Growth, Development and Reproduction of Meadow Moth Loxostege sticticalis Fed on Pea Seedlings Grown Under Elevated CO₂

Abstract

To comprehensively investigate the impacts of increased atmospheric CO₂ concentrations on the growth, development and reproduction of the meadow moth Loxostege sticticalis when fed on pea plants (Pisum sativum), in this experiment, we simulated the two CO₂ conditions: ambient CO₂ (i.e., 400 μL/L designated as aCO₂) and elevated CO₂ (i.e., 800 μL/L designated as eCO₂) by using light-CO₂ climate chambers. Subsequently, the changes in several key nutrients and defensive compounds present in pea seedlings were assessed. Moreover, we assessed the growth, development, reproduction and changes in the nutritional components and enzyme activities of L. sticticalis as they fed on pea seedlings grown under aCO₂ and eCO₂. The results showed that the CO₂ level significantly affected the measured indexes of pea seedlings and L. sticticalis. Host pea seedlings grown under eCO₂ exhibited significant increases in soluble sugar (SS), soluble protein (SP) and total amino acid (TAA) contents by 42.52%, 77.06% and 62.50%, respectively, relative to those grown under aCO₂. In addition, total phenol (TP), ethylene (ET) and jasmonic acid (JA) contents grown under eCO₂ increased significantly by 20.60%, 71.72% and 36.22%, respectively, under eCO₂ compared to aCO₂. Furthermore, the duration of egg, larva and pupa of L. sticticalis was significantly shortened by 16.63%, 10.66% and 10.12%, respectively, while the adult longevity was significantly prolonged by 19.07% after feeding on pea seedlings grown under eCO₂ in contrast to aCO₂. Furthermore, for L. sticticalis, the content of SS, SP, TAA and free fatty acid was significantly increased, by 60.02%, 77.06%, 91.67% and 27.19%, respectively. Moreover, the enzyme activity of CAT, POD, CarE and GST was also enhanced by 56.70%, 63.89%, 128.08% and 93.45%, respectively, as they fed on pea seedlings grown under eCO₂ in contrast to aCO₂. The findings of our study revealed that eCO₂ not only improved the nutritional quality but also altered the defensive compounds in the seedlings, which in turn affected the growth, development, reproduction and changes in the physiology of L. sticticalis.

1. Introduction

The current climate situation is severe, with climate change characterized by increasing atmospheric carbon dioxide (CO₂) concentration that directly affects human living environments and socio-economic development (IPCC, 2023). According to the annual reports from the U.S. National Oceanic and Atmospheric Administration (NOAA), atmospheric CO₂ concentration is rising annually, especially at an accelerating rate. As of 2023, the atmospheric carbon dioxide levels have been increasing by over 2 ppm for twelve consecutive years [1]. Results from multiple observation stations indicate that the average atmospheric CO₂ concentration in April 2024 reached 426.57 ppm, which is 53% higher than the pre-industrial level (about 278 ppm in 1750), and it could potentially double by the end of the 21st century.

For plants, CO₂ serves as a substrate for photosynthesis. The increase in atmospheric CO₂ concentration can directly enhance the photosynthetic rate, carbon (C) assimilation rate, carbon-to-nitrogen (N) ratio (i.e., C/N ratio) and crop yield while reducing N-content in plant tissues, which in turn leads to the assimilation and redistribution of C- and N-sources within plant tissues, further influencing the production and allocation of primary and secondary metabolites, and ultimately altering the content and composition of plant nutrients [2,3,4,5]. Qian et al. [6] reported an increase in soluble sugar (SS), soluble protein (SP) and free fatty acid (FFA) in kidney bean (Phaseolus vulgaris) plants grown under elevated CO₂ (eCO₂) compared to ambient CO₂ (aCO₂), indicating a shift in the plant’s metabolic condition. Li et al. [7] further found that doubling the CO₂ concentration led to a significant increase in SS and polyphenol, along with a significant decrease in FFA and caffeine in tea seedlings, demonstrating the diverse responses of plant metabolites to eCO₂. Goufo et al. [8] reported that the rice (Oryza sativa) plants grown under eCO₂ conditions exhibited increased contents of non-structural carbohydrates (including total phenols and flavonoids) throughout the growth period as compared to the rice plants grown under aCO₂ conditions. The increased photosynthetic rate due to eCO₂ can allocate excess C sources in plants and facilitate the synthesis of structural compounds (e.g., pectin and cellulose) and C-based secondary metabolites (e.g., phenols and terpenes), thus affecting plant metabolism [9]. Robinson et al. [10] found that eCO₂ enhanced the content of total phenols, tannins and flavonoids in plant tissues by 19%, 22% and 27%, respectively, while reducing the content of terpenes by 13%, indicating significant implications for the plant defense mechanisms. Regarding the plant defense mechanisms, the induced defense is mainly regulated by three signaling pathways, i.e., jasmonic acid (JA), salicylic acid (SA) and ethylene (ET). These pathways are involved in regulating diverse defensive compounds. Total phenols encompass phenolic compounds that contribute to antioxidant properties and defensive functions [11]. Additionally, saponins categorized as secondary metabolites can act as a defensive shield against pests and pathogens [12]. Ethylene plays an instrumental role in implicating both defense mechanisms and developmental processes. Salicylic acid is crucial for plant defense against necrotrophic pathogens, while jasmonic acid is critical for plant defense against insect herbivores [13]. Elevated CO₂ can affect these three types of plant hormone-dependent induced defense signaling pathways [14]. Sun et al. [15] found that eCO₂ upregulated SA defense genes and downregulated JA and ET defense genes in Arabidopsis (Arabidopsis thaliana), showing a modulation of the plant’s defense response. Such alterations in plant chemical composition can have cascading effects on herbivorous insects, thereby influencing their growth, development, reproduction and feeding and oviposition behaviors [16,17].

Numerous studies have vividly illustrated that eCO₂ adversely influenced the growth, development and reproduction of chewing insects, such as Lepidopterans. Xie et al. [18] found that eCO₂ significantly delayed the growth and development of the Asian corn borer (Ostrinia furnacalis), accompanied by a reduction in body weight and an increase in mortality, suggesting a negative impact on its fitness. Li et al. [19] found that eCO₂ reduced the body weight of the 5th instar larvae of rice leafroller (Cnaphalocrocis medinalis) by 16.1%, shortened the pupal duration by 28.7% and reduced the pupal weight by 9.9%, which further emphasized the developmental disruptions caused by the increased CO₂ concentration. Zhang et al. [20] noted a prolonged development duration and increase in body weight of the Tobacco cutworm (Spodoptera litura) under eCO₂, highlighting the complex and species-specific responses to eCO₂. Satishchandra et al. [21] found that eCO₂ delayed the larval development of American tomato moth (Tuta absoluta). The research also highlighted a significant increase in the larval mortality rate as well as fecundity of the American tomato moth, suggesting a significant shift in the pest’s life history strategy and population regulatory mechanisms under eCO₂. These collective findings from previous research provide a comprehensive framework that justifies and shapes our current investigation into the interactions between plants and insects under the influence of eCO₂.

The meadow moth (Loxostege sticticalis), a species belonging to the family Crambidae of the order Lepidoptera, is a major global agricultural pest, mainly distributed in the broad region between 36 °N and 55 °N [22]. In China, it primarily causes severe damage in the North, Northeast and Northwest regions, collectively referred to as the “Three North” regions of China. The polyphagous larvae of L. sticticalis consume a wide variety of host plants, specifically 259 species belonging to 48 distinct families [23]. In the latest edition of the “List of the First-Class Crop Pests” released by the State Ministry of Agriculture and Rural Affairs of China in 2023, L. sticticalis is ranked as the 3rd most significant pest. Currently, L. sticticalis has entered into a 4th outbreak cycle in China, which poses a severe threat to food security [24]. The majority of the studies emphasize the influence of environmental factors such as temperature [25] and humidity [22] and their impacts on the growth, development and reproduction of L. sticticalis. Despite the importance of pea plants as a vegetable crop, there are limited studies investigating the alterations in nutrient contents and defensive compounds in these plants when grown in eCO₂ conditions. Additionally, the impacts of these changes in host pea plants on the growth, development and reproduction of L. sticticalis also remain scarcely explored.

In order to clarify the impacts of eCO₂ on the growth, development and reproduction of L. sticticalis fed on pea plants under global climate change, the ambient and elevated CO₂ were simulated by using light-CO₂ culture chambers. The changes in nutrients and defensive compounds in pea seedlings, as well as the growth, development, reproduction and changes in the nutritional components and enzyme activities of L. sticticalis fed on pea seedlings grown under aCO₂ and eCO_2, were also measured. This study aims to clarify the outbreak patterns and population dynamics of L. sticticalis under future high CO₂ climate conditions, providing scientific support and a practical basis for the long-term prediction and integrated management of L. sticticalis.

2. Materials and Methods

2.1. Pea Plants

The broad-leaf green pea (Pisum sativum) was used, and the pea plants were grown in double-layer hydroponic seedling trays (length:width:height = 32 cm:24 cm:3.5 cm) with nutrients solutions recommended for the hydroponic cultivation (components of hydroponic nutrient solution: 0.7 g/L of potassium nitrate, 0.0006 g/L of boric acid, 0.7 g/L of calcium nitrate, 0.0006 g/L of manganese sulfate, 0.8 g/L of superphosphate, 0.0006 g/L of zinc sulfate, 0.28 g/L of magnesium sulfate, 0.0006 g/L of copper sulfate, 0.12 g/L of iron sulfate and 0.0006 g/L of ammonium molybdate). Before germination, the pea seeds were covered with seed germination paper and sprayed with water daily to promote sprouting. After germination, the seed germination paper was removed, and the nutrient solution was replaced every 5 days. The pea plants were grown in light-CO₂ climate chambers (GDN-400D-4-CO₂, Ningbo Southeast Instrument, Ningbo, China) under controlled CO₂ concentrations of 400 μL/L (i.e., the current atmospheric CO₂ concentration as control of ambient CO₂ (aCO₂)) and 800 μL/L (i.e., the projected CO₂ concentration by the end of the 21st century as elevated CO₂ (eCO₂)) [26], respectively. The required CO₂ concentration in the light-CO₂ climate chambers was controlled with CO₂ gas supplied from a CO₂ cylinder. The growth conditions in the climate chambers were set to a temperature of (22 ± 1) °C, relative humidity of (75 ± 5)% and photoperiod of 16 L:8 D, with a light intensity of 20,000 lx. After 20 days of seedling growth, the leaves and stems of pea plants were collected and stored in liquid nitrogen for the following testing.

2.2. Meadow Moths

The larvae of tested meadow moth L. sticticalis were collected from Kangbao County, Hebei Province of China (41.85° N, 114.26° E), in Sept 2023. A total of 100 larvae were initially used to initiate the lab colony development. The collected larvae were reared and bred in the laboratory, using broad-leaf green pea (P. sativum) as food, for three generations to ensure uniformity and eliminate the influence of genetic and environmental variability from the wild population. The test colony was maintained in light-CO₂ climate chambers (GDN-400D-4-CO₂, Ningbo Southeast Instrument, Ningbo, China) under the same conditions for the used pea plants, with a temperature of (22 ± 1) °C, relative humidity of (75% ± 5) % and a photoperiod of 16 L:8 D, with a light intensity of 20,000 lx.

2.3. Determination of Nutrients and Defensive Substances in Pea Seedlings Grown Under aCO₂ and eCO₂

To clarify the changes in the contents of nutritional components and defensive substances in pea seedlings grown under different CO₂ concentrations, this experiment was set up with two CO₂ concentrations, i.e., aCO₂ and eCO₂. The pea seedlings grown under aCO₂ and eCO₂ for 20 days were randomly collected and ground into powder, and 0.1 g plant tissues were weighed and ground into a 10% homogenate with ultrapure water. The nutritional components in pea seedlings were measured according to the instructions of the assay kits for soluble sugar (A145), soluble protein (A045-2), free fatty acid (A042-2-1) and total amino acid (A026-1-1). The defensive compounds present in pea seedlings were also measured according to the instructions of the assay kits for total phenol (A143-1-1) and saponin (BR500741). All the used assay kits except that for saponin were from the Nanjing Jiancheng Bioengineering Institute in the Jiangsu Province of China, and the assay kit for saponin was supplied from Shanghai Bioleaper Bio-Technology Co., Ltd., Shanghai, China.

The hormone levels in pea seedlings were determined using enzyme-linked immunosorbent assay (ELISA) according to the instructions of the assay kits for ethylene (M133388), salicylic acid (M133352) and jasmonic acid (M133322); all these ELISA kits were purchased from Beijing MREDA Technology Co., Ltd., of Beijing, China. The absorbance (optical density, OD) of the reaction products was, respectively, measured at wavelengths of 620 (SS), 595 (SP), 546 (FFA), 650 (AA), 760 (TP), 589 (SP) and 450 (ELISA for ET, SA and JA) nm using a microplate reader, and the corresponding concentration of these compounds was calculated. Each experimental treatment was repeated three times with each replicate using a distinct 0.1 g sample of pea seedlings.

2.4. Determination of Growth, Development and Reproduction of L. sticticalis Fed on Pea Seedlings Grown Under aCO₂ and eCO₂

To clarify the effects of eCO₂ on the growth, development and reproduction of L. sticticalis, 20 female and male pairs of newly eclosed 1-day-old adults were randomly selected for each treatment of aCO₂ and eCO₂. The female and male pairs were, respectively, placed in plastic cups for mating, with cotton balls and oviposition egg strips placed in each cup. A solution with 10% glucose was added to the cotton balls at 18:00 daily to feed the moths. After egg-laying, the oviposition egg strips were removed and placed individually into glass jars (12.6 cm high, 8.3 cm in diameter), each containing 30 eggs. The jar was covered with gauze, and a moist towel was placed over it to maintain humidity. The towel was moistened daily, and the hatching status of the eggs was observed and recorded daily.

The egg duration was calculated based on the egg-laying date and the hatching date. Each glass jar was treated as a single replicate, with three replicates per treatment (N = 30). After hatching, fresh pea seedlings were provided daily at 18:00, and the developmental stages and other related characteristics of the larvae were observed and recorded daily. When the larvae stopped feeding and entered the pupal stage, they were removed from the glass jars and individually reared in test tubes. The dates of pupation and adult emergence were respectively recorded, and the developmental duration of larvae and pupae was also respectively calculated. Each test tube was considered as one single replicate, with 50 replicates per treatment. The pupae were weighed on the 3rd day after pupation using a Huachi electronic balance with a maximum capacity of 200 g, an accuracy of 0.001 g and a readability of 0.0001 g. A total of 50 pupae were weighed per treatment. After emergence, 20 pairs of newly emerged male and female moths were paired and placed into plastic cups for mating and egg-laying, and the dates of emergence and death of the moths were recorded to calculate the adult longevity. The status of female moths was observed daily, and the number of eggs laid per day was recorded until the female moths died, and the total egg count per female moth was calculated. The eggs laid on the same day were placed in glass jars to observe hatching. If no larvae were hatched for three consecutive days, it was assumed that all viable eggs had been hatched, and the number of unhatched eggs was also counted. The fecundity (number of eggs per female) and egg hatchability were calculated based on the number of larvae hatched and the number of unhatched eggs. The formulae used were as follows: number of eggs per female = number of hatched larvae + number of unhatched eggs; egg hatchability = (number of hatched larvae/number of eggs per female) × 100%.

2.5. Bioassay for Nutritional Components and Enzyme Activity in L. sticticalis Fed on Pea Seedlings Grown Under aCO₂ and eCO₂

To clarify the changes in the contents of nutritional components and enzyme activity in L. sticticalis larvae fed on pea seedlings grown under different CO₂ concentrations, five 2nd-day 5th instar larvae were randomly selected for each treatment of aCO₂ and eCO₂. After freezing the larvae with liquid nitrogen, their weights were recorded. Then, ultrapure water was added, and the larvae were ground into a 10% homogenate. The content of soluble sugar (A145), soluble protein (A045-2), free fatty acid (A042-2-1) and total amino acid (A026-1-1) in L. sticticalis larvae was measured according to the insect test kit instructions. The enzyme activity of carboxylesterase (A133-1-1), glutathione S-transferase (BC0350), acetylcholinesterase (A024-1-1), superoxide dismutase (A001-3-1), catalase (A007-1-1) and peroxidase (A084-3-1) in L. sticticalis larvae was also measured, following the respective test kit instructions. The optical density (OD) value of the reactants was measured using a microplate reader at 620 (SS), 595 (SP), 546 (FFA), 650 (AA), 450 (CarE), 340 (GST), 412 (AChE), 450 (SOD), 405 (CAT) and 420 (POD) nm wavelengths, respectively, and the corresponding concentrations of various compounds were also calculated respectively. Each experimental treatment was repeated three times, with each replicate using a distinct 0.1 g sample of larvae. All of the assay kits employed in the study, except the glutathione S-transferase assay kit, were procured from the Nanjing Jiancheng Bioengineering Institute in the Jiangsu Province of China, and the glutathione S-transferase assay kit was procured from Beijing Solarbio Bio-Technology Co., Ltd., Shanghai, China.

2.6. Data Analysis

The experimental data were summarized and analyzed using Excel 2019 software. One-way analysis of variance (ANOVA) was performed on the experimental data to identify significant differences among treatments by using SPSS 23.0 (SPSS Inc., Chicago, IL, USA), and the independent samples t-test was used for determining the significance levels between treatments at a probability level of 0.05 (i.e., p < 0.05).

3. Results

3.1. Impact of eCO₂ on the Nutritional Components and Defensive Substances of Pea Seedlings

One-way ANOVA showed that CO₂ level significantly affected the contents of nutritional components in pea seedlings, including soluble sugar (F_(1,8) = 3.958, p = 0.017), soluble protein (F_(1,8) = 2.971, p = 0.041) and total amino acid (F_(1,8) = 3.064, p = 0.038) in pea seedlings (

Table 1. The contents of nutritional components and defensive substances in pea seedlings grown under ambient CO₂ (aCO₂) and elevated CO₂ (eCO₂).

Measured Indexes		aCO2	eCO2	One-Way ANOVA (F/p Values)
Nutritional components	Soluble sugar (mg/g)	115.97 ± 9.92 a	165.28 ± 7.53 b	F (1,8) = 3.958/ p = 0.017 *
	Soluble protein (mg/g)	64.21 ± 13.3 a	113.69 ± 10.02 b	F (1,8) = 2.971/ p = 0.041 *
	Total amino acid (mg/g)	0.08 ± 0.01 a	0.13 ± 0.02 b	F (1,8) = 3.064/ p = 0.038 *
	Free fatty acid (μmol/mg)	0.55 ± 0.07 a	0.52 ± 0.01 a	F (1,8) = 0.541/ p = 0.642
Defensive substances	Total phenols (μmol/g)	7.33 ± 0.46 a	8.84 ± 0.24 b	F (1,8) = 2.920/ p = 0.027 *
	Saponin (μg/mg)	7.13 ± 0.24 a	8.09 ± 0.42 a	F (1,8) = 1.984/ p = 0.095
	Ethylene (ng/g)	1.91 ± 0.22 a	3.28 ± 0.46 b	F (1,8) = 2.688/ p = 0.036 *
	Salicylic acid (pmol/g)	5.67 ± 0.51 a	6.27 ± 0.73 a	F (1,8) = 0.632/ p = 0.547
	Jasmonic acid (pmol/g)	5.08 ± 0.35 a	6.92 ± 0.63 b	F (1,8) = 2.551/ p = 0.043 *

Note: Different lowercase letters indicate a significant difference between the treatments of aCO₂ and eCO₂ by the independent samples t-test at p < 0.05. * Indicates being significantly different at p < 0.05 for one-way ANOVA, the same as the following tables.

), but did not significantly impact the free fatty acid content (F_(1,8) = 0.541, p = 0.642; Table 1). Compared to aCO₂, eCO₂ significantly increased the contents of soluble sugar by 42.52%, soluble protein by 77.06% and total amino acid by 62.50% (p < 0.05), except that there was a slight decrease in free fatty acid content in pea seedlings grown under eCO₂, in contrast to aCO₂ (p > 0.05; Table 1). It also indicated that CO₂ levels significantly affected the contents of total phenol (F_(1,8) = 2.920, p = 0.027), ethylene content (F_(1,8) = 2.688, p = 0.036) and jasmonic acid (F_(1,8) = 2.551, p = 0.043) but had no significant impact on the contents of saponin (F_(1,8) = 1.984, p = 0.095) and salicylic acid (F_(1,8) = 0.632, p = 0.547) in pea seedlings (Table 1). Compared to aCO₂, eCO₂ significantly increased the contents of total phenol by 20.60%, ethylene by 71.72% and jasmonic acid by 36.22% (p < 0.05; Table 1). The contents of saponin and salicylic acid were also increased as they grew under eCO₂, in contrast to aCO₂, but the changes were not significant (p > 0.05; Table 1).

3.2. Impact of eCO₂ on the Growth, Development and Reproduction of L. Sticticalis

One-way ANOVA revealed that CO₂ levels significantly affected the duration of egg (F_(1,178) = 2.420, p = 0.020), larvae (F_(1,98) = 6.782, p < 0.001), pupae (F_(1,98) = 4.707, p < 0.001) and adult longevity (F_(1,78) = 2.723, p = 0.008), but did not significantly affect the number of eggs laid per female (F_(1,38) = 0.671, p = 0.512) or pupal weight (F_(1,98) = 0.287, p = 0.755) of L. sticticalis fed on pea seedlings grown under aCO₂ and eCO₂ (

Table 2. One-way ANOVAs for the effects of CO₂ level (aCO₂ vs. eCO₂) on the growth, development and reproduction of L. sticticalis, and the contents of nutritional components and enzyme activity in L. sticticalis larvae fed on pea seedlings grown under aCO₂ and eCO₂.

Measured Indexes		F Value	p Value
Duration (days)	Egg	F (1, 17 8 ) = 2.420	p = 0.020 *
	Larva	F (1,98) = 6.782	p < 0.001 ***
	Pupa	F (1,98) = 4.707	p < 0.001 ***
Adult longevity (days)		F (1,78) = 2.723	p = 0.008 **
No. of eggs laid per female		F (1,38) = 0.671	p = 0.512
Pupal weight (mg)		F (1,98) = 0.287	p = 0.755

Note: * p < 0.05, ** p < 0.01, *** p < 0.001.

). The duration of egg (aCO₂: 4.63 d; eCO₂: 3.86 d), larva (aCO₂: 25.70 d; eCO₂: 22.96 d) and pupae (aCO₂: 17.68 d; eCO₂: 15.89 d) were significantly shortened by 16.63% (p < 0.05), 10.66% (p < 0.001) and 10.12% (p < 0.001), respectively, as they fed on pea seedlings grown under eCO₂ in contrast to aCO₂ (Figure 1a–c). Moreover, the adult longevity under eCO₂ (22.42 d) was significantly prolonged by 19.07% than under aCO₂ (18.83 d; p < 0.01, Figure 1d). The number of eggs laid per female under eCO₂ slightly increased than under aCO₂, while the pupal weight slightly decreased under eCO₂ than under aCO₂ (p > 0.05; Figure 1e,f).

3.3. Impact of eCO₂ on the Nutritional Composition and Enzyme Activity in L. sticticalis Larvae

One-way ANOVA showed that the CO₂ level significantly affected the contents of soluble sugar (F_(1,8) = 7.441, p = 0.002), soluble protein (F_(1,8) = 5.036, p = 0.007), total amino acid (F_(1,8) = 11.007, p < 0.001) and free fatty acid (F_(1,8) = 2.894, p = 0.046) in L. sticticalis larvae fed on pea seedlings grown under aCO₂ and eCO₂ (

Table 3. The contents of nutritional components and enzyme activity in L. sticticalis fed on pea seedlings grown under aCO₂ and eCO₂.

Measured Indexes		aCO2	eCO2	One-Way ANOVA (F/p Values)
Nutritional components	Soluble sugar (mg/g)	120.19 ± 5.85 a	192.33 ± 7.73 b	F (1,8) = 7.441 / p = 0.002 **
	Soluble protein (mg/g)	207.54 ± 27.44 a	351.15 ± 7.75 b	F (1,8) = 5.036 / p = 0.007 **
	Total amino acid (mg/g)	0.12 ± 0.00 a	0.23 ± 0.01 b	F (1,8) = 11.007 / p < 0.001 ***
	Free fatty acid (μmol/mg)	4.34 ± 0.23 a	3.16 ± 0.35 b	F (1,8) = 2.894 / p = 0.046 *
Enzyme activity (U/mg)	Superoxide dismutase (SOD)	1.67 ± 0.02 a	2.02 ± 0.21 a	F (1,8) = 1.657 / p = 0.238
	Catalase (CAT)	327.31 ± 50.98 a	512.9 ± 16.62 b	F (1,8) = 3.461 / p = 0.026 *
	Peroxidase (POD)	102.48 ± 13.29 a	167.95 ± 5.32 b	F (1,8) = 4.573 / p = 0.010 *
	Carboxylesterase (CarE)	85.86 ± 12.29 a	195.83 ± 35.52 b	F (1,8) = 2.926 / p = 0.043 *
	Glutathione-s-transferase (GST)	15.43 ± 3.07 a	29.85 ± 0.69 b	F (1,8) = 4.581 / p = 0.037 *
	Acetylcholinesterase (AChE)	4.30 ± 0.28 a	4.40 ± 0.35 a	F (1,8) = 0.218 / p = 0.838

Note: * p < 0.05, ** p < 0.01, *** p < 0.001. Different lowercase letters indicate a significant difference between the treatments of aCO₂ and eCO₂ by the independent samples t-test at p < 0.05.

). Compared to aCO₂, eCO₂ significantly increased the contents of soluble sugar by 60.02%, soluble protein by 77.06% and total amino acid by 91.67%, and significantly decreased the free fatty acid content by 27.19% as L. sticticalis larvae fed on pea seedlings (p < 0.05; Table 3). It also indicated that the CO₂ level significantly affected the activity of catalase (CAT) (F_(1,8) = 3.461, p = 0.026), peroxidase (POD) (F_(1,8) = 4.573, p = 0.010), carboxylesterase (CarE) (F_(1,8) = 2.926, p = 0.043) and glutathione S-transferase (GST)(F_(1,8) = 4.581, p = 0.037), but did not significantly influence the activity of superoxide dismutase (SOD) (F_(1,8) = 1.657, p = 0.238) and acetylcholinesterase (AChE) (F_(1,8) = 0.218, p = 0.838) in L. sticticalis larvae fed on pea seedlings grown under aCO₂ and eCO₂, respectively (Table 3). Compared to aCO₂, eCO₂ significantly increased the activity of CAT by 56.70%, POD by 63.89%, CarE by 128.08% and GST by 93.45%, respectively (p < 0.05; Table 3). Moreover, the activity of SOD and AChE also slightly increased as L. sticticalis larvae fed on pea seedlings grown under eCO₂ in contrast to aCO₂, respectively, but the changes were not significant (p > 0.05; Table 3).

4. Discussion and Conclusions

The impact of increased atmospheric CO₂ concentration in the environment on plants primarily manifests as changes in their chemical composition [27]. Previous studies have consistently demonstrated that eCO₂ leads to a higher C:N ratio in plants, which subsequently alters the nutrient and chemical composition within plant tissues [28,29]. In this study, eCO₂ significantly augmented the contents of soluble sugar, soluble protein and total amino acid, while simultaneously leading to a slight reduction in the free fatty acid content in pea seedlings compared with aCO₂. This can be attributed to the enhanced photosynthetic and nitrogen metabolism under eCO₂. The increased carbon dioxide availability stimulates photosynthesis for greater carbohydrate accumulation and enables more efficient utilization and storage of nitrogenous compounds due to altered nitrogen metabolism. The slight decrease in free fatty acid content might be a consequence of the redirection of carbon skeletons towards the synthesis of sugars and proteins, which are energetically and functionally more crucial under the new metabolic regime. These findings are consistent with previous research findings, which demonstrated that eCO₂ markedly increased the content of soluble sugars, soluble proteins and total amino acids in kidney bean (P. vulgaris) [6] while simultaneously decreasing the free fatty acid content in tea seedlings [7].

Secondary metabolites, such as phenols and saponins, form a complex defense system in plants to resist harmful organisms (e.g., insect pests) during evolution. The strong antioxidant capacity of total phenols can reduce oxidative stress induced by the damage of insect pests, thereby enhancing the pest resistance of plants [30]. In our study, eCO₂ significantly elevated the total phenol content in pea seedlings, corroborating the conclusions of Ryan et al. [31]. In their comprehensive review of 343 studies conducted in 2010, it was indicated that over half of the studies reported an increase in total phenol content in response to eCO₂. Saponins, a class of secondary metabolites widely found in legumes, were also found to increase in broad bean leaves under eCO₂ in contrast to aCO₂, as observed by Li [32]. Our study also found that eCO₂ significantly increased the saponin content in pea seedlings compared with aCO₂. Ethylene, salicylic acid (SA) and jasmonic acid (JA) are three key plant hormones involved in regulating plant defense mechanisms. Many documents have shown that eCO₂ can influence the growth and development of herbivorous insects by altering the plant hormone-mediated induced pest-resistance response mechanisms [33]. For instance, Sun et al. [15] found that eCO₂ enhanced the salicylic acid-induced defense pathway in Arabidopsis (A. thaliana) while reducing the JA-induced defense pathway, thereby affecting the expression of downstream resistance genes and promoting aphid population outbreaks. In this study, eCO₂ significantly increased the contents of ethylene and JA in pea seedlings, with SA content also slightly increased compared with aCO₂. The divergence from Sun et al. [15] might result from the metabolic pathway differences between legumes and cruciferous plants under eCO₂. Guo et al. [34] found that in Medicago truncatula, the jasmonate pathway is involved in promoting nodule symbiosis while suppressing host plant defense, which highlights the specialized and unique nature of hormone regulation in legumes. Considering these differences in hormonal mechanisms compared to other plant families like cruciferous plants, it is likely that legumes have their own characteristic hormone synthesis and signaling regulatory mechanisms, which result in their disparate responses when exposed to elevated CO₂ levels.

Regarding the role of these compounds, the increased soluble sugars, proteins and amino acids in pea seedlings grown under eCO₂ offer more resources for L. sticticalis larvae. They fuel the energy-consuming processes of growth and metamorphosis, but a rushed development might cause some larvae to miss out on full utilization. For the defensive compounds, higher total phenols and saponins in pea seedlings grown under eCO₂ force L. sticticalis larvae to allocate resources to counteract the stress and maintain gut integrity, which can disrupt normal development rhythms.

Changes in biochemical substances in plant tissues can affect the nutritional quality of food for herbivorous insects, which in turn influences the growth, development, reproduction, nutrition content and enzyme activity in the bodies of these insect pests [18,21,33,35,36]. Pang [37] found that prolonged exposure to high CO₂ concentrations accelerated the population growth of cotton bollworms and significantly shortened the mean generation time. In this study, eCO₂ significantly shortened the duration of eggs, larvae and pupae of L. sticticalis while slightly reducing the pupal weight. The reduction in pupal weight, consistent with Li et al. [19], though not statistically significant, may result from a trade-off between rapid development and resource allocation. Under eCO₂, the larvae might prioritize energy expenditure toward accelerated growth and metamorphosis rather than towards building substantial pupal mass. This could be due to the fact that the enhanced nutrient availability in pea seedlings grown under eCO₂ triggers a physiological response in L. sticticalis larvae to hasten their development, with the consequence that the resources allocated to pupal formation are relatively limited. The contents of soluble sugars, soluble proteins and total amino acids in L. sticticalis larvae were significantly increased as they fed on pea seedlings grown under eCO₂ in contrast to aCO₂. This indicates that eCO₂ not only increased the nutrient content in pea seedlings but also enhanced the uptake of these essential nutrients by L. sticticalis larvae, thereby accelerating its generational turnover. However, it is speculated that the shortened developmental period may lead to reduced growth resources for some individuals during the larval stage, potentially affecting their development quality. In this study, eCO₂ significantly prolonged adult longevity. This could be mainly due to the enhanced nutritional quality of pea seedlings grown under eCO₂. The increased levels of soluble sugars, proteins and amino acids in the seedlings provided adults with a more stable and sufficient energy and nutrient supply, better maintaining their physiological functions and potentially enhancing stress resistance, thus contributing to the extended lifespan. While the observed trends in adult longevity might have certain implications, it is premature to conclude that eCO₂ may positively influence the dispersal and reproduction of L. sticticalis and potentially exacerbate their threat to agricultural ecosystems, as other factors in the field environment, such as natural predators, parasites and abiotic stresses, which were not considered in this study, could have a significant impact on the population dynamics of L. sticticalis and might mitigate any potential increase in their threat. Although the increase in the number of eggs laid per female under eCO₂ was not statistically significant, it could potentially be related to the improved nutritional quality of pea seedlings. The elevated levels of soluble sugars, proteins and amino acids in the seedlings might provide a more favorable nutritional environment for the female insects. This could enhance their physiological condition and reproductive capacity to some extent, leading to a marginal increase in egg production. However, the complex interplay of various factors in the ecosystem, such as the potential changes in plant defense mechanisms and the insects’ own physiological regulatory systems, may limit the magnitude of this effect and prevent it from reaching statistical significance.

Enzymes, such as CarE, GST and AchE, are important detoxification enzymes in insects, playing a crucial role in breaking down exogenous toxins and maintaining normal physiological metabolism [38]. Studies have demonstrated that CarE plays a critical role in the metabolism of plant secondary metabolites in various insects [39]. In this particular study, it could be pivotal in handling the altered chemical composition of pea seedlings under eCO₂. For GST, its activity is often associated with the detoxification of xenobiotics and maintaining redox homeostasis [40]. The increased production of reactive oxygen species (ROS) in L. sticticalis might lead to a compensatory increase in GST activity to prevent oxidative damage under eCO₂. SOD, CAT and POD are vital protective enzymes in insects. SOD promotes the generation of O₂, removes excess superoxide anion radicals in the body and ultimately produces H₂O₂, which CAT and POD can decompose into H₂O [41]. Therefore, detoxification and protective enzymes are of great significance to the growth and development of herbivorous insects. Particularly, SOD, as the foremost defense against oxidative stress, plays a crucial role in scavenging superoxide anions under eCO₂. [42]. The upregulation of SOD activity under eCO₂ could be a response to the perturbed intracellular environment. Subsequently, CAT and POD will work in coordination to further break down the H₂O₂ produced by SOD [43]. The coordinated action of these enzymes is essential for the proper development and survival of L. sticticalis larvae. Research shows that under high CO₂ conditions, the activities of detoxification enzymes (GST, CarE) and protective enzymes (SOD, CAT, POD) in Frankliniella occidentalis and F. intonsa generally trend upwards [44]. In this study, the activities of protective enzymes (CAT, POD) and detoxification enzymes (CarE, GST) in L. sticticalis larvae significantly increased as they fed on pea seedlings grown under eCO₂ in contrast to aCO₂, and the activities of SOD and AchE in L. sticticalis larvae also showed a slight rise as they fed on pea seedlings grown under eCO₂ in contrast to aCO₂, which is consistent with previous research findings. Biologically, the enhanced enzyme activities in L. sticticalis larvae might improve their survival and growth by facilitating the synthesis of metabolic compounds in pea seedlings grown under eCO₂. At the organism level, it could affect the larvae’s physiological state, development and potential reproductive capacity. In terms of population, the increased enzyme activities may reduce the effectiveness of traditional pest control and require new strategies to manage L. sticticalis, as its population growth might be enhanced under eCO₂, posing a greater threat to agricultural ecosystems.

The results of this study indicate that eCO₂ can improve the nutritional quality of pea seedlings, promoting the growth and development of L. sticticalis. However, in terms of reproductive performance, although there were some changes in the number of eggs laid per female and pupal weight under eCO₂ compared to aCO₂, these differences were not statistically significant, suggesting that the impact of eCO₂ on the reproduction of L. sticticalis may be limited or requires further investigation. Moreover, the changes in the contents of defensive substances in pea seedlings further stimulated the changes in enzyme activity in L. sticticalis larvae, altering its environmental adaptability. Our study provides a comprehensive understanding of the impact of eCO₂ on the interaction between pea plants and L. sticticalis, which adds to the growing body of research on the ecological consequences of climate change. Also, we combined the changes in the contents of nutrient components and defensive substances in pea seedlings with the changes in growth, development, and reproduction of L. sticticalis, as well as changes in the nutritional substances and enzyme activities in its body (seen in Figure 2), to predict that the changes in plant nutrients and defensive compounds will shorten the developmental period of L. sticticalis, increase its generational output and improve its adaptability to environmental changes, thereby increasing the risk of L. sticticalis outbreaks and their migration and dispersal under the continuous rise in atmospheric CO₂ concentrations in the future. Nevertheless, it is essential to acknowledge the limitations of our current study. We have only considered the singular impact of eCO₂ on pea seedlings and meadow moths, without accounting for the interactions with other environmental factors (e.g., temperature, humidity, light, etc.). These interactions could potentially modify or even reverse the observed effects, thereby restricting the generalizability of our findings. Future research should strive to incorporate a more comprehensive environmental context to better understand the complex ecological dynamics. Additionally, our study has not delved into the intricate molecular mechanisms underlying the changes in defensive substances in pea seedlings. Future investigations could leverage molecular biology techniques to dissect the effects of eCO₂ on plant-defensive signaling pathways. This would not only deepen our understanding of the fundamental processes but also provide valuable insights for the development of more effective pest management strategies in the context of climate change. Future research should incorporate molecular biology approaches to explore the effects of eCO₂ on various plant-defensive signaling pathways.