The impact of carbon NPs on the accumulation of storage proteins and the generation advancement of the polyphagous insect pest tobacco cutworm Spodoptera litura (Fabricius)

Abstract

Spodoptera litura (Lepidoptera: Noctuidae) is globally considered one of the most important agricultural pests. It is a highly prevalent insect pest that severely damages several vegetables and crops, including cotton, castor, tobacco, beet, soybean, and cabbage. The implementation of integrated pest management (IPM) practices has led to a slight controlling of its population in the field; however, these practices are always linked with the high economic and environmental costs for eradication. In the last decade, many researchers have reported the control of this devastating insect via the utilization of nanoparticles (NPs); however, the mechanism behind its toxicity is still a gap area. In our study, we investigated the toxic impact of carbon nanoparticles (CNPs) on S. litura when administered orally. A range of immunomodulatory responses were observed, including a distorted morphology (such as abnormal pupa, distorted wings in insects, etc.) and reproductive physiology, weight reduction, and even insect death. Mass spectrometric analysis of differentially expressed proteins suggests significant downregulation of storage proteins in the larval hemolymph, which in turn resulted in an altered expression/synthesis of developmental and reproductive proteins, including vitellogenin (the major egg-filling protein required for developing embryo nourishment), in the treated insects. The poor accumulation of vitellogenin in developing eggs led to a disrupted life cycle and restricted population growth. This is the first study that provides insights into the molecular mechanism behind the toxicity offered by these tiny carbon particles.

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Environmental significance

This study envisaged the use of carbon nanoparticles (CNPs) derived from waste candle soot as a substitute to chemical insecticides for controlling the globally significant agricultural pest Spodoptera litura. The research demonstrates that the oral administration of CNPs disrupts the growth, physiology, and reproductive behavior of S. litura, leading to a distorted morphology, reduced larval weight, and disrupted reproductive physiology. The study elucidates the molecular mechanisms behind this toxicity, emphasizing the downregulation of storage proteins and the subsequent disruption of vital reproductive proteins. Notably, this study advocates the environment friendly nature of candle soot-derived CNPs and suggests their potential role as a sustainable candidate for pest control. The findings encourage further exploration of nanotechnology-based approaches in integrated pest management, promoting a more ecologically responsible agricultural future.

1. Introduction

Agriculture is the backbone of many countries, and the problem of field insect pests in agriculture is as old as agriculture itself. Biting and chewing insect pests have become a serious threat to crop productivity. Among them, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), commonly known as cotton leafworm or tobacco cutworm, is a major polyphagous insect pest, with a strong migratory ability that has seen it widely distributed all through the Middle East, east Asia, Oceania, and Pacific islands.¹ This together with its high reproductive rates are the major reasons behind the repeated incidences of outbreaks around the globe.² During the period 2008–2009, an outbreak of S. litura on soybeans in the Maharashtra and Rajasthan states of India caused a fiscal loss of around USD 300 million and 64 million, respectively.³ Several integrated pest management (IPM) practices have been followed to minimize the impact of this voracious feeder.⁴ Unfortunately, they all have witnessed limited success, except for the use of chemical pesticides, the hazards of which are well documented. The frequent practice of insecticide application has led to the rapid development of resistance against many insecticides (organophosphate, carbamate, pyrethroids, indoxacarb, abamectin, emamectin benzoate, and chlorantraniliprole), which in turn has resulted in periodic outbreaks of this devastating pest.⁵ Hence, there is an urgent need to develop an alternative method that can efficiently bring the population of S. litura under control.

Nanotechnology is an emerging branch of science that deals with the manipulation of matter in a size ranging from 1 nm to 100 nm.⁶ The alteration of any matter within this size offers unique physical and chemical properties that become the reason for the wide application of nanotechnology in various fields of science and technology, including agriculture.⁷ Nanoparticles (NPs) are used for innumerable purposes in agriculture, including pest control, plant growth and enhanced productivity, and numerous studies have focused on the use of NPs for insect pest management.⁸ Several metals and carbon NPs have been reported to have insecticidal properties against various groups of insect pests such as Lepidopterans, Hemipterans, and Dipterans.⁹ The most damaging genera that come under the order Lepidoptera are Helicoverpa spp., Spodoptera spp., and Plutella spp. They cumulatively cause up to 305–40% yield loss in cotton, 205–40% in tomato, and 22.135–46.83% in other vegetables.¹⁰ Though a variety of metal and carbon NPs have been tested for their effectiveness against these insect pests, few of them, typically silica, silver, carbon NPs, and their oxides, are repeatedly tested by many scientists around the globe. CNPs, carbon nanofibers (CNFs), carbon nanotubes (CNTs), and graphene/graphene oxide (GO) have previously demonstrated their utility for pest management.¹¹ AgNPs synthesized either from a plant source or via a chemical process exhibit a range of toxicity. For example, AgNPs from the leaf extract of Leonotis nepetifolia and Ocimum basilicum, and seed and peel extract of Glycine max and Punica granatum caused increased antioxidant activity and larval mortality (56–100%) in S. litura.^12–15 AgNPs and AuNPs prepared from various plant sources reduced the larval weight and caused mortality in the larva and pupa of Helicoverpa armigera (Hübner) and Plutella xylostella (Linnaeus).^16–22 SiNPs generated from various plant sources caused 85–100% mortality of H. armigera, S. litura, and P. xylostella larvae by disruption of the cuticle, resulting in dehydration along with spiracle and tracheal blockage.^23–26 Carbon nanoparticles in the form of fly ash have been used to control the lepidopteran pest.²⁷ The ingestion of GO and multiwalled CNTs (MWCNTs) was shown to significantly reduce the fecundity and fertility of Spodoptera frugiperda (J.E. Smith).²⁸ Waste candle soot-derived CNPs showed somewhat similar effects on the reproductive behavior of H. armigera.²⁹ A few other carbon nanomaterials, such as CNFs and CNPs, were reported to lower larval survival rates, causing developmental abnormality and a poor reproductive rate in Drosophila melanogaster.^29,30

In addition to the toxicity assessment, researchers have also tried to investigate the underlying mechanism behind the toxicity offered by these tiny particles.³¹ In general, NPs induce immune responses that lead to the altered expression of genes/proteins, resulting in the disruption of metabolic processes with increased cellular toxicity. Other common responses include the disruption of nutrition intake, formation of reactive oxygen species, and altered metabolic activities.³² All these cumulatively affect the growth, development, and reproductive ability of the pest insects. Further, the extent of toxicity is highly dependent on the type and physical properties of the selected NPs, while the mode of toxicity is reliant on the route of administration (ingestion/inhalation/physical contact).³³ However, knowledge of the molecular mechanism of NP-mediated toxicity, including the implication of an altered protein profile in insect pests, is still not clear.

In this study, we investigated the toxic impact of carbon nanoparticles (CNPs) against S. litura upon oral administration and found them to be very effective for controlling the target insect pests. We investigated the underlying NPs-mediated toxicity in insects using differential proteomics of the hemolymph (larvae, pupae) and eggs, in order to identify the potential proteins and their role in CNP-induced toxicity in insect pests. Our study demonstrates the remarkable efficacy of carbon nanoparticles in controlling S. litura through oral administration, elucidating the underlying toxicological mechanisms via hemolymph proteomics.

2. Experimental section

2.1 Materials

All of the chemicals utilized in this study were of analytical grade and were used without additional modification. Agar powder, ammonium bicarbonate (ABC), sodium deoxycholate, acetonitrile (ACN), dithiothreitol (DTT), iodoacetamide (IAA), phenylmethylsulfonyl fluoride (PMSF), and acetone were purchased from Sigma-Aldrich (CA, USA). Casein, ascorbic acid, sorbic acid, yeast extract powder, cholesterol, streptomycin sulfate, methyl powder, and formaldehyde were obtained from HiMedia Laboratories Pvt. Ltd. Trypsin Gold Mass Spec Grade was procured from Promega, (San Luis Obispo, CA, USA). Protease inhibitor cocktails (PIC) were procured from Roche Diagnostic GmbH, Manheim, Germany. Glutathione reduced (GSH, 99.5%), alpha-naphthyl acetate extra pure AR (99%), 1-chloro-2, 4 dinitrobenzene extra pure (CDNB, 99%), and fast blue RR salt were obtained from SRL Pvt. Ltd. C18 Zip-tip was procured from Thermo Fisher Scientific (Waltham, MA). Chickpea powder, multivitamin and vitamin E capsules, and candles (for nanoparticle synthesis) were purchased from the local market.

2.2 Synthesis of the nanoparticles

CNPs were synthesized according to our previous studies. The CNPs are believed to contain approximately 90% carbon elements.³⁴ In brief, the soot from candle flames was collected. Other contaminants were removed by treating with alternate cycles of 10 M nitric acid and ethyl alcohol. In the next step, the CNPs were washed with Milli-Q water several times until the pH dropped down near 7.0 and were then vacuum dried in an oven at 150 °C for 8 h. Now, the CS-CNPs were able to form colloids in water and ethanol. After drying, glittery CNPs were obtained and used as an experimental material for testing on S. litura.

2.3 Insect rearing and bioassay

S. litura larvae were reared on an artificial diet optimized in our laboratory at CSIR-National Botanical Research Institute, Lucknow, India. The dietary composition included 140 g of chickpea powder, 13 g of yeast extract powder, 4 g of ascorbic acid, 3 g of methyl powder, 0.8 g of sorbic acid, 250 mg of streptomycin sulfate, 1 capsule of vitamin E 1, and 1 capsule of multivitamin, all solubilized in 600 ml of deionized water (ddH₂O). Additionally, 17 g agar-agar powder was dissolved in 400 ml ddH₂O. The components, along with the agar powder, were thoroughly mixed, poured into Petri plates, and allowed to solidify. The culture was maintained at a temperature of 27 ± 2 °C and 65 ± 5% relative humidity with a 16 L : 8 D hour photoperiod cycle. To assess the toxicity of the candle-soot-derived nanoparticles (CS-CNPs), a cohort of neonates (N = 30 in each treatment and control) was individually housed in a sterile, small ventilated specimen tube. A stock solution of CNPs (5 mg ml⁻¹) was prepared by dissolving it in 1× PBS. Further, it was used for the preparation of treatments containing 250, 500, and 1000 μg CNPs gm⁻¹ of diet. Here 1× PBS along with diet was used as the control. The diet was changed after every 48 h. The larval weight, longevity, mortality, unconsumed diet, and fecal matter were systematically recorded at 48 h intervals throughout the experiment. The whole experiment was replicated three times.

2.4 Fecundity assessment

Newly emerged male moths were paired with female moths of the same treatment group and age. For the study of the reproductive parameters, three mating pairs from each of the three groups (250, 500, and 1000 μg gm⁻¹) along with the control were taken. Each pair was transferred to a ventilated cylindrical plastic beaker (12 D × 15 H cm) for oviposition and fed on a liquid diet (5% sucrose containing 5% honey solution) soaked in cotton fiber. Three random patches were taken for the study of the oviposition period, egg-laying pattern, no. of eggs laid per unit area, and larval emergence.

2.5 Insect's ovary dissection

Mated female moths (developed from larvae, fed on all three concentrations of CNPs and the control) were used for the detailed microscopic study of the ovary. For this, the female moths were anesthetized using diethyl ether followed by a slit made on the ventral side of the abdomen. The ovary was pulled out with the help of forceps and kept on a notched glass slide for microscopic examination under a stereomicroscope (Leica M205) to study the morphological changes.

2.6 Protein isolation from eggs and hemolymph (larvae and pupae)

Late last instar larvae and pupae were washed with 1× PBS and immobilized on ice for 20 mins. Hemolymph were collected from the larvae and pupae according to the following protocol along with some modification.³⁵ A slit was made on the ventral side of the larva and pupa and kept separately in a 1.5 ml Eppendorf tube containing a filter column. The tube containing the sample was centrifuged at 12 000 rpm for 20 min at 4 °C. The filtrate contained the upper layer of fat bodies and the lower layer of proteins, which were further collected for the follow-up studies. This was diluted in a 1 : 3 ratio with 1× PBS. The proteins from the eggs (laid by mated flies of the control and treatment groups) were extracted by crushing in a 1 : 10 ratio of 1× PBS in a 1.5 ml tube using a manual homogenizer. The homogenate was incubated on ice for 30 min and centrifuged at 4 °C for 15 min at 12 000 rpm. To suppress protease activity in the lysate, the supernatant was supplemented with 1× protease inhibitor cocktail (PIC) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Furthermore, the isolated proteins were resolved by SDS PAGE.

2.7 Sample preparation for mass spectrometric analysis

The differential proteins resolved by SDS PAGE were marked and processed for mass spectrometric analysis according to the following protocol.³⁶ For this, the Coomassie brilliant blue (CBB)-stained protein bands were excised from the gel and chopped with the help of a scalpel blade under sterile conditions. The gel pieces were destained with destaining solution containing 45% methanol and 10% glacial acetic acid to remove the remaining CBB stain. After destaining, the gel pieces were washed three times with alternate cycles of 25 mM ammonium bicarbonate (ABC) and a 2 : 1 ratio of acetonitrile (ACN) : 50 mM ABC. For the reduction of proteins, gel pieces were incubated in 5 mM DTT for 30 min at 60 °C. After incubation, the DTT solution was removed and the gel pieces were incubated with 15 mM iodoacetamide for 30 min in the dark. After that, the alkylating solution was removed and the gel pieces were washed twice with 25 mM ABC and further digested with trypsin in the ratio of 1 : 10, and overlaid with 50 mM ammonium bicarbonate buffer (pH 8.0) at 37 °C for overnight. The digested peptides were extracted in a solution containing (80% ACN and 0.1% TFA) two to three times. Using a vacuum evaporator, the extracted peptide samples were lyophilized. The lyophilized samples were suspended in 0.1% formic acid solution. C18 zip-tips were used to purify the peptides. The peptide analysis was done with a Nanoflow HPLC instrument (EASY-nLC 1200 system; Thermo Fisher Scientific, Waltham, MA, USA) coupled to a Q-Exactive mass spectrometer with a nano electrospray ion source. A 2-column system was used for the chromatography. The peptide mixtures were separated at a 35 min gradient with a flow rate of 300 nL min⁻¹ (0–10% 2 min, 10–45% 13 min, 45–70% 10 min, 70–95% 5 min, 95–95% 5 min).

2.8 Protein identification and network analysis

The Proteome Discoverer 2.2 proteomics platform was used to process the data (Proteome Discoverer, version-2.2; Thermo Fisher Scientific, Waltham, MA, USA). By setting the precursor mass tolerances set at 10 ppm, we examined these fragmentation spectra against the Spodoptera database (https://www.ncbi.nlm.nih.gov/genome/?term=Spodoptera+litura). The carbamidomethyl (C) was added as a fixed modification along with the oxidation (M), acetylation (protein N-terminal), and other modifications. The false discovery rate for peptide identification was set at 1%. KEGG analysis was done for the systematic analysis of gene function, including cellular component, biological process, and molecular function. The network analysis of the proteins involved in various pathways was carried out by the STRING algorithm (https://string-db.org).

2.9 Extraction of the larval homogenates for the enzymatic assays

The 3rd and 5th instar larvae of S. litura from all the treatments and the control were used for the evaluation of the selected enzymes that are reported to be involved in detoxification and anti-oxidative mechanisms. The larvae were washed with distilled water, cold immobilized, and crushed in liquid N₂. The obtained powder was homogenized in 1× PBS. The protein concentration was determined by Bradford assay. To ensure consistency in the initial protein concentration, a stock of 100 μg mL⁻¹ lysate was prepared and subsequently utilized for the enzyme assays.

2.9.1. Carboxyl esterase (Car E) assay. The carboxyl esterase (Car E) assay was done following the protocol described previously.³⁷ Larval homogenate (150 μL) was mixed with 1.35 mL of 0.27 mM of alpha-naphthyl acetate and incubated for 25 min at 30 °C. The reaction was stopped using a mixture of 500 μL of fast blue RR salt and 10 μL of 5% sodium dodecyl sulfate. The mixture was incubated at RT for 15 min until the color was developed. At 600 nm, the absorbance was measured. The enzyme activity was expressed in unit mg⁻¹ min⁻¹ with an extinction coefficient of 2.2 mM⁻¹ cm⁻¹.³⁸

2.9.2. Glutathione-S-transferase (GST) assay. The glutathione-S-transferase (GST) enzyme assay was performed as previously described.³⁹ In the GST assay, CDNB was used as a substrate. In brief, 100 μL of larval homogenate was mixed with 1 mM CDNB, 1 mM glutathione reduced, and 1× PBS. The absorbance was measured at 340 nm at a time interval of 30 s for up to 5 min. The enzyme activity was expressed in unit mg⁻¹ min⁻¹ with an extinction coefficient of 9.6 mM⁻¹ cm⁻¹.

2.9.3. Catalase (CAT) assay. The catalase (CAT) assay was done according to the protocol described in ref. 40 along with some modifications. The reaction mixture (1 ml) consisted of 20 μL larval homogenate with 970 μL of 50 mM KPO₄. The reaction was initiated by adding 10 μL of 1% H₂O₂. The absorbance was measured at 240 nm at a time interval of 10 s for 5 min. The enzyme activity was expressed in unit mg⁻¹ min⁻¹ with an extinction coefficient of 39.4 mM⁻¹ cm⁻¹.

2.9.4. Ascorbate peroxidase (APX) assay. The ascorbate peroxidase (APX) activity was driven by H₂O₂-mediated ascorbic acid oxidation.⁴¹ The assay mixture contained (1 ml) 50 mM KPO₄ (pH 7.0), 0.6 mM ascorbic acid, and 100 μL larval homogenate. The enzyme assay was started by adding 10 μL of 10% H₂O₂. The oxidation rate was measured at 290 nm with a time interval of 30 s for up to 3 min. The enzyme activity was expressed in unit mg⁻¹ min⁻¹ with an extinction coefficient of 2.8 mM⁻¹ cm⁻¹.

2.10 Statistical analysis

Statistical analysis of the insects' average weight was performed by eliminating two upper and two lower outliners from each of the treatment and control groups. One-way ANOVA, followed by Dunnett test using GraphPad Instat were used to analyze the average insect weight, average number of pupa and moth, and enzyme activity data. Here, p value < 0.01(**) and p value < 0.05(*) were considered statistically significant.

3. Results and discussion

3.1 Physicochemical characterization of the CNPs

CNPs prepared from waste candle soot are glittery powder with particle size ranging around 40–50 nm, and a fractal-like structure. Since most of the physicochemical characterizations were discussed thoroughly in prior work, they are not discussed in detail in the present work.³⁴ In short, the presence of multiple surface functional groups, namely –C–H group of alkene/aromatic carbon, –CO group of an alcoholic group, –CO group of carboxylic acid, –CH group of aliphatic carbon, and –O–H group of the adsorbed moisture phase, makes them highly reactive and are responsible for most of their physiochemical properties.

3.2 CNPs significantly impaired insect morphology and development

We observed a significant weight reduction ranging from 30–60 % in the 3rd, 4th, 5th, and 6th instar larvae. The weight loss was directly proportional to the tested concentrations of CNPs (Fig. 1a). Unlike the minimum tested concentration (250 μg gm⁻¹), we observed that the CNPs at higher concentrations (500 and1000 μg gm⁻¹) cause a major problem in late larval, pupal, pupa–adult interface, and in moths. At the highest tested concentrations, ∼25% of pupae could not eclose into adulthood (Fig. 1b and c), and ∼30% of the population was restricted to the pupa–adult interface. Among the moths, ∼25% were found to be abnormal (Fig. 1c and d). In this way, ∼80% of the population was severely impacted and showed visible deleterious effects on growth and development (Fig. 1). Several studies indicated the effect of nanomaterials on the development of insects.⁴² As we know, the quality of the food greatly affects the life cycle, basic physiology, and behavior of insects. Any negative impact on insect biology may either be because of the bad nutritional quality or poor digestion/absorption or the taste of the food. Here, in this study, it seems that the problems that incurred in insect development and physiology were because of the poor digestion and/or absorption of the food as there was no difference in the amount of the diet consumed between the treatments and the control groups.

Fig. 1 Effect of carbon nanoparticles on the physiology of S. litura. (a) Average weight of 3rd, 4th, 5th and 6th larval instar. Significant weight reduction is observed in 500 and 1000 μg gm⁻¹. Error bars represent the standard mean of error (N = 30). Stars above the error bars denote significant differences between treatments in comparison to control (** = p value < 0.01, Dunnett test). (b) Average number of normal and abnormal pupae. (c) Morphological deformities in pupae and flies. Wings are not properly develop and are abnormal in size & shape. (d) Average number of normal, abnormal and not emerged moths.

We also measured the larval, pupal, and adult longevity, larval movement, male–female ratio, and consistency of excreta, and found no significant differences compared with the control group.

3.3 CNPs significantly reduced the fecundity and caused various abnormalities in eggs

Successful mating was seen in all the treated and control insect groups. Egg laying occurred 3–4 days after mating. In general, leaf armyworms lay eggs in compact patches, with each cluster containing several hundred eggs, covered with hairy scales (provided by the female, also called abdominal hair). This hairy covering provides protection and heat to the developing eggs. However, notable differences were observed in the egg patches when compared to the control, such as egg quantity, covered area, thickness, and the presence of hair-like scales. There was a complete absence of hair scales on the egg patches laid by the treated moths (at all three conc.) (Fig. 2a). An increased deterioration in egg shape, texture, and color (pigmentation) were observed with the increased concentration of CNPs. The eggs were highly distorted in shape, shrunken, pigmented (blackening), and fragile in nature compared to the control (Fig. 2b). No larval emergence was observed from the eggs laid by flies fed on CNPs at 500 and 1000 μg gm⁻¹ (Fig. 2c). Only 30% of the eggs (laid by moths, fed on CNPs 250 μg gm⁻¹) hatched into larvae but were not able to pupate and/or eclose into adults (Fig. 2b). Studies published by other researchers also reported decreased S. litura fecundity upon the oral administration of NPs, but none of them reported zero larval emergences. This is the first report where CNPs completely checked the population buildup of S. litura in one–two generations. The significant reduction in egg laying and the absence of larval emergence promotes the use of waste CS-derived CNPs for the field control of S. litura.

Fig. 2 Impact assessment of the carbon nanoparticles on the oviposition & number of egg and larval emergence. (a) Panels representing the egg laying pattern of the fly fed on control and on 3 different concentrations of CNPs respectively. A significant reduction in the egg count was observed in case of the treatments (more on CNP 1000 μg gm⁻¹ fed flies). (b) Impact of carbon nanoparticles on the egg count and larval hatching. A significant reduction in the number of egg and larval hatching was observed (continuously decreasing with increased concentration of treatment). In CNPs 250 μg gm⁻¹ 30% larval emergence was observed. In CNPs 500 and CNPs 1000 μg gm⁻¹, zero larval emergence was observed.

3.4 Microscopic examination of the ovaries revealing poor and abnormal egg filling

The poor egg laying further raised the question of whether this was due to a compromised genital (ovary) development or the absence of nutrition/proteins required for proper egg filling and development. Microscopic examination of the ovaries revealed poor and abnormal egg filling along with patchy, shrunken, and dark-colored eggs in the CNPs-fed moths, at all three concentrations (Fig. 3). The filling, development, and maturation of the eggs inside the ovary require adequate feeding, a proper development of ovarian structures, and mating. These are highly regulated through various environmental factors, proteins, and hormones in insects.

Fig. 3 Microscopic evaluation of the ovary. A number of morphological changes were observed in the ovary of CNPs fed flies. Very poor egg filling was observed at the maximum tested conc of CNPs (1000 μg gm⁻¹). Zonal darkness and heavy pigmentation in the eggs was recorded in the ovaries of flies fed on CNPs 1000 μg gm⁻¹ and CNPs 500 μg gm⁻¹.

Disruption of any one of these factors can cause highly compromised egg filling and development. The decreased fecundity of the CNPs-fed insects might be influenced by one or more of the aforementioned factors. Additionally, the pesticides commonly employed in the IPM programs target decreasing the fecundity in insects. In this respect, hindrance in reproductive progression of an insect by CS-CNPs may come up as a better alternative to harmful insecticides.

3.5 CNPs caused deleterious impacts on insect development and reproduction by altering candidate protein molecules

As we have already discussed, the process of egg development and maturation is regulated by many environmental and intracellular factors. We excluded any influence of abiotic factors on the onset of deformities in the process of egg development and maturation because all experiments were conducted under controlled lab conditions and according to the guidelines for insect culture and bioassay. Instead, we chose to investigate involvement of proteins (intracellular factors), if any, in the process of deformities. Therefore, we performed gel-based differential proteomics of larvae, pupae, and eggs from the moths that could be developed following the treatment.

Comparative profiling of the proteins showed significant differences in the amount of the proteins present in the treatment moths with respect to the controls (Fig. 4b and c). The majority of the proteins were found absent and/or significantly reduced in the treatment groups (the reduction was directly proportional to the administered concentration of NPs). Mass spectrometric analysis of the bands representing differential proteins (Fig. 4b and c) suggested that the majority of downregulated proteins were involved in the process of growth and development, energy metabolism, reproduction, cell signaling, and transportation, while the proteins related to stress and immune response were found to be upregulated (Table 1). The said pathways also get altered in other insect pests (H. armigera, and Spodoptera) after exposure to harmful insecticides (azadirachtin-A, pyriproxyfen, and camptothecin), indicating the susceptibility of proteins related to the pathways described earlier toward toxicants.

Fig. 4 Protein isolation and gel profile in different stages of treated and control (PBS) insects. (a) Pigmentation in protein of treated eggs as compared to control. The highest dose of concentration of eggs exhibited more pigmentation. (b–d) Gel profile of last instar larval, pupal hemolymph and egg proteins respectively. The differential bands are marked, excised and processed for further proteomic analysis.

Table 1 List of the differentially expressed proteins and their functions

S. no.	Protein name	Occurred/found in	Biological process	Cellular component	Molecular function	Description	Upregulated/downregulated	Accession number
Reproduction
1.	Basic juvenile hormone suppressible protein 1-like	Larval, pupal hemolymph	Reproduction and metamorphosis	Extracellular region	Nutrient reservoir activity	Expressed in the last larval instar. It suppresses the juvenile hormone and makes its titer low, thus facilitating molting, metamorphosis, cuticle production, and insect development		1274116313
2.	Vitellogenin	Pupal hemolymph, egg protein	Reproduction	Extracellular space	Egg-yolk proteins are sources of nutrients during embryonic development	Reproduction-related protein. Vitellogenesis is a process in which vitellogenin (Vg) is predominantly generated in the fat body cells and transferred into developing oocytes via hemolymph. Vg is required for adult egg maturation, oviposition, and embryonic growth		XP_022835047.1
3.	Vitellogenin like	Pupal hemolymph, egg protein	Reproduction	Extracellular space	Precursor of the egg-yolk proteins that are sources of nutrients during embryonic development	Peculiar feature of female insect reproduction. Largely generated in the fat body cells and transferred into developing oocytes via hemolymph. Vg is required for adult egg maturation, oviposition, and embryonic growth		1274135864
4.	Ezrin/radixin/mesorin homologue (ERM)	Larval hemolymph	Reproduction and development	Cytoskeleton, plasma membrane	Cytoskeletal proteins and are essential for embryonic development	Found in fat body cells of insects and generally takes part in the reproductive processes of insects		1879241660
Stress and immune response
5.	Ferritin	Egg protein	Immune response	Plasma membrane	Iron transport, cellular iron ion homeostasis	Stress-related protein. Its expression is positively correlated with the levels of ROS in cells. Found in the larval and adult hemolymph and eggs		XP_022819215.1
6.	Small heat shock proteins (sHSPs)	Egg protein	Response to stimulus	Protein containing complex	Protein complex oligomerization	sHsps expression has been shown to be particularly sensitive to any type of biological stress, and it has been considered a measurable indicator of wellness, which is expressed in the hemolymph of different stages of insects		301070156
Macromolecular metabolism
7.	Fatty acid synthase	Pupal hemolymph	Fatty acid synthesis	Cytoplasm, plasma membrane	RNA binding	It is responsible for the insect lipid synthesis pathway. Expressed in the hemolymph, fat body, and flight muscles of insects		1274134351
8.	Aldehyde dehydrogenase mitochondrial	Pupal hemolymph	Carbohydrate metabolic process	Extracellular space, mitochondria	Carboxylic ester hydrolase activity	It is responsible for the synthesis of multiple fatty acids and fatty aldehydes, and hence plays an important role in immunity, growth, and development. Found predominantly in insect hemolymph		1274139732
9.	Fructose-1,6-bisphosphatase 1	Pupal hemolymph	Carbohydrate metabolic process	Extracellular space, mitochondria, cytoplasm	Transcription activator binding	It is a key player in carbohydrate and energy metabolism and extracellular immunity. It plays a central role in glycolysis. It is expressed in larval fat body		1274121303
10.	Cystathionine gamma-lyase	Pupal hemolymph	Cellular amino acid metabolic process, lipid metabolic process	Cytosol	DNA binding transcription factor activity	Protein involved in amino acid metabolism		1274112743
11.	Glutamine synthetase 2 cytoplasmic like	Pupal hemolymph	Glutathione biosynthetic process	Cytoplasm	ATP binding	Involved in glutamine synthesis and responsible for various physiological process. Expression is in the midgut and fat body tissues		1274144177
Cellular signaling, transport, and other metabolic processes
12.	Transferrin	Larval hemolymph	Iron ion transport	Extracellular space, endoplasmic reticulum	Ferric ion binding	Helps in iron delivery. This protein also functions to reduce oxidative stress found in fat bodies		1274127134
13.	Membrane alanyl aminopeptidase like	Larval hemolymph	Cell differentiation	Extracellular space, plasma membrane	Peptidase activity, signaling receptor activity	A major component in the insect gut epithelial membrane with a primary function to cleave N-terminal amino acids		1274121765
14.	Aminopeptidase like	Larval hemolymph	Cell differentiation, circulatory system development	Extracellular space, plasma membrane, secretory vesicle	Peptidase activity, signaling receptor activity	The midgut brush border of insects contains a lot of APNs, which are proteins. APNs are proteolytic enzymes that perform a variety of tasks, including digestion and defense responses		1274121759
15.	Fructose-1,6-bisphosphatase 1	Pupal hemolymph	Cell growth	Extracellular space, mitochondria, cytoplasm	Transcription activator binding	Present in fat body and involved in the synthesis of glucose		1274121303
16.	Ornithine aminotransferase mitochondrial like	Larval hemolymph	Cellular amino acid metabolic process, sensory perception	Cytoplasm, mitochondria	Ornithine transaminase activity	Ornithine aminotransferase (OAT), which is present predominantly in the fat body and flight muscle tissues of insects, plays a key role in ornithine synthesis, which is the main substrate for the synthesis of proline, polyamines, and citrulline. These amino acids play a role in DNA replication and cell cycle progression		1274113107
17.	Elongation factor	Pupal hemolymph	Translation elongation, glutathione metabolic process	Nucleus, membrane	Cadherin binding	A key regulatory factor for translation in plants, bacteria, fungi, animals, and insects. A highly conserved protein that has a significant role in peptide elongation during translation and is hence required for protein biosynthesis. The transcripts are expressed in the fat body and whole body at different levels from fifth instar larvae to pupae		1274125611
Growth and development
18.	Arylphorin subunit alpha-like	Larval hemolymph	Growth development	Extracellular region	Source of aromatic amino acids for protein synthesis during metamorphosis	Storage proteins are synthesized and released by the fat body of feeding larvae/nymphs and play a key role in growth and developmental pathways		1274115394
19.	Moderately methionine-rich storage protein	Larval hemolymph	Growth and reproduction	Extracellular region	Nutrient reservoir for egg	Synthesized and released by the fat body of feeding larvae and required for growth		5869985
20.	Actin, cytoplasmic A3a	Larval hemolymph	Immune system, developmental process, protein metabolic process, cell cycle	Extracellular region, nucleus, chromosome, cytoskeleton	Structural molecule activity, cytoskeletal protein binding	Actin, a protein well known for its diverse function, has a key role in growth and developmental pathways. Present in adult flies' brain and pupa		1879287523
21.	Xanthine dehydrogenase	Pupal hemolymph	Signaling and developmental process	Cytosol, nucleus, endomembrane system	Small ion binding, catalytic activity	Plays an important role in purine metabolism and is present in high amounts in the fat body of insects		1274140180
22.	Chitooligosaccharidolytic beta-N-acetylglucosaminidase	Pupal hemolymph	Molting and wing development	Extracellular space	Receptor activity	Chitooligosaccharidolytic beta-N-acetylglucosaminidase is a major protein that is involved in the molting and wing development of insects. Present in chitin rich tissues as epidermis		1274139746
23.	Fructose-bisphosphate aldolase A	Larval hemolymph	Developmental process	Extracellular space, cytosol, cytoplasm, cytoskeleton	Cytoskeletal protein binding	Fructose bis-phosphate aldolase is involved in the synthesis of glucose, which is required for the proper functioning of elongation factor. Present in larval fat body and ovary		111360439
24.	Riboflavin binding hexamerins	Larval, pupal hemolymph	Developmental process	Extracellular region	Transporter activity, molecular transducer activity	Synthesized and released by the fat body of feeding larvae and required for growth. Expression is in the last instar larval and pupal hemolymph and fat bodies		1342770239

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In the case of larval proteomics, one of the major bands at 60–70 kDa was identified as a group of storage proteins (basic juvenile hormone suppressible proteins, methionine-rich storage proteins, arylphorin, and riboflavin binding hexamerin). These, all high-molecular-weight proteins that are cumulatively called storage proteins, play a very important role in many vital biological processes, including metamorphosis (basic juvenile hormone suppressible proteins and methionine-rich proteins) and reproduction (arylphorins and hexamerins).⁴³ The compromised expression of these proteins can lead to developmental abnormalities and reproductive failures. In another study, it was reported that bacterial challenge on eri-silk worm led to the downregulation of storage proteins, which in turn resulted in compromised metamorphosis.⁴⁴ We could also observe similar results in our study (Fig. 1 and 2). Here, ∼80% of the larvae could not reach phenotypically healthy adulthood due to incomplete metamorphosis, which might be because of the significant down-expressions of the proteins like basic juvenile hormone suppressible proteins, methionine-rich proteins and others. The role of storage proteins in the generation advancement of insects has been well established by other researchers.⁴⁵ Furthermore, we also found a nearly complete absence of other high kDa proteins, such as vitellogenin and vitellogenin-like proteins in the eggs. This yolk protein is produced by the process of vitellogenesis in the fat body cells from storage proteins and transported into developing oocytes via hemolymph through receptor-mediated transportation. Following oviposition, Vg is necessary for egg maturation and embryonic growth.⁴⁶ The lower availability/synthesis of vitellin/vitellogenin in developing eggs leads to lowered fecundity and egg hatchability in insects, like the cotton leaf worms in our study (Fig. 4d) and also in other insects, like house cricket, red palm weevil, and warehouse moth.^47,48 It is noteworthy that in insects, storage proteins are the major reservoir to produce proteins involved in reproductive processes, including ‘vitellogenin’.⁴⁹ The hindered synthesis of storage proteins is directly linked to the decreased production of vitellogenin and the compromised process of egg development. This study further demonstrates that CNPs contribute to the failure in synthesizing storage proteins, resulting in an inadequate accumulation and synthesis of vitellogenin. Vitellogenin, an essential female-specific egg-yolk protein, is indispensable for oogenesis.

3.6 Functional annotations and pathway analysis

Mass spectrometric analysis of the differentially appeared gel bands identified a total of 24 proteins. KEGG analysis suggested that, based on their functions, these proteins belonged to 5 major groups: reproduction, stress and immune response, macromolecular metabolism, cell signaling and transport, and growth and development. All of these proteins are known to have regulatory roles in either an insect's development and/or reproduction. A brief discussion on the role of the altered pathways on the onset of the insect deformities observed in this study is given below.

3.6.1. Reproduction. Reproduction is a key process that includes the involvement of a number of biological functions, including vitellogenesis, fertilization, embryogenesis, and oviposition. Basic juvenile hormone suppressible protein, vitellogenin, vitellogenin-like protein, and esrin/radixin/moesin (ERM) homolog have been found to be downregulated in either larva/pupae/eggs. Among these, the basic juvenile hormone suppressible proteins and ERM proteins have been found in the fat body of insects and play a significant role in embryonic development and growth. However, limited data are available to support their active involvement in said biological functions. The other two proteins, i.e., vitellogenin and vitellogenin-like proteins, are well known and extensively researched due to their crucial function in the process of reproduction.⁵⁰ The absence of these leads to defects in reproduction, as observed in this study (Fig. 2b and 4d). Vg is required for egg maturation, oviposition, and embryonic growth.⁵¹ Intoxication by either graphene oxide or thiamethoxam leads to a lowered expression of Vg and impacts ovarian development, egg laying, and fecundity in Acheta domesticus and the white-backed planthopper, respectively.^51,52 Knockdown studies of vitellogenin in the cotton boll weevil A. grandis and Rhynchophorus ferrugineus (red palm weevil) and warehouse moth Cadra cautella hampered the egg-laying capacity and egg viability of the insects.^47,48

3.6.2. Stress and immune responses. Nanoparticles-mediated stress responses in insects leading to ROS generation is a well-documented phenomenon (Fig. 6). Increased ROS generation along with inflammatory responses results in a higher production of stress-responsive proteins, like small HSPs and ferritin. The exposure of nanoparticles has been reported to elicit an increase in oxidative stress levels and also stimulate the upregulation of heat shock proteins (HSPs) within organisms. For instance, exposure to silver nanoparticles in Drosophila led to an amplified expression of HSPs and also increased oxidative stress levels.⁵³ In our study, we found a significantly higher expression of both of these proteins in pupae and eggs (Table 1). Prone to stress, sHSPs have been proven to have the potential to serve as a primary biomarker of cellular injury due to their evolutionary conservation and inducibility by a wide range of inducers.⁵⁴ For instance, an increase of HSPs70 expression in moths was found to be a reliable indicator of pesticide exposure to organophosphate and pyrethroid insecticides.⁵⁵ As sHSPs possess chaperone activity, they thereby help in regulating growth and development, including oogenesis, embryo development, and signal transduction in insects, and also NPs-induced oxidative stress, apoptosis, inflammation, cytotoxicity, and genotoxicity.^56–58 Hsp70 and other Hsps genes were significantly induced after exposure to zinc, in response to temperature, starvation and parasitism in S. litura and Ectomyelois ceratoniae.^59,60 Likewise, ferritin is also a stress-inducible protein and is elevated in response to ROS generation in cells. This elevated ferritin binds with the cellular Fe, so that it is not available to further enhance the level of ROS (Fe acts as a catalyst for the generation of ROS).^61,62 In addition to their role in oxidative stress, insect ferritins also contribute very significantly in the immune response against different pathogenic challenges.⁶³ Besides, they also play a very important role in embryonic development⁶⁴ by transporting iron in the developing eggs⁶⁵ and larval development.⁶⁶

3.6.3. Macromolecular metabolism. Cellular metabolism is an important feature of all living organism. Carbohydrates, amino acids, and fatty acids serve as the primary energy source for all living beings, and their reduced expression or absence can result in various deformities in organism. In the treatment groups, the identified proteins associated with carbohydrate and energy metabolism exhibited downregulation. Among these proteins, fructose 1,6-bisphosphatase holds a central position in glycolysis and is crucial for energy production. It catalyzes the reversible aldol condensation of dihydroxyacetonephosphate and glyceraldehyde-3-phosphate, contributing to glycolysis.⁶⁷ We found a significantly lower level of fructose 1,6-bisphosphatase in the treated insects, which may interfere in the production of glucose and energy, which are indispensable for the proper growth and development production in insects, which subsequently disrupts their development. Similarly, an important metabolic enzyme is fatty acid synthase (FAS), which takes part in various physiological function, like flight, insect development, signal transmission, energy production, hormone synthesis, cell membrane formation, and others in insects.⁶⁸ In mosquitoes, female sex pheromones are typically derived from fatty acids, which are a major regulator in an insect's mating behavior.⁶⁹ In a study on locusts and S. litura, silencing of the targeted gene of FAS through RNA interference resulted in lethality during ecdysis or after molting.^70,71 We obtained similar results (Fig. 1c), which further indicated the regulatory role of FAS in the insects' development and molting. We also found lowered expressions of cystathionine gamma lyase, aldehyde dehydrogenase, and cytoplasmic glutamine synthetase in the treated insects, which play important roles in amino acid and fatty aldehydes synthesis, which are major players in the maintenance of cellular metabolism.

3.6.4. Cell signaling and transport. Cell signaling is an intriguing characteristic found across all living organisms, as it enables cells to communicate with one another through the use of intracellular and extracellular messenger molecules. An alteration in the signaling cascade disrupts the physiological processes in organisms. In our study, treatment with CNPs led to a reduction in the expression of certain proteins/enzymes that play a significant role in facilitating these fundamental signaling process. The intricate interaction of cellular processes involves a protein that is ornithine aminotransferase (OAT), primarily found in the fat body and flight muscle tissues of insects. OAT serves a vital function in ornithine synthesis, the primary substrate involved in the synthesis of proline, polyamines, and citrulline, amino acids that are crucial for DNA replication and cell cycle progression.⁷² The observed downregulation of OAT expression in our study may be a contributing factor to the emergence of deformed flies and pupae (Fig. 1c). Another identified protein is transferrin, which plays a crucial role in iron transport and homeostasis, which is essential for various physiological purposes.⁷³ The next identified protein was elongation factor (EF), which plays a pivotal role in translation regulation across a wide range of organisms, including plants, bacteria, fungi, animals, and insects.⁷⁴ This highly conserved protein is indispensable for peptide elongation during translation, playing a crucial role in protein biosynthesis and making them indispensable for cellular functioning. This might be the reason for the lower expression of overall proteins in the treated insects (Fig. 4b–d). We also found lowered expressions of membrane alanyl aminopeptidase and aminopeptidase in the treated insects, which play vital roles in cellular signaling

3.6.5. Growth and development. Growth and development in living organisms are influenced by two primary factors: the nature and amount of the diet consumed, and the intricate machinery of proteins and enzymes that coordinate these processes of growth and development. In this study, we found lowered expressions of seven proteins that play important roles in growth and development. Three of them belonged to the group of storage proteins (arylphorin, moderately methionine-rich protein, and riboflavin binding proteins).⁷⁵ Lowered expressions of these proteins may be linked with the lethality and poor reproductive abilities of S. litura in our study. Another downregulated protein was actin, a protein well known for its versatile functions, which plays a significant role in growth and development.⁷⁶ In insect embryogenesis, which is characterized by intense mitotic activity and cell movements, a substantial amount of cytoplasmic actin is required;⁷⁷ the absence of which may lead to the development of fragile and shrunken eggs, as we observed in our study. The other lowered expressed protein was xanthine dehydrogenase (XDH), which holds a crucial position in purine metabolism and is abundantly present in the fat body of insects.⁷⁸ XDH plays a pivotal role in the production of uric acid in Drosophila. Some other documented roles suggest that impaired XDH activity causes male sterility in silkworm.³⁴ Though the exact role of XDH is not known in S. litura, it seems to play regulatory roles in insect growth. Similarly, chitooligosaccharidolytic beta-N-acetylglucosaminidase is a major protein that is involved in the molting and wing development of insects. The absence of which leads to abnormal molting (molting intermediates were observed) and deteriorated wing development (Fig. 1c).

3.7 Network analysis of the differentially expressed proteins

Network analysis using the STRING algorithm suggested the involvement of two major phenomena behind the toxicity imparted by CS-derived CNPs that ultimately hampered the generation advancement in insects (Fig. 5). The first pertained to the alteration in basic metabolic processes, like the synthesis of amino acids, carbohydrates, and fatty acids, while the other one indicated alterations in the pathways (proteins or genes) involved in reproduction. These cumulatively impaired the growth, development, and reproduction of the target insect. Network analysis suggested that fatty acid synthase (FAS) is linked with aldehyde dehydrogenase (ALDH). Since these two enzymes are responsible for the synthesis of multiple fatty acids and fatty aldehydes, they hence play an important role in immunity, reproduction, growth, and development.^70,71 In this study, we found that the downregulation of these two enzymes was linked with compromised metabolism and immunity, abnormal reproduction, and impaired growth and development. An energy-generating pathway called glycolysis was also disrupted upon the oral administration of CNPs. The downregulation of the enzymes involved in glycolysis, that is fructose 1,6-bisphosphatase and fructose bis-phosphate aldolase, resulted in the poor production of glucose, which is required for the proper functioning of elongation factor-2 (a protein that cannot be overlooked for the translation of proteins). As we earlier mentioned, the fact that the amount of overall protein content was significantly less in the treatment compared with the control might be because of the improper functioning of elongation factor 2 and other players of key metabolic processes. It is reported, that in insects, the storage proteins are the major reservoir to produce the proteins involved in reproductive processes, including ‘vitellogenin’.⁵⁰ This is an irreplaceable female-specific egg-yolk protein and has a crucial role in oogenesis. The process of oogenesis is also regulated by a fat body protein, namely transferrin, which is required for the transfer of vitellin and iron for providing nutrition and mechanical strength to developing oocytes. The overall downregulation of these three proteins resulted in hindered generation advancement in S. litura (Fig. 5b).

Fig. 5 Protein–protein interaction networks. The functional interaction network of CS derived CNPs regulated proteins were created by the STRING algorithm. (a) The network of the proteins involved in metabolic activities. (b) The network of the proteins involved in reproduction.

3.8 Enzymatic assays

Detoxification enzymes in organisms have generally been proven to be the defense against foreign compounds and play significant roles in maintaining normal physiological functions. The primary mechanisms responsible for the resistance in insects are alterations in the detoxification rate of any substance. In our study, we tested the esterase and glutathione-S-transferase enzymes for evaluation of the impact of the CS-CNPs. Glutathione-S-transferase (GST) belongs to the family of an enzyme that is essential for cellular detoxification. Relative to the control groups, all three concentrations of CS-CNPs demonstrated a statistically significant increase in GST activity within the 3rd and 5th instar larvae (Fig. 6a). As far as carboxyl-esterases (Car E) are concerned, they play a role in the emergence of insect resistance. The activity of Car E increased significantly in the 3rd instar larvae at each of the three concentrations examined in our study, while the 5th instar larvae exhibit a significant increase at 500 and 1000 μg gm⁻¹ of CS-CNPs (Fig. 6b). The fact that the detoxifying enzyme levels were rising strongly suggested that the CS-CNPs were toxic to insects and were having a detrimental effect on them. ROS generation is a highly studied phenomenon that is considered to be behind the toxicity facilitated by nanomaterials in insects.⁴² The primary enzymes catalase and ascorbate peroxidase are responsible for scavenging reactive oxygen species generated by NPs, thereby reducing oxidative stress.⁷⁹ In the 3rd and 5th instar larvae, we observed a substantial increase in catalase activity at the highest tested concentration of CS-CNPs (Fig. 6c). An increase in the catalase activity in insects is also linked with a poor reproductive ability. However, we found a substantial difference in ascorbate peroxidase activity in the 5th instar larvae (250 g gm⁻¹ of CS-CNPs) (Fig. 6d). The presence of reactive oxygen species in the highest dose is an indication of ROS generation by CS-CNPs that might be responsible for the impairment of the population advancement of S. litura (Fig. 7).

Fig. 6 Effect of various concentrations of CS derived CNPs on enzymes related to oxidative stress and defense mechanism. (a) GST. (b) Car E. (c) CAT. (d) APX activity in 3rd and 5th instar larval stages (insect developmental stages). Error bars representing the standard error (N = 3). Stars above the error bars denote significant differences between treatments in comparison to control (* = p value < 0.05 and ** = p value < 0.01, Dunnett test).

Fig. 7 Schematic illustration of the summary of study.

Conclusion

The present study shows that CS-derived CNPs can have a substantial negative impact on the growth, physiology, and reproductive behavior of S. litura. The CNPs tested here had a significant deleterious impact on the larval weight, as well as the moth's reproductive ability (Fig. 7). It affected the insect physiology, defense mechanisms, and generation advancement adversely even at the lowest tested concentration. CNPs-induced cytotoxicity was indicated by an elevation in the activity of the enzymes involved in oxidative stress and defense. Microscopic examination of ovaries isolated from mated female moths fed CNPs revealed several deformities, including poor egg filling, egg darkening (pigmentation), and abnormal egg development. Our understanding of the disruption of generational advancement was made clear by proteomic studies of the differential proteins. The finding revealed that a certain hindrance in the synthesis and/or accumulation of storage proteins during the larval stages impeded normal insect metamorphosis and thereby curbed generation advancement. The downregulation of storage proteins was responsible for the absence of reproductive proteins, that is, vitellogenin. It is noteworthy that the generation advancement (life cycle) of S. litura (a polyphagous voracious crop insect pest) could be completely disrupted by the CNPs. Furthermore, previous research has shown that CS-derived CNPs are safe and/or have a low reproductive toxicity in cell lines and Drosophila models. The insights from the study may be used for the development of an alternative to conventional insecticide in the management of the destructive insect pest S. litura.

Author contributions

Manisha Mishra: conceptualized the study, evaluate the results and prepared the draft, Rashmi Pandey, Ranjana Chauhan: performed the experiments, analyzed the results and prepared the draft, Sharad Saurabh, Anoop Kumar Shukla: maintain the insect culture facility, perform the bioassay experiment and edit the manuscript, Sheelendra Pratap Singh: mass spectrometric analysis of the protein, Pradhyumna Kumar Singh and Farrukh Jamal: provide critical suggestion and reviewed the manuscript.

Conflicts of interest

The authors declare that there is no conflict of interest.

Acknowledgements

The authors are thankful to the Department of Science and Technology, Government of India for research grants (DST/INSPIRE/04/2016/001616/GAP-353) and Council of Scientific and Industrial Research, Govt. of India, New Delhi for Cotton Mission Project (HCP-0023). Authors are thankful to the Director CSIR-National Botanical Research Institute, Lucknow for their support. CSIR–NBRI communication number for this manuscript is CSIR-NBRI_MS/2023/05/12.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3en00939d

‡ Authors contributed equally to this work.

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