Data_Sheet_1_Moving Beyond the Host: Unraveling the Skin Microbiome of Endangered Costa Rican Amphibians.docx

Some neotropical amphibians, including a few species in Costa Rica, were presumed to be “extinct” after dramatic population declines in the late 1980s but have been rediscovered in isolated populations. Such populations seem to have evolved a resistance/tolerance to Batrachochytrium dendrobatidis (Bd), a fungal pathogen that causes a deadly skin disease and is considered one of the main drivers of worldwide amphibian declines. The skin microbiome is an important component of the host’s innate immune system and is associated with Bd-resistance. However, the way that the bacterial diversity of the skin microbiome confers protection against Bd in surviving species remains unclear. We studied variation in the skin microbiome and the prevalence of putatively anti-Bd bacterial taxa in four co-habiting species in the highlands of the Juan Castro Blanco National Park in Costa Rica using 16S rRNA amplicon sequencing. Lithobates vibicarius, Craugastor escoces, and Isthmohyla rivularis have recently been rediscovered, whereas Isthmohyla pseudopuma has suffered population fluctuations but has never disappeared. To investigate the life stage at which the protective skin microbiome is shaped and when shifts occur in the diversity of putatively anti-Bd bacteria, we studied the skin microbiome of tadpoles, juveniles and adults of L. vibicarius. We show that the skin bacterial composition of sympatric species and hosts with distinct Bd-infection statuses differs at the phyla, family, and genus level. We detected 94 amplicon sequence variants (ASVs) with putative anti-Bd activity pertaining to distinct bacterial taxa, e.g., Pseudomonas spp., Acinetobacter johnsonii, and Stenotrophomonas maltophilia. Bd-uninfected L. vibicarius harbored 79% more putatively anti-Bd ASVs than Bd-infected individuals. Although microbiome composition and structure differed across life stages, the diversity of putative anti-Bd bacteria was similar between pre- and post-metamorphic stages of L. vibicarius. Despite low sample size, our results support the idea that the skin microbiome is dynamic and protects against ongoing Bd presence in endangered species persisting after their presumed extinction. Our study serves as a baseline to understand the microbial patterns in species of high conservation value. Identification of microbial signatures linked to variation in disease susceptibility might, therefore, inform mitigation strategies for combating the global decline of amphibians.

20世纪80年代末种群骤降后，包括哥斯达黎加少数物种在内的一些新热带两栖动物曾被认为已“灭绝”，但随后在孤立种群中被重新发现。这类种群似乎已进化出对蛙壶菌（Batrachochytrium dendrobatidis, Bd）的抗性/耐受性——该真菌病原体可引发致命皮肤病，被认为是全球两栖动物种群下降的主要驱动因素之一。
皮肤微生物组是宿主先天免疫系统的重要组成部分，且与抗Bd能力相关。然而，存活物种的皮肤微生物组细菌多样性如何赋予其对抗Bd的保护力，目前仍不明确。
本研究以哥斯达黎加胡安·卡斯特罗·布兰科国家公园高地的4种同域共存两栖动物为研究对象，采用16S rRNA扩增子测序技术，分析了其皮肤微生物组的差异以及推定抗Bd细菌类群的检出情况。其中，Lithobates vibicarius、Craugastor escoces和Isthmohyla rivularis为近年重新发现的物种，而Isthmohyla pseudopuma虽经历种群波动但从未消失。
为探究保护性皮肤微生物组的形成生命阶段以及推定抗Bd细菌多样性发生变化的时间节点，本研究还对L. vibicarius的蝌蚪、幼体和成体的皮肤微生物组进行了分析。
研究结果显示，同域物种以及不同Bd感染状态宿主的皮肤细菌组成在门、科和属水平上均存在显著差异。本研究共检测到94个具有推定抗Bd活性的扩增子序列变异（amplicon sequence variants, ASVs），隶属于不同细菌类群，例如假单胞菌属（Pseudomonas）物种、约翰逊不动杆菌（Acinetobacter johnsonii）以及嗜麦芽窄食单胞菌（Stenotrophomonas maltophilia）。未感染Bd的L. vibicarius个体所携带的推定抗Bd ASVs数量较感染个体高出79%。尽管不同生命阶段的微生物组组成和结构存在差异，但L. vibicarius变态前与变态后阶段的推定抗Bd细菌多样性并无显著差异。
尽管样本量较小，本研究结果支持“皮肤微生物组具有动态性，可保护被认为已灭绝但仍存续的濒危物种免受持续存在的Bd侵害”这一观点。本研究可为理解高保护价值物种的微生物组模式提供基准。因此，鉴定与疾病易感性差异相关的微生物特征，可为对抗全球两栖动物种群下降的缓解策略提供参考依据。