Poison frogs are known for the outstanding diversity of alkaloid-based chemical defences with promising therapeutic applications. However, current knowledge about chemical defences in Dendrobatoidea superfamily has two sources of bias. First, cryptic, brown-colored species have been neglected in comparison to those conspicuously colored, and second, there has been little interest in characterizing metabolites other than alkaloids mediating defensive functions. In an effort to contribute to fill the gap of knowledge about cryptic species and broadening the spectrum of compounds analyzed we have applied head-space solid phase microextraction coupled to gas chromatography and mass spectrometry (HS-SPME/GC-MS) for extracting amphibian alkaloids and volatile organic compounds (VOCs) from Silverstoneia punctiventris. Using the skin from 8 specimens in 4 biological replicates we have found 33 different compounds. Twenty of them were classified as VOCs into 15 chemical classes including alkanes, alcohols, carbonyl compounds, methylpyridines, benzothiazoles, N-alkylpyrrolidines, pyrazines, and sesquiterpenoids, some of which were previously reported as repellents, defence compounds or defence pheromones in other organisms, and as sex pheromones in a treefrog. Interestingly, six of the remaining compounds were identified as alkaloids previously reported in other toxic/unpalatable dendrobatid frogs. This is the first report of alkaloids and VOCs found in the Silverstoneia genus, which has been assumed for decades as non-chemically defended. This study establishes HS-SPME/GC-MS as a new application for a simultaneous approach to amphibian alkaloids and VOCs in poison frogs while opens up new research questions to assess the co-occurrence of both type of compounds and to investigate the evolutionary significance of a defence gradient that includes olfactory avoidance, unpalatability, and toxicity in dendrobatids. In addition, our results show that amphibian alkaloids could have a dual function (olfactory at distance, taste by contact) never explored before neither in Silverstonaeia nor in any other dendrobatid species.

中文翻译：

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神秘石斛 Silverstoneia punctiventris 中有毒生物碱和挥发性有机化合物 (VOC) 的首次表征

毒蛙以生物碱为基础的化学防御的出色多样性和有希望的治疗应用而闻名。然而，目前关于石斛总科化学防御的知识有两个偏见来源。首先，与那些显眼的颜色相比，神秘的棕色物种被忽视了，其次，除了生物碱介导的防御功能之外，人们对表征代谢物的兴趣不大。为了填补有关隐性物种的知识空白并拓宽分析的化合物谱，我们应用顶空固相微萃取与气相色谱和质谱联用 (HS-SPME/GC-MS) 来提取两栖类生物碱和来自 Silverstoneia punctiventris 的挥发性有机化合物 (VOC)。使用 4 个生物学重复中的 8 个样本的皮肤，我们发现了 33 种不同的化合物。其中 20 种被归类为 VOC，分为 15 个化学类别，包括烷烃、醇类、羰基化合物、甲基吡啶、苯并噻唑、N-烷基吡咯烷、吡嗪和倍半萜类化合物，其中一些以前被报道为其他生物的驱虫剂、防御化合物或防御信息素，和作为树蛙的性信息素。有趣的是，剩余的化合物中有 6 种被鉴定为先前在其他有毒/难吃的树蛙蛙中报道的生物碱。这是在银石属植物中发现的生物碱和 VOC 的第一份报告，几十年来一直被认为是非化学防护的。本研究将 HS-SPME/GC-MS 作为同时检测毒蛙中两栖类生物碱和 VOC 的新应用，同时开辟了新的研究问题，以评估两种类型的化合物的共存情况并研究一种防御梯度，包括嗅觉回避、可口性和 dendrobatids 的毒性。此外，我们的结果表明，两栖类生物碱可能具有双重功能（远距离嗅觉，接触味觉），这在 Silverstonaeia 和任何其他石斛属物种中都从未探索过。