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Experiments exploring the role of juvenile hormone during the life cycle of firebrat insects provide clues about the evolution of metamorphosis.Experiments exploring the role of juvenile hormone during the life cycle of firebrat insects provide clues about the evolution of metamorphosis.

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.

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 Krúñíóüüüppel 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. Significance MicroRNAs are short, single-stranded RNAs that bind to target mRNAs and block their translation. Five years ago we observed in the cockroach Blattella germanica that general depletion of microRNAs prevents metamorphosis. This observation led to two key questions: Which microRNAs are involved in this action, and which target do they act on? The results reported herein show that the microRNAs involved are those of an miR-2 family (miR-2, miR-13a, and miR-13b), and the target is the transcription factor Krúñíóüppel homolog 1, a master repressor of insect metamorphosis. The data presented indicate that miR-2 microRNAs rapidly clear Krúñíóüüppel homolog 1 transcripts in the last nymphal instar, a process that is crucial for proper metamorphosis. This reveals the elegant mechanism of an miRNA family leading metamorphosis to its correct conclusion.

Juvenile Hormone (JH) represses metamorphosis of young instars in insects. One of the main players in hormonal signalling is Methoprene-tolerant (Met), which plays the role of JH receptor. Using the Polyneopteran insect Blattella germanica as the model and RNAi for transcript depletion, we have confirmed that Met transduces the antimetamorphic signal of JH in young nymphs and plays a role in the last nymphal instar moult in this species. Previously, the function of Met as the JH receptor had been demonstrated in the Eumetabola clade, with experiments in Holometabola (in the beetle Tribolium castaneum) and in their sister group Paraneoptera (in the bug Pyrrhocoris apterus). Our result shows that the function of Met as JH receptor is also conserved in the more basal Polyneoptera. The function of Met as JH transducer might thus predate the evolutionary innovation of metamorphosis. Moreover, expression of Met was also found in last nymphal instar of B. germanica, when JH is absent. Depletion of Met in this stage provoked deficiencies in wing growth and ecdysis problems in the imaginal moult. Down-regulation of the ecdysone-inducible gene E75A and Insulin-Like-Peptide 1 in these Met-depleted specimens suggest that Met is involved in the ecdysone and insulin signalling pathways in last nymphal instar, when JH is virtually absent.

Insects are the only known animals in which sexual differentiation is controlled by sex-specific splicing. The doublesex transcription factor produces distinct male and female isoforms, which are both essential for sex-specific development. dsx splicing depends on transformer, which is also alternatively spliced such that functional Tra is only present in females. This pathway has evolved from an ancestral mechanism where dsx was independent of tra and expressed and required only in males. To reconstruct this transition, we examined three basal, hemimetabolous insect orders: Hemiptera, Phthiraptera, and Blattodea. We show that tra and dsx have distinct functions in these insects, reflecting different stages in the changeover from a transcription-based to a splicing-based mode of sexual differentiation. We propose that the canonical insect tra-dsx pathway evolved via merger between expanding dsx function (from males to both sexes) and narrowing tra function (from a general splicing factor to dedicated regulator of dsx).

Insect metamorphosis occurs in two main forms, hemimetaboly (simple) and holometaboly (complete), both regulated by hormonal and genetic pathways involving the transcription factors Krüppel homolog 1 (Kr-h1), Broad-Complex (BR-C), and Ecdysone-induced protein 93F (E93). The BTB–zinc finger protein Chronologically inappropriate morphogenesis (Chinmo), recently identified in the fruit fly Drosophila melanogaster , an holometabolan, as a larval state maintainer, was studied here in the German cockroach, Blattella germanica , an hemimetabolan. We also examined another BTB transcription factor, Abrupt (Ab), based on findings in another holometabolan, the red flour beetle, Tribolium castaneum , suggesting a cooperative role. We characterized chinmo expression in B. germanica and found sustained transcript levels during the N4 and N5 nymphal instars, followed by a marked decline at the final N6 instar. RNA interference (RNAi) knockdown of chinmo at N4 induced precocious metamorphosis two molts later, accompanied by reduced Kr-h1 and elevated E93 expression. Combined knockdown of chinmo and E93 revealed that Chinmo primarily represses E93 . Similarly, ab knockdown also triggered precocious metamorphosis, decreasing Kr-h1 and increasing E93 expression; double knockdown of ab and E93 indicated that Ab primarily promotes Kr-h1 expression. These results expand the MEKRE93 pathway by identifying Chinmo and Ab as additional regulators that help maintain the juvenile state in both hemimetabolan and holometabolan insects. Holometaboly likely evolved from hemimetabolan ancestors through the embryonic internalization of wing primordia into imaginal cells, which enabled the emergence of distinct larval forms. Key regulatory factors like Kr-h1, Chinmo, Ab, BR-C, and E93, already present in hemimetabolan lineages, were conserved and rewired in holometabolans. Crucial shifts in this evolutionary transition include Chinmo-mediated inhibition of BR-C and an inversion in the juvenile hormone effect on BR-C , from activation to repression.

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.

The three modes of insect postembryonic development are ametaboly, hemimetaboly and holometaboly, the latter being considered the only significant metamorphosis mode. However, the emergence of hemimetaboly, with the genuine innovation of the final moult, represents the origin of insect metamorphosis and a necessary step in the evolution of holometaboly. Hemimetaboly derives from ametaboly and might have appeared as a consequence of wing emergence in Pterygota, in the early Devonian. In extant insects, the final moult is mainly achieved through the degeneration of the prothoracic gland (PG), after the formation of the winged and reproductively competent adult stage. Metamorphosis, including the formation of the mature wings and the degeneration of the PG, is regulated by the MEKRE93 pathway, through which juvenile hormone precludes the adult morphogenesis by repressing the expression of transcription factor E93, which triggers this change. The MEKRE93 pathway appears conserved in extant metamorphosing insects, which suggest that this pathway was operative in the Pterygota last common ancestor. We propose that the final moult, and the consequent hemimetabolan metamorphosis, is a monophyletic innovation and that the role of E93 as a promoter of wing formation and the degeneration of the PG was mechanistically crucial for their emergence. This article is part of the theme issue ‘The evolution of complete metamorphosis’.

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.

Transmembrane glycerol transport is typically facilitated by aquaglyceroporins in Prokaryota and Eukaryota. In holometabolan insects however, aquaglyceroporins are absent, yet several species possess polyol permeable aquaporins. It thus remains unknown how glycerol transport evolved in the Holometabola. By combining phylogenetic and functional studies, here we show that a more efficient form of glycerol transporter related to the water-selective channel AQP4 specifically evolved and multiplied in the insect lineage, resulting in the replacement of the ancestral branch of aquaglyceroporins in holometabolan insects. To recapitulate this evolutionary process, we generate specific mutants in distantly related insect aquaporins and human AQP4 and show that a single mutation in the selectivity filter converted a water-selective channel into a glycerol transporter at the root of the crown clade of hexapod insects. Integration of phanerozoic climate models suggests that these events were associated with the emergence of complete metamorphosis and the unparalleled radiation of insects.