Metabolite Changes in Orange Dead Leaf Butterfly Kallima inachus during Ontogeny and Diapause

Holometabolism is a form of insect development which includes four life stages: egg, larva, pupa, and imago (or adult). The developmental change of whole body in metabolite levels of holometabolous insects are usually ignored and lack study. Diapause is an alternative life-history strategy that can occur during the egg, larval, pupal, and adult stages in holometabolous insects. Kallima inachus (Lepidoptera: Nymphalidae) is a holometabolous and adult diapausing butterfly. This study was intended to analyze metabolic changes in K. inachus during ontogeny and diapause through a non-targeted UPLC-MS/MS (ultra-performance liquid chromatograph coupled with tandem mass spectrometry) based metabolomics analysis. A variety of glycerophospholipids (11), amino acid and its derivatives (16), and fatty acyls (nine) are crucial to the stage development of K. inachus. 2-Keto-6-acetamidocaproate, N-phenylacetylglycine, Cinnabarinic acid, 2-(Formylamino) benzoic acid, L-histidine, L-glutamate, and L-glutamine play a potentially important role in transition of successive stages (larva to pupa and pupa to adult). We observed adjustments associated with active metabolism, including an accumulation of glycerophospholipids and carbohydrates and a degradation of lipids, as well as amino acid and its derivatives shifts, suggesting significantly changed in energy utilization and management when entering into adult diapause. Alpha-linolenic acid metabolism and ferroptosis were first found to be associated with diapause in adults through pathway analyses. Our study lays the foundation for a systematic study of the developmental mechanism of holometabolous insects and metabolic basis of adult diapause in butterflies.

Automated summary

This study analyzed the metabolic changes in K. inachus during different stages and diapause using a non-targeted metabolomics analysis. Glycerophospholipids, amino acids and their derivatives, and fatty acyls were found to be crucial in the developmental stages of K. inachus. Significant changes in energy utilization and management were observed during adult diapause, including the accumulation of glycerophospholipids and carbohydrates, degradation of lipids, and shifts in amino acid and its derivatives. Pathway analyses also revealed the association of alpha-linolenic acid metabolism and ferroptosis with adult diapause.