The Occurrence of the Holometabolous Pupal Stage Requires the Interaction between E93, Krüppel-Homolog 1 and Broad-Complex

Abstract

Complete metamorphosis (Holometaboly) is a key innovation that underlies the spectacular success of holometabolous insects. Phylogenetic analyses indicate that Holometabola form a monophyletic group that evolved from ancestors exhibiting hemimetabolous development (Hemimetaboly). However, the nature of the changes underlying this crucial transition, including the occurrence of the holometabolan-specific pupal stage, is poorly understood. Using the holometabolous beetle Tribolium castaneum as a model insect, here we show that the transient up-regulation of the anti-metamorphic Krüppel-homolog 1 ( TcKr-h1) gene at the end of the last larval instar is critical in the formation of the pupa. We find that depletion of this specific TcKr-h1 peak leads to the precocious up-regulation of the adult-specifier factor TcE93 and, hence, to a direct transformation of the larva into the adult form, bypassing the pupal stage. Moreover, we also find that the TcKr-h1-dependent repression of TcE93 is critical to allow the strong up-regulation of Broad-complex ( TcBr-C), a key transcription factor that regulates the correct formation of the pupa in holometabolous insects. Notably, we show that the genetic interaction between Kr-h1 and E93 is also present in the penultimate nymphal instar of the hemimetabolous insect Blattella germanica, suggesting that the evolution of the pupa has been facilitated by the co-option of regulatory mechanisms present in hemimetabolan metamorphosis. Our findings, therefore, contribute to the molecular understanding of insect metamorphosis, and indicate the evolutionary conservation of the genetic circuitry that controls hemimetabolan and holometabolan metamorphosis, thereby shedding light on the evolution of complete metamorphosis.

Author Summary

Complete metamorphosis is an evolutionary innovation that has been critical for the success of insects. Phylogenetic relationships reveal that holometabolous insects evolved from ancestors displaying hemimetabolous development. Yet, little is known about the molecular nature of the changes required for such transition, including the evolution of the holometabolan-specific pupal stage. Here, by using Tribolium castaneum, we report that the crosstalk between Krüppel-homolog 1 ( Kr-h1), E93 and Broad-Complex genes at the end of the larval development has been a key event underlying the formation of the pupa. Interestingly, we show that the interaction between Kr-h1 and E93 is also present in hemimetabolous insects, suggesting that the pupal stage has evolved by the co-option of regulatory mechanisms already present in hemimetabolous insects.