To gain insights into how juvenile hormone (JH) came to regulate insect metamorphosis, we studied its function in the ametabolous firebrat, Thermobia domestica. Highest levels of JH occur during late embryogenesis, with only low levels thereafter. Loss-of-function and gain-of-function experiments show that JH acts on embryonic tissues to suppress morphogenesis and cell determination and to promote their terminal differentiation. Similar embryonic actions of JH on hemimetabolous insects with short germ band embryos indicate that JH's embryonic role preceded its derived function as the postembryonic regulator of metamorphosis. The postembryonic expansion of JH function likely followed the evolution of flight. Archaic flying insects were considered to lack metamorphosis because tiny, movable wings were evident on the thoraces of young juveniles and their positive allometric growth eventually allowed them to support flight in late juveniles. Like in Thermobia, we assume that these juveniles lacked JH. However, a postembryonic reappearance of JH during wing morphogenesis in the young juvenile likely redirected wing development to make a wing pad rather than a wing. Maintenance of JH then allowed wing pad growth and its disappearance in the mature juvenile then allowed wing differentiation. Subsequent modification of JH action for hemi- and holometabolous lifestyles are discussed.

• Klíčová slova
• developmental biology, differentiation, ecdysone, juvenile hormone, metamorphosis, myoglianin, precocene, thermobia domestica,
• MeSH
• biologická proměna * fyziologie   MeSH
• hmyz MeSH
• juvenilní hormony * MeSH
• morfogeneze MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• juvenilní hormony * MeSH

To gain insights into how juvenile hormone (JH) came to regulate insect metamorphosis, we studied its function in the ametabolous firebrat, Thermobia domestica. Highest levels of JH occur during late embryogenesis, with only low levels thereafter. Loss-of-function and gain-of-function experiments show that JH acts on embryonic tissues to suppress morphogenesis and cell determination and to promote their terminal differentiation. Similar embryonic actions of JH on hemimetabolous insects with short germ band embryos indicate that JH's embryonic role preceded its derived function as the postembryonic regulator of metamorphosis. The postembryonic expansion of JH function likely followed the evolution of flight. Archaic flying insects were considered to lack metamorphosis because tiny, movable wings were evident on the thoraces of young juveniles and their positive allometric growth eventually allowed them to support flight in late juveniles. Like in Thermobia, we assume that these juveniles lacked JH. However, a postembryonic reappearance of JH during wing morphogenesis in the young juvenile likely redirected wing development to make a wing pad rather than a wing. Maintenance of JH then allowed wing pad growth and its disappearance in the mature juvenile then allowed wing differentiation. Subsequent modification of JH action for hemi- and holometabolous lifestyles are discussed.

• Klíčová slova
• differentiation, ecdysone, juvenile hormone, metamorphosis, myoglianin, precocene,
• Publikační typ
• časopisecké články MeSH
• preprinty MeSH

TAIMAN (TAI), the only insect ortholog of mammalian Steroid Receptor Coactivators (SRCs), is a critical modulator of ecdysone and juvenile hormone (JH) signaling pathways, which govern insect development and reproduction. The modulatory effect is mediated by JH-dependent TAI's heterodimerization with JH receptor Methoprene-tolerant and association with the Ecdysone Receptor complex. Insect hormones regulate insect physiology and development in concert with abiotic cues, such as photo- and thermoperiod. Here we tested the effects of JH and ecdysone signaling on the circadian clock by a combination of microsurgical operations, application of hormones and hormone mimics, and gene knockdowns in the linden bug Pyrrhocoris apterus males. Silencing taiman by each of three non-overlapping double-strand RNA fragments dramatically slowed the free-running period (FRP) to 27-29 hours, contrasting to 24 hours in controls. To further corroborate TAIMAN's clock modulatory function in the insect circadian clock, we performed taiman knockdown in the cockroach Blattella germanica. Although Blattella and Pyrrhocoris lineages separated ~380 mya, B. germanica taiman silencing slowed the FRP by more than 2 hours, suggesting a conserved TAI clock function in (at least) some insect groups. Interestingly, the pace of the linden bug circadian clock was neither changed by blocking JH and ecdysone synthesis, by application of the hormones or their mimics nor by the knockdown of corresponding hormone receptors. Our results promote TAI as a new circadian clock modulator, a role described for the first time in insects. We speculate that TAI participation in the clock is congruent with the mammalian SRC-2 role in orchestrating metabolism and circadian rhythms, and that TAI/SRCs might be conserved components of the circadian clock in animals.

• MeSH
• buněčná membrána MeSH
• cirkadiánní hodiny * genetika   MeSH
• cirkadiánní rytmus genetika   MeSH
• ekdyson genetika   MeSH
• hmyz MeSH
• juvenilní hormony genetika   MeSH
• savci MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ekdyson MeSH
• juvenilní hormony MeSH

Juvenile hormones (JHs) control insect metamorphosis and reproduction. JHs act through a receptor complex consisting of methoprene-tolerant (Met) and taiman (Tai) proteins to induce transcription of specific genes. Among chemically diverse synthetic JH mimics (juvenoids), some of which serve as insecticides, unique peptidic juvenoids stand out as being highly potent yet exquisitely selective to a specific family of true bugs. Their mode of action is unknown. Here we demonstrate that, like established JH receptor agonists, peptidic juvenoids act upon the JHR Met to halt metamorphosis in larvae of the linden bug, Pyrrhocoris apterus. Peptidic juvenoids induced ligand-dependent dimerization between Met and Tai proteins from P. apterus but, consistent with their selectivity, not from other insects. A cell-based split-luciferase system revealed that the Met-Tai complex assembled within minutes of agonist presence. To explore the potential of juvenoid peptides, we synthesized 120 new derivatives and tested them in Met-Tai interaction assays. While many substituents led to loss of activity, improved derivatives active at sub-nanomolar range outperformed hitherto existing peptidic and classical juvenoids including fenoxycarb. Their potency in inducing Met-Tai interaction corresponded with the capacity to block metamorphosis in P. apterus larvae and to stimulate oogenesis in reproductively arrested adult females. Molecular modeling demonstrated that the high potency correlates with high affinity. This is a result of malleability of the ligand-binding pocket of P. apterus Met that allows larger peptidic ligands to maximize their contact surface. Our data establish peptidic juvenoids as highly potent and species-selective novel JHR agonists.

• Klíčová slova
• hormone receptor, juvenile hormone, ligand-binding pocket, metamorphosis, oogenesis,
• MeSH
• hmyz metabolismus   MeSH
• juvenilní hormony * metabolismus   MeSH
• larva MeSH
• ligandy MeSH
• methopren * metabolismus   MeSH
• peptidy farmakologie   MeSH
• rozmnožování MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• juvenilní hormony * MeSH
• ligandy MeSH
• methopren * MeSH
• peptidy MeSH

Methyl farnesoate (MF) plays hormonal regulatory roles in crustaceans. An epoxidated form of MF, known as juvenile hormone (JH), controls metamorphosis and stimulates reproduction in insects. To address the evolutionary significance of MF epoxidation, we generated mosquitoes completely lacking either of the two enzymes that catalyze the last steps of MF/JH biosynthesis and epoxidation, respectively: the JH acid methyltransferase (JHAMT) and the P450 epoxidase CYP15 (EPOX). jhamt-/- larvae lacking both MF and JH died at the onset of metamorphosis. Strikingly, epox-/- mutants, which synthesized MF but no JH, completed the entire life cycle. While epox-/- adults were fertile, the reproductive performance of both sexes was dramatically reduced. Our results suggest that although MF can substitute for the absence of JH in mosquitoes, it is with a significant fitness cost. We propose that MF can fulfill most roles of JH, but its epoxidation to JH was a key innovation providing insects with a reproductive advantage.

• Klíčová slova
• Aedes aegypti, corpora allata, juvenile hormone, methyl farnesoate, reproduction,
• MeSH
• Aedes enzymologie   genetika   MeSH
• biologická proměna MeSH
• genetická zdatnost * MeSH
• juvenilní hormony biosyntéza   MeSH
• molekulární evoluce * MeSH
• nenasycené mastné kyseliny metabolismus   MeSH
• rozmnožování MeSH
• seskviterpeny metabolismus   MeSH
• sexuální chování zvířat MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• juvenilní hormony MeSH
• methyl farnesoate MeSH Prohlížeč
• nenasycené mastné kyseliny MeSH
• seskviterpeny MeSH

Evidence accumulates that the functional plasticity of insulin and insulin-like growth factor signaling in insects could spring, among others, from the multiplicity of insulin receptors (InRs). Their multiple variants may be implemented in the control of insect polyphenism, such as wing or caste polyphenism. Here, we present a comprehensive phylogenetic analysis of insect InR sequences in 118 species from 23 orders and investigate the role of three InRs identified in the linden bug, Pyrrhocoris apterus, in wing polymorphism control. We identified two gene clusters (Clusters I and II) resulting from an ancestral duplication in a late ancestor of winged insects, which remained conserved in most lineages, only in some of them being subject to further duplications or losses. One remarkable yet neglected feature of InR evolution is the loss of the tyrosine kinase catalytic domain, giving rise to decoys of InR in both clusters. Within the Cluster I, we confirmed the presence of the secreted decoy of insulin receptor in all studied Muscomorpha. More importantly, we described a new tyrosine kinase-less gene (DR2) in the Cluster II, conserved in apical Holometabola for ∼300 My. We differentially silenced the three P. apterus InRs and confirmed their participation in wing polymorphism control. We observed a pattern of Cluster I and Cluster II InRs impact on wing development, which differed from that postulated in planthoppers, suggesting an independent establishment of insulin/insulin-like growth factor signaling control over wing development, leading to idiosyncrasies in the co-option of multiple InRs in polyphenism control in different taxa.

• Klíčová slova
• decoy of insulin receptor, gene structure, insects, insulin receptor, insulin signaling, wing polyphenism,
• MeSH
• biologická evoluce * MeSH
• duplikace genu MeSH
• Heteroptera genetika   růst a vývoj   MeSH
• hmyz anatomie a histologie   genetika   MeSH
• křídla zvířecí anatomie a histologie   růst a vývoj   MeSH
• receptor inzulinu genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• receptor inzulinu MeSH

Insect metamorphosis boasts spectacular cases of postembryonic development when juveniles undergo massive morphogenesis before attaining the adult form and function; in moths or flies the larvae do not even remotely resemble their adult parents. A selective advantage of complete metamorphosis (holometaboly) is that within one species the two forms with different lifestyles can exploit diverse habitats. It was the environmental adaptation and specialization of larvae, primarily the delay and internalization of wing development, that eventually required an intermediate stage that we call a pupa. It is a long-held and parsimonious hypothesis that the holometabolous pupa evolved through modification of a final juvenile stage of an ancestor developing through incomplete metamorphosis (hemimetaboly). Alternative hypotheses see the pupa as an equivalent of all hemimetabolous moulting cycles (instars) collapsed into one, and consider any preceding holometabolous larval instars free-living embryos stalled in development. Discoveries on juvenile hormone signalling that controls metamorphosis grant new support to the former hypothesis deriving the pupa from a final pre-adult stage. The timing of expression of genes that repress and promote adult development downstream of hormonal signals supports homology between postembryonic stages of hemimetabolous and holometabolous insects. This article is part of the theme issue 'The evolution of complete metamorphosis'.

• Klíčová slova
• evolution, hormone receptor, juvenile hormone, metamorphosis, signal transduction, transcription factor,
• MeSH
• biologická proměna * MeSH
• hmyz klasifikace   růst a vývoj   MeSH
• juvenilní hormony metabolismus   MeSH
• kukla růst a vývoj   MeSH
• signální transdukce * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• juvenilní hormony MeSH

The CRISPR/Cas9 technique is widely used in experimentation with human cell lines as well as with other model systems, such as mice Mus musculus, zebrafish Danio reiro, and the fruit fly Drosophila melanogaster. However, publications describing the use of CRISPR/Cas9 for genome editing in non-model organisms, including non-model insects, are scarce. The introduction of this relatively new method presents many problems even for experienced researchers, especially with the lack of procedures to tackle issues concerning the efficiency of mutant generation. Here we present a protocol for efficient genome editing in the non-model insect species Pyrrhocoris apterus. We collected data from several independent trials that targeted several genes using the CRISPR/Cas9 system and determined that several crucial optimization steps led to a remarkably increased efficiency of mutant production. The main steps are as follows: the timing of embryo injection, the use of the heteroduplex mobility assay as a screening method, in vivo testing of sgRNA efficiency, and G0 germline mosaicism screening. The timing and the method of egg injections used here need to be optimized for other species, but other here-described optimization solutions can be applied immediately for genome editing in other insect species.

• Klíčová slova
• CRISPR/Cas9, efficiency optimization, genetic mosaicism, genome editing, non-model insect,
• Publikační typ
• časopisecké články MeSH