Natural communities of microbes inhabiting amphibian skin, the skin microbiome, are critical to supporting amphibian health and disease resistance. To enable the pro-active health assessment and management of amphibians on Army installations and beyond, we investigated the effects of acute (96h) munitions exposures to Rana pipiens (leopard frog) tadpoles and the associated skin microbiome, integrated with RNAseq-based transcriptomic responses in the tadpole host. Tadpoles were exposed to the legacy munition 2,4,6-trinitrotoluene (TNT), the new insensitive munition (IM) formulation, IMX-101, and the IM constituents nitroguinidine (NQ) and 1-methyl-3-nitroguanidine (MeNQ). The 96h LC50 values and 95% confidence intervals were 2.6 (2.4, 2.8) for ΣTNT and 68.2 (62.9, 73.9) for IMX-101, respectively. The NQ and MeNQ exposures caused no significant impacts on survival in 96h exposures even at maximum exposure levels of 3560 and 5285 mg/L, respectively. However, NQ and MeNQ, as well as TNT and IMX-101 exposures, all elicited changes in the tadpole skin microbiome profile, as evidenced by significantly increased relative proportions of the Proteobacteria with increasing exposure concentrations, and significantly decreased alpha-diversity in the NQ exposure. The potential for direct effects of munitions exposure on the skin microbiome were observed including increased abundance of munitions-tolerant phylogenetic groups, in addition to possible indirect effects on microbial flora where transcriptional responses suggestive of changes in skin mucus-layer properties, antimicrobial peptide production, and innate immune factors were observed in the tadpole host. Additional insights into the tadpole host's transcriptional response to munitions exposures indicated that TNT and IMX-101 exposures significantly enriched transcriptional expression within type-I and type-II xenobiotic metabolism pathways, where dose-responsive increases in expression were observed. Significant enrichment and increased transcriptional expression of heme and iron binding functions in the TNT exposures served as likely indicators of known mechanisms of TNT toxicity including hemolytic anemia and methemoglobinemia. The significant enrichment and dose-responsive decrease in transcriptional expression of cell cycle pathways in the IMX-101 exposures was consistent with previous observations in fish, while significant enrichment of immune-related function in response to NQ exposure were consistent with potential immune suppression at the highest NQ exposure concentration. Finally, the MeNQ exposures elicited significantly decreased transcriptional expression of keratin 16, type I, a gene likely involved in keratinization processes in amphibian skin. Overall, munitions showed the potential to alter tadpole skin microbiome composition and affect transcriptional profiles in the amphibian host, some suggestive of potential impacts on host health and immune status relevant to disease susceptibility.

居住在两栖动物皮肤上的自然微生物群落，即皮肤微生物组，对于支持两栖动物的健康和抗病能力至关重要。为了主动评估和管理两栖动物在陆军装置及其他地方的健康状况，我们调查了急性（96h）弹药暴露于小林蛙的影响（豹蛙）t和相关的皮肤微生物组，并与host宿主中基于RNAseq的转录组反应相结合。暴露于传统的2,4,6-三硝基甲苯（TNT）弹药，新的不敏感弹药（IM）配方IMX-101以及IM成分硝基胍（NQ）和1-甲基-3-硝基胍（MeNQ） 。ΣTNT的96h LC50值和95％置信区间分别为2.6（2.4，2.8）和IMX-101为68.2（62.9，73.9）。NQ和MeNQ暴露量即使分别达到3560和5285 mg / L的最大暴露量，对96h暴露量的存活率也没有明显影响。但是，NQ和MeNQ以及TNT和IMX-101暴露都引起了skin皮肤微生物组的变化，事实证明，随着暴露浓度的增加，变形杆菌相对比例显着增加，NQ暴露中的α多样性显着降低。观察到了弹药暴露对皮肤微生物组的直接影响的潜力，包括增加了耐弹药的系统发生群体的数量，此外还对微生物菌群产生了间接影响，其中转录反应提示皮肤粘液层性质的变化，抗菌肽的产生，在host宿主中观察到了先天免疫因子。into宿主对弹药暴露的转录反应的其他见解表明，TNT和IMX-101暴露显着丰富了I型和II型异源生物代谢途径中的转录表达，观察到剂量反应性表达增加。TNT暴露中血红素和铁结合功能的大量富集和转录表达的增加，可能是已知的TNT毒性机制（包括溶血性贫血和高铁血红蛋白血症）的指标。IMX-101暴露中细胞周期途径转录表达的显着富集和剂量响应性降低与先前在鱼类中观察到的一致，而响应NQ暴露的免疫相关功能的显着富集与在鱼体内的潜在免疫抑制相一致。 NQ暴露浓度最高。最后，MeNQ暴露引起角蛋白16（I型）的转录表达显着降低，该基因可能与两栖动物皮肤角化过程有关。总体，