Effects of Si and N addition on Oryza sativa and its invasive grazer apple snails (Ampullariidae): resistance traits and feeding metrics

Effects of Si and N addition on Oryza sativa and its invasive grazer apple snails (Ampullariidae): resistance traits and feeding metrics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of Si and N addition on Oryza sativa and its invasive grazer apple snails (Ampullariidae): resistance traits and feeding metrics Wei Li, Jingyi Zhao, Hua Yu, Wen-Hong Dai, Yao-Bin Song, Ming Dong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7154936/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Silicon, as a crucial element of plants, may contribute to plants’ resistance to biotic stresses like herbivore feeding. In tropical and subtropical Asia, apple snail (Ampullariidae) is an invasive aquatic herbivorous snail in agricultural and wetland ecosystems, like paddy field, which suffer from heavy eutrophication of nitrogen. However, little is known about the potential effects of feeding rice leaves treated with silicon and nitrogen on the feeding metrics of apple snails. We conducted a greenhouse experiment to examine the effect of silicon and nitrogen addition on rice seedlings as grazed by apple snails, in which two levels (0 and 1.5 mM) of silicon addition and three levels (0.72, 1.44 and 5.76 mM) of nitrogen addition were used. We measured plant growth, leaf element contents and leaf defense characteristics of rice. We determined the snail feeding metrics after feeding in different rice leaves for one week. Silicon addition increased plant mass in the low and high nitrogen addition and increased the C/N ratio only in the middle nitrogen addition. Silicon addition significantly decreased the growth of apple snails while significantly increasing flavonoid content and the force of fracture of rice leaves in all the nitrogen levels. Silicon addition increased the tannin content of rice leaves in the middle nitrogen addition but decreased in high nitrogen treatment. Moreover, silicon addition increased the leaf sulfur content of rice at all three levels of nitrogen addition. Silicon addition could improve the defense of rice against invasive herbivory by apple snails, which sheds insights on the protection of wetland crops in the context of control of eutrophication and biological invasion. biological invasion herbivory plant defense rice silicon Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Apple snail (Ampullariidae), the freshwater snail native to South America and invasive in Asia, is listed among the top 100 of the world's worst invasive species (Lowe et al., 2000 ). This snail primarily feeds on aquatic plants and crops (Hayes et al., 2008 ). This snail negatively affects the biomass of wild macrophytes, water quality, and algal production in the invasive range (Carlsson et al., 2004 ; Carlsson & Lacoursière, 2005 ). The snail has caused enormous damage to wetland ecosystems and agricultural production, especially the rice planting area ((Horgan et al., 2021 ; Jiang et al., 2022 ); Constantine et al., 2023 ). The snail primarily consumes vegetables and aquatic plants, which can lead to a decrease in the shoot biomass and vegetation cover of the communities it invades (O'Neil et al., 2023 ). This activity could result in a 50% reduction in agricultural production (Panda et al., 2021 ), ultimately diminishing the ecosystem services of wetlands and farmlands. This snail causes significant economic losses in its non-native habitat, including China (Yang et al., 2019 ). Due to China's diverse geographical landscapes and varied climatic conditions, the country is vulnerable to invasions by apple snail (Wan & Yang, 2016 ). In China, apple snail has invaded the main areas of rice plantations ranging from the Yangtze River Basin, leading to huge crop production cuts (Horgan et al., 2014 ; Yang et al., 2018 ; Xiao et al., 2023). By 2006, the area affected by apple snails in rice fields had exceeded 300,000 hectares, resulting in a direct loss of up to 12.5 million dollars (Li et al., 2009 ). The snail caused the most damage to 21-day-old transplanted rice seedlings, which can result in up to 100% damage (Sin, 2003 ). The snail can constantly damage the rice. After the rice is harvested, snails can conceal themselves in the mud, posing a threat to the rice crop in the following year (Yao et al., 2024 ). Unfortunately, massive physical and chemical measures used to eradicate apple snail in invaded ranges have shown limited efficiency (Yam et al., 2016 ; Wang et al., 2022 ). Wetlands serve as reservoirs abundant in amorphous silicon, playing a significant role in shaping the global biogeochemical cycle of this element (Struyf & Conley, 2009 ; Struyf et al., 2009 ). Notably, the concentration of silicon found in the tissues of wetland plants frequently surpasses 1%, potentially exerting an influence on the nitrogen cycling processes within these plants (Schoelynck et al., 2010 ; Schaller et al., 2016 ). Silicon is recognized as a beneficial element for plants, particularly grasses, not only due to its high dry-weight concentration (Yamamoto et al., 2012 ), but also for its vital role in mitigating abiotic and biotic stress (Reynolds et al., 2009 ; Song et al., 2020 ; de Tombeur et al., 2023 ). Silicon can help plants mitigate damage caused by herbivores in multiple ways. Firstly, silicon can enhance plant defense with the deposition of inorganic amorphous silicon oxide (SiO 2 ) phytoliths in the epidermis of plant tissues (Ma, 2004 ). For example, Johnson et al. ( 2020 ) found feeding plants with silicon resulted in a 50% reduction in the growth rate of an aphid ( Rhopalosiphum padi ). On the other hand, silicon can also activate the jasmonate acid and salicylic acid metabolism defense pathways (Ye et al., 2013 ). Jasmonate acid metabolic defense pathways are often associated with leaf damage by herbivores (Browse & Howe, 2008 ). Moreover, silicon supports the production of defensive secondary metabolites in plants and boosts the activity of oxidation-related enzymes (Gomes et al., 2005 ). This results in a toxic impact on herbivores and lowers their preference for these plants. In addition, silicon can improve the release of volatile organic compounds by plants, which can attract the natural enemies of herbivores (Kvedaras et al., 2010 ; Liu et al., 2017 ) and interfere with the recognition of the host plants by herbivores (Veromann et al., 2013 ). Therefore, we expect silicon could help rice alleviate the herbivory stress of animal pests such as invasive apple snail. Nitrogen is essential for plant growth and reproduction (Chen et al., 2008 ). Nitrogen plays a critical role in herbivores. The growth of herbivores is significantly affected by the consumption of high-nitrogen food (Tao & Hunter, 2015 ). Due to overloading reactive nitrogen from food production and energy production (Galloway et al., 2004 ), most ecosystems suffer from eutrophication (Xiao et al., 2019 ). Previous studies found that excess nitrogen deposition may reduce defense costs (Jamieson et al., 2012 ; Xiao et al., 2019 ). An increase in nitrogen availability can lead to changes in their secondary metabolites (Tao & Hunter, 2012 ), which are associated with plant resistance to herbivores. Jamieson & Bowers ( 2012 ) found that the growth of Calophasia lunula in Linaria dalmatica was promoted by nitrogen enrichment. Plants have evolved to balance the trade-offs among growth/, reproduction and defense (Karasov et al. 2017 ). Moreover, available nitrogen concentration could significantly affect the silicon uptake of plants because silicon is considered a cheap defensive substance (Johnson et al., 2021 ). Therefore, a trade-off exists between silicon and nitrogen accumulation in Si-accumulating plant species, potentially having significant implications for their defense mechanisms against insect herbivores (Wu et al., 2017 ). However, we did not know if there are interactive effects of silicon and nitrogen on the growth of rice and its invasive feeder apple snails. In this study, we conducted a four-month cultural experiment in which rice plants experienced three levels of nitrogen supply combined with two levels of silicon addition. Then, we fed the apple snails with rice leaves in different treatments for a week in the laboratory. We propose the following scientific questions: (1) Does silicon addition under different nitrogen levels affect the growth and development of rice? (2) Will altering nitrogen levels affect silicon's ability to defend rice against apple snail when feeding on silicon-treated rice leaves? 2. Materials and methods 2.1 Experimental design (1) Experiment with N and Si additions From March 15 to July 2, 2022, we conducted preliminary plant cultivation in the greenhouse of the Xiasha Campus of Hangzhou Normal University (30°32′N, 120°40′E), Hangzhou, Zhejiang, China. The greenhouse has an average temperature of 25 ℃. Rice seeds from the Chinese Academy of Agricultural Sciences were disinfected with alcohol. We then placed the seeds on wet filter paper until they began to take root. Once the seeds were rooted, we transferred them to a seedling box filled with peat soil. When the seedlings had grown to approximately 10 cm in height, we planted them in pots filled with two liters of agricultural field soil. The experiment employed a factorial design with six treatments comprising three nitrogen levels via urea (0.72, 1.44, and 4.76 mM for low, medium, and high nitrogen, respectively) combined with and without 1.5 Mm Si addition supplied as sodium silicate, each treatment replicated 12 times using a total of 72 rice seedlings (Wu et al., 2017 ). The treatments without silicon addition received the corresponding concentration of sodium chloride to ensure a consistent soil osmotic pressure (Johnson et al., 2021 ). We added sodium chloride to the group without silicon addition to ensure the consistency of sodium ions. The soil was consistently submerged below the water surface during the experiment. Silicon and nitrogen were added once a week for 2 months. Two months of treatment cultures were performed to prevent the negative effects of short treatment duration. We harvested them all two months after planting when the rice seedlings had developed 6 to 8 leaves. Six seedlings from each treatment group were utilized to determine plant indicators. The remaining six seedlings were exclusively used for the snail feeding experiment. (2) Snail feeding experiment A snail-feeding experiment was conducted from July 2 to July 8. The apple snails were fed with leaves of the harvested rice plants, which had previously experienced the six treatments in the plant growth experiment. One month before the feeding experiment started, apple snails of similar size were collected from the stream in Hangzhou, Zhejiang, China (30°32′N, 120°1′E) and then placed in the laboratory culture with lettuce as food. The provision of lettuce was reduced a week before and stopped two days before the feeding experiment started. At the beginning of the experiment, we selected 36 healthy adult apple snails with similar size. Each snail was carefully wiped clean to remove moisture before being weighed. The snails were then randomly divided into six groups, with each group containing six animals. Each snail was placed in its own circular container, resulting in a total of 36 containers being used for the experiment. Each group was then given six treated rice leaves to feed on for a week. Timely replaced the rice leaves every day and recorded leaf loss, and then weighed again to ensure an increase in apple snails’ weight. After that, all apple snails were stored in a -80 ℃ refrigerator until determining the cellulase activity of apple snail. 2.2 Plant sample collection and analysis The relative chlorophyll content (SPAD) of the rice leaves was measured using a portable chlorophyll fluorometer (Minolta SPAD-502Plus) when the rice reached six to eight leaves. We picked three well-grown rice leaves for every plant to assess the relative chlorophyll content. The mean chlorophyll value from these three leaves was considered as the plant's relative chlorophyll content. Then half of the rice plants were harvested and the fresh weight of their different parts was determined in the laboratory. The harvested rice was rinsed with water, and then the surface moisture of the rice was absorbed using paper towels. The rice was divided into aboveground and underground parts. We used an electronic universal material testing machine (Instron 3343) to determine the force of fracture of rice leaves. The rice leaves with similar physiological conditions were fixed vertically to the machine and pulled at a constant speed from both ends until they broke. The maximum force at fracture was recorded, and overall leaf toughness was estimated using blade thickness, width, and the maximum force at fracture (Johnson et al., 2019 ). The plant samples were dried in an oven set at 60°C until they reached a constant weight. For each treatment group, the biomass of the six rice plants was measured by an electronic balance. The dried rice leaves were ground using a mixer ball mill (MM400, Retsch, Germany). The ground leaves were subjected to determinants of carbon (C), nitrogen (N), and sulfur (S) content using an element analyzer (vario PYRO cube; Elemental, Germany) (Hu et al., 2022 ). Flavonoid content was extracted from about 0.02 g of rice leaves with 60% ethanol and the corresponding content by using a spectrophotometer using the method of Lin et al. ( 2014 ). The amount of flavone in the extract was measured using a spectrophotometer with the sodium nitrite-aluminum nitrate method. Tannin content was extracted from 0.1 g of rice leaves using ultrapure water at 80 ℃, to determine the tannin content according to the method by Makkar ( 2003 ). The tannin content in the extract was determined using the Folin-Ciocalteu colorimetric method and measured with a spectrophotometer. The total phenols of rice leaves were extracted from about 0.1 g using 60% ethanol to determine the total phenolic content according to the method of Cheng et al.(2013). Total phenol in the extract was determined using the Folin-Ciocalteu colorimetric method and measured with a spectrophotometer. Plant silicon content was determined by using sodium hydroxide solution at high temperature, according to the determination of rice leaf silicon content by the molybdenum blue colorimetric method (Kraska & Breitenbeck, 2010 ). For each element or organic compound, we used six rice plants for each treatment group and conducted the experiment in three replicates. 2.3 Snail sample analysis The apple snails were placed in an ultra-low temperature refrigerator and thawed in an ice water bath, out of its intestines and stomach, using a mortar for grinding homogenate on ice. Cellulase of the snail's intestines and stomach was extracted using a sodium citrate buffer containing the protease inhibitors. Cellulase activity was measured according to the method of Kim et al. ( 2014 ). Relative growth rate (RGR) was defined for the snails by Johnson et al. ( 2021 ): $$\:\text{RGR(}\text{mg}\text{}{mg}^{-1}\text{}{day}^{-1}\text{)=}\left(\frac{\text{F}\text{i}\text{n}\text{a}\text{l}\:\text{m}\text{a}\text{s}\text{s}\:\text{a}\text{f}\text{t}\text{e}\text{r}\:\text{f}\text{e}\text{e}\text{d}\text{i}\text{n}\text{g}-\text{I}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\:\text{m}\text{a}\text{s}\text{s}\:\text{b}\text{e}\text{f}\text{o}\text{r}\text{e}\:\text{f}\text{e}\text{e}\text{d}\text{i}\text{n}\text{g}}{\text{I}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\:\text{m}\text{a}\text{s}\text{s}\:\text{b}\text{e}\text{f}\text{o}\text{r}\text{e}\:\text{f}\text{e}\text{e}\text{d}\text{i}\text{n}\text{g}\bullet\:\text{d}\text{a}\text{y}\text{s}}\right)$$ 2.4 Data analyses Two-way ANOVA was used to test the effects of nitrogen concentration and silicon imposed on plant biomass, tannin content, flavonoid content, phenol content, silicon content, plant mechanical force, leaf loss, and cellulase activity, principal effect analysis. We conducted multiple comparisons to explore the significant differences between different treatment groups subjected to silicon addition and nitrogen treatment. Duncan's Multiple Range Test was utilized to pinpoint significant differences between silicon additions and nitrogen treatments, establishing a significance threshold at a p-value of less than 0.05. All analyses were performed in SPSS 20.0. We utilized a structural equation model through linear regression to investigate the impact of silicon and nitrogen addition on leaf resistance, leaf element content, and snail feeding metrics. This model could examine the effects of various treatments on plant growth, leaf resistance, rice element content, and the influence of leaf resistance and element content on snail feeding metrics in apple snails. However, we excluded several indicators due to collinearity in constructing the structural equation model, including all rice plant growth, leaf sulfur content and leaf C/N ratio. To obtain a p -value greater than 0.05 for the entire model, weight gain and leaf consumption of apple snails were excluded. The structural equation model was constructed using the lavaan package (Rosseel, 2012 ) in R version 4.3.3 (R Core Team, 2024). 3 Results 3.1 Leaf resistance characteristics under silicon and nitrogen addition Silicon addition significantly affected flavonoid content ( p < 0.001) and force of fracture ( p < 0.001) and nitrogen levels significantly affected total phenolic content ( p = 0.011), tannin content ( p = 0.003) and force of fracture ( p < 0.001). Silicon addition and nitrogen levels showed an interaction effect on tannin content ( p < 0.001) (Table 1 ). Silicon addition increased the tannin content in middle nitrogen levels while showing the opposite results in high nitrogen levels (Fig. 1 a). Silicon addition significantly increased the flavonoid content in all nitrogen levels while the medium showed the lowest flavonoid content (Fig. 1 b). There were no significant effects of silicon addition on total phenolic content (Fig. 1 c). Silicon addition and high nitrogen levels significantly increased the force of fracture in rice leaves (Fig. 1 d). With the increasing of nitrogen levels, there were significant increase in total phenolic content and force of fracture (Fig. 1 c and 1 d). Table 1 Leaf resistance characteristics, snail feeding metrics, leaf element content, and plant growth responses to silicon addition and nitrogen levels from Two-way ANOVA. Bold font indicates p < 0.05. Silicon (Si) Nitrogen (N) Si × N Related variables F p F p F p Leaf resistance characteristics Total phenolic content 1.149 0.292 5.254 0.011 0.129 0.897 Tannin content 0.478 0.495 7.030 0.003 12.363 < 0.001 Flavonoid content 37.217 < 0.001 2.074 0.143 1.262 0.298 Force of fracture 47.416 < 0.001 12.676 < 0.001 0.793 0.462 Snail feeding metrics Leaf consumption 5.477 0.026 1.698 0.200 1.361 0.272 Relative growth rate 22.227 < 0.001 3.114 0.059 2.742 0.081 Weight gain 13.637 0.001 2.664 0.086 2.907 0.070 Cellulase activity 19.946 < 0.001 11.96 < 0.001 2.708 0.083 Leaf element content Leaf carbon content 16.604 < 0.001 0.153 0.895 0.949 0.398 Leaf nitrogen content 23.120 < 0.001 4.482 0.020 0.192 0.826 Leaf sulfur content 25.508 < 0.001 0.351 0.707 0.753 0.480 Leaf silicon content 23.823 < 0.001 22.407 < 0.001 6.150 0.006 Leaf C/N ratio 4.737 0.038 2.132 0.136 0.374 0.691 Plant growth Relative chlorophyll content (SPAD) 50.203 < 0.001 56.252 < 0.001 2.081 0.142 Total mass 94.289 < 0.001 47.264 < 0.001 25.937 < 0.001 Shoot mass 60.645 < 0.001 28.744 < 0.001 16.007 < 0.001 Root mass 625.700 < 0.001 386.760 < 0.001 201.200 < 0.001 3.2 Snail feeding metrics under silicon and nitrogen addition Silicon addition affected leaf consumption ( p = 0.026), RGR ( p < 0.001), weight gain ( p = 0.001) and cellulase activity ( p < 0.001) of snails. Nitrogen levels significantly affected cellulase activity ( p < 0.001), and did not significantly affect other feeding metrics. There was no significant interaction between nitrogen levels and silicon addition on snail feeding metrics (Table 1 ). Silicon addition significantly decreased leaf consumption, RGR and weight gain of apple snails in low and high nitrogen levels (Fig. 2 a, b, c). Silicon addition decreased the cellulase activity of apple snails, and low and high nitrogen levels increased the cellulase activity of apple snails (Fig. 2 d). 3.3 Leaf element contents under silicon and nitrogen addition Silicon addition significantly affected leaf N ( p < 0.001), C ( p < 0.001), S ( p < 0.001), Si ( p < 0.001) and leaf C/N ratio ( p = 0.038) while nitrogen levels significantly affected leaf N ( p = 0.020) and leaf Si ( p < 0.001) and only showed interacted effects on leaf Si ( p = 0.006) (Table 1 ). Silicon addition significantly decreased leaf carbon and nitrogen content (Fig. 3 a, b), but significantly increased leaf sulfur content in all nitrogen levels (Fig. 3 c). Silicon addition significantly increased leaf silicon content in low and medium nitrogen levels, and high nitrogen levels showed the lowest leaf silicon content (Fig. 3 d). Silicon addition increased the leaf C/N ratio in medium nitrogen levels (Fig. 4 e). 3.4 Plant growth under silicon and nitrogen addition Silicon addition and nitrogen level significantly affected relative chlorophyll content, total mass, shoot mass and root mass of rice (all p < 0.001). There were significant interactions between nitrogen levels and silicon addition on the total mass, shoot mass and root mass of rice (all p < 0.001), but relative chlorophyll content ( p = 0.142) (Table 1 ). Silicon addition and nitrogen levels significantly increased relative chlorophyll content of rice in all nitrogen levels (Fig. 4 a). Silicon addition significantly increased total mass, shoot mass and root biomass in low and high nitrogen levels, but not in medium nitrogen level (Fig. 4 b, c, d). Generally, relative chlorophyll content total mass, shoot mass and root mass of rice increased with the increasing nitrogen level, 3.5 Effect of silicon and nitrogen addition on leaf resistance characteristics and snail feeding metrics Both silicon addition (standardized path coefficient β = 0.73, p < 0.001, Fig. 5 ) and nitrogen addition ( β = 0.42, p < 0.001, Fig. 5 ) showed a positive effect on the force of fracture. The combined effects of silicon, nitrogen additions, and leaf silicon content jointly explained 70% of fracture of force. Nitrogen addition (standardized path coefficient β = 0.39, p < 0.05, Fig. 5 ) showed a positive effect on the total phenolic content. Silicon addition ( β = 0.74, p < 0.001, Fig. 5 ) positively affected the flavonoid content. Moreover, silicon addition, nitrogen addition and leaf silicon content are jointly explained 51% of flavonoid content. Silicon addition ( β = -0.38, p < 0.05, Fig. 5 ) played a negative effect on tannin content while nitrogen addition ( β = 0.39, p < 0.05, Fig. 5 ) and leaf silicon content ( β = 0.63, p < 0.01, Fig. 5 ) played a positive effect. Silicon addition ( β = 0.46, p < 0.001, Fig. 5 ) showed a positive effect on leaf silicon content while nitrogen addition ( β = -0.53, p < 0.001, Fig. 5 ) showed a negative effect. Silicon addition and nitrogen jointly explained 50% of leaf silicon content. Force of fracture ( β = -0.38, p < 0.05, Fig. 5 ) played a negative effect on the RGR of apple snails. In addition, leaf silicon showed negative effects on the RGR ( β = -0.36, p < 0.05, Fig. 5 ) and cellulase activity ( β = -0.67, p < 0.001, Fig. 5 ) of apple snails. 4 Discussion Silicon additions led to a change in plant physical traits and the levels of the secondary metabolites proceeded to impact the ability of plants to defend against insects. The results obtained here indicated that the resistance of rice to invasive snail was affected by silicon and nitrogen addition. 4.1 Effects of silicon on rice growth in nitrogen treatments Many studies have shown that silicon is essential in plant response to biotic and abiotic stresses (Reynolds et al., 2009 ; Song et al., 2020 ; de Tombeur et al., 2023 ). Lack of nitrogen is a common stress in plants, exacerbated by human activity, leading to high nitrogen levels in plants (Dentener et al., 2006 ). Silicon significantly improved the rice biomass both at low and high nitrogen levels in the results. Nitrogen is one of the most essential mineral elements that plants require for growth and to achieve high crop yield (Chen et al., 2020 ). It has been reported that silicon can increase nitrogen uptake in an unexpected nitrogen addition in rice (Deus et al., 2020 ). In addition, silicon addition can up-regulated N-assimilation genes (e.g., OsGS2 , OsFd-GOGAT , OsNADH-GOGAT2 , OsGDH2 , and OsNR1 ) (Wu et al., 2017 ). However, the silicon content in plant leaves is affected by the amount of nitrogen supplied to the plants, as shown in our results. Wu et al. ( 2017 ) found that high nitrogen levels have a negative effect on the transcript levels of OsLsi1 and OsLsi2 , which directly affect the absorption of silicon. Moreover, in this experiment, silicon addition remarkably improves the leaf sulfur content in rice, while sulfur can improve the utilization of nitrogen in plants (Bloem et al., 2007 ). Increasing nitrogen could induce soil acidification, resulting in asynchronous responses across micronutrients in both plants and soil. Sodium metasilicate increases soil pH (Li et al., 2012 ), which can relieve soil acidification and asynchrony responses across micronutrients by silicon addition in rice. In addition, high nitrogen lowers the leaf silicon concentration in rice, while silicon relieves the effect. However, in middle nitrogen, silicon did not affect plant biomass, while silicon improved the leaf C/N ratio in rice. The C/N ratio is commonly used as an indicator of plant growth and flowering potential, and a high C/N ratio is believed to have a better nitrogen use efficiency (Gren, 2004 ). This implies that silicon increases the growth of rice, which could be explained by two potential mechanisms. On the one hand, silicon addition impacted the relative chlorophyll content of rice leaves under all nitrogen conditions, consequently improving the photosynthetic rate. On the other hand, silicon addition increased the leaf sulfur content, leading to an improvement in nitrogen availability, making up for the decrease in leaf nitrogen concentration. 4.2 Effects of silicon in rice resistance under nitrogen treatments To resist to herbivores, plants evolved diverse mechanisms to fight against herbivores in various chemical and physical traits (Aljbory & Chen, 2018 ). Silicon usually helps plants against herbivores in mechanical barriers and shows a high correlation with biochemical attributes (Fawe et al., 1998 ). Silicon is deposited in leaf hairs, trichomes and spines, which could influence the physical traits of the leaves (Hartley et al., 2015 ). In the results, silicon addition improved the force of fracture in rice leaves. Another research showed that an increase in silicon content in leaves resulted in an increased force required for a needle to penetrate the sheath (Schurt et al., 2012 ). By covalently crosslinking with hemicelluloses, silicon addition enhanced the mechanical properties of cells (He et al., 2015 ). Moreover, it has been approved that silicon can activate the jasmonate acid signaling pathway, which is associated with plant defense against herbivores, leading to an improvement in defense-related capabilities. Previous studies reported that silicon could promote the production of defense-related enzymes polyphenol oxidase (PPO), superoxide dismutase (SOD), peroxidase (POD) and so on (Peter et al., 1985 ). These enzymes play an essential role in plant defense which can directly injure herbivores or result in an increase of secondary metabolic compounds such as flavonoids and phenolics which are harmful or toxic to herbivores (Han et al., 2016 ). Sulfur is a necessary trace element for plant growth and development (Aziz et al., 2016 ). It is commonly found in sulfur in proteins, peptides, metabolites, and enzymes (Falk et al., 2007 ). Sulfur-containing metabolites and proteins in plant heavy metal detoxification, antioxidants, and maintenance of protein-reducing states play an indispensable role. Recent research showed silicon played a positive effect in the sulfur transporter HvST1;1 led to a higher uptake of SO 4 2− in Hordeum vulgare cv. Irina is under collective stress (Maillard et al., 2018 ). Another study demonstrated that silicon increased sulfur uptake and utilization in basil ( Ocimum basilicum ) (Barreto et al., 2022 ). This research showed silicon improved sulfur accumulation in rice in sulfur-rich conditions. Silicon addition significantly increases the production of total phenolic, flavonoid, and force of fracture at all nitrogen levels. Additionally, rice supplemented with silicon in high nitrogen levels shows a reduction in tannin content. The environment is more likely to affect the tannin concentration in plant leaves, and the temperature and soil nitrogen concentration significantly affect the tannin content (Fine et al., 2006 ). This study provided experimental evidence that silicon addition improved the defense level of rice with different nitrogen levels. 4.3 Effects of feeding rice leaves experiencing silicon and nitrogen addition on apple snails Silicon decreases the growth of apple snails and the weight of the leaves that the apple snail eats. The acquisition of nitrogen is the most critical trait for herbivores' growth, while intake in leaves at high silicon reduced leaf digestibility to limit the nitrogen acquisition in Spodoptera exempta (Massey & Hartley, 2009 ). The growth of apple snails was determined by the plant nutrients, especially nitrogen (Qiu et al., 2011 ). Silicon addition decreased leaf nitrogen content at all nitrogen levels, as indicated by our results. In addition, foods with a low carbon-to-nitrogen ratio can promote the growth of snails (Sharfstein & Steinman, 2001 ). According to our findings, Silicon addition resulted in a higher carbon-to-nitrogen ratio in rice leaves across all nitrogen levels. Silicon can also disrupt the integrity of epidermal cells in the gut of herbivores, affecting insect digestion (dos Santos et al., 2015 ). Moreover, silicon addition increases the activity and production of a variety of enzymes such as proteinase and lipoxygenases (Singh et al., 2020 ). Up-regulating defense-related genes results in an increase in the activity of phytohormones and the production of defense-related secondary metabolites such as phenolics, tannins and flavonoids (Hall et al., 2019 ). The phenol in green plants is an important trait of plant defense against herbivores (Dixit et al., 2017 ). Phenolic compounds can bind with proteins, leading to the damage of biological enzymes which can reduce the absorption of amino acids by herbivores as well as protein hydrolysis (Appel & Schultz, 1992 ). Phenolics in plants play a crucial role in the growth of the snail. Consuming plant leaves containing high levels of phenolics will inhibit snail growth and may cause death (Qiu et al., 2011 ). Tannins act as insect repellents for herbivores by containing toxins that can affect their growth and survival (Mazid et al., 2011 ). Tannins significantly decreased the growth of herbivores by limiting protein digestion efficiencies (Feeny, 1968 ). Flavonoids are an effective means of plant defense against generalists and are important in plant host recognition and inhibition of herbivore feeding (Simmonds, 2001 ). At the results from structural equation modeling analysis, it was determined that flavones and tannins had no significant impact on apple snails' RGR and cellulase activity. The growth of apple snails is significantly impacted by phenolic content. In this case, the content of tannin and flavonoid did not significantly impact the cellulase activity of apple snails. On the other hand, the feeding experiment was conducted for only one week. The impact of tannin content and flavonoid on the RGR of apple snails has not been proven yet. In the results, silicon addition decreased the activity of cellulase of apple snails, which is a group of hydrolytic enzymes that can digest the plant cell wall and decompose the biological macromolecules into digestible, nutritive oligomers and monomers (Pauchet et al., 2010 ). In addition, silicon addition increased phenolic, flavonoid content, and force of fracture in rice with all nitrogen, leading to a reduction in the leaf consumption of apple snails and showing a negative effect on the growth of apple snails. 4.4 Implications for control of apple snail damage in invaded rice agricultural systems Rice is a staple crop providing food for almost half of the world's population (Zhong et al., 2019 ). Ensuring the ordinary production of rice is crucial for global food security. However, biological invasions cause great damage to farmland ecosystems, resulting in a dramatic decline in food production (Bradshaw et al., 2016 ; Paini et al., 2016 ). The invasion of the apple snail in Southeast Asian countries has significantly reduced rice production. The invasive snail prefers to feed on rice seedlings, as young plants are more consistent with their diet (Naylor, 1996 ). The snail feeds on the base of rice leaves before cutting down the entire plant, ultimately killing the rice seedling (Schnorbach, 1995 ). Therefore, controlling biological invasions is crucial for maintaining high rice yields and promoting sustainable rice agriculture systems. This study found that silicon can effectively reduce the negative effects caused by apple snail invasions in rice agriculture by promoting rice growth and defense. The concentration of silicon is crucial for plant growth and development. However, there were significant differences in the silicon uptake capacity of plants, both in terms of species and genotypes (Ma & Yamaji, 2006 ). Selecting rice varieties that have a high capacity to absorb silicon is an essential step (Meharg & Meharg, 2015 ). The role of silicon in the rice ecosystem, both in terms of input and output, is crucial and cannot be overlooked. Slag that contains a high amount of silicon has been utilized as a fertilizer for rice crops (Zhu et al., 2019 ). There is a significant amount of silicon in rice stalks and other parts. Therefore, returning these parts to the rice agriculture systems is necessary to maintain the silicon cycle (Meharg & Meharg, 2015 ). Future research should focus on identifying suitable rice varieties and scientifically managing the silicon cycle in rice fields. This approach will contribute significantly to the effective control of apple snail damage in invaded rice agricultural systems. 5. Conclusions Silicon addition promoted the growth of the rice but inhibited the growth of the snails. Silicon addition increased the production of secondary metabolites in rice, leading to enhanced resistance. The addition of silicon reduces the nitrogen content and improves the phenolic levels of rice leaves. Apple snails thrive on food high in nitrogen and low in phenolics. Nitrogen addition increases the force of fracture and the content of phenolics. However, nitrogen addition limited the rice’s absorption of silicon, and there was an apparent trade-off between the rice’s absorption of nitrogen and silicon. On the other hand, nitrogen addition increases leaf nitrogen content, which may lead to an increase in the growth of the apple snail. Our results suggest that silicon supply is expected to become a more environment-friendly way to alleviate the harm of the invasive snail effectively. Moreover, using nitrogen fertilizer legitimately in rice fields is essential for controlling the apple snail. Declarations Acknowledgments This work was supported by NSFC (grant no. 32371580). We thank Y. Y. Shen, Y. Chen, J. Yang, K. Shi, W. Q. Li, J. J. Shi and H. J. Zou for their kind help with the experiment. We are especially grateful to Z. Q. Cao for his help with statistical analyses. 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P., Zeng, B., & Dong, M. (2019). Nutrient enhancement of allelopathic effects of exotic invasive on native plant species. Plos One, 14, e0206165. https://doi.org/10.1371/journal.pone.0206165 Yam, R. S. W., Fan, Y. T., & Wang, T. T. (2016). Importance of macrophyte quality in determining life-history traits of the apple snails Pomacea canaliculata : Implications for bottom-up management of an invasive herbivorous rest in constructed wetlands. Int. J. Environ. Res. Public Health, 13, 248. https://doi.org/10.3390/ijerph13030248 Yamamoto, T., Nakamura, A., Iwai, H., Ishii, T., Ma, J. F., Yokoyama, R., Nishitani, K., Satoh, S., & Furukawa, J. (2012). Effect of silicon deficiency on secondary cell wall synthesis in rice leaf. J. Plant Res., 125, 771-779. https://doi.org/10.1007/s10265-012-0489-3 Yang, Q. Q., Liu, S. W., Li, J. N., Wang, D., & Yu, X. P. (2019). Microsatellite evidence for multiple paternity in non-native populations of Pomacea canaliculata (Caenogastropoda: Ampullariidae) in China. Aquat. Invasions, 14, 656-666. https://doi.org/10.3391/ai.2019.14.4.06 Yang, Q. Q., Liu, S. W., He, C., & Yu, X. P. (2018). Distribution and the origin of invasive apple snails, Pomacea canaliculata and P. maculata (Gastropoda: Ampullariidae) in China. Sci Rep, 8, 1185. https://doi.org/10.1038/s41598-017-19000-7 Yao, F. C., Chen, Y. T., Liu, J. M., Zhang, J. E., Xiao, Z. H., Shi, Z. J., Chen, C., Qin, Z. (2024). Strategies of invasive snail Pomacea canaliculata during hibernation in rice fields of south China: Effects of body size, sex, and soil depth. Pest Manage. Sci. https://doi.org/10.1002/ps.8327 Ye, M., Song, Y. Y., Long, J., Wang, R. L., Baerson, S. R., Pan, Z. Q., Zhu-Salzman, K., Xie, J. F., Cai, K. Z., Luo, S. M., & Zeng, R. S. (2013). Priming of jasmonate-mediated antiherbivore defense responses in rice by silicon. Proc. Natl. Acad. Sci. U. S. A., 110, E3631-E3639. https://doi.org/10.1073/pnas.1305848110 Zhong, Z. H., Lin, L. Y., Chen, M. L., Lin, L. L., Chen, X. F., Lin, Y. H., Chen, X., Wang, Z. H., Norvienyeku, J., & Zheng, H. K. (2019). Expression divergence as an evolutionary alternative mechanism adopted by two rice subspecies against rice blast infection. Rice, 12, 12. https://doi.org/10.1186/s12284-019-0270-5 Zhu, D. D., Xue, B., Jiang, Y. S., & Wei, C. D. (2019). Using chemical experiments and plant uptake to prove the feasibility and stability of coal gasification fine slag as silicon fertilizer. Environ. Sci. Pollut. Res., 26, 5925-5933. https://doi.org/10.1007/s11356-018-4013-8 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7154936","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":509479565,"identity":"5de0d2cf-51e3-47ed-9a99-96b674e87e60","order_by":0,"name":"Wei Li","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Li","suffix":""},{"id":509479567,"identity":"6042e9eb-3524-48b4-adba-da5d76d359d1","order_by":1,"name":"Jingyi Zhao","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Jingyi","middleName":"","lastName":"Zhao","suffix":""},{"id":509479569,"identity":"5cadf09a-6542-4332-ae59-d6866f90a1dc","order_by":2,"name":"Hua Yu","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Yu","suffix":""},{"id":509479571,"identity":"540627ff-8bed-4f7f-9ec4-55ca133c8ace","order_by":3,"name":"Wen-Hong Dai","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Wen-Hong","middleName":"","lastName":"Dai","suffix":""},{"id":509479573,"identity":"daed753b-26d0-47ca-a63e-e88423998d8a","order_by":4,"name":"Yao-Bin Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYDACCTBpkwDhsRGvJY10LYdJ0GJwu/3h44Jf5/PkZ/cYMHwoO8zAP7uBgJY7Z4yNZ/bdLmacc8aAcca5wwwSdw4Q0HIjh02at+d2YrNEjgEzb9thBgOJBEJa0p8BtZxLbANp+UuclgQzaZ4fBxJ7QFoYidEiCfILb0Ny4gyJtIKDPefSeSRuENDCBwoxnj92ifNnJG988KPMWo5/BgEtCgeABGMbhANi8+BXDwTyDSDyD0F1o2AUjIJRMJIBAICfRWZgLi2KAAAAAElFTkSuQmCC","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":true,"prefix":"","firstName":"Yao-Bin","middleName":"","lastName":"Song","suffix":""},{"id":509479574,"identity":"c8e27210-c822-4d64-873b-f28b44275586","order_by":5,"name":"Ming Dong","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Dong","suffix":""}],"badges":[],"createdAt":"2025-07-18 07:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7154936/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7154936/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90679181,"identity":"c4102bc2-fa45-4e89-a571-5e0f827c3112","added_by":"auto","created_at":"2025-09-05 15:12:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":222900,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of silicon addition and nitrogen levels on tannin content (a), flavonoid content (b), total phenolic content (c) and force of fracture (d) of rice leaves. Values are presented as means ± SE. Bars with different letters indicate significant difference (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). The legends: Si addition refers to the addition of silicon while control indicates no silicon addition.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7154936/v1/b02fc46cd731de641fcd85af.png"},{"id":90679523,"identity":"6b977d53-946a-4842-8ef0-69fa3a44931b","added_by":"auto","created_at":"2025-09-05 15:20:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":213087,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of silicon addition and nitrogen levels on leaf consumption (a), RGR (b), weight gain (c) and cellulase activity (d) of apple snails. Values are presented as means ± SE. Bars with different letters indicate significant difference (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). The legends: Si addition refers to the addition of silicon while control indicates no silicon addition.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7154936/v1/a4d3c0fe9037a7101c436002.png"},{"id":90679182,"identity":"33f88d9e-3a86-4fcb-a169-9807e8091346","added_by":"auto","created_at":"2025-09-05 15:12:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":279498,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of silicon addition and nitrogen levels on leaf carbon content (a), leaf nitrogen content (b), leaf sulfur content (c), leaf silicon content (d) and leaf C/N ratio (e) of rice leaves. Values are presented as means ± SE. Bars with different letters indicate significant difference (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). The legends: Si addition refers to the addition of silicon while control indicates no silicon addition.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7154936/v1/9e5af6dc2500cd4bb6a8aaea.png"},{"id":90679183,"identity":"e819e1bf-9991-426e-9a30-1173538d7ac7","added_by":"auto","created_at":"2025-09-05 15:12:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":199805,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of silicon and nitrogen additions on relative chlorophyll content (a), total biomass (b) shoot mass (c) and root mass (d) of rice. Values are presented as means ± SE. Bars with different letters indicate significant difference (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05). The legends: Si addition refers to the addition of silicon while control indicates no silicon addition.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7154936/v1/a612f3e36867e00b8de010f8.png"},{"id":90679186,"identity":"093d3fac-5982-4444-9921-3bbcc823eb78","added_by":"auto","created_at":"2025-09-05 15:12:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":216975,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of silicon addition and nitrogen addition on rice's resistance characteristics and leaf element content, as well as the RGR and cellulase activity of\u003cem\u003e \u003c/em\u003eapple snails, were analyzed using a structural equation model. Red and green arrows represent significant positive and negative pathways, and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. R\u003csup\u003e2\u003c/sup\u003e represents the proportion of variance that is explained by each dependent variable in the model. The model's Chi-square value was 29.605, with a \u003cem\u003ep\u003c/em\u003e-value of 0.057. The AIC value was 797.9 and the GFI was 0.97.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7154936/v1/2cfc56fdbc8ddeb019588287.png"},{"id":93917736,"identity":"5c5cc1ed-f91c-444b-bd41-d21eb723dd64","added_by":"auto","created_at":"2025-10-20 09:08:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2299465,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7154936/v1/4056c6fd-f3e6-4885-bc85-a98d0dc11fd0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Si and N addition on Oryza sativa and its invasive grazer apple snails (Ampullariidae): resistance traits and feeding metrics","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eApple snail (Ampullariidae), the freshwater snail native to South America and invasive in Asia, is listed among the top 100 of the world's worst invasive species (Lowe et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). This snail primarily feeds on aquatic plants and crops (Hayes et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). This snail negatively affects the biomass of wild macrophytes, water quality, and algal production in the invasive range (Carlsson et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Carlsson \u0026amp; Lacoursi\u0026egrave;re, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The snail has caused enormous damage to wetland ecosystems and agricultural production, especially the rice planting area ((Horgan et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Jiang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e); Constantine et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The snail primarily consumes vegetables and aquatic plants, which can lead to a decrease in the shoot biomass and vegetation cover of the communities it invades (O'Neil et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This activity could result in a 50% reduction in agricultural production (Panda et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), ultimately diminishing the ecosystem services of wetlands and farmlands. This snail causes significant economic losses in its non-native habitat, including China (Yang et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Due to China's diverse geographical landscapes and varied climatic conditions, the country is vulnerable to invasions by apple snail (Wan \u0026amp; Yang, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In China, apple snail has invaded the main areas of rice plantations ranging from the Yangtze River Basin, leading to huge crop production cuts (Horgan et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xiao et al., 2023). By 2006, the area affected by apple snails in rice fields had exceeded 300,000 hectares, resulting in a direct loss of up to 12.5\u0026nbsp;million dollars (Li et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The snail caused the most damage to 21-day-old transplanted rice seedlings, which can result in up to 100% damage (Sin, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The snail can constantly damage the rice. After the rice is harvested, snails can conceal themselves in the mud, posing a threat to the rice crop in the following year (Yao et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Unfortunately, massive physical and chemical measures used to eradicate apple snail in invaded ranges have shown limited efficiency (Yam et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWetlands serve as reservoirs abundant in amorphous silicon, playing a significant role in shaping the global biogeochemical cycle of this element (Struyf \u0026amp; Conley, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Struyf et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Notably, the concentration of silicon found in the tissues of wetland plants frequently surpasses 1%, potentially exerting an influence on the nitrogen cycling processes within these plants (Schoelynck et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Schaller et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Silicon is recognized as a beneficial element for plants, particularly grasses, not only due to its high dry-weight concentration (Yamamoto et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), but also for its vital role in mitigating abiotic and biotic stress (Reynolds et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; de Tombeur et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Silicon can help plants mitigate damage caused by herbivores in multiple ways. Firstly, silicon can enhance plant defense with the deposition of inorganic amorphous silicon oxide (SiO\u003csub\u003e2\u003c/sub\u003e) phytoliths in the epidermis of plant tissues (Ma, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). For example, Johnson et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) found feeding plants with silicon resulted in a 50% reduction in the growth rate of an aphid (\u003cem\u003eRhopalosiphum padi\u003c/em\u003e). On the other hand, silicon can also activate the jasmonate acid and salicylic acid metabolism defense pathways (Ye et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Jasmonate acid metabolic defense pathways are often associated with leaf damage by herbivores (Browse \u0026amp; Howe, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Moreover, silicon supports the production of defensive secondary metabolites in plants and boosts the activity of oxidation-related enzymes (Gomes et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). This results in a toxic impact on herbivores and lowers their preference for these plants. In addition, silicon can improve the release of volatile organic compounds by plants, which can attract the natural enemies of herbivores (Kvedaras et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and interfere with the recognition of the host plants by herbivores (Veromann et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Therefore, we expect silicon could help rice alleviate the herbivory stress of animal pests such as invasive apple snail.\u003c/p\u003e\u003cp\u003eNitrogen is essential for plant growth and reproduction (Chen et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Nitrogen plays a critical role in herbivores. The growth of herbivores is significantly affected by the consumption of high-nitrogen food (Tao \u0026amp; Hunter, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Due to overloading reactive nitrogen from food production and energy production (Galloway et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), most ecosystems suffer from eutrophication (Xiao et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Previous studies found that excess nitrogen deposition may reduce defense costs (Jamieson et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Xiao et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). An increase in nitrogen availability can lead to changes in their secondary metabolites (Tao \u0026amp; Hunter, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), which are associated with plant resistance to herbivores. Jamieson \u0026amp; Bowers (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) found that the growth of \u003cem\u003eCalophasia lunula\u003c/em\u003e in \u003cem\u003eLinaria dalmatica\u003c/em\u003e was promoted by nitrogen enrichment. Plants have evolved to balance the trade-offs among growth/, reproduction and defense (Karasov et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, available nitrogen concentration could significantly affect the silicon uptake of plants because silicon is considered a cheap defensive substance (Johnson et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, a trade-off exists between silicon and nitrogen accumulation in Si-accumulating plant species, potentially having significant implications for their defense mechanisms against insect herbivores (Wu et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, we did not know if there are interactive effects of silicon and nitrogen on the growth of rice and its invasive feeder apple snails.\u003c/p\u003e\u003cp\u003eIn this study, we conducted a four-month cultural experiment in which rice plants experienced three levels of nitrogen supply combined with two levels of silicon addition.\u003c/p\u003e\u003cp\u003eThen, we fed the apple snails with rice leaves in different treatments for a week in the laboratory. We propose the following scientific questions: (1) Does silicon addition under different nitrogen levels affect the growth and development of rice? (2) Will altering nitrogen levels affect silicon's ability to defend rice against apple snail when feeding on silicon-treated rice leaves?\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Experimental design\u003c/h2\u003e\u003cp\u003e\u003cb\u003e(1) Experiment with N and Si additions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFrom March 15 to July 2, 2022, we conducted preliminary plant cultivation in the greenhouse of the Xiasha Campus of Hangzhou Normal University (30\u0026deg;32\u0026prime;N, 120\u0026deg;40\u0026prime;E), Hangzhou, Zhejiang, China. The greenhouse has an average temperature of 25 ℃. Rice seeds from the Chinese Academy of Agricultural Sciences were disinfected with alcohol. We then placed the seeds on wet filter paper until they began to take root. Once the seeds were rooted, we transferred them to a seedling box filled with peat soil. When the seedlings had grown to approximately 10 cm in height, we planted them in pots filled with two liters of agricultural field soil. The experiment employed a factorial design with six treatments comprising three nitrogen levels via urea (0.72, 1.44, and 4.76 mM for low, medium, and high nitrogen, respectively) combined with and without 1.5 Mm Si addition supplied as sodium silicate, each treatment replicated 12 times using a total of 72 rice seedlings (Wu et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The treatments without silicon addition received the corresponding concentration of sodium chloride to ensure a consistent soil osmotic pressure (Johnson et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We added sodium chloride to the group without silicon addition to ensure the consistency of sodium ions. The soil was consistently submerged below the water surface during the experiment. Silicon and nitrogen were added once a week for 2 months. Two months of treatment cultures were performed to prevent the negative effects of short treatment duration. We harvested them all two months after planting when the rice seedlings had developed 6 to 8 leaves. Six seedlings from each treatment group were utilized to determine plant indicators. The remaining six seedlings were exclusively used for the snail feeding experiment.\u003c/p\u003e\u003cp\u003e\u003cb\u003e(2) Snail feeding experiment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA snail-feeding experiment was conducted from July 2 to July 8. The apple snails were fed with leaves of the harvested rice plants, which had previously experienced the six treatments in the plant growth experiment. One month before the feeding experiment started, apple snails of similar size were collected from the stream in Hangzhou, Zhejiang, China (30\u0026deg;32\u0026prime;N, 120\u0026deg;1\u0026prime;E) and then placed in the laboratory culture with lettuce as food. The provision of lettuce was reduced a week before and stopped two days before the feeding experiment started.\u003c/p\u003e\u003cp\u003eAt the beginning of the experiment, we selected 36 healthy adult apple snails with similar size. Each snail was carefully wiped clean to remove moisture before being weighed. The snails were then randomly divided into six groups, with each group containing six animals. Each snail was placed in its own circular container, resulting in a total of 36 containers being used for the experiment. Each group was then given six treated rice leaves to feed on for a week. Timely replaced the rice leaves every day and recorded leaf loss, and then weighed again to ensure an increase in apple snails\u0026rsquo; weight. After that, all apple snails were stored in a -80 ℃ refrigerator until determining the cellulase activity of apple snail.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Plant sample collection and analysis\u003c/h2\u003e\u003cp\u003eThe relative chlorophyll content (SPAD) of the rice leaves was measured using a portable chlorophyll fluorometer (Minolta SPAD-502Plus) when the rice reached six to eight leaves. We picked three well-grown rice leaves for every plant to assess the relative chlorophyll content. The mean chlorophyll value from these three leaves was considered as the plant's relative chlorophyll content. Then half of the rice plants were harvested and the fresh weight of their different parts was determined in the laboratory. The harvested rice was rinsed with water, and then the surface moisture of the rice was absorbed using paper towels. The rice was divided into aboveground and underground parts. We used an electronic universal material testing machine (Instron 3343) to determine the force of fracture of rice leaves. The rice leaves with similar physiological conditions were fixed vertically to the machine and pulled at a constant speed from both ends until they broke. The maximum force at fracture was recorded, and overall leaf toughness was estimated using blade thickness, width, and the maximum force at fracture (Johnson et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The plant samples were dried in an oven set at 60\u0026deg;C until they reached a constant weight. For each treatment group, the biomass of the six rice plants was measured by an electronic balance.\u003c/p\u003e\u003cp\u003eThe dried rice leaves were ground using a mixer ball mill (MM400, Retsch, Germany). The ground leaves were subjected to determinants of carbon (C), nitrogen (N), and sulfur (S) content using an element analyzer (vario PYRO cube; Elemental, Germany) (Hu et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Flavonoid content was extracted from about 0.02 g of rice leaves with 60% ethanol and the corresponding content by using a spectrophotometer using the method of Lin et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The amount of flavone in the extract was measured using a spectrophotometer with the sodium nitrite-aluminum nitrate method. Tannin content was extracted from 0.1 g of rice leaves using ultrapure water at 80 ℃, to determine the tannin content according to the method by Makkar (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The tannin content in the extract was determined using the Folin-Ciocalteu colorimetric method and measured with a spectrophotometer. The total phenols of rice leaves were extracted from about 0.1 g using 60% ethanol to determine the total phenolic content according to the method of Cheng et al.(2013). Total phenol in the extract was determined using the Folin-Ciocalteu colorimetric method and measured with a spectrophotometer. Plant silicon content was determined by using sodium hydroxide solution at high temperature, according to the determination of rice leaf silicon content by the molybdenum blue colorimetric method (Kraska \u0026amp; Breitenbeck, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). For each element or organic compound, we used six rice plants for each treatment group and conducted the experiment in three replicates.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Snail sample analysis\u003c/h2\u003e\u003cp\u003eThe apple snails were placed in an ultra-low temperature refrigerator and thawed in an ice water bath, out of its intestines and stomach, using a mortar for grinding homogenate on ice. Cellulase of the snail's intestines and stomach was extracted using a sodium citrate buffer containing the protease inhibitors. Cellulase activity was measured according to the method of Kim et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Relative growth rate (RGR) was defined for the snails by Johnson et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e):\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{RGR(}\\text{mg}\\text{}{mg}^{-1}\\text{}{day}^{-1}\\text{)=}\\left(\\frac{\\text{F}\\text{i}\\text{n}\\text{a}\\text{l}\\:\\text{m}\\text{a}\\text{s}\\text{s}\\:\\text{a}\\text{f}\\text{t}\\text{e}\\text{r}\\:\\text{f}\\text{e}\\text{e}\\text{d}\\text{i}\\text{n}\\text{g}-\\text{I}\\text{n}\\text{i}\\text{t}\\text{i}\\text{a}\\text{l}\\:\\text{m}\\text{a}\\text{s}\\text{s}\\:\\text{b}\\text{e}\\text{f}\\text{o}\\text{r}\\text{e}\\:\\text{f}\\text{e}\\text{e}\\text{d}\\text{i}\\text{n}\\text{g}}{\\text{I}\\text{n}\\text{i}\\text{t}\\text{i}\\text{a}\\text{l}\\:\\text{m}\\text{a}\\text{s}\\text{s}\\:\\text{b}\\text{e}\\text{f}\\text{o}\\text{r}\\text{e}\\:\\text{f}\\text{e}\\text{e}\\text{d}\\text{i}\\text{n}\\text{g}\\bullet\\:\\text{d}\\text{a}\\text{y}\\text{s}}\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Data analyses\u003c/h2\u003e\u003cp\u003eTwo-way ANOVA was used to test the effects of nitrogen concentration and silicon imposed on plant biomass, tannin content, flavonoid content, phenol content, silicon content, plant mechanical force, leaf loss, and cellulase activity, principal effect analysis. We conducted multiple comparisons to explore the significant differences between different treatment groups subjected to silicon addition and nitrogen treatment. Duncan's Multiple Range Test was utilized to pinpoint significant differences between silicon additions and nitrogen treatments, establishing a significance threshold at a p-value of less than 0.05. All analyses were performed in SPSS 20.0.\u003c/p\u003e\u003cp\u003eWe utilized a structural equation model through linear regression to investigate the impact of silicon and nitrogen addition on leaf resistance, leaf element content, and snail feeding metrics. This model could examine the effects of various treatments on plant growth, leaf resistance, rice element content, and the influence of leaf resistance and element content on snail feeding metrics in apple snails. However, we excluded several indicators due to collinearity in constructing the structural equation model, including all rice plant growth, leaf sulfur content and leaf C/N ratio. To obtain a \u003cem\u003ep\u003c/em\u003e-value greater than 0.05 for the entire model, weight gain and leaf consumption of apple snails were excluded. The structural equation model was constructed using the \u003cem\u003elavaan\u003c/em\u003e package (Rosseel, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) in R version 4.3.3 (R Core Team, 2024).\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Leaf resistance characteristics under silicon and nitrogen addition\u003c/h2\u003e\u003cp\u003eSilicon addition significantly affected flavonoid content (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and force of fracture (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and nitrogen levels significantly affected total phenolic content (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011), tannin content (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) and force of fracture (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Silicon addition and nitrogen levels showed an interaction effect on tannin content (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Silicon addition increased the tannin content in middle nitrogen levels while showing the opposite results in high nitrogen levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Silicon addition significantly increased the flavonoid content in all nitrogen levels while the medium showed the lowest flavonoid content (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). There were no significant effects of silicon addition on total phenolic content (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Silicon addition and high nitrogen levels significantly increased the force of fracture in rice leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). With the increasing of nitrogen levels, there were significant increase in total phenolic content and force of fracture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eLeaf resistance characteristics, snail feeding metrics, leaf element content, and plant growth responses to silicon addition and nitrogen levels from Two-way ANOVA. Bold font indicates \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eSilicon (Si)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eNitrogen (N)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eSi \u0026times; N\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRelated variables\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLeaf resistance characteristics\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal phenolic content\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.149\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.292\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.254\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.011\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.897\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTannin content\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.478\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.495\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.030\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.003\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12.363\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFlavonoid content\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e37.217\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.074\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.143\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.262\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.298\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eForce of fracture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e47.416\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.676\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.793\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.462\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSnail feeding metrics\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeaf consumption\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.477\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.026\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.698\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.361\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.272\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRelative growth rate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e22.227\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.114\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.059\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.742\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.081\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWeight gain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13.637\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.664\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.086\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.907\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.070\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCellulase activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19.946\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.708\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.083\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLeaf element content\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeaf carbon content\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16.604\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.153\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.895\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.949\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.398\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeaf nitrogen content\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23.120\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.482\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.020\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.192\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.826\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeaf sulfur content\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25.508\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.351\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.707\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.753\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.480\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeaf silicon content\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23.823\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e22.407\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.150\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.006\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeaf C/N ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.737\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.038\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.132\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.136\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.374\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.691\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePlant growth\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRelative chlorophyll content (SPAD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50.203\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56.252\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.081\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.142\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal mass\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e94.289\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e47.264\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e25.937\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eShoot mass\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60.645\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.744\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16.007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRoot mass\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e625.700\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e386.760\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e201.200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Snail feeding metrics under silicon and nitrogen addition\u003c/h2\u003e\u003cp\u003eSilicon addition affected leaf consumption (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026), RGR (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), weight gain (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and cellulase activity (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) of snails. Nitrogen levels significantly affected cellulase activity (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and did not significantly affect other feeding metrics. There was no significant interaction between nitrogen levels and silicon addition on snail feeding metrics (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Silicon addition significantly decreased leaf consumption, RGR and weight gain of apple snails in low and high nitrogen levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b, c). Silicon addition decreased the cellulase activity of apple snails, and low and high nitrogen levels increased the cellulase activity of apple snails (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Leaf element contents under silicon and nitrogen addition\u003c/h2\u003e\u003cp\u003eSilicon addition significantly affected leaf N (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), C (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), S (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), Si (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and leaf C/N ratio (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.038) while nitrogen levels significantly affected leaf N (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.020) and leaf Si (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and only showed interacted effects on leaf Si (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Silicon addition significantly decreased leaf carbon and nitrogen content (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b), but significantly increased leaf sulfur content in all nitrogen levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Silicon addition significantly increased leaf silicon content in low and medium nitrogen levels, and high nitrogen levels showed the lowest leaf silicon content (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Silicon addition increased the leaf C/N ratio in medium nitrogen levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Plant growth under silicon and nitrogen addition\u003c/h2\u003e\u003cp\u003eSilicon addition and nitrogen level significantly affected relative chlorophyll content, total mass, shoot mass and root mass of rice (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). There were significant interactions between nitrogen levels and silicon addition on the total mass, shoot mass and root mass of rice (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but relative chlorophyll content (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.142) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Silicon addition and nitrogen levels significantly increased relative chlorophyll content of rice in all nitrogen levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Silicon addition significantly increased total mass, shoot mass and root biomass in low and high nitrogen levels, but not in medium nitrogen level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c, d). Generally, relative chlorophyll content total mass, shoot mass and root mass of rice increased with the increasing nitrogen level,\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Effect of silicon and nitrogen addition on leaf resistance characteristics and snail feeding metrics\u003c/h2\u003e\u003cp\u003eBoth silicon addition (standardized path coefficient \u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.73, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and nitrogen addition (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.42, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) showed a positive effect on the force of fracture. The combined effects of silicon, nitrogen additions, and leaf silicon content jointly explained 70% of fracture of force. Nitrogen addition (standardized path coefficient \u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.39, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) showed a positive effect on the total phenolic content. Silicon addition (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.74, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) positively affected the flavonoid content. Moreover, silicon addition, nitrogen addition and leaf silicon content are jointly explained 51% of flavonoid content. Silicon addition (\u003cem\u003eβ\u003c/em\u003e = -0.38, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) played a negative effect on tannin content while nitrogen addition (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.39, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and leaf silicon content (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.63, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) played a positive effect. Silicon addition (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.46, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) showed a positive effect on leaf silicon content while nitrogen addition (\u003cem\u003eβ\u003c/em\u003e = -0.53, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) showed a negative effect. Silicon addition and nitrogen jointly explained 50% of leaf silicon content. Force of fracture (\u003cem\u003eβ\u003c/em\u003e = -0.38, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) played a negative effect on the RGR of apple snails. In addition, leaf silicon showed negative effects on the RGR (\u003cem\u003eβ\u003c/em\u003e = -0.36, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and cellulase activity (\u003cem\u003eβ\u003c/em\u003e = -0.67, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) of apple snails.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eSilicon additions led to a change in plant physical traits and the levels of the secondary metabolites proceeded to impact the ability of plants to defend against insects. The results obtained here indicated that the resistance of rice to invasive snail was affected by silicon and nitrogen addition.\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Effects of silicon on rice growth in nitrogen treatments\u003c/h2\u003e\u003cp\u003eMany studies have shown that silicon is essential in plant response to biotic and abiotic stresses (Reynolds et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; de Tombeur et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Lack of nitrogen is a common stress in plants, exacerbated by human activity, leading to high nitrogen levels in plants (Dentener et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Silicon significantly improved the rice biomass both at low and high nitrogen levels in the results. Nitrogen is one of the most essential mineral elements that plants require for growth and to achieve high crop yield (Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It has been reported that silicon can increase nitrogen uptake in an unexpected nitrogen addition in rice (Deus et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, silicon addition can up-regulated N-assimilation genes (e.g., \u003cem\u003eOsGS2\u003c/em\u003e, \u003cem\u003eOsFd-GOGAT\u003c/em\u003e, \u003cem\u003eOsNADH-GOGAT2\u003c/em\u003e, \u003cem\u003eOsGDH2\u003c/em\u003e, and \u003cem\u003eOsNR1\u003c/em\u003e) (Wu et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, the silicon content in plant leaves is affected by the amount of nitrogen supplied to the plants, as shown in our results. Wu et al. (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) found that high nitrogen levels have a negative effect on the transcript levels of \u003cem\u003eOsLsi1\u003c/em\u003e and \u003cem\u003eOsLsi2\u003c/em\u003e, which directly affect the absorption of silicon. Moreover, in this experiment, silicon addition remarkably improves the leaf sulfur content in rice, while sulfur can improve the utilization of nitrogen in plants (Bloem et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIncreasing nitrogen could induce soil acidification, resulting in asynchronous responses across micronutrients in both plants and soil. Sodium metasilicate increases soil pH (Li et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), which can relieve soil acidification and asynchrony responses across micronutrients by silicon addition in rice. In addition, high nitrogen lowers the leaf silicon concentration in rice, while silicon relieves the effect. However, in middle nitrogen, silicon did not affect plant biomass, while silicon improved the leaf C/N ratio in rice. The C/N ratio is commonly used as an indicator of plant growth and flowering potential, and a high C/N ratio is believed to have a better nitrogen use efficiency (Gren, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). This implies that silicon increases the growth of rice, which could be explained by two potential mechanisms. On the one hand, silicon addition impacted the relative chlorophyll content of rice leaves under all nitrogen conditions, consequently improving the photosynthetic rate. On the other hand, silicon addition increased the leaf sulfur content, leading to an improvement in nitrogen availability, making up for the decrease in leaf nitrogen concentration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Effects of silicon in rice resistance under nitrogen treatments\u003c/h2\u003e\u003cp\u003eTo resist to herbivores, plants evolved diverse mechanisms to fight against herbivores in various chemical and physical traits (Aljbory \u0026amp; Chen, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Silicon usually helps plants against herbivores in mechanical barriers and shows a high correlation with biochemical attributes (Fawe et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Silicon is deposited in leaf hairs, trichomes and spines, which could influence the physical traits of the leaves (Hartley et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the results, silicon addition improved the force of fracture in rice leaves. Another research showed that an increase in silicon content in leaves resulted in an increased force required for a needle to penetrate the sheath (Schurt et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). By covalently crosslinking with hemicelluloses, silicon addition enhanced the mechanical properties of cells (He et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Moreover, it has been approved that silicon can activate the jasmonate acid signaling pathway, which is associated with plant defense against herbivores, leading to an improvement in defense-related capabilities. Previous studies reported that silicon could promote the production of defense-related enzymes polyphenol oxidase (PPO), superoxide dismutase (SOD), peroxidase (POD) and so on (Peter et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). These enzymes play an essential role in plant defense which can directly injure herbivores or result in an increase of secondary metabolic compounds such as flavonoids and phenolics which are harmful or toxic to herbivores (Han et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSulfur is a necessary trace element for plant growth and development (Aziz et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It is commonly found in sulfur in proteins, peptides, metabolites, and enzymes (Falk et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Sulfur-containing metabolites and proteins in plant heavy metal detoxification, antioxidants, and maintenance of protein-reducing states play an indispensable role. Recent research showed silicon played a positive effect in the sulfur transporter \u003cem\u003eHvST1;1\u003c/em\u003e led to a higher uptake of SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e in \u003cem\u003eHordeum vulgare\u003c/em\u003e cv. Irina is under collective stress (Maillard et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Another study demonstrated that silicon increased sulfur uptake and utilization in basil (\u003cem\u003eOcimum basilicum\u003c/em\u003e) (Barreto et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This research showed silicon improved sulfur accumulation in rice in sulfur-rich conditions. Silicon addition significantly increases the production of total phenolic, flavonoid, and force of fracture at all nitrogen levels. Additionally, rice supplemented with silicon in high nitrogen levels shows a reduction in tannin content. The environment is more likely to affect the tannin concentration in plant leaves, and the temperature and soil nitrogen concentration significantly affect the tannin content (Fine et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). This study provided experimental evidence that silicon addition improved the defense level of rice with different nitrogen levels.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Effects of feeding rice leaves experiencing silicon and nitrogen addition on apple snails\u003c/h2\u003e\u003cp\u003eSilicon decreases the growth of apple snails and the weight of the leaves that the apple snail eats. The acquisition of nitrogen is the most critical trait for herbivores' growth, while intake in leaves at high silicon reduced leaf digestibility to limit the nitrogen acquisition in \u003cem\u003eSpodoptera exempta\u003c/em\u003e (Massey \u0026amp; Hartley, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The growth of apple snails was determined by the plant nutrients, especially nitrogen (Qiu et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Silicon addition decreased leaf nitrogen content at all nitrogen levels, as indicated by our results. In addition, foods with a low carbon-to-nitrogen ratio can promote the growth of snails (Sharfstein \u0026amp; Steinman, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). According to our findings, Silicon addition resulted in a higher carbon-to-nitrogen ratio in rice leaves across all nitrogen levels. Silicon can also disrupt the integrity of epidermal cells in the gut of herbivores, affecting insect digestion (dos Santos et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Moreover, silicon addition increases the activity and production of a variety of enzymes such as proteinase and lipoxygenases (Singh et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Up-regulating defense-related genes results in an increase in the activity of phytohormones and the production of defense-related secondary metabolites such as phenolics, tannins and flavonoids (Hall et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe phenol in green plants is an important trait of plant defense against herbivores (Dixit et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Phenolic compounds can bind with proteins, leading to the damage of biological enzymes which can reduce the absorption of amino acids by herbivores as well as protein hydrolysis (Appel \u0026amp; Schultz, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Phenolics in plants play a crucial role in the growth of the snail. Consuming plant leaves containing high levels of phenolics will inhibit snail growth and may cause death (Qiu et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Tannins act as insect repellents for herbivores by containing toxins that can affect their growth and survival (Mazid et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Tannins significantly decreased the growth of herbivores by limiting protein digestion efficiencies (Feeny, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1968\u003c/span\u003e). Flavonoids are an effective means of plant defense against generalists and are important in plant host recognition and inhibition of herbivore feeding (Simmonds, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). At the results from structural equation modeling analysis, it was determined that flavones and tannins had no significant impact on apple snails' RGR and cellulase activity. The growth of apple snails is significantly impacted by phenolic content. In this case, the content of tannin and flavonoid did not significantly impact the cellulase activity of apple snails. On the other hand, the feeding experiment was conducted for only one week. The impact of tannin content and flavonoid on the RGR of apple snails has not been proven yet. In the results, silicon addition decreased the activity of cellulase of apple snails, which is a group of hydrolytic enzymes that can digest the plant cell wall and decompose the biological macromolecules into digestible, nutritive oligomers and monomers (Pauchet et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In addition, silicon addition increased phenolic, flavonoid content, and force of fracture in rice with all nitrogen, leading to a reduction in the leaf consumption of apple snails and showing a negative effect on the growth of apple snails.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e4.4 Implications for control of apple snail damage in invaded rice agricultural systems\u003c/h2\u003e\u003cp\u003eRice is a staple crop providing food for almost half of the world's population (Zhong et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Ensuring the ordinary production of rice is crucial for global food security. However, biological invasions cause great damage to farmland ecosystems, resulting in a dramatic decline in food production (Bradshaw et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Paini et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The invasion of the apple snail in Southeast Asian countries has significantly reduced rice production. The invasive snail prefers to feed on rice seedlings, as young plants are more consistent with their diet (Naylor, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). The snail feeds on the base of rice leaves before cutting down the entire plant, ultimately killing the rice seedling (Schnorbach, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Therefore, controlling biological invasions is crucial for maintaining high rice yields and promoting sustainable rice agriculture systems. This study found that silicon can effectively reduce the negative effects caused by apple snail invasions in rice agriculture by promoting rice growth and defense. The concentration of silicon is crucial for plant growth and development. However, there were significant differences in the silicon uptake capacity of plants, both in terms of species and genotypes (Ma \u0026amp; Yamaji, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Selecting rice varieties that have a high capacity to absorb silicon is an essential step (Meharg \u0026amp; Meharg, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The role of silicon in the rice ecosystem, both in terms of input and output, is crucial and cannot be overlooked. Slag that contains a high amount of silicon has been utilized as a fertilizer for rice crops (Zhu et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). There is a significant amount of silicon in rice stalks and other parts. Therefore, returning these parts to the rice agriculture systems is necessary to maintain the silicon cycle (Meharg \u0026amp; Meharg, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Future research should focus on identifying suitable rice varieties and scientifically managing the silicon cycle in rice fields. This approach will contribute significantly to the effective control of apple snail damage in invaded rice agricultural systems.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eSilicon addition promoted the growth of the rice but inhibited the growth of the snails. Silicon addition increased the production of secondary metabolites in rice, leading to enhanced resistance. The addition of silicon reduces the nitrogen content and improves the phenolic levels of rice leaves. Apple snails thrive on food high in nitrogen and low in phenolics. Nitrogen addition increases the force of fracture and the content of phenolics. However, nitrogen addition limited the rice\u0026rsquo;s absorption of silicon, and there was an apparent trade-off between the rice\u0026rsquo;s absorption of nitrogen and silicon. On the other hand, nitrogen addition increases leaf nitrogen content, which may lead to an increase in the growth of the apple snail. Our results suggest that silicon supply is expected to become a more environment-friendly way to alleviate the harm of the invasive snail effectively. Moreover, using nitrogen fertilizer legitimately in rice fields is essential for controlling the apple snail.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by\u0026nbsp;NSFC (grant no. 32371580). We thank Y. Y. Shen, Y. Chen, J. Yang, K. Shi, W. Q. Li, J. J. Shi and H. J. Zou for their kind help with the experiment. We are especially grateful to Z. Q. Cao for his help with statistical analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by Natural Science Foundation of China (Grant numbers [32371580]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAljbory, Z., \u0026amp; Chen, M. S. (2018). Indirect plant defense against insect herbivores: A review. Insect Sci., 25(1), 2-23. https://doi.org/10.1111/1744-7917.12436\u003c/li\u003e\n\u003cli\u003eAppel, H. M., \u0026amp; Schultz, J. C. (1992). 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Res., 26, 5925-5933. https://doi.org/10.1007/s11356-018-4013-8 \u003cbr\u003e \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"biological invasion, herbivory, plant defense, rice, silicon","lastPublishedDoi":"10.21203/rs.3.rs-7154936/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7154936/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSilicon, as a crucial element of plants, may contribute to plants\u0026rsquo; resistance to biotic stresses like herbivore feeding. In tropical and subtropical Asia, apple snail (Ampullariidae) is an invasive aquatic herbivorous snail in agricultural and wetland ecosystems, like paddy field, which suffer from heavy eutrophication of nitrogen. However, little is known about the potential effects of feeding rice leaves treated with silicon and nitrogen on the feeding metrics of apple snails. We conducted a greenhouse experiment to examine the effect of silicon and nitrogen addition on rice seedlings as grazed by apple snails, in which two levels (0 and 1.5 mM) of silicon addition and three levels (0.72, 1.44 and 5.76 mM) of nitrogen addition were used. We measured plant growth, leaf element contents and leaf defense characteristics of rice. We determined the snail feeding metrics after feeding in different rice leaves for one week. Silicon addition increased plant mass in the low and high nitrogen addition and increased the C/N ratio only in the middle nitrogen addition. Silicon addition significantly decreased the growth of apple snails while significantly increasing flavonoid content and the force of fracture of rice leaves in all the nitrogen levels. Silicon addition increased the tannin content of rice leaves in the middle nitrogen addition but decreased in high nitrogen treatment. Moreover, silicon addition increased the leaf sulfur content of rice at all three levels of nitrogen addition. Silicon addition could improve the defense of rice against invasive herbivory by apple snails, which sheds insights on the protection of wetland crops in the context of control of eutrophication and biological invasion.\u003c/p\u003e","manuscriptTitle":"Effects of Si and N addition on Oryza sativa and its invasive grazer apple snails (Ampullariidae): resistance traits and feeding metrics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-05 15:12:15","doi":"10.21203/rs.3.rs-7154936/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"89786ccc-9eba-49ac-a799-28a99cc98fa5","owner":[],"postedDate":"September 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T09:08:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-05 15:12:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7154936","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7154936","identity":"rs-7154936","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}