Insect metamorphosis is regulated by ecdysteroids, which induce molts, and juvenile hormone (JH), which inhibits metamorphic changes. The molecular action of ecdysteroids has been thoroughly studied, but that of JH is poorly understood, with data currently only being available for holometabolous species, like Drosophila melanogaster and Tribolium castaneum. We studied the function of Krüppel homolog 1 (Kr-h1) in Blattella germanica, a hemimetabolous model. Kr-h1 is a Zn finger transcription factor whose function as transductor of the antimetamorphic action of JH has recently been demonstrated in D. melanogaster and T. castaneum. The RNAi experiments reported herein indicated that Kr-h1 transduces the antimetamorphic action of JH also in B. germanica, thereby suggesting that this role is an ancestral condition that has been conserved in insect evolution from hemimetabolous to holometabolous species.

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.

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.insect metamorphosis | microRNA | juvenile hormone | evolution of metamorphosis | insect hormones M icroRNAs (miRNAs) are endogenous, ca. 22-nt, singlestrand, noncoding RNAs that regulate gene expression by acting posttranscriptionally through basepairing between the socalled seed sequence of the miRNA (nucleotides 2-8 at its 5′ end) and the complementary seed match sequence in the target mRNA (1). Since miRNAs were first discovered in the nematode Caenorhabditis elegans in the 1990s (2) and subsequently detected in other species (3), a remarkable diversity of them has been reported in a variety of organisms, including insects, plants, viruses, and vertebrates (4). Information available indicates that miRNAs are key players in gene regulatory networks, driving cell differentiation, conferring robustness, and channeling the development of multicellular organisms (5-8). Furthermore, the escalation in complexity in early bilaterian evolution has been correlated with a strong increase in the number of miRNAs (9-11). In insects, miRNAs have been shown to be involved in fine-tuning a number of biological processes such as cell proliferation, apoptosis and growth, oogenesis, and development, as well as response to biological stress (12).In 2009 we first looked at whether miRNAs might have a relevant role in the regulation of insect metamorphosis. Only a few published studies had considered the contribution of miRNAs to this process, but these focused on specific aspects, such as wing formation and neuromuscular development (13-16). They also used the fly Drosophila melanogaster as a model, a species that follows the holometabolan mode of metamorphosis, in which the juvenile stages are extre...