The role of Broad in the development ofTribolium castaneum:implications for the evolution of the holometabolous insect pupa

Abstract

The evolution of complete metamorphosis in insects is a key innovation that has led to the successful diversification of holometabolous insects, yet the origin of the pupa remains an enigma. Here, we analyzed the expression of the pupal specifier gene broad (br), and the effect on br of isoform-specific, double-stranded RNA-mediated silencing, in a basal holometabolous insect, the beetle Tribolium castaneum. All five isoforms are weakly expressed during the penultimate instar and highly expressed during the prepupal period of the final instar. Application of hydroprene, a juvenile hormone analog, during the penultimate instar caused a repeat of the penultimate br expression patterns, and the formation of supernumerary larvae. Use of dsRNA against the br core region, or against a pair of either the br-Z2 or br-Z3 isoform with the br-Z1 or br-Z4 isoform, produced mobile animals with well-differentiated adult-like appendages, but which retained larval-like urogomphi and epidermis. Disruption of either the br-Z2 or the br-Z3 isoform caused the formation of shorter wings. Disruption of both br-Z1 and br-Z4 caused the appearance of pupal traits in the adults, but disruption of br-Z5 had no morphological effect. Our findings show that the br isoform functions are broadly conserved within the Holometabola and suggest that evolution of br isoform expression may have played an important role in the evolution of the pupa in holometabolous insects.

Broad-Complex acts downstream of Met in juvenile hormone signaling to coordinate primitive holometabolan metamorphosis

“…These data suggested that the moderate levels of BR-C mRNAs, detectable during premetamorphic stages in both species, had no essential role. This scenario would agree with the fact that zygotic BR-C function is not required in Drosophila BR-C null nonpupariating mutants until the onset of metamorphosis (Belyaeva et al, 1980;Kiss et al, 1988), and with recent data on Tribolium (Suzuki et al, 2008). However, as neither RNAi nor the likely hypomorphic TcBR-C KS342 allele present a complete loss-of-function situation, we cannot exclude a possibility that BR-C plays some additional role, not visualized by our phenotypes.…”

“…BR-C in both cases promotes the pupal fate (Zhou and Riddiford, 2002), and therefore JH must regulate BR-C differently in larvae and in pupae. In lepidopteran (Zhou et al, 1998;Reza et al, 2004), as well as in Tribolium (Suzuki et al, 2008) larvae, JH prevents BR-C expression until the onset of metamorphosis, and presumably that is how JH prevents pupal differentiation. Conversely, removal of the JH source (allatectomy) causes both BR-C misexpression and precocious pupal development.…”

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle

“…Therefore, our mosaic analysis of broad suggests that the larval epidermal cells are not pupally committed in the absence of broad, and thus they repeat larval-larval molts, although the hosts metamorphosed into pupae and adults. These results clearly indicate that broad is essential for the juvenile-adult transition in Bombyx, as found in other holometabolous insects (19,21,(41)(42)(43)(44)(45). Given the results of mosaic analysis, we further examined the expression of broad mRNAs in JH-deficient and JH signalingdeficient mutant Bombyx (Fig.…”

Juvenile hormone analogue, fenoxycarb, modulates ecdysone-triggered transcriptional hierarchy during programmed cell death of midgut in silkworm, Bombyx mori (Lepidoptera: Bombycidae)

“…In contrast, fenoxycarb treatment induced the expression of BR-C Z2, BR-C Z4 and BHR3 at the same stage of development. According to previous studies on holometabolous insects, high levels of JH are correlated with a low expression of BR-C and a prolonged expression of them in treated insects during the late prepupal stage may due to high levels of 20E in the presence of JH (Zhou et al, 1998;Suzuki et al, 2008). According to these results, the patterns in the expression of BRC isoforms are similar to that recorded in M. sexta, D. melanogaster and A. aegypti (Wu et al, 2006) in which the application of juvenile hormone analogues causes their re-expression.…”