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Insights into larval development and protein biochemical alterations of Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae) following Beauveria bassiana and Solanum lycopersicum treatments

Chemical and Biological Technologies in Agriculture volume 11, Article number: 164 (2024) Cite this article

Abstract

Background

The polyphagous notorious pest, black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), cause significant production losses due to its distinctive feeding and hiding behavior, making it particularly challenging to control it with conventional methods. Therefore, sustainable agriculture demands more effective and environmentally safe pest control solutions. This study aimed to investigate the toxicity of two insecticide alternatives, the entomopathogenic fungus (EPF) Beauveria bassiana and Solanum lycopersicum extract (Tomato plant crude extract, TPCE), using two bioassay methods: the poisoned bait method and the leaf dipping method. In addition, the impact of these biological tools on larval development and protein profiles was evaluated.

Results

The bait application of both tested materials exhibited higher toxicity than the leaf dipping method, as indicated by the toxicity index. The LC50 values for B. bassiana were 1.6 × 10⁸ and 1.8 × 10⁶ conidia ml−1 using the leaf dipping method and poisoned baits method, respectively. For TPCE, the LC50 values were 4.35 and 1.51 mg ml−1 for the same methods, respectively. In addition, sublethal concentrations of both materials altered the larval and pupal durations. B. bassiana significantly reduced the concentration of larval hemolymph protein. A maximum of 12 protein bands in the control sample, with molecular weights (Mw) ranging between 35 and 120 kDa, were detected by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). In B. bassiana-treated larvae, ten bands were detected with Mw ranging from 35 to 120 kDa. At least seven bands were detected in TPCE-treated larvae, with Mw ranging from 35 to 97 kDa.

Conclusions

The findings of this study can be integrated into management programs for A. ipsilon. In addition, the availability of B. bassiana and TPCE in Egypt and their cost-effectiveness as insecticide alternatives support their use in the management programs of this critical pest. These methods are particularly effective when applied in bait form.

Graphical Abstract

Background

The black cutworm, Agrotis ipsilon (Hufnagel), is a major pest of crops like maize, rice, okra, cotton, tomato, cabbage, potato, and coffee [1,2,3,4,5,6,7]. Damage from A. ipsilon in maize is estimated to range from 30 to 40% under moderate infestation and can reach up to 90% in heavily infested fields [8]. A single A. ipsilon larva at the cutting age, 4th instar or older per meter of row can result in significant plant loss (27%) and a yield reduction of 2900 kg/ha during the coleoptile to a one-leaf stage of maize development [9]. These yield losses rendered the black cutworm a primary concern for farmers and agricultural authorities [10]. In addition, the nocturnal behavior of the black cutworm complicates control efforts, as they feed under cover of darkness and retreat into the soil during daylight hours [11]. This nocturnal habit makes them challenging to detect and manage. Consequently, effective pest management strategies should encompass a range of tools and methods to address the feeding behaviors of this pest.

Chemical management through insecticide has been noted for its success in controlling the black cutworm A. ipsilon [12,13,14,15]. However, the indiscriminate use of these insecticides has resulted in adverse effects on non-target organisms, environmental pollution, and resistance development [16,17,18,19].

Alternatively, biological control agents (BCAs) may play a crucial role in sustainable crop production by offering a pest management approach with minimal environmental impact [20]. Among these BCAs, entomopathogenic fungi (EPF) have managed A. ipsilon effectively under certain conditions [21, 22]. For example, the fungus Beauveria bassiana has demonstrated notable efficacy against various insect pests, particularly lepidopteran larvae [23,24,25,26,27,28]. In addition, it shows versatility in its application, demonstrating efficacy under different environmental conditions and against various target insect species [29].

However, the effectiveness of B. bassiana is influenced by factors such as environmental conditions (temperature, humidity, and UV radiation), which can impact its ability to infect and kill target pests. Optimal efficacy is often observed under moderate temperatures and high humidity, while extreme temperatures or prolonged UV exposure can reduce its efficacy [30, 31]. Formulation type and application methods also play a role, as some provide better adherence to insect cuticles or improved environmental persistence [32]. In addition, due to thinner cuticles, early larval stages are more susceptible [33]. The success of B. bassiana can be affected by its interaction with other pest management strategies, including chemical insecticides [34]. Ongoing research seeks to optimize its use in integrated pest management programs (IPM).

Other BCAs and natural insecticides, such as Lavandula multifida [35], Cymbopogon citratus [36], neem products [37], methanolic extract from Melia azedarach fruits [38], ethanolic extract of Nigella sativa [39], and extract of Tephrosia nubica [40], have proven highly effective in managing A. ipsilon. Furthermore, species within the Solanaceae plant family, which are economically and ecologically significant, produce a diverse range of compounds that affect a broad spectrum of insect taxa, predominantly herbivorous and pestiferous species. These compounds, primarily exhibiting insecticidal properties, demonstrate activities such as molluscicides, acaricides, nematicides, bactericides, and fungicides due to their specific biological actions [41]. These natural insecticides offer environmentally friendly alternatives to conventional chemical pesticides.

Integrated Pest Management (IPM) strategies that combine these biological control agents with selective chemical methods can enhance sustainability by minimizing environmental impacts and reducing the risk of resistance development in pest populations. For example, the use of B. bassiana or natural plant extracts, such as those from Solanum lycopersicum (tomato), can be integrated with selective chemical insecticides that are less harmful to non-target organisms, including natural predators and pollinators. This integrated approach can improve pest control efficiency while preserving beneficial insect populations and maintaining ecological balance.

However, there are challenges in implementing such IPM strategies effectively. One challenge is the variability in the efficacy of biological agents due to environmental conditions (e.g., temperature and humidity) or pest life stages, which can affect the consistency of results. In addition, the resistance development for biological control agents and natural insecticides. Resistance to B. bassiana can develop due to the repeated use of the same strain, leading to selection pressure on pest populations [42]. Similarly, resistance can also develop against natural insecticides, particularly if they are derived from single active compounds or used repeatedly without rotation [43]. Furthermore, the potential for interactions between biological agents and chemical treatments, such as antagonistic or synergistic effects, must be carefully considered and managed [7]. There may also be economic constraints, as biological control agents and natural insecticides sometimes have higher upfront costs or require more frequent applications than conventional chemical pesticides.

Applying toxic substances can significantly impact the toxicity outcomes, especially for insects like A. ipsilon that reside below the soil surface. The challenge of fully exposing these insects to insecticides through spraying can reduce the effectiveness of pest control strategies [44]. Furthermore, Shakur et al. [45] found that incorporating lanate and indoxacarb insecticides with baits was more effective in managing A. ipsilon than foliar application of the same insecticides. However, He et al. [43] suggested that using a combination of artificial diets with insecticides might act as a preventive measure. This approach disrupts insects' essential nutrient intake for regular growth and metabolic function, reducing survival rates, developmental progress, and reproduction within the A. ipsilon population post-insecticide exposure. This highlights the importance of selecting the most suitable treatment method based on the specific insect pest, and the type of pesticide used.

Apart from the immediate lethal effects of toxic substances on pests, chemical applications can indirectly impact the survival of insects exposed to the sublethal doses [46,47,48]. These effects can significantly influence pest population dynamics over time. For instance, exposure to sublethal doses can lead to behavioral changes, such as altered feeding or movement patterns, which may affect the pest’s ability to find food, mate, or avoid predators, potentially reducing overall fitness [49, 50]. However, sublethal exposure can also trigger compensatory mechanisms, such as increased reproduction rates or faster development, allowing pests to recover their populations quickly and even exceed pre-exposure levels.

Moreover, sublethal effects can contribute to resistance development in pest populations. Insects exposed to sublethal doses may survive and pass on genetic traits that confer partial resistance to the toxic substance. Over time, repeated exposure can lead to the selection of resistant individuals, ultimately increasing the frequency of resistance alleles within the population [51]. In addition, sublethal exposure can enhance detoxification enzyme activities or other physiological adaptations, further accelerating resistance development [52, 53]. To mitigate these effects, integrated pest management (IPM) strategies should consider the potential long-term consequences of sublethal exposures and resistance development. Incorporating diverse control methods, rotating insecticides with different modes of action, and monitoring pest populations for signs of resistance can help maintain control effectiveness and delay resistance onset.

Hence, chemical application in pest control is a complex process with direct and indirect effects, including the proteins, which are intricate compounds in all viable cells. Among these, nucleoproteins play a crucial role in cell division, while hormones and enzymes regulate numerous chemical reactions in cellular metabolism. Therefore, studying the separation and identification of insect proteins post-treatment with toxic substances holds promise for understanding protein modulation. This knowledge can be leveraged for pest management strategies [54].

This study aimed to evaluate the toxicity of B. bassiana and tomato leaf extract on black cutworm (A. ipsilon) larvae through two different bioassay methods, including leaf dipping and poisoned baits. In addition, we investigated their sublethal effects on larval and pupal durations based on the toxicity data obtained from these bioassay methods. Our experiments were guided by the hypothesis that, besides causing direct mortality, the applied agents could decrease pest fitness, thus improving pest suppression. Furthermore, we analyzed the changes in the protein profiles of treated larvae hemolymph using the SDS–PAGE technique.

Materials and methods

Insect culture

A culture of black cutworm, A. ipsilon, was sourced from the Agriculture Research Center, Egypt. This culture was maintained on castor leaves (Ricinus communis) for more than ten generations under laboratory conditions of 27 ± 2 °C and 65 ± 5% relative humidity, without insecticide exposure. The larvae were reared in 500 ml glass jars until reaching the third instar stage, with a layer of sawdust at the base for humidity absorption, covered with muslin secured by a rubber band. To prevent cannibalism, fourth instar larvae were individually placed in small cups and provided with castor plant leaves until they reached the pupation stage [55, 56]. Newly emerged moths were paired in large glass jars, with a cotton piece soaked in a 10% sugar solution provided as nourishment. Black net strips were included in the jars to facilitate egg laying. Fourth-instar larvae of similar weight (5.0 ± 0.4 mg of the mean weight), and size were selected for subsequent experiments.

Fungus culture

An isolate of Beauveria bassiana (Balsamo) (Deuteromycota: Hyphomycetes) (3873AUMC) was acquired from the culture collection of the Mycological Center at Assiut University. This specific isolate was chosen based on recent publications demonstrating its pathogenicity to various pest species [57, 58]. A preliminary bioassay was performed on A. ipsilon.

The fungal culture was cultivated on sabouraud dextrose yeast agar (SDYA), comprising 20 g of glucose, 15 g of agar, 10 g of peptone, and 5 g of yeast extract. After autoclaving at 120 °C for 20 min, the mixture was poured into sterile glass Petri dishes (9 cm in diameter) and incubated for 15 days at 25 °C in darkness [25].

A conidia suspension was prepared by collecting conidia from the Petri dishes into a sterile 0.1% tween 80 aqueous solution [59]. This suspension underwent sonication for 5 min, and a single layer of linen served as a filter to eliminate mycelia.

The resulting conidial stock suspension was assessed using an improved Neubauer bright line hemocytometer (Neubauer improved HBG, Germany) under a ZEISS light microscope at 400X magnification [28].

Conidial viability was determined before suspension preparation by spreading 0.2 mL of 1 × 105 conidia/mL on the SDAY medium. Conidial germination was evaluated under a microscope after 24 h, with a germination percentage exceeding 95% for all experiments [60].

Tomato plant leaves collection and extraction

Fresh leaves of the tomato plant (Solanum lycopersicum, variety Ellisa), aged 2 months, were gathered in the early morning from an unsprayed field in Meet el Amel village, Aga City, Dakahlia Governorate, Egypt (30°53′43.8 "N 31°19′35.8" E). These leaves were washed with water and air-dried at room temperature (26 ± 2 °C) for 2 weeks. Subsequently, the dried leaves were manually ground into a fine powder.

Following the method of Abd-Allah [61], the leaf powder was immersed in a flask containing an equal mixture of ethanol, acetone, and hexane (1:1:1) for approximately 1 week. Afterward, the flask was agitated on a shaker, and the contents were filtered using filter paper. The solvents were then evaporated using a vacuum rotary evaporator under reduced pressure at 40 °C. The residue was allowed to cool at room temperature to eliminate any remaining solvent traces. Finally, the crude extract was collected, weighed, and stored in a deep freezer until required.

GC–MS analysis

The chemical composition analysis of the tomato plant crude extract (TPCE) was carried out using a Trace GC1310-ISQ mass spectrometer (Thermo Scientific, based in Austin, TX, USA). The setup and adjustments of the apparatus followed the procedures outlined by Awad et al. [56].

Bioassays

Two bioassay methods were employed to assess the effects of the B. bassiana suspension and the TPCE on fourth-instar A. ipsilon larvae: the leaf dipping technique and the poisoned bait technique.

For the B. bassiana conidial suspensions, fresh preparations were made by diluting the conidia with 0.1% tween 80 to achieve final concentrations of 2.2 × 105, 2.2 × 106, 2.2 × 107, and 2.2 × 108 conidia ml−1, measured using a Malassez chamber. These concentrations were selected based on a preliminary bioassay on A. ipsilon larvae. The presence of B. bassiana in deceased larvae was confirmed using the method outlined by Resquín-Romero et al. [25]. In summary, the deceased larvae were collected daily, sterilized with 1% sodium hypochlorite, rinsed twice with distilled water, placed on wet filter paper in Petri dishes sealed with parafilm, and maintained at room temperature (26 ± 2 °C). Five days after treatment, the larvae cuticles were examined under a light microscope to detect fungal growth.

The stock concentration of TPCE was prepared based on the crude weight after solvent evaporation and the volume of distilled water added (w/v). Tween 80 served as an emulsifier at a concentration of 0.05%. Four concentrations were derived from the stock concentration following a primary bioassay on A. ipsilon larvae: 0.5, 1, 5, and 10 mg ml−1.

Leaf dipping technique

The leaf dipping bioassay technique utilizing castor oil plant leaf discs was employed to determine the lethal concentration (LC) values of the B. bassiana suspension and TPCE on A. ipsilon fourth-instar larvae. Castor leaf discs (50 mm diameter) were punched using a cork borer, dipped into each concentration of B. bassiana suspension and TPCE for 20 s, then dried at room temperature for 1 h. Forty fourth-instar larvae for each tested concentration were placed individually in small cups (63 × 80 mm) containing a 1.5-inch layer of fine sawdust, covered with a clean muslin cloth, and divided into four replicates (10 larvae/replicate). The larvae were starved for 4 h before feeding and were allowed to feed on the treated leaf discs for 5 days. Control larvae were treated with leaf discs immersed in sterile water with 0.1% tween 80 for the B. bassiana suspension and 0.05% tween 80 for the tomato crude extract. Larval mortality was recorded at 1, 3, and 5 days.

Baits method

The baits were prepared following the method described by Balevski et al. [62] with slight modifications. A mixture of 1.5 kg of wheat bran, 0.5 g molasses (black honey) as an attractant, and approximately 0.5 L of water was thoroughly combined and left overnight in a warm, dark place to ferment. Subsequently, 10 g of the bait was mixed with 10 ml of each tested concentration of B. bassiana suspension and TPCE. The poisoned baits were then equally divided among each treatment and provided to individual larvae.

Each concentration was tested on 40 fourth-instar larvae (160 larvae per treatment), with larvae placed individually in small cups covered with a muslin cloth. The larvae were divided into four replicates, each containing 10 larvae. The bait for the control larvae was mixed with distilled water; 0.1% tween 80 was used for the B. bassiana suspension, and 0.05% tween 80 was used for the TPCE.

Biological parameters

The impact of the B. bassiana suspension at concentrations of 2.2 × 105 and 2.2 × 106 conidia ml−1 was assessed using the leaf dipping method on the durations of the fifth- and sixth-instar larvae, pre-pupal, and pupal stages. In addition, the same parameters were evaluated at a concentration of 2.2 × 105 conidia ml⁻1 using the bait method. The effect of TPCE at concentrations of 0.5 and 1 mg ml−1 was evaluated on the same parameters using the leaf dipping method and at a concentration of 0.5 mg ml−1 using the bait method.

For each tested concentration, the experiment was conducted with four replicates, each containing 10 larvae housed in small cups as previously described. After a 5-day exposure period to the respective concentrations, the surviving larvae were individually transferred into new, clean cups containing an untreated diet for further observation and study.

Biochemical study (protein profile)

Insect protein extraction

Fourth-instar larvae were exposed to the LC50 values of the B. bassiana suspension and TPCE. After 5 days, hemolymph samples were collected from live larvae by removing the forelegs and transferring the hemolymph into Eppendorf tubes coated with a layer of crystalline phenyl thiourea to prevent melanization [63]. To minimize protein degradation, all procedures were conducted on ice, and a few crystals of protease inhibitor, i.e., p-aminobenzamidine, were added to the hemolymph samples immediately upon collection. Three hemolymph replicates were collected for each treatment. The collected hemolymph was centrifuged at 1190 g at 4 ℃ for 6 min and stored at – 20 ℃ for subsequent analysis.

Bradford's method [64] was employed to assess the total protein concentration in the hemolymph of A. ipsilon larvae, utilizing Coomassie brilliant blue dye and bovine serum albumin as standards. All chemicals utilized for protein determination were of analytical grade and sourced from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA).

Gel preparation and running

The protein fractionation process was carried out using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS–PAGE), adhering to the method outlined by Laemmli [65]. This involved preparing a separating gel comprising 15% acrylamide in 1.5 M Tris–HCl buffer (pH 8.8) and a stacking gel with 3% acrylamide in 0.5 M Tris–HCl buffer (pH 6.8). Protein samples were prepared from 10 μL aliquots of hemolymph, which were resuspended in 5 mL of 5X SDS–PAGE loading buffer (containing 60 mM Tris–HCl at pH 6.8, 3.75 mM EDTA, 2.0% SDS, 25% glycerol, 14.4 mM β-mercaptoethanol, and 0.1% bromophenol blue). To prevent degradation, the samples were boiled in a water bath for 2 min, quickly cooled in ice water, and loaded onto the gel.

Electrophoresis was performed at a steady 100 V for 6 h at 2 mA per well. Post-electrophoresis, the gel was stained using Coomassie brilliant blue R250 (comprising 1.25 g of dye in a 450 mL solution of methanol: H2O in a 1:1 v/v ratio and 50 mL of glacial acetic acid). The gel was then destained in a methanol/acetic acid solution (90 mL of methanol: H₂O in a 1:1 v/v ratio and 10 mL of acetic acid) and placed on an orbital shaker until the protein bands were colorless. Following the destaining, the molecular weights of the protein bands were ascertained by comparing them with a set of standards ranging from 5 kilodaltons (kDa) to 250 kDa.

Data analysis

The data were tested to ensure they met the assumptions of parametric tests. Continuous variables were subjected to the Shapiro–Wilk and Kolmogorov–Smirnov tests for normality. Mortality data were corrected for untreated controls [66]. The LC-values were subjected to Probit analysis [67] using the LdP LineTM software (Ehabsoft, http://www.ehabsoft.com/ldpline/). ANOVA analyses were performed for the experimental groups for the recorded developmental parameters. Data were presented as the mean and standard deviation. Tukey’s multiple comparisons were conducted to compare the control group with B. bassiana and TPCE applied using the leaf dipping or poisoned bait bioassay methods. P values were considered significant at < 0.05. Data visualization was carried out when needed using GraphPad Software Inc., San Diego, California, USA.

Results

Toxicity assays of Beauveria bassiana

The mortality percentage of A. ipsilon larvae in response to Beauveria bassiana using two different bioassay methods (leaf dipping and poisoned baits) at 1-, 3-, and 5-day post-treatment is presented in Fig. 1. In addition, the LC values of Beauveria bassiana (conidia ml−1) on A. ipsilon fourth-instar larvae 5-day post-treatment using both bioassay methods are presented in Table 1.

Fig. 1
figure 1

Mortality (%) of Agrotis ipsilon larvae in response to Beauveria bassiana exposure using two different bioassay methods: (A) leaf dipping and (B) poisoned baits. Tukey’s multiple comparison test was performed with the data expressed as mean (± S.D) (significantly P < 0.05)

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Table 1 LC values of Beauveria bassiana (conidia ml−1) on Agrotis ipsilon fourth-instar larvae 5-day post-treatment using leaf dipping and baits bioassay methods
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The LC values of B. bassiana using the leaf dipping method were 1.6 × 108 and 1.4 × 1012 conidia ml−1 for LC50 and LC90, respectively, while these values were 1.8 × 106 and 6.1 × 108 conidia ml−1, respectively, using the poisoned baits method.

GC analysis of tomato plant crude extract (TPCE)

The chemical composition of the tomato plant crude extract is listed in Table 2. The main bioactive constituents of the extract were octacosane (36.27% area) and 15-nonacosanone (27.97% area) (Fig. 2).

Table 2 Chemical composition of tomato plant crude extract (TPCE)
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Fig. 2
figure 2

Chemical configuration of the main bioactive constituents of tomato plant crude extract (TPCE)

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Toxicity assays of TPCE

The percentage mortality in A. ipsilon larvae in response to TPCE using the leaf dipping and poisoned baits methods at 1-, 3-, and 5-day post-treatment is presented in Fig. 3. The LC values of TPCE (mg ml−1) against A. ipsilon larvae 5-day post-treatment for both bioassay methods are presented in Table 3. The LC values were 4.35 and 83.1 mg ml−1 for LC50 and LC90, respectively, using the leaf dipping method, while these values were 1.51 and 24.2 mg ml−1, respectively, using the poisoned baits method.

Fig. 3
figure 3

Mortality (%) of Agrotis ipsilon larvae in response to TPCE treatment using two different bioassay methods: (A) leaf dipping and (B) poisoned baits. Tukey’s multiple comparison test was performed with the data expressed as mean (± S.D) (significantly P < 0.05)

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Table 3 LC values of TPCE (mg ml−1) on Agrotis ipsilon fourth-instar larvae 5-day post-treatment using leaf dipping and baits bioassay methods
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Effect of Beauveria bassiana on larval and pupal durations of Agrotis ipsilon

The effects of B. bassiana on the larval and pupal durations of A. ipsilon using the leaf dipping method are shown in Table 4.

Table 4 Impact of Beauveria bassiana on the larval and pupal durations of Agrotisipsilon using the leaf dipping and poisoned baits methods
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A concentration of 2.2 × 106 conidia ml−1 led to a significant increase in the fifth- and sixth-instar larval duration, extending to 9.52 and 9.00 days, respectively, compared to the control group's 7.02 days. Similarly, a concentration of 2.2 × 105 conidia ml−1 significantly prolonged the fifth-instar larval duration to 8.00 days, compared to the control group with 7.05 days larval duration. Both concentrations of 2.2 × 105 and 2.2 × 106 conidia ml−1 notably extended pupal durations to 11.5 and 11 days, respectively, compared to the control group’s 10 days.

The effects of B. bassiana on A. ipsilon’s larval and pupal durations, as evidenced by the poisoned baits method, are illustrated in Table 4. A noteworthy decrease in the durations of the fifth- and sixth-instar larvae, pre-pupal, and pupal stages was observed with a concentration of 2.2 × 105 conidia ml−1, compared to the control group.

Effect of TPCE on the larval and pupal durations of Agrotis ipsilon

The TPCE impact on the larval and pupal durations of A. ipsilon using the leaf dipping method is outlined in Table 5.

Table 5 Impact of TPCE on the larval and pupal durations of A. ipsilon using the leaf dipping method
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The concentrations of 0.5 and 1 mg ml−1 notably decreased the fifth- and sixth-instar larvae duration to 4.75 and 6.00 days and 5.04 and 6.05 days, respectively, compared to the control group's durations of 7.05 and 7.02 days. In addition, these concentrations significantly reduced pupal durations to 8.00 and 8.91 days, respectively, compared to the control group’s 10.0 days.

The impacts of TPCE on A. ipsilon's larval and pupal durations, as shown in Table 5 via the poisoned baits method, revealed a significant reduction in larval and pupal durations when a concentration of 0.5 mg ml−1 was used compared to the control group.

Changes in hemolymph total protein

The alterations in the total protein levels of A. ipsilon 4th larval instar hemolymph following a 5-day treatment with B. bassiana and TPCE are depicted in Fig. 4. The treatment with B. bassiana resulted in a significant decrease in protein concentration [F (2,6) = 9.93*, P = 0.012], whereas TPCE showed an insignificant decrease compared to the control group.

Fig. 4
figure 4

The total protein contents of Agrotis ipsilon larval hemolymph after 5 days of treatment with B. bassiana suspension and TPCE. Tukey's multiple comparison test was conducted with the data expressed as mean (± S.D). Mean values with different letters are significantly different (P ≤ 0.05)

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Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) banding pattern

The protein electrophoretic profile of untreated larvae, larvae exposed to the LC50 of B. bassiana, and larvae treated with TPCE is presented in Table 6. The control sample exhibited a maximum of 12 bands, with Rf values ranging from 0.73 for a band with a molecular weight (Mw) of 35 kDa to 0.23 for a band with an Mw of 120 kDa. In contrast, B. bassiana-treated larvae displayed ten bands with Mw ranging from approximately 120 to 35 kDa, and tomato leaf extract-treated larvae showed a minimum of seven bands with Mw ranging from approximately 35–97 kDa.

Table 6 Molecular weight (KDa) and relative fragmentation (Rf) of hemolymph protein pattern of A. ipsilon larvae exposed to the LC50 value of Beauveria bassiana and tomato plant crude extract (TPCE)
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Notably, three unique bands were observed: two in the control samples (C) with Rf values of 0.35 and 0.46 and Mw of 90 and 68 kDa, respectively, and one in the samples of TPCE-treated larvae with an Rf value of 0.31 and Mw of 97 kDa.

Discussion

In response to concerns regarding environmental impact and genetic resistance from repeated pesticide use [68, 69], alternative insect management strategies have gained considerable attention [70]. This study evaluated the toxicity of bio-insecticide alternatives, specifically the entomopathogenic fungus B. bassiana and a botanical extract from S. lycopersicum (tomato plant crude extract, TPCE), against A. ipsilon using two exposure methods.

In our investigation, lower LC50 and LC90 values for B. bassiana and tomato plant crude revealed the greater efficacy of the poisoned baits method in delivering active compounds to A. ipsilon larvae, resulting in increased toxicity. The reduced amounts of insecticidal agents needed for desired pest mortality levels, as indicated by the lower LC50 and LC90 values, can be attributed to the delivery system of the active compounds through direct ingestion by the pests upon bait consumption, facilitating quicker and more efficient absorption [71].

However, additional factors may also contribute to the higher effectiveness observed with poisoned baits. Environmental conditions, such as humidity and temperature, could affect the stability and persistence of the active compounds in the bait, potentially enhancing their efficacy. The bait medium may also provide a more stable environment for the active compounds, preventing degradation that might occur on the leaf surface. Furthermore, behavioral aspects of the larvae, such as feeding preferences and movement patterns, could increase the likelihood of consuming the bait and thus directly affect the toxicity outcomes [72].

Incorporating insecticidal agents into bait increases the likelihood of pests ingesting lethal doses directly while potentially reducing environmental impact by minimizing insecticide usage and decreasing exposure of non-target organisms to treatment [73]. However, future studies under realistic field conditions are needed to evaluate the effects of these biopesticides on non-target organisms, particularly beneficial insects. Such assessments will be crucial to ensure that these biopesticides can be safely integrated into pest management programs without unintended ecological consequences.

The discovery that poisoned baits surpass leaf dipping in efficacy for B. bassiana, and TPCE carries substantial implications for integrated pest management (IPM) strategies. IPM aims to employ the most efficient and environmentally sustainable pest control methods [74, 75]. Integration of poisoned baits can enhance the effectiveness of biological control agents (BCAs) such as B. bassiana and natural plant extracts, potentially diminishing reliance on synthetic chemical pesticides. For instance, poisoned baits have been successfully used in IPM programs to control several pests, demonstrating their practicality and scalability in different agricultural contexts [76, 77]. However, several practical challenges need to be considered for successful implementation. These challenges include the formulation of baits to maintain the stability and viability of active compounds, ensuring attractiveness to the target pest while minimizing non-target effects, and developing cost-effective and scalable production methods [71]. In addition, there may be challenges related to the acceptance of bait strategies by farmers and other stakeholders, who may have concerns about costs, ease of use, and compatibility with existing pest management practices [78, 79]. Addressing these challenges through targeted research, extension services, and farmer education could enhance the feasibility and adoption of poisoned bait methods in IPM.

In many insect species, the epicuticular layer comprises a thin waxy coating rich in lipids such as hydrocarbons, phospholipids, fatty acids, glycolipids, and wax [80]. These components shield insects from desiccation and microbial threats. While B. bassiana produces lipid-degrading enzymes like cytochrome P450 monooxygenases that can break down insect hydrocarbons [81], the method of application plays a critical role in its efficacy. In our study, we employed leaf dipping and poisoned bait methods, which may not directly target the epicuticle but effectively deliver the fungal pathogen through ingestion or contact during feeding. B. bassiana has been demonstrated to be toxic to numerous lepidopteran pests [23, 24, 82], and its sublethal effects across different modes of exposure have also been well-documented [83].

Comparing the two application methods of B. bassiana on the same insect could yield valuable insights into optimizing its use for pest control. Such a comparison may help determine the most effective and efficient method for deploying B. bassiana against specific lepidopteran pests, enhancing its practical application in integrated pest management programs.

On the other hand, TPCE has been demonstrated to possess insecticidal and miticidal properties [84, 85]. In the present study, GC–MS analysis revealed that the major components of TPCE are octacosane and 15-nonacosanone, with respective areas of 36.27 and 27.97%. These compounds were identified as major components in various tomato leaf cultivars in a study by El-Badawy et al. [86]. Their significant presence in the extract suggests their critical role in its bioactivity. The hydrophobic nature of these compounds can disrupt insect cuticles, leading to desiccation and mortality, particularly when TPCE is applied using the leaf dipping bioassay method.

In our investigation, both B. bassiana and TPCE adversely affected the larval and pupal durations and adult longevity of A. ipsilon by altering larval and pupal stages. When administered as poisoned baits, both treatments notably reduced these durations. This indicates that larvae consuming the baits received higher doses of B. bassiana or TPCE, leading to rapid physiological disruption or mortality. Consistent with our findings, Russo et al. [26] reported that B. bassiana inoculated with a preferred food of Helicoverpa gelotopoeon (Dyar) (Lepidoptera: Noctuidae) similarly resulted in a shortened life cycle duration.

This reduction in the larval and pupal durations of A. ipsilon under TPCE treatment suggests the presence of compounds within the extract that disrupt insect growth. Various researchers have reported similar observations, noting reduced larval durations following treatment with plant leaf extracts [87,88,89]. In addition, it has been proposed that these disruptions in insect development exposed to plant-derived compounds may result from hormonal system disturbances, induced by these substances [90, 91].

In contrast to our other findings, applying B. bassiana using the leaf dip method extended the duration of the fifth- and sixth-larval instars and the pupal stage. This suggests that fungal infection may hinder larval development by impeding the physiological processes crucial for molting and metamorphosis. The prolonged development could stem from sub-lethal stress induced by the pathogen, slowing down larvae as they struggle to cope with the infection [92]. The varied responses of the same insect to the same pathogen likely arise from differences in exposure method, highlighting the importance of application methods in determining the efficacy of BCAs.

Insect hemolymph is crucial in transporting hormones and nutrients, facilitating development, storing amino acids, and providing innate immunity, thus influencing various physiological processes [93]. Variations in protein concentration within larval hemolymph can be attributed to the diverse impacts of different treatments on physiological processes [94]. Consequently, differences in protein patterns between control and treated larvae may reflect adaptations and modifications within the organism to cope with the treatments.

In our study, treatment with the LC50 value of B. bassiana significantly decreased the protein concentration in treated larvae compared to the control. This observation aligns with Gabarty et al. [95], who reported a 55% decline in protein concentration in S. littoralis 4 days after treatment with the LC50 value of B. bassiana. The reduction in total protein and the depletion of soluble protein from the host's hemolymph during parasitism may be attributed to proteolytic enzymes released into the insect’s hemocoel, breaking down host proteins [96].

Our study’s qualitative assessment of hemolymph protein using SDS–PAGE revealed alterations in protein bands across various treatments. The appearance and disappearance of specific protein bands during larval development suggest that certain proteins are synthesized to meet immediate physiological needs [97]. In addition, these alterations indicate stress or metabolic changes in the larvae. Interestingly, larvae treated with TPCE exhibited fewer protein bands than control larvae and those treated with B. bassiana, with the appearance of a new band with a molecular weight of 97 kDa. The presence of a new band in the treated larvae might indicate the formation of a new protein, which could be related to one of the detoxification enzymes, as [54] suggested. However, the absence of protein bands in the hemolymph of insects treated with TPCE suggests a disruption in protein synthesis or degradation of existing proteins, likely caused by the bioactive compounds present in the extract. This indicates that TPCE significantly impacts the insect's physiological processes, potentially impeding its development and survival.

Conclusion

Both B. bassiana and TPCE significantly affect the mortality and development of A. ipsilon larvae, with poisoned baits demonstrating superior effectiveness as a delivery method. Moreover, alterations in hemolymph protein levels indicate physiological stress induced by these treatments. Future research should prioritize optimizing application methods and concentrations to enhance efficacy while mitigating potential environmental impacts. Field studies are crucial to validate these findings in real-world conditions and to assess the long-term viability of integrating these bio-insecticides into holistic pest management strategies.

Availability of data and materials

No datasets were generated or analysed during the current study.

Abbreviations

EPF:

Entomopathogenic fungi

TPCE:

Tomato plant crude extract

GC–Mass:

Gas chromatography–mass spectroscopy

SDS:

Sodium dodecyl sulfate

BCAs:

Biological control agents

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Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

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Authors and Affiliations

  1. Plant Protection Research Institute, Agricultural Research Center, Dokki, Giza, Egypt

    Ghada E. Abd-Allah

  2. Department of Economic Entomology and Pesticides, Faculty of Agriculture, Cairo University, Giza, 12613, Egypt

    Moataz A. M. Moustafa & Fatma S. Ahmed

  3. Zoology & Entomology Department, Faculty of Science, Al-Azhar University (Girls Branch), Cairo, Egypt

    Eman El-said, Enayat M. Elqady, Lina A. Abou El-Khashab & Hend H. A. Salem

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  1. Ghada E. Abd-Allah
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  2. Moataz A. M. Moustafa
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Contributions

All authors contributed 100% participation. The authors G. E. A., M.A. M. M., F. S. A., E. E., E. M. E., L. A. A., and H.H. A. S. contributed the following: suggesting and putting the idea, preparing the manuscript writing and finishing the paper, and data analysis. The authors read and approved the final manuscript.

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Correspondence to Fatma S. Ahmed.

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Abd-Allah, G.E., Moustafa, M.A.M., Ahmed, F.S. et al. Insights into larval development and protein biochemical alterations of Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae) following Beauveria bassiana and Solanum lycopersicum treatments. Chem. Biol. Technol. Agric. 11, 164 (2024). https://doi.org/10.1186/s40538-024-00692-9

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