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The effects of lead (Pb) and pest damage on soil enzyme activities, pakchoi and Spodoptera litura performance

Published online by Cambridge University Press:  19 September 2024

Huiyang Liu ,
Yimeng Shi ,
Yuxuan Zou ,
Zaiya Song ,
Huai Tian ,
Xianjun Yang  and
Xiaohong Li Open the ORCID record for Xiaohong Li [Opens in a new window]
Huiyang Liu
Affiliation:
College of Agriculture and Forestry Ecology, Shaoyang University, Shaoyang, China
Yimeng Shi
Affiliation:
College of Food and Chemical Engineering, Shaoyang University, Shaoyang, China
Yuxuan Zou
Affiliation:
College of Agriculture and Forestry Ecology, Shaoyang University, Shaoyang, China
Zaiya Song
Affiliation:
College of Agriculture and Forestry Ecology, Shaoyang University, Shaoyang, China
Huai Tian
Affiliation:
College of Agriculture and Forestry Ecology, Shaoyang University, Shaoyang, China
Xianjun Yang
Affiliation:
College of Agriculture and Forestry Ecology, Shaoyang University, Shaoyang, China
Xiaohong Li*
Affiliation:
College of Agriculture and Forestry Ecology, Shaoyang University, Shaoyang, China College of Food and Chemical Engineering, Shaoyang University, Shaoyang, China
*
Corresponding author: Xiaohong Li; Email: xiaohongli86@126.com
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Abstract

Plant–soil interactions have bottom–up and top–down effects within a plant community. Heavy metal pollution can change plant–soil interactions, directly influence bottom–up effects and indirectly affect herbivores within the community. In turn, herbivores can affect plant–soil interactions through top–down effects. However, the combined effects of heavy metals and herbivores on soil enzymes, plants and herbivores have rarely been reported. Therefore, the effects of lead (Pb), Spodoptera litura and their combined effects on soil enzyme activities, pakchoi nutrition, defence compounds and S. litura fitness were examined here. Results showed that Pb, S. litura and their combined effects significantly affected soil enzymes, pakchoi and S. litura. Specifically, exposure to double stress (Pb and S. litura) decreased soil urease, phosphatase and sucrase activities compared with controls. Furthermore, the soluble protein and sugar contents of pakchoi decreased, and the trypsin inhibitor content and antioxidant enzyme activity increased. Finally, the S. litura development period was extended, and survival, emergence rates and body weight decreased after exposure to double stress. The combined stress of Pb and S. litura significantly decreased soil enzyme activities. Heavy metal accumulation in plants may create a superposition or synergistic effect with heavy metal-mediated plant chemical defence, further suppressing herbivore development. Pb, S. litura and their combined effects inhibited soil enzyme activities, improved pakchoi resistance and reduced S. litura development. The results reveal details of soil–plant–herbivore interactions and provide a reference for crop pest control management in the presence of heavy metal pollution.

Keywords

bottom–up effectsheavy metalsplant–herbivore interactionsplant–soil interactionstop–down effects
Type
Research Paper
Information
Bulletin of Entomological Research , Volume 114 , Issue 4 , August 2024 , pp. 473 - 481
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Copyright
Copyright © The Author(s), 2024. Published by Cambridge University Press

Introduction

Soil provides moisture and nutrients to plants, which can alter the growth and nutritional quality of plant shoot systems (Wang et al., Reference Wang, Ruan, Kostenko, Carvalho, Hannula, Mulder, Bu, van der Putten and Bezemer2019; Han et al., Reference Han, Lavoir, Rodriguez-Saona and Desneux2022). These changes of plant shoot systems can subsequently affect the performance of foliar herbivores, thereby influencing the ecological dynamics via bottom–up effects (Pineda et al., Reference Pineda, Zheng, van Loon, Pieterse and Dicke2010; Wang et al., Reference Wang, Ruan, Kostenko, Carvalho, Hannula, Mulder, Bu, van der Putten and Bezemer2019; Han et al., Reference Han, Lavoir, Rodriguez-Saona and Desneux2022). Plants produce vegetative litter and rhizospheric secretions that alter the physicochemical properties and microbial communities of the soil (Orwin et al., Reference Orwin, Buckland, Johnson, Turner, Smart, Oakley and Bardgett2010; Cantarel et al., Reference Cantarel, Pommier, Desclos-Theveniau, Diquélou, Dumont, Grassein, Kastl, Grigulis, Laîné, Lavorel, Lemauviel-Lavenant, Personeni, Schloter and Poly2015; Carrillo et al., Reference Carrillo, Ingwell, Li and Kaplan2019; Huberty et al., Reference Huberty, Choi, Heinen and Bezemer2020, Reference Huberty, Steinauer, Heinen, Jongen, Hannula, Choi and Bezemer2022). Furthermore, herbivorous insects affect the concentrations of primary and secondary plant root tissue compounds, which can directly affect interactions between the roots and soil organisms; therefore, top–down effects, such as plant–herbivore interactions, can impact plant–soil interactions (Martijn et al., Reference Martijn Bezemer, van der Putten, Martens, van de Voorde, Mulder and Kostenko2013; Huberty et al., Reference Huberty, Choi, Heinen and Bezemer2020; Han et al., Reference Han, Lavoir, Rodriguez-Saona and Desneux2022).

Heavy metal pollution seriously impacts global agricultural ecosystems (Abdu et al., Reference Abdu, Abdullahi and Abdulkadir2017; Lin et al., Reference Lin, Wang, Liu and Dong2022a). Soil enzymes play a decisive role in metabolic processes and the overall balance of soil (Nurzhan et al., Reference Nurzhan, Tian, Nuralykyzy and He2022; Tan et al., Reference Tan, He, Guo, Wang, Nie, Ye, He and Shen2023). Heavy metals can influence soil enzyme activities, although the degree of influence depends on different factors, such as heavy metal type and concentration, and soil enzyme type (Gao et al., Reference Gao, Zhou, Mao, Zhi and Shi2010; Aponte et al., Reference Aponte, Meli, Butler, Paolini, Matus, Merino, Cornejo and Kuzyakov2020; Tang et al., Reference Tang, Xu, Song, Ge and Yue2022). Herbivore feeding can also affect the production of plant root secretions and indirectly affect soil enzyme activity (Classen et al., Reference Classen, DeMarco, Hart, Whitham, Cobb and Koch2006; Huang et al., Reference Huang, Liu, Chen, Chen, Chen, Li and Hu2013; Song et al., Reference Song, Lee, Hong, Choi, Hong, Bae, Mysore, Park and Ryu2015; Hoysted et al., Reference Hoysted, Bell, Lilley and Urwin2018; Long et al., Reference Long, Liu and Li2022).

Following damage by herbivores, a plant may respond by exhibiting induced resistance through long-distance signal conduction and integration (Karban, Reference Karban2011; Li, Reference Li2016). The formation of herbivore-induced compounds requires certain nutrients and energy, which ultimately reduces the plant's nutrient content (Karban and Baldwin, Reference Karban and Baldwin1997; Zavala et al., Reference Zavala, Patankar, Gase and Baldwin2004; Dicke, Reference Dicke2015; Li, Reference Li2016). Herbivore-induced plant compounds include defence proteins and toxic secondary metabolites (Paudel et al., Reference Paudel, Lin, Foolad, Ali, Rajotte and Felton2019; Li et al., Reference Li, Yang, Huang and Wu2021). These compounds destroy digestion and the absorption system of herbivorous insects, subsequently interfering with nutrient absorption and utilisation by insects (Li, Reference Li2016; Zhao et al., Reference Zhao, Li, Leng, Wang and Li2019; Li et al., Reference Li, Yang, Huang and Wu2021). This is detrimental to the developmental performance of insects and may result in their death (Karban and Baldwin, Reference Karban and Baldwin1997; Paudel et al., Reference Paudel, Lin, Foolad, Ali, Rajotte and Felton2019; Zhao et al., Reference Zhao, Li, Leng, Wang and Li2019; Li et al., Reference Li, Yang, Huang and Wu2021). For example, when Helicoverpa zea consumes tomatoes, the chemical content of the leaf increases together with an increase in the trypsin protease inhibitor (TPI) content (Paudel et al., Reference Paudel, Lin, Foolad, Ali, Rajotte and Felton2019). TPI can inhibit the digestion, absorption and utilisation of food proteins by insects (Bhattacharyya et al., Reference Bhattacharyya, Leighton and Babu2007; Li, Reference Li2016; Zhao et al., Reference Zhao, Li, Leng, Wang and Li2019). Herbivore-induced plant chemical defences significantly alter plant sugars and nitrogenous compounds (Watanabe and Kitagawa, Reference Watanabe and Kitagawa2000; Li et al., Reference Li, Fang, Lv and Zhang2008; Long et al., Reference Long, Liu and Li2022); therefore, plant-induced resistance inhibits herbivorous insects from acquiring nutrients from plants (Hu, Reference Hu2004; Li et al., Reference Li, Fang, Lv and Zhang2008; Li, Reference Li2016).

Heavy metals are transferred and enriched through the soil–plant–herbivorous insect food chain and affect the plant nutrient content by activating plant antioxidant enzyme systems and causing plant resistance (Jiang et al., Reference Jiang, Wang, Dong and Yan2018, Reference Jiang, Wang and Yan2020; Sang et al., Reference Sang, Xu, Bashir and Ali2018; Tibbett et al., Reference Tibbett, Green, Rate, De Oliveira and Whitaker2021). The antioxidant enzyme system in plants protects them from heavy metal damage (Bhaduri and Fulekar, Reference Bhaduri and Fulekar2012; Sidhu et al., Reference Sidhu, Bali, Bhardwaj, Singh, Batish and Kohli2018). For example, the nutrient content of Chenopodium murale decreases when the lead (Pb) content is excessively high, whereas the activities of superoxide dismutase (SOD), polyphenol oxidase (POD), and catalase (CAT) significantly increase (Sidhu et al., Reference Sidhu, Bali, Bhardwaj, Singh, Batish and Kohli2018).

Heavy metals trigger bottom–up effects and affect pest populations (Butler and Trumble, Reference Butler and Trumble2008; Han et al., Reference Han, Lavoir, Rodriguez-Saona and Desneux2022). Therefore, herbivores feeding on heavy metal-stressed plants are not only subject to the direct toxic effects of the heavy metal(s) (elemental defence) but also to a possible heavy metal-mediated plant defence response (chemical defence) (Boyd, Reference Boyd2012; Sahu et al., Reference Sahu, Dutta, Ray, Pradhan and Dandapat2018; Chen et al., Reference Chen, Wang and Shu2020; Jiang et al., Reference Jiang, Wang and Yan2020; Yactayo-Chang et al., Reference Yactayo-Chang, Tang, Mendoza, Christensen and Block2020; Lin et al., Reference Lin, Zhu, He, Xie, Li, Han, Li, Yang, Liu and Zhu2022b). Insect larvae usually show adverse effects, such as weight loss; they undergo a prolonged development period and have a decreased emergence rate (Chen et al., Reference Chen, Wang and Shu2020; Jiang et al., Reference Jiang, Tan, Guo and Yan2021; Lin et al., Reference Lin, Zhu, He, Xie, Li, Han, Li, Yang, Liu and Zhu2022b). Heavy metal accumulation in plants and the heavy-metal-mediated plant defence response have a superimposed or synergistic effect that inhibits insect growth and development (Boyd, Reference Boyd2012; Cheruiyot et al., Reference Cheruiyot, Boyd and Moar2015; Sahu et al., Reference Sahu, Dutta, Ray, Pradhan and Dandapat2018; Chen et al., Reference Chen, Wang and Shu2020; Jiang et al., Reference Jiang, Wang and Yan2020; Yactayo-Chang et al., Reference Yactayo-Chang, Tang, Mendoza, Christensen and Block2020; Lin et al., Reference Lin, Zhu, He, Xie, Li, Han, Li, Yang, Liu and Zhu2022b). Heavy-metal-driven plant defence mechanisms are inconsistent with herbivore-induced defence mechanisms; however, their effects on herbivores are similar when inducing plant defence expression (Sidhu et al., Reference Sidhu, Bali, Bhardwaj, Singh, Batish and Kohli2018; Zhang et al., Reference Zhang, Zhou and Wang2022). Therefore, these two mechanisms simultaneously regulate plant defences against herbivores (Huang et al., Reference Huang, Chen, Huang and Yu2021).

Plant–soil interactions have bottom–up and top–down effects (Huberty et al., Reference Huberty, Choi, Heinen and Bezemer2020, Reference Huberty, Steinauer, Heinen, Jongen, Hannula, Choi and Bezemer2022; Han et al., Reference Han, Lavoir, Rodriguez-Saona and Desneux2022). For example, high concentrations of cadmium (Cd) or copper (Cu) in contaminated soil enhance maize resistance to Spodoptera frugiperda and significantly prolong its larval development period (Winter et al., Reference Winter, Borkowski, Zeier and Rostás2012). Soil urease and phosphatase activities decrease following soybean consumption by S. litura (Long et al., Reference Long, Liu and Li2022). Several studies have shown that heavy metal and herbivore damage can induce bottom–up and top–down effects, respectively; however, the combined effects of heavy metals and herbivores on soil, plants and herbivorous insects have rarely been studied.

This study investigated the bottom–up effects triggered by Pb, top–down effects triggered by S. litura, and combined effects of Pb and S. litura on the soil enzyme activity and fitness of pakchoi and S. litura. The results reveal details of soil–plant–herbivore interactions and provide a reference for crop pest control management in the presence of heavy metal pollution.

Materials and methods

Soil and plants

The soils used in this study were collected at a depth of 0–20 cm in a fallow field within a suburb of Shaoyang City, China (26.9°N, 111.3°E). The soil was of medium fertility, with a pH of 6.13 and a Pb content of 4.02 mg kg−1. The total nitrogen, total phosphorus, total potassium and organic matter contents were 1.25, 0.52, 21.08 and 20.23 g kg−1, respectively, and the alkali-hydrolytic nitrogen, available phosphorus and available potassium contents were 113.81, 6.05 and 155.28 mg kg−1, respectively. The soils were air-dried and filtered through a 20-mesh sieve. Pb(NO3)2 was then dissolved in tap water and sprayed evenly on the soil at a concentration of 400 mg kg−1 (dry weight: Pb[NO3]2/soil). The Pb-treated soil was aged for 15 days. Control soil was treated with tap water and aged for 15 days.

Pakchoi (‘Shouhe’) seeds were sown in both treated and controlled soils. Following germination, robust seedlings were selected for transplantation into plastic pots (diameter: 20 cm, height: 18 cm) filled with either treated or control soils. Each pot was planted with one seedling. Pakchoi seedlings were planted in sequential batches every 5 days from 10 May to 20 June 2022. To ensure an adequate number of plants, each treatment batch was grown in 24 pots. Pots were covered with gauze netting to prevent infestation by other organisms. The plants were maintained in the field and were used in the experiments after they reached the six-leaf stage.

Herbivores

In 2021, S. litura individuals were collected from tobacco fields in the suburbs of Shaoyang City. Larvae were reared on artificial diets in incubators at 26 ± 1 °C, 60 ± 10% relative humidity and with a 14:10 (light: dark) photoperiod. Adult moths were supplied with 10% honey solution on a cotton ball, which was refreshed daily.

Experimental setup

Four treatments were set up in this experiment as follows: pakchoi plants planted in control soil, without any treatment (CK group); pakchoi plants planted in Pb-treated soil (Pb stress group); pakchoi plants planted in control soil with three 2nd instar S. litura larvae placed on each leaf (the 2nd‒5th leaf from the bottom of the pakchoi plant). When approximately 1/3rd of the leaf area was consumed, the larvae were removed from the plants (herbivore treatment group). In addition, pakchoi plants were planted in Pb-treated soil and three 2nd instar S. litura larvae were placed on each leaf (the 2nd‒5th leaf from the bottom of the pakchoi plant). When approximately 1/3rd of the leaf area was consumed, the larvae were removed from the plants (double stress group, Pb + herbivore). Each pakchoi plant was used in experiments immediately after treatment.

Ten pakchoi plants were randomly selected from each treatment. Subsequently, 10 g (wet weight) of rhizosphere soil (0‒1 cm outside the pakchoi root system) was collected from each pakchoi plant and air-dried separately to measure phosphatase activity (disodium phenyl phosphate colorimetric method), urease (sodium phenate‒sodium hypochlorite colorimetric method) and sucrose (3,5-dinitrosalicylic acid colorimetric method) (n = 10 per treatment) (Guan, Reference Guan1986).

Tissue samples from the 3rd leaf (from the top) of each pakchoi plant (n = 10 per treatment) were flash-frozen in liquid nitrogen. The samples were then stored at −80 °C until further analysis. Soluble protein, soluble sugar, Pb and TPI contents were determined by Coomassie brilliant blue staining, the anthrone method, an atomic absorption spectrophotometer (AA-6880; Shimadzu) and the spectrophotometric method (DR6000; Hach), respectively (Bradford, Reference Bradford1976; Li, Reference Li2016; Zhang et al., Reference Zhang, Zong, Fang, Huang, Li, Xu, Wang and Liu2020; Winiarska-Mieczan et al., Reference Winiarska-Mieczan, Jachimowicz, Kwiecień, Krusiński, Kislova, Sowińska, Zasadna and Yanovych2023).

POD, SOD and CAT activities were determined using guaiacol, nitroblue tetrazolium photoreduction and potassium permanganate titration methods, respectively (Chance and Maehly, Reference Chance and Maehly1955; Gupta et al., Reference Gupta, Webb, Holaday and Allen1993).

Two neonate S. litura were raised in an insect-rearing box (diameter: 5 cm, height: 3 cm) containing moisturising filter paper and provided with fresh leaves excised from treated plants daily (CK group, Pb stress group, herbivore treatment group and double stress group, respectively). The neonate larvae in each treatment group were counted, and the pupated larvae were regarded as one repetition (n = 30 per treatment). The larval survival rate (30/total tested larvae per treatment), developmental period (from newly hatched larvae to pupae), emergence rate and adult dry weight (measured after adults had been dried in an 80 °C thermostatic drying box for 48 h) were recorded.

Data analysis

Data were checked for homogeneity of variance, and soluble sugar content and S. litura adult dry weight were logarithmically transformed. Two-way analysis of variance (ANOVA) was used to determine the effects of Pb stress and S. litura feeding on soil enzyme activities and the fitness of pakchoi and S. litura. A logistic generalised linear model was used for the analysis because the survival and emergence rates of S. litura were presented as binary data. Multiple comparisons were performed using Tukey's honestly significant difference test. All analyses were performed using R (R Development Core Team, 2022).

Results

Soil enzyme activities

Soil urease activity was significantly affected by Pb stress (F 1, 56 = 98.22, P < 0.05), herbivore treatment (F 1, 56 = 27.83, P < 0.05) and their combined effects (F 1, 56 = 4.24, P < 0.05). Compared with the controls, soil urease activity was the lowest in the double stress group (Pb + herbivore group, 47.86% decrease), followed by the Pb stress group (34.19% decrease) and then the herbivore treatment group (21.37% decrease). There was a significant difference among treatments (P < 0.05) (fig. 1a).

Figure 1. (a) Urease, (b) phosphatase and (c) sucrase activities of soil. Soil enzyme activity under Pb stress, herbivore stress (Spodoptera litura), Pb and herbivore double stress and the control (CK). Data are mean values ± standard error, and different lowercase letters indicate statistically significant differences as determined using Tukey's honestly significant difference.

Soil phosphatase activity was significantly affected by Pb stress (F 1, 56 = 578.65, P < 0.05), herbivore treatment (F 1, 56 = 78.21, P < 0.05) and their combined effects (F 1, 56 = 4.62, P < 0.05). Compared with the controls, soil phosphatase activity was the lowest in the double stress group (46.81% decrease), followed by the Pb stress group (31.21% decrease) and then the herbivore treatment group (9.57% decrease). There was a significant difference among treatments (P < 0.05) (fig. 1b).

Soil sucrase activity was significantly affected by Pb stress (F 1,56 = 151.72, P < 0.05), herbivore treatment (F 1, 56 = 68.12, P < 0.05) and their combined effects (F 1, 56 = 1.90, P < 0.05). Compared with the controls, soil sucrase activity was the lowest in the double stress group (36.93% decrease), followed by the Pb stress group (19.59% decrease) and then the herbivore treatment group (12.36% decrease). There were significant differences among treatments (P < 0.05) (fig. 1c).

Pakchoi analysis

The soluble protein content was significantly affected by Pb stress (F 1, 56 = 7.42, P < 0.05), herbivore treatment (F 1, 56 = 12.22, P < 0.05) and their combined effects (F 1, 56 = 5.24, P < 0.05). Compared with that in the controls, the soluble protein content was the lowest in the double stress group (45.05% decrease), followed by the herbivore treatment group (31.53% decrease) and then the Pb stress group (27.03% decrease). There was a significant difference between the double stress group and the other treatment groups (P < 0.05) (fig. 2a).

Figure 2. Leaf defensive chemicals and nutrient content in pakchoi plants under Pb stress, herbivore stress (Spodoptera litura), Pb and herbivore double stress and the control (CK). (a) Soluble protein content, (b) soluble sugar content, (c) trypsin protease inhibitor (TPI), (d) peroxidase (POD) activity, (e) superoxide dismutase (SOD) activity, (f) catalase (CAT) activity and (g) Pb content in pakchoi plants. Data are mean values ± standard error, and different lowercase letters indicate statistically significant differences as determined using Tukey's honestly significant difference.

The soluble sugar content was significantly affected by Pb stress (F 1, 56 = 31.53, P < 0.05), herbivore treatment (F 1, 56 = 27.25, P < 0.05) and their combined effects (F 1, 56 = 8.75, P < 0.05). Compared with that in the controls, the soluble sugar content was the lowest in the double stress group (41.46% decrease), followed by the Pb stress group (16.03% decrease) and then the herbivore treatment group (14.52% decrease). There was a significant difference between the double stress group and the other treatment groups (P < 0.05) (fig. 2b).

TPI content was significantly affected by Pb stress (F 1, 56 = 177.64, P < 0.05), herbivore treatment (F 1, 56 = 333.39, P < 0.05) and their combined effects (F 1, 56 = 21.23, P < 0.05). Compared with that in the controls, the TPI content was the highest in the double stress group (1.03-fold increase), followed by the herbivore treatment group (0.75-fold increase) and then the Pb stress group (0.59-fold increase). There were significant differences among treatments (P < 0.05) (fig. 2c).

POD activity was significantly affected by Pb stress (F 1, 56 = 146.14, P < 0.05), herbivore treatment (F 1, 56 = 109.22, P < 0.05) and their combined effects (F 1, 56 = 8.40, P < 0.05). Compared with that in the controls, POD activity was the highest in the double stress group (3.25-fold increase), followed by the Pb stress group (1.33-fold increase) and then the herbivore treatment group (1.09-fold increase). There was no significant difference between the Pb and herbivore stress groups (P > 0.05) (fig. 2d).

SOD activity was significantly affected by Pb stress (F 1, 56 = 111.28, P < 0.05), herbivore treatment (F 1, 56 = 61.98, P < 0.05) and their combined effects (F 1, 56 = 17.03, P < 0.05). Compared with that in the controls, SOD activity was the highest in the double stress group (0.88-fold increase), followed by the Pb stress group (0.31-fold increase) and then the herbivore treatment group (0.18-fold increase). There was no significant difference between the Pb and herbivore stress groups (P > 0.05) (fig. 2e).

CAT activity was significantly affected by Pb stress (F 1, 56 = 149.62, P < 0.05), herbivore treatment (F 1, 56 = 109.80, P < 0.05) and their interaction (F 1, 56 = 16.20, P < 0.05). Compared with that in the controls, CAT activity was the highest in the double stress group (1.73-fold increase), followed by the Pb stress group (0.63-fold increase) and then the herbivore treatment group (0.49-fold increase). There was no significant difference between the Pb and herbivore stress groups (P > 0.05) (fig. 2f).

The leaf Pb content was significantly affected by Pb stress (F 1, 56 = 479.24, P < 0.05) and herbivore treatment (F 1, 56 = 0.048, P < 0.05); however, there was no effect from their combined effects (F 1, 56 = 0.07, P > 0.05). The herbivore treatment group had the lowest leaf Pb content (5.6 × 10−3 ± 4.56 × 10−4 mg kg−1), and the double stress group had the highest leaf Pb content (0.37 ± 0.03 mg kg−1), with a significant difference (P < 0.05) (fig. 2g).

Herbivore performance

The S. litura survival rate was significantly affected by Pb stress (logistic ANOVA, χ 2 = 12.47, P < 0.05), herbivore treatment (logistic ANOVA, χ 2 = 9.42, P < 0.05) and their combined effects (logistic ANOVA, χ 2 = 1.84, P < 0.05). Compared with that in the controls, survival rates decreased by 33.7, 29.69 and 49.36% in the Pb, herbivore and double stress groups, respectively. There was no significant difference between the Pb and herbivore treatment groups (P > 0.05) (fig. 3a).

Figure 3. (a) Survival rate, (b) larval development period, (c) pupa eclosion rate and (d) adult body weight of Spodoptera litura larvae fed with pakchoi and under Pb stress, herbivore stress, Pb and herbivore double stress and the control (CK). Data are mean values ± standard error, and different lowercase letters indicate statistically significant differences as determined using Tukey's honestly significant difference.

The larval development time was significantly affected by Pb stress (F 1, 116 = 408.03, P < 0.05), herbivore treatment (F 1, 116 = 234.44, P < 0.05) and their combined effects (F 1, 116 = 22.64, P < 0.05). Compared with that in the controls, the larval development time was prolonged by 3.85, 2.63 and 8.86 days in the Pb, herbivore and double stress groups, respectively. There were significant differences among treatments (P < 0.05) (fig. 3b).

The S. litura emergence rate was significantly affected by Pb stress (logistic ANOVA, χ 2 = 8.22, P < 0.05), herbivore treatment (logistic ANOVA, χ 2 = 4.29, P < 0.05) and their combined effects (logistic ANOVA, χ 2 = 0.12, P < 0.05). Compared with that in the controls, the emergence rate decreased by 16.67, 10 and 40% in the Pb, herbivore and double stress groups, respectively. There was no significant difference between the Pb and herbivore treatment groups (P > 0.05) (fig. 3c).

The adult dry weight was significantly affected by Pb stress (F 1, 80 = 123.02, P < 0.05), herbivore treatment (F 1, 80 = 82.11, P < 0.05) and their combined effects (F 1, 80 = 4.65, P < 0.05). Compared with that in the controls, the adult dry weight decreased by 6.1, 5.27 and 14.26 mg in the Pb, herbivore and double stress groups, respectively. There was no significant difference between the Pb and herbivore treatment groups (P > 0.05) (fig. 3d).

Discussion

This study showed that Pb, S. litura and their combined effects significantly affected soil enzymes, pakchoi and S. litura. Compared with the control groups, the following was observed: soil urease, phosphatase and sucrase activities decreased; the soluble protein and soluble sugar content of pakchoi plants decreased; the TPI content and POD, SOD and CAT activities increased; and S. litura growth and development were inhibited. Pb adversely affected soil–plant–herbivore interactions from bottom to top. It was shown that S. litura feeding affected plant–soil interactions from top to down and induced plant resistance. The combined effects of Pb and S. litura increased the plant defence response.

Soil enzyme activity is an important index that reflects soil characteristics (Long et al., Reference Long, Liu and Li2022). Soil urease activity can directly reflect the soil nitrogen supply, and phosphatase activity affects soil organophosphorus decomposition and conversion (Huang et al., Reference Huang, Liu, Chen, Chen, Chen, Li and Hu2013; Long et al., Reference Long, Liu and Li2022). Sucrase activity can promote sugar hydrolysis and accelerate the soil carbon cycle (Jin et al., Reference Jin, Sleutel, Buchan, De Neve, Cai, Gabriels and Jin2009; Adetunji et al., Reference Adetunji, Lewu, Mulidzi and Ncube2017). Liang et al. (Reference Liang, Ding, Shen, Liu, Li and Xi2018) found that high Pb concentrations inhibited soil urease and phosphatase activities. Herbivory can directly or indirectly affect the composition and functioning of soil communities via changes in plant nutrients or defence compounds. For example, Classen et al. (Reference Classen, DeMarco, Hart, Whitham, Cobb and Koch2006) found that damage to Pinus edulis caused by Matsucoccus acalyptus significantly reduced soil enzyme activities. These findings that Pb or S. litura stress reduced soil enzyme activities agree with the results of this study. The present study also explored the influence of the combined effects of Pb and S. litura on enzyme activity. The results showed that soil enzyme activities were lower under double stress than under single stress, probably because the combined stress of Pb and S. litura exacerbated the changes in the soil environment and adversely affected enzyme activities related to the soil carbon, nitrogen and phosphorus cycles.

Herbivores feeding on plants produce herbivore-induced compounds that decrease the nutritional content of the plant, and these changes are detrimental to the growth and development of herbivorous insects and may lead to their death (Lou and Baldwin, Reference Lou and Baldwin2003; Sznajder and Harvey, Reference Sznajder and Harvey2003; Li, Reference Li2016; Li et al., Reference Li, Yang, Huang and Wu2021). Yin et al. (Reference Yin, Feng, Zhang, Li, Lin, Hu and Chen2012) found that the protein and glucose contents decreased in mustard, cabbage and kale after damage by Plutella xylostella. Moreover, the TPI content in soybean leaves increased following S. litura and Spodoptera exigua larval feeding; this was detrimental to the subsequent S. litura (Li, Reference Li2016). Dowd and Lagrimini (Reference Dowd and Lagrimini2006) found that Manduca sexta and Heliothis virescens larvae fed tobacco with a high POD content exhibited delayed growth and the associated harm to the plant was reduced. The present study showed that S. litura stress reduced the nutrient content of pakchoi leaves, increased defence levels and inhibited S. litura growth and development.

Soil-mediated plant quality changes can alter aboveground plant–insect interactions (Kos et al., Reference Kos, Tuijl, de Roo, Mulder and Bezemer2015). Heavy metals in soil are absorbed by plant roots and transported to different tissues through various transporter proteins (Maksymiec et al., Reference Maksymiec, Wianowska, Dawidowicz, Radkiewicz, Mardarowicz and Krupa2005; Lei et al., Reference Lei, Sun, Sun, Zhu, Li and Zheng2020). Excessively high heavy metal accumulation inhibits plant growth and development (Maksymiec et al., Reference Maksymiec, Wianowska, Dawidowicz, Radkiewicz, Mardarowicz and Krupa2005; Lei et al., Reference Lei, Sun, Sun, Zhu, Li and Zheng2020). Mao et al. (Reference Mao, Nan, Cao, Gao, Guo, Meng and Yang2018) found that the protein content of soybean leaves decreased with increasing mercury (Hg) concentrations; however, SOD, POD and CAT activities first increased and then decreased. Antioxidant enzyme activity is inhibited when heavy metal concentrations exceed the plant's capacity. Heavy metals enter directly into the food chain and are toxic to insects; however, they also enhance the defence response of plants (Winter et al., Reference Winter, Borkowski, Zeier and Rostás2012). Cd-treated plants were more resistant to herbivorous insects than Cd-treated artificial feeds, indicating that Cd accumulation may activate plant defence responses (Li et al., Reference Li, Chen, Jin, Li, Wang and Shu2018). The present study showed that Pb stress reduced the soluble protein and soluble sugar contents and increased the defence levels of pakchoi plants. Pb inhibited S. litura growth and development, which may have resulted from synergistic effects of the elemental and chemical defences. In the Pb and S. litura double stress groups, Pb accumulation induced plant chemical defence, and pakchoi resistance induced by S. litura produced a superimposed or synergistic effect, resulting in an overall increase in the total defence level of pakchoi plants. In addition, the level of plant chemical defence is regulated under environmental stress by the allocation of plant resources (Boyd, Reference Boyd2012; Cheruiyot et al., Reference Cheruiyot, Boyd and Moar2015; Sahu et al., Reference Sahu, Dutta, Ray, Pradhan and Dandapat2018; Chen et al., Reference Chen, Wang and Shu2020; Jiang et al., Reference Jiang, Wang and Yan2020). This system can be considered in the context of the optimal defence theory (Barto and Cipollini, Reference Barto and Cipollini2005; Kooyers et al., Reference Kooyers, Blackman and Holeski2017). Pakchoi plants may incur a level of pest damage, such that the produced chemical defence compounds exceed the growth and development of the plant. Thus, the plant utilises significant resources to synthesise defence-related compounds, thereby enhancing its chemical defence ability (Karban and Baldwin, Reference Karban and Baldwin1997; Zavala et al., Reference Zavala, Patankar, Gase and Baldwin2004; Li, Reference Li2016).

In conclusion, the bottom–up effects of Pb on soil–pakchoi–S. litura interactions were analysed and the results showed that the simultaneous presence of elemental and chemical defences enhanced the insect resistance of pakchoi plants. In addition, the top–down effects of S. litura larval feeding on pakchoi, soil enzyme activity and subsequent S. litura infestation were explored. The results of this study provide novel information about the joint effects of Pb and herbivores on soil–plant–herbivorous insect interactions and management of crop pest control under heavy metal pollution. However, short-term toxicological tests on Pb were conducted here, and the cumulative effects and long–term nature of these effects were not considered. Further studies are, therefore, necessary to determine whether the effects of heavy metals can be extended to the progeny of herbivorous insects and their natural enemies at higher trophic levels.

Acknowledgements

This research was supported by the Natural Science Foundation of Hunan Province (grant number: 2022JJ40409), the Scientific Research Project of Hunan Education Department (grant number NSFC-23A0539), and Aid Program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province.

Author contributions

X. L. and X. Y. designed the study; H. L., Y. S. and Y. Z. performed the experiments; Z. S. and H. T. analysed the results and produced the figures; and X. L. wrote the manuscript. All authors have discussed and approved the final manuscript draft.

Competing interests

None.

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Figure 0

Figure 1. (a) Urease, (b) phosphatase and (c) sucrase activities of soil. Soil enzyme activity under Pb stress, herbivore stress (Spodoptera litura), Pb and herbivore double stress and the control (CK). Data are mean values ± standard error, and different lowercase letters indicate statistically significant differences as determined using Tukey's honestly significant difference.

Figure 1

Figure 2. Leaf defensive chemicals and nutrient content in pakchoi plants under Pb stress, herbivore stress (Spodoptera litura), Pb and herbivore double stress and the control (CK). (a) Soluble protein content, (b) soluble sugar content, (c) trypsin protease inhibitor (TPI), (d) peroxidase (POD) activity, (e) superoxide dismutase (SOD) activity, (f) catalase (CAT) activity and (g) Pb content in pakchoi plants. Data are mean values ± standard error, and different lowercase letters indicate statistically significant differences as determined using Tukey's honestly significant difference.

Figure 2

Figure 3. (a) Survival rate, (b) larval development period, (c) pupa eclosion rate and (d) adult body weight of Spodoptera litura larvae fed with pakchoi and under Pb stress, herbivore stress, Pb and herbivore double stress and the control (CK). Data are mean values ± standard error, and different lowercase letters indicate statistically significant differences as determined using Tukey's honestly significant difference.