Broad-complex functions in postembryonic development of the cockroach Blattella germanica shed new light on the evolution of insect metamorphosis Jia-Hsin Huang 1 , Jesus Lozano, Xavier Belles ⁎ Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), Passeig Marítim de la Barceloneta 37, 08003 Barcelona, Spain abstract article info Article history: Received 1 August 2012 Received in revised form 27 September 2012 Accepted 28 September 2012 Available online 3 October 2012 Keywords: Insect metamorphosis Juvenile hormone Ecdysone Evolution of holometaboly Drosophila Tribolium Background: Insect metamorphosis proceeds in two modes: hemimetaboly, gradual change along the life cycle; and holometaboly, abrupt change from larvae to adult mediated by a pupal stage. Both are regulated by 20-hydroxyecdysone (20E), which promotes molts, and juvenile hormone (JH), which represses adult morphogenesis. Expression of Broad-complex (BR-C) is induced by 20E and modulated by JH. In holometab- olous species, like Drosophila melanogaster, BR-C expression is inhibited by JH in young larvae and enhanced in mature larvae, when JH declines and BR-C expression specifies the pupal stage. Methods: Using Blattella germanica as a basal hemimetabolous model, we determined the patterns of expres- sion of BR-C mRNAs using quantitative RT-PCR, and we studied the functions of BR-C factors using RNA inter- ference approaches. Results: We found that BR-C expression is enhanced by JH and correlates with JH hemolymph concentration. BR-C factors appear to be involved in cell division and wing pad growth, as well as wing vein patterning. Conclusions: In B. germanica, expression of BR-C is enhanced by JH, and BR-C factors appear to promote wing growth to reach the right size, form and patterning, which contrast with the endocrine regulation and com- plex functions observed in holometabolous species. General significance: Our results shed new light to the evolution from hemimetaboly to holometaboly regard- ing BR-C, whose regulation and functions were affected by two innovations: 1) a shift in JH action on BR-C expression during young stages, from stimulatory to inhibitory, and 2) an expansion of functions, from reg- ulating wing development, to determining pupal morphogenesis. © 2012 Elsevier B.V. All rights reserved. 1. Introduction The origin and evolution of insect metamorphosis poses one of the most enigmatic conundrums in evolutionary biology. In his “On the Origin of Species”, Charles Darwin already complained about the difficulty of integrating insect metamorphosis (due to the striking difference between the morphologies and life styles of larvae and adults of the same species) into his theory of species evolution by natural selection [1]. However, it is clear that insect metamorphosis has been a key innovation in insect evolution as most of the present biodiversity on Earth is composed of metamorphosing insects, with approximately 1 million species described, and 10–30 million still to be discovered [2,3]. The first systematic studies on insect metamorphosis were carried out by Renaissance entomologists, who established that post-embryonic changes are most spectacular in insects like butterflies, beetles and flies, which undergo a dramatic morphological transformation from larva to pupa and adult, a phenomenon now known as holometaboly. Other insects, such as locusts and cockroaches, also metamorphose from the last nymphal instar to adult, although the change of form is not as radical given that the nymphs are similar to the adults. However, they undergo qualitative metamorphic changes, such as formation of mature wings and external genitalia in a type of metamorphosis known as hemimetaboly [4,5]. Metamorphosis evolved from hemimetaboly to holometaboly, and the latter innovation was most successful because more than 80% of present insects are holometabolous species (including the “big four” orders: Lepidoptera, Coleoptera, Diptera and Hymenop- tera) [2,3]. Therefore, explaining the evolutionary transition from hemimetaboly to holometaboly may give a new look to explain how this amazing biodiversity originated, and the study of the processes regulating metamorphosis shall surely provide important clues for such a goal [6]. Insect metamorphosis is regulated by two hormones, the molting hormone, which promotes molting, and the juvenile hormone (JH), which represses metamorphosis and, thus determines the molt type: to an immature stage when it is present, or to the adult when it is absent [4,6,7]. Although the molecular action of JH is still poorly understood [8], we know that an important transducer of the JH signal is Methoprene tolerant (Met), a transcription factor that was discovered in Drosophila Biochimica et Biophysica Acta 1830 (2013) 2178–2187 ⁎ Corresponding author. Tel.: +34 932309636; fax: +34 932211011. E-mail address: xavier.belles@ibe.upf-csic.es (X. Belles). 1 Permanent address: Department of Entomology, National Taiwan University, Taipei 106, Taiwan. 0304-4165/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagen.2012.09.025 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen