Toxicity of Pb continuous and pulse exposure on intestinal anatomy, bacterial diversity, and metabolites of Pelophylax nigromaculatus in pre-hibernation

Amphibians are often exposed to pulse pollution due to fluctuational inputs of pollutants in water. Traditional ecotoxicology research is mainly performed under constant exposure conditions, which is not consistent with the true environmental pollution. Frogs are sensitive to changes in water pollutants in pre-hibernation. Thus, to understand the toxicity difference to continuous and pulse exposure in environmental concentrations of Pb (100 μg/L), Pelophylax nigromaculatus adults were exposed to short-term treatments (8 days) in pre-hibernation. Individual mortality, intestinal anatomical structure, bacterial diversity, and metabolites were measured in a control group (CON), a Pb continuous treatment group (CEPb) and a Pb pulse treatment group (PEPb). The results showed that PEPb significantly increased individual mortality, compared to the control group and CEPb. PEPb induced pathological changes in the small intestinal tissues, such as mucosal erosion, swollen and distorted villi, large vacuoles, and the proliferation of goblet cells. In addition, PEPb altered the structure and diversity of intestinal bacteria, resulting in an increase in some pathogenic bacteria (e.g. Bacteroides and Ruminococcus) and a decrease in beneficial bacteria (e.g. Cetobacterium and Akkermansia). Both CEPb and PEPb significantly changed intestinal metabolites and metabolic pathways. Moreover, PEPb has a significant effect on the metabolism of amino acids by increasing the content of 5-Aminopentanoic acid, cis-4-Hydroxy-l-proline, Glycocholic acid, l-Alanine, and l-Isoleucine. We concluded that PEPb may lead to intestine impairment of P. nigromaculatus in pre-hibernation by inducing intestinal structural integrity destruction, bacterial imbalance, and metabolic dysfunction, resulting in a significant increase in mortality. The study provides new insights for understanding the intestinal responses of frogs to pulse metal exposure.