Abstract:

addy field soils are important artificial wetland ecosystems related to the development of agriculture and environment. China is the largest rice producer and consumer in the world. However, rice cultivation regions in Southern China face a dual environmental challenge: soil heavy metals (e.g., Cd, Cu, Ni, and Zn) contamination and the widespread invasion of Pomacea canaliculata, which together heighten risks to agricultural safety and public health. Sources of heavy metals in paddy field soils could be anthropogenic activities, such as mining, smelting, and urbanization. Heavy metals contamination in paddy soils not only inhibit rice growth but also threaten food security and ecological integrity, through bioaccumulation in the food chain. Meanwhile, the invasive snail Pomacea canaliculata damages rice seedlings in Southern China and acts as an intermediate host for parasites such as Angiostrongylus cantonensis. Despite the widespread application of soil amendments such as biochar and lime to immobilize heavy metals and reduce their bioavailability, previous research has focused on soil-plant systems, overlooking the long-term physiological impacts on invasion snail Pomacea canaliculata. In this study, a field experiment was conducted in an acidic paddy soil contaminated with heavy metals in Hangzhou, Zhejiang Province. Treatments included control (CK), swine manure biochar (BC, 4.5 t·ha⁻¹), lime (LM, 1.0 t·ha⁻¹), and swine manure biochar combined with lime (BL, 4.5 t·ha⁻¹+1.0 t·ha⁻¹). The concentrations of bioavailable heavy metals in soil and total elements in nail muscle tissues were determined by inductively coupled plasma atomic emission spectrometry. The intestinal microbiota community structure of Pomacea canaliculata was characterized using 16S rRNA high-throughput sequencing, with functional profiling and heavy metals resistance gene prediction performed through PICRUSt2 and BacMet databases. Results showed that the application of biochar and lime significantly increased soil pH and EC values (p<0.05). Compared with the control, the treatments BC, LM and BL increased the soil pH by 0.48, 0.56, and 0.64 units, respectively. Among them, the combined application of biochar and lime was more effective at increasing soil pH than either biochar or lime applied individually. Compared with the control, BC treatment reduced the concentrations of bioavailable Cd, Cu, and Ni in soil by 26.45%, 70.91%, and 91.20%, respectively, while BL treatment reduced the concentrations of bioavailable Cd, Cu, Ni, and Zn by 43.20%, 75.50%, 56.44%, and 85.85%, respectively. Notably, BL most effectively immobilized Cd, correlating with the greatest soil pH increase. LM and BL treatments markedly inhibited Zn bioavailability (>85% reduction). Although both LM and BL treatments reduced the concentrations of bioavailable heavy metals in soil, their effects on heavy metal accumulation in snail tissues differed: the concentrations of Cd, Ni, Cu and Zn in the muscle tissue of Pomacea canaliculata in the control were 0.09, 0.70, 162.97 and 48.13 mg·kg⁻¹ after 30 days of culture. The concentrations of Cd, Ni, Cu and Zn in muscle tissue of Pomacea canaliculata treated with BC treatment were 0.13, 0.53, 214.88 and 48.69 mg·kg⁻¹. The concentrations of Cd, Ni, Cu and Zn in muscle tissue of Pomacea canaliculata in LM treatment were 0.07, 0.29, 124.98 and 40.09 mg·kg⁻¹, respectively. The concentrations of Cd, Ni, Cu and Zn in muscle tissue of Pomacea canaliculata treated with BL were 0.11, 0.37, 183.78 and 42.85 mg·kg⁻¹, respectively. The concentrations of Cd and Cu in muscle tissue of Pomacea canaliculata treated with BL increased by 20.83% and 12.77%, respectively, relative to the control. All the treatments raised the concentration of Ca in snails, but had no significant effect on the concentrations of K, Mg, and Na. There were significant differences in Shannon and Pielou indexes of intestinal flora of Pomacea canaliculata under BC, LM, and BL treatments. For BL treatment, the relative abundances of Planctomycetes and Verrucomicrobia decreased significantly, while the relative abundances of Firmicutes and Actinobacteria increased significantly. LEfSe analysis results showed that the relative abundance of a variety of potentially harmful bacteria in treatments LM and BC was higher. For example, the LM treatment contained potential pathogens in the genera of Geobacillus and Flavobacteriaceae, thereby increasing the potential infection risk of Pomacea canaliculata. Functional prediction showed that all treatments decreased the relative abundance of membrane transport, energy metabolism and carbohydrate metabolism pathways, indicating that the overall metabolic activity of functional groups of intestinal microbes in Pomacea canaliculata was significantly inhibited. BL treatment significantly increased the abundance of Cd- and Zn-resistance genes (e.g., czcB, czcC, and czcD). These findings suggest that, despite a reduction in the bioavailability of heavy metals in the soil following the application of biochar and lime, the accumulation of heavy metals in the muscle tissue of Pomacea canaliculata increased. This may be due to the interaction between the feeding habits of Pomacea canaliculata and the intestinal microenvironment. Pomacea canaliculata may directly ingest biochar particles that adsorbed heavy metals while feeding on soil. In the acidic intestinal environment, pH reduction facilitates the re-release of heavy metals from the biochar surface, thereby enhancing their bioavailability. As a pivotal component of host physiological regulation, alterations in gut microbial composition reflect the impact of environmental stress. An increase in Firmicutes and Actinobacteria may represent an adaptive response by the host to environmental stressors; however, the decline in specific degradative bacterial taxa suggests a reduction in nutrient absorption capacity and energy metabolism efficiency in Pomacea canaliculata. Functional gene analysis further confirmed that the application of biochar and lime aggravated the toxic stress of heavy metals on Pomacea canaliculata, resulting in its metabolic disorder. The study highlights that the application of biochar and lime can intensify heavy metal stress on invasive species of Pomacea canaliculata, thereby disrupting their gut microbiome composition and the associated metabolic functions. This work provides novel insights to support the integrated management of soil heavy metal contamination and invasive species control in paddy soil ecosystems. To elucidate the mechanisms by which biochar affects heavy metal accumulation and intestinal microbiota in invasive Pomacea canaliculata, future research should employ controlled experiments. We suggest future research areas including: 1) the type and application rate of biochar from various biomass feedstocks; 2) the influence of pyrolysis temperature on biochar's physicochemical properties and functional effects; and 3) the variation in response across different snail life stages.

Key words: Pomacea canaliculata, gut microbiota, heavy metal, biochar, lime, invasive species

摘要：

中国南方水稻种植区中土壤不仅存在重金属污染，其中广泛分布的入侵物种福寿螺同时加剧农业安全生产风险。当前土壤改良剂在稻田重金属污染治理中应用广泛，但其是否能影响福寿螺生理代谢从而抑制其生长尚不明确。通过野外稻田设置实验，探究生物炭（BC，4.5 t·hm⁻²）、石灰（LM，1.0 t·hm⁻²）及生物炭＋石灰复配处理（BL）施用在修复重金属污染土壤同时对福寿螺体内金属富集及肠道微生物群落的影响。结果表明，BC和BL处理使福寿螺肌肉组织中镉（Cd）的含量显著提高了40.63%和20.83%；铜（Cu）的含量显著提高了31.85%和12.77%。不同处理组下福寿螺肠道菌群的Shannon与Pielou指数均存在显著差异；NMDS分析显示BC、LM、BL组β多样性均与对照组（CK）存在差异。在BL处理下，肠道浮游菌门（Planctomycetes）、疣微菌门（Verrucomicrobia）相对丰度显著降低，而厚壁菌门（Firmicutes）、放线菌门（Actinobacteria）相对丰度显著升高。功能注释显示，所有处理组均使膜运输、能量代谢和碳水化合物代谢通路相对丰度降低，表明福寿螺肠道微生物功能类群的整体代谢活性受到了显著抑制，同时BL处理显著提升了Cd、锌（Zn）抗性基因的相对丰度。生物炭与石灰施用显著增强福寿螺肌肉组织重金属积累水平，影响其肠道微生物组成及功能基因丰度变化。

关键词: 福寿螺, 肠道微生物, 重金属, 生物炭, 石灰, 生物入侵