Of the approximately one million described insect species, ground beetles (Coleoptera: Carabidae) have long captivated the attention of evolutionary biologists due to the diversity of defensive compounds they synthesize. Produced using defensive glands in the abdomen, ground beetle chemicals represent over 250 compounds including predator-deterring formic acid, which has evolved as a defensive strategy at least three times across Insecta. Despite being a widespread method of defense, formic acid biosynthesis is poorly understood in insects. Previous studies have suggested that the folate cycle of one-carbon (C1) metabolism, a pathway involved in nucleotide biosynthesis, may play a key role in defensive-grade formic acid production in ants. Here, we report on the defensive gland transcriptome of the formic acid-producing ground beetle Harpalus pensylvanicus. The full suite of genes involved in the folate cycle of C1 metabolism are significantly differentially expressed in the defensive glands of H. pensylvanicus when compared to gene expression profiles in the rest of the body. We also find support for two additional pathways potentially involved in the biosynthesis of defensive-grade formic acid, the kynurenine pathway and the methionine salvage cycle. Additionally, we have found an array of differentially expressed genes in the secretory lobes involved in the biosynthesis and transport of cofactors necessary for formic acid biosynthesis, as well as genes presumably involved in the detoxification of secondary metabolites including formic acid. We also provide insight into the evolution of the predominant gene family involved in the folate cycle (MTHFD) and suggest that high expression of folate cycle genes rather than gene duplication and/or neofunctionalization may be more important for defensive-grade formic acid biosynthesis in H. pensylvanicus. This provides the first evidence in Coleoptera and one of a few examples in Insecta of a primary metabolic process being co-opted for defensive chemical biosynthesis. Our results shed light on potential mechanisms of formic acid biosynthesis in the defensive glands of a ground beetle and provide a foundation for further studies into the evolution of formic acid-based chemical defense strategies in insects.

在描述的大约一百万种昆虫中，地甲虫（鞘翅目：Carabidae）长期以来一直吸引着进化生物学家的注意力，因为它们合成的防御性化合物的多样性。地甲虫的化学物质使用腹部的防御腺体产生，代表了 250 多种化合物，包括阻止捕食者的甲酸，甲酸在昆虫纲中作为防御策略至少进化了 3 次。尽管是一种广泛使用的防御方法，但昆虫中对甲酸生物合成知之甚少。先前的研究表明，单碳 (C1) 代谢的叶酸循环是一种参与核苷酸生物合成的途径，可能在蚂蚁的防御级甲酸生产中发挥关键作用。在这里，我们报告了产甲酸地甲虫的防御腺转录组Harpalus pensylvanicus。参与 C1 代谢叶酸循环的全套基因在H. pensylvanicus的防御腺体中显着差异表达与身体其他部位的基因表达谱相比。我们还发现了对可能参与防御级甲酸生物合成的另外两条途径的支持，即犬尿氨酸途径和甲硫氨酸回收循环。此外，我们还在分泌叶中发现了一系列差异表达的基因，这些基因涉及甲酸生物合成所必需的辅因子的生物合成和转运，以及可能涉及包括甲酸在内的次生代谢物解毒的基因。我们还深入了解了参与叶酸循环 (MTHFD) 的主要基因家族的进化，并表明叶酸循环基因的高表达而不是基因复制和/或新功能化可能对防御级甲酸生物合成更重要。H. pensylvanicus。这提供了鞘翅目中的第一个证据，也是昆虫纲中初级代谢过程被用于防御性化学生物合成的几个例子之一。我们的研究结果阐明了地甲虫防御腺体中甲酸生物合成的潜在机制，并为进一步研究昆虫中基于甲酸的化学防御策略的进化奠定了基础。