, indicating a significant difference.

Table 2 Population growth parameters (mean ± SE) of Phthorimaea absoluta on Gibberellin + Vermiwash (GV) treated plants and control.
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In Fig. 1, the survival rates of different stages (sxj) are illustrated. This curve depicts the likelihood of egg survival at age x and stage j. The survival curves extend up to day 48 and 42 for the GV treatment and control group, respectively. Additionally, this figure demonstrates overlapping stages throughout the developmental period.

Fig. 1
figure 1

Age-stage-specific survival rate (sxj) of Phthorimaea absoluta on treated tomato plants. The control set of data are the same as in our previously published work23.

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In Fig. 2, the age-stage-specific life expectancy (exj) of P. absoluta is depicted under two distinct conditions: GV treatment and the control. It is observed that as the age advances, their life expectancy (exj) diminishes accordingly. This decline in life expectancy is primarily attributed to the natural process of aging, as no other factors contributing to mortality were identified within the study parameters.

Fig. 2
figure 2

Age-stage-specific life expectancy (exj) of Phthorimaea absoluta on treated tomato plants. The control set of data are the same as in our previously published work23.

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Figure 3 presents the age-stage reproductive value (vxj), a measure indicating the reproductive contribution of individual P. absoluta specimens with specific fertility (fx), under both GV treatment and control conditions. This metric offers insights into the relative reproductive potential of individuals across various age and developmental stages. By comparing the reproductive values of subjects from both treatment groups, we gain a comprehensive understanding of how GV treatment influences the reproductive capacity of P. absoluta compared to untreated counterparts.

Fig. 3
figure 3

Age-stage-specific reproductive values (vxj) of Phthorimaea absoluta on treated tomato plants. The control set of data are the same as in our previously published work23.

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Figure 4 presents an analysis of age-specific survival rates (lx), age-specific fecundity (mx), and age-specific maternity (lxmx) in two distinct experimental settings: GV treatment and control. The lx curve highlights an interesting observation, indicating a relatively slower decline in pre-adult survival rates within the control group compared to those treated with GV. Specifically, 53% and 80% of insects advanced to the adult stage in the GV and control groups, respectively, shedding light on the differential impacts of treatment on developmental progression. Furthermore, the examination of peak mean daily fecundity unveils intriguing differences between the two groups, with GV-treated individuals exhibiting peak fecundity at day 38, whereas those in the control group reached their maximum fecundity at day 32.

Fig. 4
figure 4

Age-specific survivorship (lx), age-stage-specific fecundity (fxj), age-specific fecundity (mx) and age-specific maternity (lxmx) of Phthorimaea absoluta on treated tomato plants. The control set of data are the same as in our previously published work23.

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Antioxidant system activity

According to the obtained results, the activity level of the enzyme POX in 4th instar larvae of P. absoluta showed a decreasing trend with GV treatment (0.001 ± 0.0001) compared to the control (0.002 ± 0.0001), which was statistically significant. In contrast, the activity level of the enzyme SOD in larvae fed on plants containing GV (0.809 ± 0.011) showed a significant increase compared to the control (0.745 ± 0.009). Similarly, the activity of the enzyme CAT also increased with GV treatment (0.979 ± 0.018) compared to the control (0.0906 ± 0.005). Data obtained from the enzyme GPDH activity did not show a significant difference between the GV treatment and control. The results are presented in Table 3.

Table 3 Antioxidant enzyme functions observed in the 4th developmental stage larvae of Phthorimaea absoluta on treated plants.
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Detoxifying enzymes

The results of measuring the detoxifying enzyme activities are presented in Table 4. The activity of the GST enzyme with both substrates, DCNB and CDNB, did not show a significant difference between the GV treatment and control groups. The amount of cytochrome P450 enzyme in insects fed with treated plants (0.325 ± 0.006) was significantly higher than the control group (0.264 ± 0.004). The activities of general esterase enzymes were measured, both of which demonstrated an increasing trend. The activity of α-naphthyl esterase enzyme in the GV treatment (0.606 ± 0.015) was significantly higher than that in the control (0.542 ± 0.009). Additionally, the activity of β-naphthyl esterase enzyme in the GV treatment (0.584 ± 0.007) was higher than in the control (0.520 ± 0.009), with this difference being statistically significant.

Table 4 Detoxifying enzyme functions observed in the 4th developmental stage larvae of Phthorimaea absoluta on treated plants.
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Plant metabolites

Important metabolites of tomato plants were measured under the influence of spraying solutions containing GV and a control. Total sugar and anthocyanin levels in plants sprayed with GV solution and the control did not show a significant difference. The total phenol content in plants treated with GV (15.16 ± 0.67) was higher than in control plants (13.61 ± 1.17), and the difference between treatments was statistically significant. Additionally, the measured flavonoid level in plants sprayed with GV solution was significantly higher than in control plants, which were 1.85 ± 0.04 and 1.07 ± 0.12, respectively. The tannin content in GV-treated plants (31.97 ± 1.85) was higher than in the control (25.39 ± 1.69). The difference in tannin levels between treatments was statistically significant. The results of metabolite measurements are presented in Fig. 5.

Fig. 5
figure 5

The level of primary and secondary metabolites (a): Flavonoid, (b): Total phenol content, (c): Anthocyanin, (d): Total suger, (e): Tannin in tomato plants treated with GV and control. The control set of data are the same as in our previously published work23.

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Discussion

Farmers, aiming to increase agricultural productivity, use chemical fertilizers and pesticides, which have a negative impact on the environment and human health46. There is a new approach to sustainable agriculture that offers maximal benefits to farmers while minimizing environmental impact47. One such approach could be the use of fertilizers of organic origin (vermiwash) and an organic plant growth regulator (gibberellin). Our results in the present work aimed to prove this attitude by utilizing a combination of vermiwash + gibberellin on the biology of tomato leaf miner. The combination of these two substances interestingly proved to be quite satisfactory and impaired the growth, reproductive values, population parameters, as well as physiology of the target insect.

The preadult duration of the tomato leaf miner increased with the application of vermiwash and gibberellin. Furthermore, the use of vermiwash and gibberellin resulted in a decrease in the pre-adult survival rate. Additionally, treatment with vermiwash and gibberellin led to an increase in the lifespan of the tomato leaf miner. This indicates that plant growth regulators influence insects in diverse ways. Some researchers have proposed that the detrimental effects of gibberellic acid (GA3) on various insect life parameters might stem from its chemical resemblance to the juvenile hormone (JH)48,49. Numerous researches have focused on the pest-suppression properties of organic fertilizers, such as vermicompost. Edwards et al., showed that the life stages of Tetranychus urticae Koch, Pseudococcus sp., and Meyzus persicae (Sulzer) were severely affected after using vermicompost, a bioproduct of earthworms50. Moreover, other studies have shown that vermicompost can effectively mitigate infestations of sucking insects such as aphids and mites in peanut crops51. This indicates that vermicompost not only improves soil fertility but also aids in better pest management in sustainable agricultural systems. The results of Mardani et al., demonstrated that treatment with vermicompost resulted in a longer developmental period for M. persicae nymphs, meaning that the use of this natural fertilizer is unfavorable for this insect52. Another study by Yassen et al., reported a reduction in M. persicae populations feeding on Calendula officinalis plants cultivated with vermicompost53. Taken together, these studies collectively emphasize the potential of vermicompost in managing aphid populations, thus enhancing plant tolerance against pest attacks. Getnet and Raja, demonstrated that increasing doses of vermicompost led to a decrease in Brevicoryne brassicae L. populations on cabbage plants54. It has been established that phenolic compounds present in plants can significantly impact insect survival rates and impede their growth and reproductive capacities55,56. According to Ravi et al. and Koul, the suppression of harmful populations by vermicompost might be attributed to the presence of phenolic acid compounds within them57,58. Additionally, it has been noted that organically grown plants tend to contain higher levels of phenolic compounds compared to those grown with inorganic fertilizers59.

In the present study, the values of r and λ in insects fed on plants treated with vermiwash and gibberellin showed a significant decrease compared to the control group. The parameter λ (the finite rate of increase) is a crucial demographic indicator utilized to assess and compare the fitness of populations under varying climatic conditions and diverse food availability scenarios. This metric provides valuable insights into how different environments and nutritional resources impact the reproductive success and growth rates of populations60. The r (the intrinsic rate of increase) is a fundamental demographic parameter in population ecology. It represents the rate at which a population increases in size per individual, per unit of time, under ideal environmental conditions with unlimited resources61. Numerous studies have documented that the shift towards organic methods, which often involve the use of natural supplements and soil health-enhancing techniques, appears to create conditions less favorable for these pests and pathogens, thereby decreasing their potential to cause damage50,62,63. Ramesh et al., found that organic crops, including rice, exhibit greater tolerance and resistance to insect attacks compared to conventionally grown crops. This increased toughness is attributed to the organic crops’ production of thicker cell walls and reduced levels of free amino acids. The thicker cell walls provide a physical barrier against insect penetration, while the lower levels of free amino acids make the plants less attractive and nutritious to pests. These findings highlight the benefits of organic farming practices in enhancing the natural defenses of crops against insect infestations64.

Plant Growth Regulators (PGRs) represent a new generation of agricultural chemicals used as foliar fertilizers, modifying natural plant growth from seed germination to maturity in crop plants. However, economically, producing these chemicals is not cost-effective, and determining optimal conditions for their application is challenging65. Therefore, considering the properties of these chemicals, the demand for naturally derived fertilizers has increased. Research has shown that earthworms produce significant amounts of plant hormones in their secretions66. Vermiwash, the extract obtained from vermicompost containing a combination of earthworm secretions and excreta of Eisenia fetida (Savigny) earthworms, along with micronutrients and organic soil molecules, is valuable for plant growth and can be used for foliar spray63. Studies have also indicated that the application of vermiwash not only improves plant growth but also enhances plant yield and resistance to pests and diseases. Researchers suggest that this could be due to the presence of organic acids such as humic acid, folic acid, growth regulators, amino acids, vitamins, and enzymes such as proteases, amylases, and urease67.

Glutathione S-transferases play a major role as a detoxifying agent against various foreign substances entering insect bodies by either metabolizing or sequestering these compounds. Additionally, other detoxifying enzymes such as general esterases can also contribute to resistance by altering enzyme production or activity through quantitative or qualitative changes68. The activity of some detoxifying enzymes involved in the metabolism of foreign compounds (e.g. catalase, esterases, glutathione S-transferase, peroxidase, superoxide dismutase, and acid phosphatase) can be influenced by PGRs, either increasing or decreasing69. These compounds can also serve as alternatives to conventional pesticides for managing economically significant pests70.

In the present study, a significant increase in the activity of alpha and beta-naphthyl esterases was demonstrated in those larvae that were fed by a mixture of vermiwash-gibberellin-treated leaves compared to the control. However, no change was observed in the activity of glutathione S-transferase enzyme. It has been previously shown that foliar spraying of a PGR (cytokinin) enhanced the activity of esterases in second instar nymphs of Lipaphis erysimi, indicating the PGRs’ crucial role in enhancing plant resistance71.

Antioxidants are stable compounds that neutralize free radicals through electron donation, reducing the damage caused by free radical activity72. Our results showed an increased activity level of antioxidant enzymes, including catalase and superoxide dismutase, in insects fed on treated plants compared with the control. Meanwhile, a decreasing trend in peroxidase enzyme activity was observed. Another study also reported an increasing trend in the activity of esterase, catalase, and superoxide dismutase enzymes in the last instar larvae of G. mellonella fed with gibberellic acid, indicating physiological compatibility to compensate for stress induced by gibberellic acid. In fact, this compound acts as a source of free radicals, activating the insect’s antioxidant system73. Additionally, an increase in antioxidant enzyme activity was reported in adult M. persicae fed on leaves treated with vermicompost by Mardani et al.74.

The increase in SOD enzyme activity may be due to the induction of this enzyme by superoxide radicals to protect larvae against oxidative stress75. Also, increased CAT enzyme activity may be associated with the response to H2O2 removal, hence related to oxidative stress76. Among the detoxifying enzymes, cytochrome P450 monooxygenases have been studied more extensively due to their key role in plant-insect interactions. This enzymatic system not only has a protective role that shields insects from inadvertent chemical contact but also metabolizes physiological hormones, lipids, and other internal chemicals77. The production of secondary metabolites in plants can be induced by insect feeding, and these metabolites, in turn, alter the activity of insect detoxifying enzymes78. Similarly, it was shown that a diet containing secondary metabolites from pine, such as flavonoids, significantly increased the activity of P450 and GSTs enzymes in the second instar larvae of Lymantria dispar L. Plants defend themselves against herbivores by producing toxic secondary metabolites such as cyanides, alkaloids, and some terpenoids79. In the present study, foliar spray solution along with tomato plant leaves with gibberellin hormone and vermiwash significantly increased secondary metabolites such as phenols, flavonoids, and tannins in treated plants compared to the control. Suthar68 also reported an increase in protein, sugar, and starch levels as a result of foliar spraying vermiwash in Cyamopsis tertagonoloba L. and Trigonella foenum-graecum L. plants. Additionally, it was observed that plant hormones such as cytokinins, salicylic acid, jasmonic acid, etc., induce secondary metabolite production in plants80. The total content of phenols and anthocyanins increased in Brassica juncea L. plants using jasmonic acid supplements81.

This study demonstrates the potential of using gibberellin and vermiwash as part of an integrated pest management strategy for P. absoluta in tomato cultivation. The combined treatment significantly affected the pest’s life cycle and enzymatic activities, indicating its viability as a pest control method. Specifically, the GV treatment extended the developmental stages and total lifespan of P. absoluta while reducing key population growth parameters, suggesting a potential reduction in pest proliferation. Additionally, alterations in antioxidant and detoxifying enzyme activities in treated larvae highlight the physiological impacts of GV treatment. These findings indicate that further field-scale research is necessary to optimize the use of gibberellin and vermiwash in agriculture, enabling their effective application in integrated pest management while also minimizing environmental risks and potential health hazards associated with chemical pesticides. Integrating such biocontrol measures can contribute to more sustainable and resilient agricultural systems.