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 hormone (JH), a sesquiterpenoid produced by the insect corpus allatum gland (CA), is a key regulator of insect metamorphosis, reproduction, caste differentiation, and polyphenism. The first part of JH biosynthesis occurs via the universal eukaryotic mevalonate pathway. The final steps involve epoxidation and methylation. However, the sequence of these steps might not be conserved among all insects and Crustacea. Therefore, we used available genomic and transcriptomic data and identified JH acid methyltransferase (JHAMT), analyzed their genomic duplications in selected model organisms, and reconstructed their phylogeny. We have further reconstructed phylogeny of FAMeT proteins and show that evolution of this protein group is more complicated than originally appreciated. The analysis delineates important milestones in the evolution of several JH biosynthetic enzymes in arthropods, reviews major literature data on the last steps of JH synthesis, and defines questions and some hypotheses worth pursuing experimentally.

• Klíčová slova
• Alternative splicing, Epoxidase, Evolution, Gene duplication, Juvenile hormone, Methyl transferase,
• MeSH
• corpora allata MeSH
• hmyz genetika   metabolismus   MeSH
• hmyzí proteiny metabolismus   MeSH
• juvenilní hormony * metabolismus   MeSH
• seskviterpeny * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hmyzí proteiny MeSH
• juvenilní hormony * MeSH
• seskviterpeny * 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

Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JH receptor [JHR]), a ligand-activated complex of transcription factors consisting of the JH-binding protein methoprene-tolerant (MET) and its partner taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly because of the difficulty of obtaining purified and functional JHR proteins. Here, we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle T. castaneum and the mosquito Aedes aegypti. Recombinant JHR subunits from each species were coexpressed in an insect cell line using a baculovirus system. MET-TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex in greater detail. Biochemical analyses and MS confirmed that T. castaneum JHR was a 1:1 heterodimer consisting of MET and Taiman proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the MET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of MET. Our present characterization of the recombinant JHR is an initial step toward understanding JHR structure and function.

• Klíčová slova
• PAS domain, basic helix–loop–helix/transcription factor, hormone receptor, insect, juvenile hormone, ligand-binding protein, methoprene, nuclear translocation, protein phosphorylation, protein purification,
• MeSH
• Aedes genetika   metabolismus   MeSH
• fosforylace MeSH
• hmyzí proteiny genetika   metabolismus   MeSH
• juvenilní hormony metabolismus   MeSH
• posttranslační úpravy proteinů * MeSH
• receptory buněčného povrchu genetika   metabolismus   MeSH
• Sf9 buňky MeSH
• Spodoptera MeSH
• Tribolium genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hmyzí proteiny MeSH
• juvenilní hormony MeSH
• receptory buněčného povrchu 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

The juvenile hormones (JHs) are a group of sesquiterpenoids synthesized by the corpora allata. They play critical roles during insect development and reproduction. To study processes that are controlled by JH, researchers need methods to identify and quantify endogenous JHs and tools that can be used to increase or decrease JH titers in vitro and in vivo. The lipophilic nature of JHs, coupled with the low endogenous titers, make handling and quantification challenging. JH titers in insects can easily be increased by the topical application of JH analogs, such as methoprene. On the other hand, experimentally reducing JH titers has been more difficult. New approaches to modulate JH homeostasis have been established based on advances in RNA interference and CRISPR/Cas9-based genome editing. This review will summarize current advances in: (1) the detection and quantification of JHs from insect samples; (2) approaches to manipulating JH titers; and (3) next-generation tools to modulate JH homeostasis.

• Klíčová slova
• JH signaling, JH titer, juvenile hormone,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

This study reports the development and application of a liquid chromatography method coupled to electrospray tandem mass spectrometry (LC-MS/MS) for the identification and quantification of the five most common juvenile hormone (JH) homologs and methyl farnesoate (MF). The protocol allows the simultaneous analysis in a single LC run of JH I, JH II, JH III, JH III bisepoxide (JHB3) and JH III skipped bisepoxide (JHSB3). The identification of JHs is based on multiple reaction monitoring (MRM), using two of the most abundant fragmentation transitions for each hormone. Addition of deuterated JH III as an internal standard permits the absolute quantification of the different JHs. The JH homologs common structural features led to similar chromatographic behavior, as well as related fragmentation patterns, which facilitated the simultaneous detection of all the homologs in a single LC-MS/MS run. The protocol detects JHs in the low femtomole range, allowing often the analysis of JH in individual insects. Fragmentation of each of the JH homologs generates unique diagnostic ions that permitted the identification and quantification of JHs from samples of different species of Diptera, Lepidoptera, Heteroptera and Hymenoptera. Having a simple protocol, which can undisputedly determine the identity of the homologs present in a particular species, provides us with the opportunity to identify and quantify JHs existing in insects that are pests, vector of diseases or important research models.

• Klíčová slova
• Homologs, Juvenile hormone, Liquid chromatography, MRM, Quantification,
• MeSH
• chromatografie kapalinová * MeSH
• Diptera chemie   MeSH
• Heteroptera chemie   MeSH
• Hymenoptera chemie   MeSH
• juvenilní hormony analýza   chemie   MeSH
• Lepidoptera chemie   MeSH
• tandemová hmotnostní spektrometrie * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• juvenilní hormony 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