Global Metabolomics of Fireflies (Coleoptera: Lampyridae) Explore Metabolic Adaptation to Fresh Water in Insects

SIMPLE SUMMARY: To date, little is known about the molecular mechanisms of aquatic adaptation in invertebrates, particularly in insects, which are the most abundant and diverse species. How aquatic insects adapt to freshwater environments remains largely unknown. Fireflies have terrestrial and aquatic lineages according to the habits of their larvae, and provides a good opportunity to explore aquatic adaptation of insects. We generated adult and larval metabolomes of two firefly species (aquatic Aquatica leii and terrestrial Lychnuris praetexta), and then a set of metabolites and their pathways involved in freshwater adaptation of aquatic firefly species were investigated by intraspecific and interspecific metabolomics comparisons, as well as by functional enrichment analysis. These molecules and pathways were primarily involved in oxidative stress/xenobiotics/immunity response, energy metabolism, sense function, and morphological attributes related to the freshwater lifestyle of aquatic A. leii. This study suggests that abundance-level changes in metabolites contributed to freshwater adaptation of fireflies, and provides insights into the metabolic mechanisms of aquatic adaptation in insects. ABSTRACT: Aquatic insects are well-adapted to freshwater environments, but metabolic mechanisms of such adaptations, particularly to primary environmental factors (e.g., hypoxia, water pressure, dark light, and abundant microbes), are poorly known. Most firefly species (Coleoptera: Lampyridae) are terrestrial, but the larvae of a few species are aquatic. We generated 24 global metabolomic profiles of larvae and adults of Aquatica leii (freshwater) and Lychnuris praetexta (terrestrial) to identify freshwater adaptation-related metabolites (AARMs). We identified 110 differentially abundant metabolites (DAMs) in A. leii (adults vs. aquatic larvae) and 183 DAMs in L. praetexta (adults vs. terrestrial larvae). Furthermore, 100 DAMs specific to aquatic A. leii larvae were screened as AARMs via interspecific comparisons (A. leii vs. L. praetexta), which were primarily involved in antioxidant activity, immune response, energy production and metabolism, and chitin biosynthesis. They were assigned to six categories/superclasses (e.g., lipids and lipid-like molecules, organic acids and derivatives, and organoheterocyclic compound). Finally, ten metabolic pathways shared between KEGG terms specific to aquatic fireflies and enriched by AARMs were screened as aquatic adaptation-related pathways (AARPs). These AARPs were primarily involved in energy metabolism, xenobiotic biodegradation, protection of oxidative/immune damage, oxidative stress response, and sense function (e.g., glycine, serine and threonine metabolism, drug metabolism-cytochrome P450, and taste transduction), and certain aspects of morphology (e.g., steroid hormone biosynthesis). These results provide evidence suggesting that abundance changes in metabolomes contribute to freshwater adaptation of fireflies. The metabolites identified here may be vital targets for future work to determine the mechanism of freshwater adaptation in insects.