Reduction of embryonic E93 expression as a hypothetical driver of the evolution of insect metamorphosis.

The early embryo of the cockroach Blattella germanica exhibits high E93 expression. In general, E93 triggers adult morphogenesis during postembryonic development. Here we show that E93 is also crucial in early embryogenesis in the cockroach, as a significant number of E93-depleted embryos are unable to develop the germ band under maternal RNAi treatment targeting E93. Moreover, transcriptomic analysis indicates that E93 depletion results in important gene expression changes in the early embryo, and many of the differentially expressed genes are involved in development. Then, using public databases, we gathered E93 expression data in embryo and preadult stages, finding that embryonic expression of E93 is high in hemimetabolan species (whose juveniles, or nymphs, are similar to the adult) and low in holometabolans (whose juveniles, or larvae, are different from the adult). E93 expression is also low in Thysanoptera and in Hemiptera Sternorrhyncha, hemimetabolans with postembryonic quiescent stages, as well as in Odonata, the nymph of which is very different from the adult. In ametabolans, such as the Zygentoma Thermobia domestica, E93 transcript levels are very high in the early embryo, whereas during postembryonic development they are medium and relatively constant. We propose the hypothesis that during evolution, a reduction of E93 expression in the embryo of hemimetabolans facilitated the larval development and the emergence of holometaboly. Independent decreases of E93 transcripts in the embryo of Odonata, Thysanoptera, and different groups of Hemiptera Sternorrhyncha would have allowed the development of modified juvenile stages adapted to specific ecophysiological conditions.

Regulation of metamorphosis in neopteran insects is conserved in the paleopteran Cloeon dipterum (Ephemeroptera).

In the Paleozoic era, more than 400 Ma, a number of insect groups continued molting after forming functional wings. Today, however, flying insects stop molting after metamorphosis when they become fully winged. The only exception is the mayflies (Paleoptera, Ephemeroptera), which molt in the subimago, a flying stage between the nymph and the adult. However, the identity and homology of the subimago still is underexplored. Debate remains regarding whether this stage represents a modified nymph, an adult, or a pupa like that of butterflies. Another relevant question is why mayflies have the subimago stage despite the risk of molting fragile membranous wings. These questions have intrigued numerous authors, but nonetheless, clear answers have not yet been found. By combining morphological studies, hormonal treatments, and molecular analysis in the mayfly Cloeon dipterum, we found answers to these old questions. We observed that treatment with a juvenile hormone analog in the last nymphal instar stimulated the expression of the Kr-h1 gene and reduced that of E93, which suppress and trigger metamorphosis, respectively. The regulation of metamorphosis thus follows the MEKRE93 pathway, as in neopteran insects. Moreover, the treatment prevented the formation of the subimago. These findings suggest that the subimago must be considered an instar of the adult mayfly. We also observed that the forelegs dramatically grow between the last nymphal instar, the subimago, and the adult. This necessary growth spread over the last two stages could explain, at least in part, the adaptive sense of the subimago.

E93-depleted adult insects preserve the prothoracic gland and molt again.

Insect metamorphosis originated around the middle Devonian, associated with the innovation of the final molt; this occurs after histolysis of the prothoracic gland (PG; which produces the molting hormone) in the first days of adulthood. We previously hypothesized that transcription factor E93 is crucial in the emergence of metamorphosis, because it triggers metamorphosis in extant insects. This work on the cockroach Blattella germanica reveals that E93 also plays a crucial role in the histolysis of PG, which fits the above hypothesis. Previous studies have shown that the transcription factor FTZ-F1 is essential for PG histolysis. We have found that FTZ-F1 depletion towards the end of the final nymphal instar downregulates the expression of E93, whereas E93-depleted nymphs molt to adults that retain a functional PG. Interestingly, these adults are able to molt again, which is exceptional in insects. The study of insects able to molt again in the adult stage may reveal clues about how nymphal epidermal cells definitively become adult cells, and whether it is possible to reverse this process.

Flight or protection: the genes Ultrabithorax and apterous in the determination of membranous and sclerotized wings in insects.

Present-day pterygote insects have two pairs of wings, one in the mesothorax (T2), the other in the metathorax (T3), and both have diverged in structure and function in different groups. Studies in endopterygote and paraneopteran species have shown that the gene Ultrabithorax (Ubx) specifies the identity and wing structure in T3, whereas the gene apterous (ap) significantly contributes to forming modified T2 wings. We wondered whether these Ubx and ap mechanisms operate in the lineage of polyneopterans. To explore this possibility, we used the cockroach Blattella germanica (Polyneoptera and Blattodea), in which the T2 wings are sclerotized (tegmina), whereas those of the T3 are membranous. We found that Ubx determines the structure of T3 and the membranous wing, while ap significantly contributes to form the sclerotized T2 tegmina. These results along with the studies carried out on the beetle Tribolium castaneum by Tomoyasu and collaborators suggest that ap plays an important role in the sclerotization and melanization of the T2 wings in neopteran groups that have sclerotized forewings. In turn, the sclerotizing properties of ap demonstrated in beetles and cockroaches suggest that the origin of this function goes back to the emergence of Neoptera, in the mid Devonian.

Tergal and pleural wing-related tissues in the German cockroach and their implication to the evolutionary origin of insect wings.

The acquisition of wings has facilitated the massive evolutionary success of pterygotes (winged insects), which now make up nearly three-quarters of described metazoans. However, our understanding of how this crucial structure has evolved remains quite elusive. Historically, two ideas have dominated in the wing origin debate, one placing the origin in the dorsal body wall (tergum) and the other in the lateral pleural plates and the branching structures associated with these plates. Through studying wing-related tissues in the wingless segments (such as wing serial homologs) of the beetle, Tribolium castaneum, we obtained several crucial pieces of evidence that support a third idea, the dual origin hypothesis, which proposes that wings evolved from a combination of tergal and pleural tissues. Here, we extended our analysis outside of the beetle lineage and sought to identify wing-related tissues from the wingless segments of the cockroach, Blattella germanica. Through detailed functional and expression analyses for a critical wing gene, vestigial (vg), along with re-evaluating the homeotic transformation of a wingless segment induced by an improved RNA interference protocol, we demonstrate that B. germanica possesses two distinct tissues in their wingless segments, one with tergal and one with pleural nature, that might be evolutionarily related to wings. This outcome appears to parallel the reports from other insects, which may further support a dual origin of insect wings. However, we also identified a vg-independent tissue that contributes to wing formation upon homeotic transformation, as well as vg-dependent tissues that do not appear to participate in wing formation, in B. germanica, indicating a more complex evolutionary history of the tissues that contributed to the emergence of insect wings.

Juvenile hormone signaling in short germ-band hemimetabolan embryos.

The role of juvenile hormone (JH) in insect embryos is far from understood, especially in short germ-band hemimetabolan species. To shed light on this issue, we depleted the mRNA levels of Krüppel homolog 1, Methoprene-tolerant and JH acid O-methyltransferase, key elements of JH signaling, in embryos of the short germ-band hemimetabolan species Blattella germanica This precluded the formation of the germ-band anlage in a group of embryos. Hatchability was also reduced, which might have been caused by premature upregulation of laccase 2, a promoter of cuticle tanning. In other cases, development was interrupted in mid embryogenesis, involving defects related to dorsal closure and appendage formation. These phenotypes possibly result from the low levels of Broad-complex (BR-C) produced under JH-depleted conditions. This contrasts with holometabolan species, in which JH does not promote BR-C expression, which remains low during embryo development. Possibly, the stimulatory role of JH on BR-C expression and the morphogenetic functions of BR-C in hemimetabolan embryos were lost in holometabolan species. If so, this might have been a key driver for the evolution of holometabolan metamorphosis.

Myoglianin triggers the premetamorphosis stage in hemimetabolan insects.

Insect metamorphosis is triggered by a decrease in juvenile hormone (JH) in the final juvenile instar. What induces this decrease is therefore a relevant question. Working with the cockroach Blattella germanica, we found that myoglianin (Myo), a ligand in the TGF-ß signaling pathway, is highly expressed in the corpora allata (CA, the JH-producing glands) and the prothoracic gland [(PG), which produce ecdysone] during the penultimate (fifth) nymphal instar (N5). In the CA, high Myo levels during N5 repress the expression of juvenile hormone acid methyl transferase, a JH biosynthesis gene. In the PG, decreasing JH levels trigger gland degeneration, regulated by the factors Krüppel homolog 1, FTZ-F1, E93, and inhibitor of apoptosis-1. Also in the PG, a peak of myo expression in N5 indirectly stimulates the expression of ecdysone biosynthesis genes, such as neverland, enhancing the production of the metamorphic ecdysone pulse in N6. The Myo expression peak in N5 also represses cell proliferation, which can enhance ecdysone production. The data indicate that Myo triggers the premetamorphic nymphal instar in B. germanica and possibly in other hemimetabolan insects.-Kamsoi, O., Belles, X. Myoglianin triggers the premetamorphosis stage in hemimetabolan insects.

Krüppel homolog 1 and E93: The doorkeeper and the key to insect metamorphosis.

Insect metamorphosis is regulated by two main hormones: ecdysone (20E), which promotes molting, and juvenile hormone (JH), which inhibits adult morphogenesis. The transduction mechanisms for the respective hormonal signals include the transcription factors Krüppel homolog 1 (Kr-h1) and E93, which are JH- and 20E-dependent, respectively. Kr-h1 is the main effector of the antimetamorphic action of JH, while E93 is a key promoter of metamorphosis. The ancestral regulatory axis of metamorphosis, which operates in insects with hemimetabolan (gradual) metamorphosis and is known as the MEKRE93 pathway, is based on Kr-h1 repression of E93. In the last juvenile stage, when the production of JH dramatically decreases, Kr-h1 expression is almost completely interrupted, E93 becomes upregulated and metamorphosis proceeds. The holometabolan (complete) metamorphosis mode of development includes the peculiar pupal stage, a sort of intermediate between the final larval instar and the adult stage. In holometabolan species, Broad-Complex (BR-C) transcription factors determine the pupal stage and E93 stimulates the expression of BR-C in the prepupa. The MEKRE93 pathway is conserved in holometabolan insects, which have added the E93/BR-C interaction loop to the ancestral (hemimetabolan) pathway during the evolution from hemimetaboly to holometaboly.

Diversity of piRNA expression patterns during the ontogeny of the German cockroach.

The Piwi-interacting RNA (piRNA) system is an evolutionarily conserved mechanism involved in the control of transposable elements and maintenance of genomic stability, especially in germ line cells and in early embryo stages. However, relevant particularities, both in mechanism and function, exist across species among metazoans and even within the insect class. As a member of the scarcely studied hemimetabolan group, Blattella germanica can be a suitable reference model to study insect evolution. We present the results of a stringent process of identification and study of expressed piRNAs for B. germanica across 11 developmental stages, ranging from unfertilized egg to nymphs and adult female. Our results confirm the dual origin of piRNA in this species, with a majority of them being generated from the primary pathway, and a smaller but highly expressed set of sequences participating in the secondary ("ping-pong") reamplification pathway. An intriguing partial complementarity in expression is observed between the piRNA of the two biogenesis pathways, with those generated in the secondary pathway being quite restricted to early embryo stages. In addition, many piRNAs are exclusively expressed in late embryo and nymphal stages. These observations point at piRNA functions beyond the role of transposon control in early embryogenesis. Our work supports the view of a more complex scenario, with different sets of piRNAs acting in different times and having a range of functions wider than previously thought.

Remodeling of the juvenile hormone pathway through caste-biased gene expression and positive selection along a gradient of termite eusociality.

The evolution of division of labor between sterile and fertile individuals represents one of the major transitions in biological complexity. A fascinating gradient in eusociality evolved among the ancient hemimetabolous insects, ranging from noneusocial cockroaches through the primitively social lower termites-where workers retain the ability to reproduce-to the higher termites, characterized by lifetime commitment to worker sterility. Juvenile hormone (JH) is a prime candidate for the regulation of reproductive division of labor in termites, as it plays a key role in insect postembryonic development and reproduction. We compared the expression of JH pathway genes between workers and queens in two lower termites (Zootermopsis nevadensis and Cryptotermes secundus) and a higher termite (Macrotermes natalensis) to that of analogous nymphs and adult females of the noneusocial cockroach Blattella germanica. JH biosynthesis and metabolism genes ranged from reproductive female-biased expression in the cockroach to predominantly worker-biased expression in the lower termites. Remarkably, the expression profile of JH pathway genes sets the higher termite apart from the two lower termites, as well as the cockroach, indicating that JH signaling has undergone major changes in this eusocial termite. These changes go beyond mere shifts in gene expression between the different castes, as we find evidence for positive selection in several termite JH pathway genes. Thus, remodeling of the JH pathway may have played a major role in termite social evolution, representing a striking case of convergent molecular evolution between the termites and the distantly related social hymenoptera.

Expansions of key protein families in the German cockroach highlight the molecular basis of its remarkable success as a global indoor pest.

The German cockroach, Blattella germanica, is a worldwide pest that infests buildings, including homes, restaurants, and hospitals, often living in unsanitary conditions. As a disease vector and producer of allergens, this species has major health and economic impacts on humans. Factors contributing to the success of the German cockroach include its resistance to a broad range of insecticides, immunity to many pathogens, and its ability, as an extreme generalist omnivore, to survive on most food sources. The recently published genome shows that B. germanica has an exceptionally high number of protein coding genes. In this study, we investigate the functions of the 93 significantly expanded gene families with the aim to better understand the success of B. germanica as a major pest despite such inhospitable conditions. We find major expansions in gene families with functions related to the detoxification of insecticides and allelochemicals, defense against pathogens, digestion, sensory perception, and gene regulation. These expansions might have allowed B. germanica to develop multiple resistance mechanisms to insecticides and pathogens, and enabled a broad, flexible diet, thus explaining its success in unsanitary conditions and under recurrent chemical control. The findings and resources presented here provide insights for better understanding molecular mechanisms that will facilitate more effective cockroach control.

MiR-2 family regulates insect metamorphosis by controlling the juvenile hormone signaling pathway.

In 2009 we reported that depletion of Dicer-1, the enzyme that catalyzes the final step of miRNA biosynthesis, prevents metamorphosis in Blattella germanica. However, the precise regulatory roles of miRNAs in the process have remained elusive. In the present work, we have observed that Dicer-1 depletion results in an increase of mRNA levels of Krüppel homolog 1 (Kr-h1), a juvenile hormone-dependent transcription factor that represses metamorphosis, and that depletion of Kr-h1 expression in Dicer-1 knockdown individuals rescues metamorphosis. We have also found that the 3'UTR of Kr-h1 mRNA contains a functional binding site for miR-2 family miRNAs (for miR-2, miR-13a, and miR-13b). These data suggest that metamorphosis impairment caused by Dicer-1 and miRNA depletion is due to a deregulation of Kr-h1 expression and that this deregulation is derived from a deficiency of miR-2 miRNAs. We corroborated this by treating the last nymphal instar of B. germanica with an miR-2 inhibitor, which impaired metamorphosis, and by treating Dicer-1-depleted individuals with an miR-2 mimic to allow nymphal-to-adult metamorphosis to proceed. Taken together, the data indicate that miR-2 miRNAs scavenge Kr-h1 transcripts when the transition from nymph to adult should be taking place, thus crucially contributing to the correct culmination of metamorphosis.

MicroRNAs and the Evolution of Insect Metamorphosis.

MicroRNAs (miRNAs) are involved in the regulation of a number of processes associated with metamorphosis, either in the less modified hemimetabolan mode or in the more modified holometabolan mode. The miR-100/let-7/miR-125 cluster has been studied extensively, especially in relation to wing morphogenesis in both hemimetabolan and holometabolan species. Other miRNAs also participate in wing morphogenesis, as well as in programmed cell and tissue death, neuromaturation, neuromuscular junction formation, and neuron cell fate determination, typically during the pupal stage of holometabolan species. A special case is the control of miR-2 over Kr-h1 transcripts, which determines adult morphogenesis in the hemimetabolan metamorphosis. This is an elegant example of how a single miRNA can control an entire process by acting on a crucial mediator; however, this is a quite exceptional mechanism that was apparently lost during the transition from hemimetaboly to holometaboly.

Smads and insect hemimetabolan metamorphosis.

In contrast with Drosophila melanogaster, practically nothing is known about the involvement of the TGF-ß signaling pathway in the metamorphosis of hemimetabolan insects. To partially fill this gap, we have studied the role of Smad factors in the metamorphosis of the German cockroach, Blattella germanica. In D. melanogaster, Mad is the canonical R-Smad of the BMP branch of the TGF-ß signaling pathway, Smox is the canonical R-Smad of the TGF-ß/Activin branch and Medea participates in both branches. In insects, metamorphosis is regulated by the MEKRE93 pathway, which starts with juvenile hormone (JH), whose signal is transduced by Methoprene-tolerant (Met), which stimulates the expression of Krüppel homolog 1 (Kr-h1) that acts to repress E93, the metamorphosis trigger. In B. germanica, metamorphosis is determined at the beginning of the sixth (final) nymphal instar (N6), when JH production ceases, the expression of Kr-h1 declines, and the transcription of E93 begins to increase. The RNAi of Mad, Smox and Medea in N6 of B. germanica reveals that the BMP branch of the TGF-ß signaling pathway regulates adult ecdysis and wing extension, mainly through regulating the expression of bursicon, whereas the TGF-ß/Activin branch contributes to increasing E93 and decreasing Kr-h1 at the beginning of N6, crucial for triggering adult morphogenesis, as well as to regulating the imaginal molt timing.

CREB-binding protein contributes to the regulation of endocrine and developmental pathways in insect hemimetabolan pre-metamorphosis.

BACKGROUND: CREB-binding protein (CBP) is a promiscuous transcriptional co-regulator. In insects, CBP has been studied in the fly Drosophila melanogaster, where it is known as Nejire. Studies in D. melanogaster have revealed that Nejire is involved in the regulation of many pathways during embryo development, especially in anterior/posterior polarity, through Hedgehog and Wingless signaling, and in dorsal/ventral patterning, through TGF-ß signaling. Regarding post-embryonic development, Nejire influences histone acetyl transferase activity on the ecdysone signaling pathway. METHODS AND RESULTS: Functional genomics studies using RNAi have shown that CBP contributes to the regulation of feeding and ecdysis during the pre-metamorphic nymphal instar of the cockroach Blattella germanica and is involved in TGF-ß, ecdysone, and MEKRE93 pathways, contributing to the activation of Kr-h1 and E93 expression. In D. melanogaster, Nejire's involvement in the ecdysone pathway in pre-metamorphic stages is conserved, whereas the TGF-ß pathway has only been described in the embryo. CBP role in ecdysis pathway and in the activation of Kr-h1 and E93 expression is described here for the first time. CONCLUSIONS: Studies in D. melanogaster may have been suggestive that CBP functions in insects are concentrated in the embryo. Results obtained in B. germanica indicate, however, that CBP have diverse and important functions in post-embryonic development and metamorphosis, especially regarding endocrine signaling. GENERAL SIGNIFICANCE: Further research into a higher diversity of models will probably reveal that the multiple post-embryonic roles of CBP observed in B. germanica are general in insects.

Comparative analysis of miRNA expression during the development of insects of different metamorphosis modes and germ-band types.

BACKGROUND: Do miRNAs contribute to specify the germ-band type and the body structure in the insect embryo? Our goal was to address that issue by studying the changes in miRNA expression along the ontogeny of the German cockroach Blattella germanica, which is a short germ-band and hemimetabolan species. RESULTS: We sequenced small RNA libraries representing 11 developmental stages of B. germanica ontogeny (with especial emphasis on embryogenesis) and the changes in miRNA expression were examined. Data were compared with equivalent data for two long germ-band holometabolan species Drosophila melanogaster and Drosophila virilis, and the short germ-band holometabolan species Tribolium castaneum. The identification of B. germanica embryo small RNA sequences unveiled miRNAs not detected in previous studies, such as those of the MIR-309 family and 54 novel miRNAs. Four main waves of miRNA expression were recognized (with most miRNA changes occurring during the embryonic stages): the first from day 0 to day 1 of embryogenesis, the second during mid-embryogenesis (days 0-6), the third (with an acute expression peak) on day 2 of embryonic development, and the fourth during post-embryonic development. The second wave defined the boundaries of maternal-to-zygotic transition, with maternal mRNAs being cleared, presumably by Mir-309 and associated scavenger miRNAs. CONCLUSION: miRNAs follow well-defined patterns of expression over hemimetabolan ontogeny, patterns that are more diverse during embryonic development than during the nymphal stages. The results suggest that miRNAs play important roles in the developmental transitions between the embryonic stages of development (starting with maternal loading), during which they might influence the germ-band type and metamorphosis mode.

A role for Taiman in insect metamorphosis.

Recent studies in vitro have reported that the Methoprene-tolerant (Met) and Taiman (Tai) complex is the functional receptor of juvenile hormone (JH). Experiments in vivo of Met depletion have confirmed this factor's role in JH signal transduction, however, there is no equivalent data regarding Tai because its depletion in larval or nymphal stages of the beetle Tribolium castaneum and the bug Pyrrhocoris apterus results in 100% mortality. We have discovered that the cockroach Blattella germanica possesses four Tai isoforms resulting from the combination of two indels in the C-terminal region of the sequence. The presence of one equivalent indel-1 in Tai sequences in T. castaneum and other species suggests that Tai isoforms may be common in insects. Concomitant depletion of all four Tai isoforms in B. germanica resulted in 100% mortality, but when only the insertion 1 (IN-1) isoforms were depleted, mortality was significantly reduced and about half of the specimens experienced precocious adult development. This shows that Tai isoforms containing IN-1 are involved in transducing the JH signal that represses metamorphosis. Reporter assays indicated that both T. castaneum Tai isoforms, one that contains the IN-1 and another that does not (DEL-1) activated a JH response element (kJHRE) in Krüppel homolog 1 in conjunction with Met and JH. The results indicate that Tai is involved in the molecular mechanisms that repress metamorphosis, at least in B. germanica, and highlight the importance of distinguishing Tai isoforms when studying the functions of this transcription factor in development and other processes.

Ecdysone signalling and ovarian development in insects: from stem cells to ovarian follicle formation.

Although a great deal of information is available concerning the role of ecdysone in insect oogenesis, research has tended to focus on vitellogenesis and choriogenesis. As such, the study of oogenesis in a strict sense has received much less attention. This situation changed recently when a number of observations carried out in the meroistic polytrophic ovarioles of Drosophila melanogaster started to unravel the key roles played by ecdysone in different steps of oogenesis. Thus, in larval stages, a non-autonomous role of ecdysone, first in repression and later in activation, of stem cell niche and primordial germ cell differentiation has been reported. In the adult, ecdysone stimulates the proliferation of germline stem cells, plays a role in stem cell niche maintenance and is needed non-cell-autonomously for correct differentiation of germline stem cells. Moreover, in somatic cells ecdysone is required for 16-cell cyst formation and for ovarian follicle development. In the transition from stages 8 to 9 of oogenesis, ecdysone signalling is fundamental when deciding whether or not to go ahead with vitellogenesis depending on the nutritional status, as well as to start border cell migration. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Chorion formation in panoistic ovaries requires windei and trimethylation of histone 3 lysine 9.

Epigenetic modifications play key roles in transcriptional regulation. Trimethylation of histone 3 lysine 9 (H3K9me3) is one of the most widely studied histone post-translational modifications, and has been linked to transcriptional repression. In Drosophila melanogaster, Windei is needed for H3K9me3 in female germ line cells. Here, we report the occurrence of a D. melanogaster Windei (Wde) ortholog in the ovary of the hemimetabolous insect Blattella germanica, which we named BgWde. Depletion of BgWde by RNAi reduced H3K9me3 in follicular cells, which triggered changes in transcriptional regulation that led to the prevention of chorion gene expression. In turn, this impaired oviposition (and the formation of the ootheca) and, therefore, prevented reproduction. Windei and H3K9me3 have already been reported in follicular cells of D. melanogaster, but this is the first time that the function of these modifications has been demonstrated in the said cells. This is also the first time that an epigenetic marker is reported as having a key role in choriogenesis.

Broad-complex functions in postembryonic development of the cockroach Blattella germanica shed new light on the evolution of insect metamorphosis.

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 holometabolous 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 expression of BR-C mRNAs using quantitative RT-PCR, and we studied the functions of BR-C factors using RNA interference 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 complex functions observed in holometabolous species. GENERAL SIGNIFICANCE: Our results shed new light to the evolution from hemimetaboly to holometaboly regarding 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 regulating wing development, to determining pupal morphogenesis.