Impact of caffeic acid on growth, development and biochemical physiology of insect pest, Spodoptera litura (Fabricius)

Abstract

The tobacco cutworm, Spodoptera litura (Fabricius) is a serious cosmopolitan pest that attacks several economically important crops such as maize, sorghum, chickpea, pigeon pea, cotton, tobacco and sunflower. It has developed resistance to most pesticides resulting in its continual outbreak. The effect of caffeic acid on second instar larvae of S. litura was evaluated by carrying out bioassays, nutritional assays, immune assays and biochemical assays with phenolic acids. Bioassays carried out with second instar larvae of S. litura showed growth inhibiting effects of various concentrations (5 ppm, 25 ppm, 125 ppm, 625 ppm and 3125 ppm) of caffeic acid on S. litura in comparison to control. A significant increase in mortality as well as an increased development time was observed with increase in the concentration of caffeic acid. A decrease in nutritional indices, including relative growth rate (RGR), relative consumption rate (RCR), efficiency of conversion of ingested food (ECI), efficiency of conversion of digested food (ECD), and approximate digestibility (AD), indicated that dietary caffeic acid also negatively impacted the nutritional physiology of S. litura larvae. Caffeic acid has a significant impact on the immunological response of S. litura larvae. As the concentration of caffeic acid increased, the overall number of hemocytes decreased. Enzymatic assays revealed a significant increase in antioxidant enzymes when S. litura larvae were given an artificial diet containing LC₅₀ concentration of phenolic acid for an interval of 24, 48, 72 and 96 h. The levels of oxidative stress markers (hydrogen peroxide, protein carbonyl and lipid peroxide) were also significantly enhanced in S. litura larvae after treatment with phenolic acid. According to our study, caffeic acid can be employed as a substitute for traditional insecticides to reduce the population of S. litura.

1. Introduction

The tobacco cutworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) is one of the many important polyphagous [1,2] insect pests plaguing a large number of economically important crops [3], having a host range of 180 plant species [4], and a huge potential to invade new areas due to its ability to adapt to new climatic and ecological conditions. In recent years, a frequent outbreak of S. litura has been observed in soybean fields [5] in the years 1997, 2005, 2008 and 2009. In 2008 approximately 1.4 m ha of soybean was damaged by S. litura in the Vidarbha region of Maharashtra and causing extensive defoliation in the plant [6]. It is broadly distributed in Asia, including tropical, subtropical, temperate areas and Oceania. It has also been recorded to attack over 112 cultivated plant species of which 60 are from India [7]. This pest has also become a new invasive species in West and Central Africa and the first outbreak was recorded in early 2016 [8].

Since management of insect pest is facing ecological as well as economic challenges by the continuous and injudicious use of synthetic chemical pesticides and because of the increased resistance rate, it has become imperative that some promising plant based compounds be identified that are sustainable, environmentally acceptable and safe. Caffeic acid is a polyphenol produced through the secondary metabolism of vegetables [[9], [10], [11], [12]], including olives, fruits propolis, beans, carrots, potatoes and coffee [9,13,14]. Caffeic acid helps plants defend themselves against predators, pests and illnesses by inhibiting the growth of insects, fungi and bacteria [13]. It also helps plant leaves protect themselves from ultraviolet radiation B (UV–B) [13,15]. Caffeic acid allows the plant to defend themselves against predators, pests, and illnesses by inhibiting the growth of insects, fungi, and bacteria [13,14]. In this study, we explored the potential of caffeic acid as a dietary pesticide for insect pest control in agriculture.

Various growth and development parameters were assessed in support of the antibiosis of these natural phenols to S. litura. Biochemical studies along with stress markers evaluation were also done to determine the enzymatic response of S. litura towards caffeic acid.

2. Material and methods

2.1. Rearing of S. litura

Fresh and clean castor leaves (Ricinus communis L.) were used to rear S. litura larvae in the Biological Oxygen Demand (BOD) incubator under the optimum conditions of 25 °C, 65 RH, and a 12:12 (D:L) photoperiod. Until adult emergence, pupae were shifted to pupation jars (15 cm × 10 cm) with 4–5 cm of moistened sand and filter paper. To promote egg laying, adults were moved to oviposition jars (15 cm × 10 cm) lined with filter paper, and a cotton swab dipped in a solution of water and honey (4:1) was provided as food.

2.2. Test compounds

For conducting the experiments, stock solution of caffeic acid (4000 ppm) was prepared in 0.5% ethanol, which was diluted to get the experimental concentrations of 5, 25, 125, 625 and 3125 ppm and an agar-based diet amended with these concentrations was prepared using the methodology suggested by Ref. [16]. A diet without compound but having 0.5% ethanol was used as control.

2.3. Bioassays

The bioassays were conducted at optimum conditions of temperature, humidity and photoperiod of 27 ± 2 °C, 65 ± 5%, L16:D8 respectively in the B·O.D incubator. For the conduction of experiments, the S. litura larvae (2nd instar) were starved for 24 h prior to experiments to clean their gut and were then transferred individually into sterilized solo cups consisting of a piece of diet (1.5 cm × 1.5 cm). The solo cups were daily cleaned and were provided with a fresh diet. The larvae were monitored until pupation to record the time taken by the larva to pupate, mortality of larvae and the number of pupae formed. The pupae were observed further for recording the total number of emerged adults. Each experiment consisted of six replicates with five larvae in each replication, giving us a total of 30 larvae per treatment.

2.4. Nutritional assays

A 3-day nutritional assay was also conducted to assess the influence of caffeic acid on the nutritional physiology of larvae as per the methodology provided by Waldbauer [17] and Koul et al. [16]. Separate experiments were performed. There were six replicates of each concentration with the control, each containing five larvae. At the conclusion of a three-day experiment conducted at 60°C within an incubator, the larvae's dry weights were measured. Similar to that, dry weight measurements were also made for diet and faeceal matter.

The dry weight data were used to measure moisture loss within controlled circumstances. The nutritional indices were calculated as RGR = G/I, RCR = C/I, ECI = 100 × G/C, ECD = G/C–F × 100, AD = C–F/C × 100 where G = change in larval dry weight/day, I = starting larval dry weight, C = change in diet dry weight/day, F = dry weight of frass/day. The experiment was conducted twice.

Where RGR = Relative growth rate, RCR = Relative consumption rate, ECI = Efficiency of conversion of ingested food, ECD = Efficiency of conversion of digested food, AD = Approximate digestibility.

2.5. Immunological response of S. litura

The experimental larvae were provided with artificial diet treated with LC₅₀ concentration of caffeic acid for 24, 48, 72, 96 and 120 h, to assess the impact of caffeic acid on immunological response in the second instar larvae of S. litura. Both the treatment and control diet-fed larvae were kept in a biological oxygen demand (B·O.D.) incubator at standard conditions. To collect the hemolymph, a sterilized needle was utilized to penetrate the prothoracic legs. The hemolymph of ten larvae from every treated group were randomly selected for each interval of time and collected. Total hemocyte count (THC) from the collected hemolymph was examined using Tauber and Yeager's [18] approach. All the trials were run twice.

2.6. Biochemical analysis of S. litura

For S. litura second instar larvae, estimation of different antioxidant enzymes was done. The larvae were given a diet augmented with LC₅₀ concentration of caffeic acid for varying time periods, i.e. 24, 48, 72, and 96 h. Each experiment was repeated twice.

2.6.1. Superoxide dismutase (SOD)

Using Kono's approach, the activity of SOD was calculated [19]. Larvae were weighed and homogenised (10% w/v) in 50 mM extraction buffer to create the extract. At 4 °C, the homogenate was centrifuged for 20 min at 10,000 rpm. 1.3 mL of 50 mM sodium carbonate buffer (pH 10), 0.5 mL of NBT, 0.1 mL of 0.6% Triton X-100, and 0.1 mL of 20 mM hydroxylamine hydrochloride were combined with the supernatant (0.5 mL) comprising the enzyme (pH 6). Japan's Shimadzu UV 1800 spectrophotometer was used to measure the change in absorbance over a 5-min period at 1-min intervals.

2.6.2. Catalase (CAT)

The Bergmeyer procedure was used to measure the CAT activity [20]. In 0.05 M potassium phosphate buffer, larvae were weighed and homogenised (5% weight-to-volume) (pH 7). Centrifugation of the homogenate at 25,00 g for 20 min at 4°C was done. In a cuvette containing 2.9 mL of H₂O₂ and 0.1 mL of supernatant, a change in absorbance at 240 nm was monitored for 3 min at intervals of 30 s at a temperature of 25 °C.

2.6.3. Ascorbate peroxidase (APOX)

Asada's method was used to measure the APOX activity [21]. Centrifugation of the treated homogenised larvae (10% w/v) at 10,000 g was done for 40 min at 4 °C in extraction buffer. 0.1 mL of supernatant, 0.6 mL of 50 mM potassium phosphate buffer, and 0.125 mL of 0.3% H₂O₂ were added in a cuvette, and the change in absorbance was recorded at 290 nm for duration of 5 min at intervals of 30 s at 25 °C.

2.6.4. Glutathione S-transferase (GST)

The Chien and Dauterman methodology was used to extract and estimate the GST [22]. In the extraction buffer (0.1 M Sodium phosphate buffer, pH 7.6) containing 0.1 mM PTU, larvae were homogenised before being subjected to centrifugation at 10,000 rpm for 30 min at 4 °C. A cuvette was filled with the homogenate (100 mL), ethanolic CDNB solution (60 mL), GSH solution (200 mL), and 0.1 M sodium phosphate buffer (7.6 pH). After adding these components, a change in absorbance at 340 nm was measured for 5 min at intervals of 1 min at 30 °C.

2.7. Oxidative stress markers

2.7.1. Lipid peroxide (LP) content

Hermes-Lima et al. [23] and Vontas et al. [24] methodology were used to calculate the LP content [71]. The larvae were homogenised (20% weight/volume) in chilled methanol, and then centrifuged at 10,000 g for 5 min. The resulting supernatant (30 μL) was combined with 520 μL of distilled water, 50 μL of sulphuric acid, 250 μL of ferrous sulphate, 100 μL of xylenol orange, and 15 μL of distilled water before being incubated at 30 °C for 15 min. The absorbance was measured at 520 nm, and a standard curve was created using cumene H₂O₂ serially diluted at concentrations between 50 and 100 mM.

2.7.2. Hydrogen peroxide (H₂O₂) content

The Green and Hill approach was employed for determining H2O2 content [24]. The larvae were subjected to a 15-min centrifuge at 3,000 g at 4 °C after being homogenised (10% weight/volume) in 50 mM Potassium phosphate buffer (pH 7). 200 μL of 4-aminoantipyrine (4 mM), 200 μL of phenol, 200 μL of peroxidase (0.4 U/mL), 200 μL of supernatant, and 200 μL of potassium phosphate buffer (50 mM) were used in the reaction mixture, which was incubated at 30 °C for 10 min. At 510 nm, the absorbance was measured. Using hydrogen peroxide serially diluted between 100 and 1000 μM, the standard curve was created.

2.7.3. Protein carbonyl content

The Levine et al. [25] method was used to calculate the protein carbonyl content [25]. Centrifugation was done at 8,000 g for 8 min at 40°C after the larvae were homogenised (10% w/v) in the extraction buffer. The mixture was centrifuged at 8,000 g for 5 min at 4°C with the supernatant (300 μL) added to 22.2 μL of 10% streptomycin sulphate produced in 50 mM HEPES buffer (pH 7). To obtain a protein pellet, recentrifugation at 8,000 g for 5 min was done after adding 100 μL of 20% trichloroacetic acid to the supernatant. The pellet received 500 μL of 7 mM DNPH produced in 2 M HCL at 37 °C. The pellet received 500 μL of 7 mM DNPH produced in 2 M HCL at 37 °C. The mixture was then left to sit in the darkness for 1 h while being shaken periodically. After adding about 750 μL of 20% trichloroacetic acid, the mixture was once more centrifuged at 8000 g for 5 min at 4 °C. The resultant pellet was vortexed on a regular basis after being rinsed three times with ethyl acetate and 95% ethanol (1:1). After being dissolved in 6 M guanidine hydrochloride (800 mL) and incubated for 5 min at 37 °C, the protein pellet was once more centrifuged at 8,000 g for 5 min at 4 °C. Japan's Shimadzu UV 1800 spectrophotometer was used to test the absorbance at 370 nm.

2.7.4. Statistical analysis

Statistical comparisons were made between means within experiments to avoid any confounding effects from variation in methods between experiments. Data were then subjected to the analysis of variance (ANOVA) and tukey's test to find significant differences between the average values using ASSISTAT and MINITAB softwares.

3. Results

3.1. Effect of caffeic acid on growth and development of S. litura

The developmental and nutritional parameters of larvae were significantly impacted with caffeic acid (Table 1, Table 2). The larval mortality was significantly increased in a concentration-dependent manner with highest mortality (63.33%) observed at 3125 ppm. The larval, pupal and the total development period also showed significant increase with concentration in comparison to control. A significant decrease in emergence of adult was observed with the increasing concentration of caffeic acid and maximum decline by 40% was noticed at 3125 ppm in comparison to control. Pupal weight also showed a significant reduction in all the treated larvae in comparison to control (Table 1). Using probit analysis, the LC₅₀ concentration (385.19 ppm) was determined from the results acquired for larval mortality.

Table 1.

Larval mortality, pupation, adult emergence (in %), Larval period, pupal period, total developmental period (in days), pupal weight (in mg) (Means ± S.E.) of S. litura when second instar larvae were fed on different concentrations (ppm) of caffeic acid.

Concentrations	Larval Mortality	Adult Emergence	Larval Period	Pupal Period	Total Development Period	Pupal weight
Control	3.33 ± 3.33a	80.00 ± 2.10a	18.70 ± 0.14a	8.78 ± 0.27a	27.45 ± 0.23a	264.26 ± 5.64a
5 ppm	23.33 ± 3.15b	66.67 ± 4.22b	19.78 ± 0.30ab	9.29 ± 0.39ab	29.29 ± 0.45ab	243.68 ± 4.21a
25 ppm	30.00 ± 4.47b	60.00 ± 4.67b	20.13 ± 0.39b	9.52 ± 0.36ab	29.72 ± 0.40ab	237.32 ± 7.31a
125 ppm	46.70 ± 4.20bc	53.33 ± 4.22bc	22.68 ± 0.31b	10.54 ± 0.78b	33.52 ± 0.45b	189.77 ± 8.28b
625 ppm	53.33 ± 4.89c	43.33 ± 3.67c	23.495 ± 0.26b	10.55 ± 0.43b	34.17 ± 0.44b	185.53 ± 8.61b
3125 ppm	63.33 ± 5.89c	40.00 ± 3.17c	23.92 ± 0.34b	12.68 ± 0.64c	36.47 ± 0.56b	179.21 ± 5.24b
F value	8.20**	12.57**	6.48**	5.48**	7.72**	26.53**

**Significant at 1%. Means followed by the same letter within the columns are not significantly different according to Tukey's test at P ≤ 0.05.

Table 2.

RGR, RCR (in mg/mg/day), ECI, ECD, AD (in %) (means ± S.E.) of S. litura when second instar larvae were fed on different concentrations (ppm) of caffeic acid.

Concentrations	RGR	RCR	ECI	ECD	AD
Control	1.43 ± 0.06a	14.65 ± 0.83a	10.73 ± 0.35a	10.84 ± 0.67a	88.89 ± 1.25a
5 ppm	0.93 ± 0.04b	12.34 ± 0.53b	9.31 ± 0.49b	8.22 ± 0.48b	84.67 ± 1.37a
25 ppm	0.76 ± 0.03bc	9.85 ± 0.36c	9.08 ± 0.45b	7.84 ± 0.71bc	82.41 ± 2.25a
125 ppm	0.61 ± 0.03cd	9.36 ± 0.24c	8.43 ± 0.71bc	6.72 ± 0.54bcd	78.34 ± 2.32ab
625 ppm	0.46 ± 0.03de	7.33 ± 0.41d	7.60 ± 0.57c	6.19 ± 0.58cd	74.16 ± 3.12b
3125 ppm	0.30 ± 0.02e	6.96 ± 0.57d	7.21 ± 0.95c	5.29 ± 0.44d	67.37 ± 2.56ab
F-value	92.63**	32.54**	12.28**	16.35**	7.97 **

**Significant at 1%. Means followed by the same letter within the columns are not significantly different according to Tukey's test at P ≤ 0.05.

3.2. Effect of caffeic acid on the nutritional physiology of S. litura

The nutritional indices of the larvae were adversely affected by the phenolic compound (Table 2). A decline was noticed in the RGR and RCR of when provided diet amended with different concentrations of caffeic acid in comparison to control (Table 2). At 3125 ppm concentration, the RGR declined significantly by 79.02% in comparison to control. A maximum decrease in the RCR (52.49%) was noticed in larvae treated with caffeic acid at 3125 ppm when compared to control. The ECI, ECD and AD were also significantly reduced with increasing concentration of caffeic acid and maximum reduction was noticed at the highest concentration of 3125 ppm in comparison to control (Table 2).

3.3. Effect of caffeic acid on the immune response of S. litura

The total hemocyte count was also significantly declined in comparison to control at all the time intervals (24 h–120 h) (Fig. 1) but the maximum reduction was recorded at 120 h treatment interval in comparison to control.

Fig. 1.

Influence of caffeic acid on total hemocyte count (means ± S.E.) of S. litura larvae.

3.4. Impact on enzymatic activity and stress markers

Catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APOX), and glutathione-S-transferase (GST) were the enzymes tested for how caffeic acid affected their activities. The results are shown in Table 3. Comparing treated larvae to the control, it was found that all antioxidant enzyme activity had significantly increased. In comparison to the control, CAT activity was significantly higher as the time interval increased. The 96 h group showed the greatest growth (t = 44.82, p ≤ 0.01). When caffeic acid was administered to larvae, SOD activity also increased in a similar manner, with the 96-h exposure group experiencing the greatest increases (t = 28.56, p ≤ 0.01). In comparison to control, APOX activity increased with increasing treatment interval, reaching its peak at 96 h (t = 18.74, p ≤≤ 0.01). GST activity was seen to rise in all treatment groups as compared to the control, reaching its peak at the 96-h treatment interval (t = 16.85, p ≤ 0.01). When compared to control, all treatment periods had significantly higher levels of LP, H₂O₂, and protein carbonyl content, according to the study of oxidative stress markers (Table 4). Overall, following caffeic acid treatment, all enzyme activities and levels as well as stress markers were increased, and the effect of treatment time was also determined to be substantial.

Table 3.

Effect of caffeic acid on antioxidant enzymes activities of in S. litura.

Enzyme activity	Concentrations	24 h	48 h	72 h	96 h	F value
Catalase (μmol/mg)	Control LC50 T-value	14.84 ± 0.30a 28.23 ± 0.41a 13.37*	15.64 ± 0.38a 36.65 ± 0.39b 15.29**	15.24 ± 0.36a 49.27 ± 0.37c 14.94**	15.44 ± 0.22a 53.17 ± 0.23c 44.82**	1.23 142.23**
SOD (μmol/mg)	Control LC50 T-value	24.26 ± 0.20a 37.56 ± 0.21a 14.96**	25.54 ± 0.38a 38.14 ± 0.31a 15.79**	26.49 ± 0.42a 47.72 ± 0.61b 17.65**	26.97 ± 0.42a 47.56 ± 0.31b 28.56**	0.22 89.26**
Ascorbate peroxidase activity (μmol/mg)	Control LC50 T-value	33.47 ± 0.39a 69.34 ± 1.72a 15.64**	34.61 ± 0.12a 70.63 ± 1.49a 17.62**	36.42 ± 0.27a 86.92 ± 1.93b 18.33**	35.27 ± 0.25a 92.39 ± 2.31b 18.74**	1.27 14.86**
GST activity (μmol/mg)	Control LC50 T-value	7.23 ± 0.43a 13.48 ± 0.42a 7.67**	7.57 ± 0.23a 15.36 ± 0.27b 12.24**	8.23 ± 0.38a 17.89 ± 0.68c 14.64**	8.49 ± 0.35a 18.50 ± 0.46c 16.85**	0.45 12.23**

The values represented as mean ± standard error. * describes the significant difference between exposed group and control group (t-test, p ≤ 0.05). Different letters a, b, c between the columns are significantly different (Tukey's test, p ≤ 0.05) and signify the effect of duration.

Table 4.

Stress markers content in S. litura after treatment with caffeic acid.

Enzyme activity	Concentrations	24 h	48 h	72 h	96 h	F value
Hydrogen peroxide content (μmol/mg)	Control LC50 T-value	19.79 ± 0.20a 27.28 ± 0.55a 13.86**	20.16 ± 0.27a 38.16 ± 0.74b 14.24**	20.25 ± 0.32a 46.65 ± 0.59c 17.49**	21.17 ± 0.22a 56.34 ± 0.22d 19.86**	2.45 35.40**
Lipid peroxide content (μmol/mg)	Control LC50 T-value	5.77 ± 0.10a 8.67 ± 0.26a 12.39**	6.09 ± 0.47a 8.85 ± 0.22a 18.51**	6.25 ± 0.37a 8.89 ± 0.36b 16.27**	6.18 ± 0.22a 9.34 ± 0.45c 17.26**	3.24 14.24**
Protein carbonyl content (μmol/mg)	Control LC50 T-value	1.52 ± 0.02a 4.24 ± 0.22a 7.62**	1.75 ± 0.02a 5.44 ± 0.33a 8.89**	1.79 ± 0.03a 6.87 ± 0.32b 12.35**	1.81 ± 0.01a 7.78 ± 0.65c 14.97**	0.45 15.72**

The values represented as mean ± standard error. * describes the significant difference between exposed group and control group (t-test, p ≤ 0.05). Different letters a, b, c between the columns are significantly different (Tukey's test, p ≤ 0.05) and signify the effect of duration.

4. Discussion

The development and nutritional physiology of S. litura larvae were adversely affected by caffeic acid. After treatment in the larvae, adult emergence and pupal weight considerably decreased, whereas larval mortality, larval period, and development period elevated. Flavonoids such as caffeic acid, chlorogenic acid and protocatechuic acid had also been recorded to have negative impact on the growth and survival of Helicoverpa armigera (Hubner) [26]. Similarly, quercetin significantly increased the mortality of larvae of Egyptian cotton leafworm, Spodoptera littoralis (Boisd.) [27]. The larval mortality and total development period of S. litura larvae was also reported to increase significantly with increasing concentration of pyrogallol and hydroquinone [28,29]. Syringic and ferulic acids present in extracts of different cotton varieties resulted in 100% mortality during the first week of treatment in H. armigera [30]. The phenolic compounds such as gallic acid, epigallocatechin gallate, p-coumaric acid, salicylic acid, catechin and quercetin in an extract of acerola bagasse flour extended the prepupal stage and increased the mortality of S. frugiperda caterpillars [31]. Vimladevi et al. [32] also observed larvicidal activities of phenolic acids of seaweed, Chaetomorpha antennina (Bory.) against the third instar larvae of yellow fever mosquito, A. aegypti.

Bhattacharya and Chenchaiah [33] also recorded that phenolic acids viz. chlorogenic acid, gallic acid and syringic acid along with hydroquinone that occurred in the red gram's seed coat extract had inhibitory effects on the growth and development of S. litura. Similar to this, S. litura larvae showed decreased emergence of adult and higher larval mortality when fed an artificial diet including phenolics such as ellagic, gallic, and ferulic acids [[34], [35], [36]]. According to Steinly and Berenbaum [37], tannin damages the Papilo polyxenes (F.), a swallowtail caterpillar, by causing regressive lesions in its intestines. The cells of the midgut showed signs of degeneration in Spodoptera eridania (Stoll) larvae fed on phenols (Salicortin and Tremulacin) [38]. Also, we have noted that pupa weight was lower in S. litura larvae fed on caffeic acid-treated diets than in controls. Summers and Felton had similarly noted a notable decrease in H. zea pupal mass following larval consumption of phenolic acids [39]. The mean larval mass and mean pupal weight of H. armigera decreased when supplemented with ferulic acid derived from wheat, according to Wang et al. [40]. Agrell et al. [41] also noted that S. littoralis pupae and larvae were smaller due to the alfalfa plant's increased apigenin concentration. Caffeic acid was found to have a significant impact on the nutritional physiology of larvae of S. litura. The RGR, RCR, ECD, ECI and AD of S. litura larvae significantly declined with an increase in the concentration of caffeic acid. Rani and Pratyusha [42] too had reported the antifeedant effect in S. litura by the increased level of phenolics in G. hirsutum. The decline in growth rate of S. litura larvae can be attributed to the decrease in consumption rate and a decline in conversion efficiencies of the larvae suggesting a toxic rather than an antifeedant effect, caused after ingestion of the compounds. ECI is the measure of the food consumed that is metabolized into biomass [16] while ECD measures the efficiency of the insect to convert digested food into growth [43]. Because S. litura larvae have low ECI and ECD values, it is possible that most of the food they ate was digested to provide energy for the detoxification of phenolic acids rather than for the larvae's growth. These results are in line with those of Wang et al. [40], who found that cotton bollworm larval and pupal weight reduced coupled with lower RGR, AD, and ECD after consuming an artificial diet containing ferulic acid. Yuan et al. [44] too had reported a decrease in the growth rate, ECI and ECD of fall webworm, Hyphantria cunea (Drury) when fed on an artificial diet containing a high concentration of tannic acid. Kopper et al. [45] found that the paper birch, B. papyrifera condensed tannins increased stadium duration, decreased RGR, ECD and ECI of white-marked tussock moth, O. leucostigma. They suggested that prolonged developmental time compensates for reduced growth rate and thus leads to an increase in total consumption. The AD also declined with increasing concentration of the phenolics in the diet of the treated larvae. AD represents the proportion of ingested food that is actually digested. The declined AD values could be due to the poor digestibility ability of the larvae.

The S. litura larvae exposed to caffeic acid experienced a substantial drop in the total hemocyte count. Following the larvae's maturity, it was discovered that the fall in hemocyte count accelerated. Ayyangar and Rao [46] reported an earlier considerable decrease in total hemocyte count in the hemolymph of final-instar S. litura larvae in response to azadirachtin exposure. Many specialized and generalist lepidopterans were observed to respond negatively to increased dietary glucosinolates, iridoid glycosides, and hydrolysable tannins [[47], [48], [49]]. Our results categorically demonstrated that caffeic acid can decrease the insect's immune response, increasing its susceptibility to attack by its allies and enemies. Phenolics on oxidation generate free radicals in herbivorous insects which are detrimental to their growth [50,51]. Superoxide is the first free radical generated by the oxidation process. The superoxide radicals undergo dismutation and get converted into H₂O₂ in a reaction catalyzed by superoxide dismutase, an antioxidant enzyme that forms the primary defense against these radicals [52]. Our results on biochemical analysis showed a significant increase in SOD activity of S. litura larvae after exposure to caffeic acid. Rani and Pratyusha [42] had also perceived an increased SOD activity in the larvae of S. litura after feeding on cotton plants having high concentrations of phenolic compounds. Increased SOD activity is evidence of increased superoxide radical production. Pristos et al. [53] highlighted the importance of SOD to defend insects from the prooxidant activity of phenolics, quercetin and xanthotoxin. These compounds often lead to the production of ROS accompanied by an elevation of SOD activity as observed in the larvae of cabbage looper, T. ni, southern armyworm, S. eridania and black swallowtail, P. polyxenes [54].

The treated S. litura larvae showed a considerable increase in catalase (CAT) activity. By changing hazardous H₂O₂ produced by SOD activity into water and oxygen (2H₂O₂ → 2H₂O + O₂), catalase (CAT) removes it from the body. In addition, S. litura larvae fed tannic acid showed an increase in CAT activity, according to Krishnan and Sehnal [55]. Several lepidopterans, such as the cabbage looper (T. ni) and the southern armyworm (S. eridania), have similarly shown an increase in CAT activity after being subjected to xanthotoxin and quercetin [56,57]. Tannic acid was added to the diet of S. littoralis larvae, and this resulted in an increase in catalase activity [51]. Phenolic compounds, xanthotoxin and quercetin were found to increase enzymatic activity of catalase in S. eridania and T. nivalis [56].

Only at low quantities [72] that are typically not scavenged by CAT can ascorbate peroxidase (APOX) remove hydrogen peroxide (ascorbic acid + H₂O₂ → dehydroascorbic acid + 2H₂O). Ascorbate is oxidised to breakdown hydrogen peroxide, while NADPH is oxidised to decontaminate oxidising agents [21,39]. The activity of APOX in S. litura larvae was increased when they were provided with caffeic acid-incorporated diet. Similarly, increased activity of APOX in the foregut and midgut of Egyptian cotton leafworm, S. littoralis larvae has also been perceived with tannic acid feeding [51].

As compared with the control group at every treatment interval, our findings of GST activity in S. litura larvae treated with caffeic acid showed significant elevation in enzyme activity. Our findings get support from the work of Rani and Pratyusha [42] who had also reported increased GST activity in S. litura larvae fed on phenolics viz. gallic acid, catechin and caffeic acid extracted from the upland cotton plant, Gossypium hirusutum. Induced GST activity has also been perceived in fruit fly, D. melanogaster after exposure to phenols [58] and in tissues of grain aphid, Sitobion avenae fed on host plants having a higher concentration of phenolics [59]. Increased GST activity was also recorded in Micromelalopha troglodyte (Graeser) larvae with tannic acid [60] and in gypsy moth, Lymantria dispar larvae, forest tent caterpillar, Malacosoma disstria and the mosquito bug, Helopeltis theiovora (Waterh.) larvae with phenolic glycosides [61].

Feeding of S. litura larvae on diet amended with caffeic acid evoked a substantial oxidative response. The stress markers (protein carbonyl, lipid peroxide and hydrogen peroxide content) increased notably with extended feeding indicating a certain deterioration of insect tissues. An increase in oxidative stress has also been reported in daidzein fed S. litura larvae [62]. Barbehenn et al. [63] reported that increased oxidative stress in the gut wall of M. disstria was linked with enhanced phenolic oxidation. An increase in PC and peroxides in S. littoralis larvae with tannic acid ingestion has also been recorded by Krishnan and Sehnal [51]. O-dihydroxy phenolics (caffeic acid and chlorogenic acid) were also linked with enhanced levels of lipid peroxidation products, oxidised protein and free iron in H. zea [64]. Enhanced levels of superoxides, peroxides and protein carbonyl were observed in S. littoralis larvae after they were fed on potato plants rich in allelochemicals [55]. Lukasik [65] too had perceived higher levels of H₂O₂ in bird cherry-oat aphid, Rhopalosiphum padi and grain aphid, S. avenae exposed to dietary phenolics viz. quercetin, caffeic acid and chlorogenic acid. Oxidative stress resulting from enhanced production of ROS. Increased levels of ROS cause oxidative damage which can severely disrupt the functioning of cells resulting in death [66,67]. Long term association of insects with their host plants can influence their susceptibilities to several insecticides [68]. Host plants allelochemicals can induce certain detoxifying enzymes in insect larvae [69]. Further, these detoxifying enzymes such as SOD, CAT, GST etc. can also result in development of insect's resistance against insecticides [70]. We have noticed an induction of detoxifying enzymatic activity in S. litura larvae after feeding on caffeic acid supplemented diet. However, increased oxidative stress and reduced immune response negatively affects the overall fitness of insect which greatly impaired the growth and development of the S. litura larvae. Induction of detoxifying enzymatic activity in S. litura provided information regarding the adaptive mechanism of insects against caffeic acid during insect plant interactions. However, further research is required to ascertain the expression of associated genes and regulatory pathways of S. litura in response to caffeic acid.

5. Conclusion

Our findings suggest that caffeic acid has insecticidal properties, impairing the growth and physiology of insects. As a result, this phenolic compound has the potential to be utilized as a biopesticide for ecofriendly insect pest control. Plant breeders may be able to use the findings to improve the expression of genes encoding phenolic compounds, which could impede the development of insect pests. Field studies are also needed before the implementation of this phenolic compound in an integrated pest management programme.

Author contribution statement

Abhay punia: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Vijay singh; Anita THAKUR: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.

Nalini Singh Chauhan: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

No data was used for the research described in the article.

Additional information

No additional information is available for this paper.

Declaration of interest's statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.