With the rapid development of industries and the extensive use of pesticides and fertilizers, environmental elements such as soil, water, and air—essential for the survival of humans and various organisms—have been severely polluted by heavy metals (Xiao, 2009; Zeng, 2022). According to statistics, the area of arable land in China has reached 1,278,666.6 km², while the area of heavy metal-polluted soil is as high as 3.33 million hm², with mercury (Hg), lead (Pb), zinc (Zn), cadmium (Cd), and other factors exacerbating heavy metal pollution in arable land and soil (Wang, 2023). Farmland is a crucial component of land that plays a vital role in agricultural development and the national economy. Therefore, soil heavy metal pollution has inevitably become a global problem that urgently needs to be solved (Wang et al., 2024).

When plants are contaminated with Cd, cell division slows down, chloroplasts are severely damaged, respiration and photosynthesis are negatively affected, the transpiration rate gradually decreases, and growth and development are inhibited (El Rasafi et al., 2022; Zhang and Lu, 2024). The content of heavy metals such as Cu, Pb, and Zn in garden plants in the Yunnan region and the same organs of different garden plants follow the order of Zn > Pb > Cu (Fan et al., 2016). Perhaps, because Zn and Cu are essential elements for plant growth and plants have a much greater demand for Zn than Cu, physiological processes such as chlorophyll synthesis and photosynthesis require Zn (Yadegari, 2017; Olivari et al., 2020).

A typical stress response of plants under abiotic stress is a sharp increase in soluble sugar content, especially glucose, sucrose, and fructose (Couée et al., 2006). Pb and Zn stress can reduce the activity of castor sugar metabolism enzymes (Gao, 2019). Meanwhile, due to the influence of heavy metals on plant amino acid metabolism, ecological plants with higher levels of proline have a higher tolerance to heavy metals such as Cu and Zn (Xu, 2018). However, not all plants accumulate proline under abiotic stress conditions; there is no significant correlation between the degree of proline accumulation in some plants and their stress tolerance (Hu and Xu, 2014). Zn is an essential component of carbonic anhydrase, a key enzymein plant chloroplasts that catalyzes the fixation of carbon dioxide. Therefore, appropriately supplementing Zn can increase the chlorophyll content in plant leaves (Xu et al., 2003). However, high concentrations of Zn can exhibit inhibitory effects on plant photosynthesis and growth and development (Wang et al., 2021). When the Cd concentration was 8.9 × 10⁻⁵ mg L⁻¹, the photosynthetic pigment content of Halophila ovalis was significantly inhibited (Ralph and Burchett, 1998). However, heavy metals such as Zn and Cd often coexist in nature, and the effects of combined pollution vary among different plants. Under the same Cd concentration treatment, the trend of chlorophyll decline is more significant when exogenous Zn is added to Lycopersicon esculentum, with all treatment groups showing lower chlorophyll levels than the control group (Zhao et al., 2020).

As is well known, the production of eggs is one of the most energy-demanding events in the adult life of female insects (Lu et al., 2019), and trehalose is the major circulating sugar used in oocyte growth, accounting for 80%–90% of the total sugar in insect hemolymph (Thompson, 2003). Trehalose metabolism not only regulates the biosynthesis of chitin in insects, thereby affecting their growth and development (Merzendorfer and Zimoch, 2003; Chen et al., 2010), but also participates in the uptake of vitellogenin (Vg) by oocytes, indirectly affecting their development (Wang et al., 2020). In eukaryotes, trehalose is only synthesized through the trehalose synthase/trehalose-6-phosphatase pathway (TPS/TPP) (Avonce et al., 2006). Trehalase (TRE) is the only enzyme that can irreversibly hydrolyze trehalose into glucose, providing energy for insect growth, development, and activity (Luo et al., 2022; Wang, 2022). In addition, heavy metal stress can affect insulin biosynthesis and secretion by inhibiting the expression of key genes in the insulin signaling pathway, thereby affecting trehalose metabolism in adult Spodoptera litura (Yang, 2021a).

Insect Vg is a high-molecular weight glycolipid complex protein composed of approximately 1%–14% carbohydrates, 6%–16% lipids, and 84% amino acids (Hiremath and Lehtoma, 1997; Tufail and Takeda, 2008). It can undergo a series of complex biochemical processes and precipitate yolk protein (YP) in crystal form (Matozzo et al., 2008; Tufail and Takeda, 2009), providing nutrients for oocyte maturation and embryonic development. Under the stress of 5,000 mg/kg Pb, the expression of the Vg gene in female Musca domestica on the first day of emergence is significantly downregulated (Liu, 2023). In addition, studies have shown that under Pb stress, the relative expression of the female Vg gene was downregulated in 5th-instar larvae and adults of Spodoptera exigua (Qian, 2016).

Vicia faba L. is an important edible legume crop that combines food, vegetables, feed, medicine, and green manure in China (Zhang et al., 2018). It is rich in nutrients and serves as an important crop for land use and intercropping, playing a vitalrole in the adjustment of planting structures. It is also one of the characteristic economic crops in northwest China (Li et al., 2015). However, due to its periodic parthenogenesis, short generation cycle, and strong reproductive and dispersal abilities, the population of Megoura crassicauda can rapidly grow in the short term (Feng, 2015), causing huge losses to leguminous crops and forage (Wu et al., 2015). Consumers at all levels can transfer and accumulate heavy metals in the food chain through predation relationships (Di, 2017). For example, after treating soil with heavy metal solution, higher levels of heavy metals were detected in eggplants, tomatoes, broad beans, mealybugs, aphids, and ladybugs than in the control group (Wang et al., 2017a; Butt et al., 2018; Naikoo et al., 2021; Li et al., 2024; Yin et al., 2024), indicating that heavy metals can flow and accumulate through the food chain. However, a large amount of research has only focused on the treatment of single heavy metal element stress, neglecting the fact that in most practical situations, it is a combination of multiple heavy metal elements (Yang, 2021b). In addition, most heavy metal stress cultures start from seedlings (Li et al., 2022; Hassani et al., 2023). Furthermore, Cd and Zn belong to the same element family, with similar chemical and environmental characteristics (Garg and Kaur, 2013), and studies have observed that Zn pollution and Cd pollution in soil could occur simultaneously (Wang et al., 2017b). Therefore, in this study, Zn and Cd were selected as stress factors, and four treatment groups were set up with single Cd and Cd–Zn with different ratios of compound stress. Heavy metal element compound exposure was applied to broad bean seeds to explore the growth and physiological adaptation mechanisms of broad beans under different levels of mixed heavy metal stress and the impact of this stress on aphids through the food chain.

Soil heavy metal pollution has become a widespread issue, significantly impacting plants directly exposed to it and the animals that feed on these plants. After in-depth research, it was found that in reality, soil pollution mostly exists in the state of composite pollution of multiple heavy metals or coexists with other pollutants (Xing, 2018; Zhao et al., 2020). Cd and Zn, which belong to the IIB group of elements in the periodic table, are classified as thiophilic elements in the chemical classification of earth elements. In addition, due to their similar chemical properties, they are always found together in nature. Their presence in soil harms plants and then enters the terrestrial food chain, eventually reaching humans and posing a threat to human health (Wang et al., 2005). When multiple metal elements interact with each other, their composite effect significantly changes the original activity or toxicity of individual heavy metals, exhibiting antagonistic or synergistic promoting effects (Wang et al., 2018).

Heavy metal pollution can affect many aspects of plant growth, including seed germination, plant height, biomass, yield, and quality (Wang et al., 2023). Under Pb–Zn composite treatment, with the increase in the Zn concentration, the growth indicators of Taiwan Paulownia plants significantly decrease, the plant leaves turn yellow and wither, and the roots and stems cannot grow normally (Liu, 2013). In this study, under Cd–Zn-combined stress treatment, the germination rate of broad beans significantly decreased, and the higher the zinc concentration, the greater the decrease (Figure 1A). Within 9–15 days, a low concentration of heavy metal stress can increase seedling height, but high concentrations of Zn can hinder the growth of broad beans (Table 1). The decrease in these growth indicators is also consistent with the phenomenon of reduced plant biomass, as stated in previous studies (Liu, 2013; Pavithra et al., 2016; De Conti et al., 2020). Therefore, according to research (Wang et al., 2010), high concentrations of Zn disrupt the normal physiological and biochemical processes of root cells by inhibiting seed germination, lateral root formation, and affecting the luster of plant leaves, thereby inhibiting the growth and development of seedlings and leading to plant growth retardation.

The level of the chlorophyll content can reflect the photosynthetic capacity of plants, indicating the degree of leaf senescence and oxidation under environmental stress (Xia and Zhu, 2013). When the Zn concentration was 30 mg L⁻¹, duckweed plants were damaged, 80% of the leaves lost their green color, and black spots appeared (Tang et al., 2010). In our experiment, there was also a phenomenon of decreased chlorophyll content under stress (Figure 1B). On one hand, it may be due to heavy metal stress that damages the structure of plant cell membranes and chloroplasts (Choudhury and Panda, 2005; Basile et al., 2012); on the other hand, it may affect chlorophyll synthesis by disrupting the enzymes required for chlorophyll synthesis, thereby inhibiting or even damaging the plant photosynthetic system (Li et al., 2012a). Alternatively, under high concentrations of heavy metal stress, Zn and Cd exhibit a synergistic effect, causing most leaves to lose their green color and chlorophyll function (Quan, 2022).

In both highland barley and wheat, the average contents of Cd and Zn varied in the order: root > leaf > stem (Tong et al., 2022). Our research results are consistent with these findings, and we found that the accumulation of Cd in broad beans is related to whether they are simultaneously infected with Zn (Figure 2). Zn can alleviate oxidative stress caused by Cd by stabilizing and protecting cell membranes, thereby slowing down the inhibition of Cd on rice growth (Hassan et al., 2005). In our study, when there was no aphid infestation, appropriately increasing the proportion of Zn under composite stress increased the Cd content in broad bean stems and leaves. Based on extensive research (Wang et al., 1999; Hassan et al., 2005; Liu et al., 2010; Li et al., 2012b; Rong et al., 2021), it is speculated that Zn weakens the damage caused by Cd in plants and stimulates the production of more carriers in roots, increasing the amount of Cd that can be fixed in plants. Due to transpiration, Cd is transported upward with water, gradually entering the upstream organs of plants and forming stable complexes. Feeding may alter the accumulation level of heavy metals in plants (Huang et al., 2021). We found that aphid infestation increases the Cd content in the roots of broad beans and the Zn content in the roots and stems but inhibits the increase in the Zn content under the leaves (Figure 2). This may be because aphid infestation leads to an increase in the concentration of Cd and Zn in plant phloem secretions (Stolpe et al., 2017a; Stolpe et al., 2017b), and the increase in the Zn concentration in soil makes Zn more competitive, allowing it to bind with zinc enzymes to maintain its original activity and prevent Cd toxicity (Wu, 2012), indirectly increasing the Cd content in roots. Otherwise, there may be another unidentified Cd absorption system in plant roots that involves systemic signaling (Plaza et al., 2015). Heavy metals can be transferred to phytophagous insects through food chains (Jiang et al., 2021), and during our research process, it was also found that aphids prefer to be densely distributed under leaves, resulting in a decrease in Cd and Zn content in the leaves.

Plant cells will accumulate a large number of osmotic regulatory substances such as proline and soluble sugar to reduce cell osmotic potential and regulate plant physiological metabolism under stress (Wu et al., 2019). Under Zn stress, the content of proline and soluble sugars in the roots and leaves of wheat seedlings significantly increased (Li et al., 2013). The content of proline in Elytrigia elongata significantly increased under the combined treatment of Cd and Zn (Tian et al., 2012). This study also found that Cd–Zn infection can significantly increase the soluble sugar content and proline content in the stems and leaves of broad beans (Figure 3). It is speculated that this is because plant cells secrete a large amount of stress-protective substances to alleviate heavy metal damage (Zhang et al., 2021). However, the accumulation of proline under Zn stress was not observed (Wan et al., 2023), and most reports on the role of proline are not consistent, highlighting the need for further study (Tanwir et al., 2021).

Trehalose, as an energy substance and structural component, can protect organisms from environmental stress under adverse conditions and play an important role in cellular energy metabolism and other processes (Thompson, 2003; Zhao et al., 2021). Trehalase belongs to glucosidase, which can catalyze the hydrolysis of one trehalose molecule to produce two glucose molecules, and it is widely present in microorganisms, insects, and plants (Shukla et al., 2015). Membrane-bound trehalase mainly exists in the muscles, while soluble trehalase mainly exists in the small intestine and brain tissue (Yang et al., 2007). In our study, under heavy metal infection, the activity of soluble trehalase in F1 and F2 adult aphids was significantly enhanced, while membrane-bound trehalase showed no significant change, resulting in a significant decrease in the trehalose content, but the phenomenon exhibited by F3 adult aphids was the opposite (Figures 4A, D, E). This may be because when aphids were initially infected with heavy metals, they obtained glucose by hydrolyzing trehalose (Figure 4B), which helped protect somatic cells and allowed them to better adapt to stress environments (Behm, 1997). After three generations, aphids gradually adapted to heavy metal environments, relying solely on glucose metabolism generated by glycogen breakdown to meet their life activities (Figure 4C). TRE and TPS are trehalase and trehalose synthase genes in insects, playing important roles in their physiological activities. In a dry environment, the expression of TPS in Drosophila melanogaster is enhanced, the expression of TRE is weakened, and the content of trehalose in the body is increased (Thorat et al., 2012). In our experiment, the F3 generation under heavy metal stress conforms to this phenomenon, while the expression of TPS and TRE is enhanced in F1 and F2 and is more significant when the Zn content is 100 mg/kg (Figure 5). It is speculated that this is because under external stimulation, trehalose metabolism in aphids is vigorous, and enzyme gene expression is also feedback-regulated (Chen et al., 2017).

Insect Vg and YP are the two main types of yolk proteins in insects, and Vg is a precursor substance of YP, which can be degraded into small-molecule peptides and amino acids during insect embryogenesis, providing nutrients and functional substances such as amino acids, fats, and carbohydrates for embryonic development (Tufail and Takeda, 2009; Yan et al., 2010). However, recent studies suggested that Vg plays an important role in other aspects of insect biology, such as the caste differentiation process in social insects, wound healing, protection against oxidative stress, immunity, and life span regulation (Kodrík et al., 2019). In addition, clarifying the important egg-laying-related gene Vg and its regulatory mechanism can provide new ideas for the prevention and control of Hemiptera insects (such as aphids), which is of great significance for effectively reducing agricultural and forestry losses (Pan et al., 2023). In our study, F1 aphids significantly accumulated trehalose under Cd–Zn-mixed stress (Figure 4A), while female aphid production showed a significant decrease (Figure 6A), which is consistent with the inference that trehalose accumulation, which usually occurs in stress responses, diminishes reproductive activity and reduces longevity (Zhou et al., 2022; Xu et al., 2023). However, partial Cd–Zn composite treatment increased the expression level of Vg (Figure 6B), which is not completely consistent with the reduction in female aphid production. It is speculated that aphids can achieve stress protection by expressing Vg, which is consistent with the inference that this gene has potentially acquired non-reproductive functions (Cardoso-Júnior et al., 2021). Therefore, it can be speculated that heavy metal stress, to some extent, inhibits female aphid production, but it may not be indirectly inhibited by affecting Vg expression. Instead, it may reduce production by regulating insect trehalose accumulation and inhibiting reproductive activity. This requires further exploration to determine whether there are other pathways of influence and regulatory mechanisms.

In summary, the results showed that Cd (12.5 mg/kg) and different levels of Cd–Zn-combined treatment promoted the growth of broad beans, increasing seedling height, but when the Zn concentration was too high, a synergistic inhibitory effect was observed. The absorption capacity and distribution of heavy metals in various components of broad beans varied, resulting in differences in the content of heavy metals in different parts. Moreover, under Cd–Zn composite treatment, aphids infesting the plants promoted the absorption of Zn through the roots and stems of broad beans. In addition, during Cd–Zn composite treatment, broad beans adapted to heavy metal complex stress by regulating total soluble sugar, proline, and chlorophyll metabolism in each plant component. Meanwhile, with intergenerational changes, different treatment groups dominate the expression levels of TRE and TPS genes in each generation. Correspondingly, the sugar content and trehalase activity in aphids respond to the expression. The number of offspring of female aphids in each group of three generations showed a decreasing trend initially, followed by an increase, with intergenerational changes, and the trend of Vg expression levels was slightly different, further confirming the close relationship between insect trehalose metabolism and reproduction, as well as the possible role of Vg in other life activities of insects. These findings suggest the need for further experiments to explore these mechanisms.