BACKGROUND: Juvenile hormone (JH) is synthesized by the corpora allata (CA) and controls development and reproduction in insects. We recently used CRISPR/Cas9 to establish a line lacking the enzyme that catalyzes the final step of JH biosynthesis in mosquitoes, a P450 epoxidase. The CA of the epox-/- mutants do not synthesize epoxidized JH III but methyl farneosate (MF), a weak agonist of the JH receptor. Female epox-/- mosquitoes have reduced JH signaling and show a substantial loss of reproductive fitness. To understand the molecular basis of this loss of fitness, we constructed ovarian mRNA libraries of Ae. aegypti of the Orlando strain wild-type (WT) and epoxidase null mutants (epox-/-) and investigated differential expression of reproductive genes. RESULTS: We performed triplicate RNA-seq analyses of female WT and epox-/- ovaries dissected at four critical stages of oogenesis: Ovaries from newly eclosed females (0h), sugar-fed females at 4 days post-eclosion (4d SF), females 16h (16h BF), and 48 h after a blood meal (48h BF). Silencing of epoxidase resulted in a drastic change in the expression of thousands of genes. CONCLUSIONS: Our results suggest that epoxidase deficiency leads to a reduction in JH signaling that has significant effects on Ae. aegypti ovarian transcriptome profiles. Ecdysteroid titers are dysregulated in the mutants, leading to a significant delay in the expression of vitelline membrane genes and other transcripts. We discovered changes in the expression of 230 long non-coding RNAs (lncRNAs) that may play an important role in the regulation of ovarian genes.

• Keywords
• Aedes aegypti, Ecdysteroids, Juvenile hormone, Ovaries, Transcriptome, lncRNA,
• MeSH
• Aedes * genetics   physiology   MeSH
• Juvenile Hormones * metabolism   MeSH
• Oogenesis * genetics   MeSH
• Ovary * metabolism   MeSH
• Signal Transduction * genetics   MeSH
• Gene Expression Profiling MeSH
• Transcriptome * MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Juvenile Hormones * MeSH

Germ cells are essential to sexual reproduction. Across the animal kingdom, extracellular signaling isoprenoids, such as retinoic acids (RAs) in vertebrates and juvenile hormones (JHs) in invertebrates, facilitate multiple processes in reproduction. Here we investigated the role of these potent signaling molecules in embryonic germ cell development, using JHs in Drosophila melanogaster as a model system. In contrast to their established endocrine roles during larval and adult germline development, we found that JH signaling acts locally during embryonic development. Using an in vivo biosensor, we observed active JH signaling first within and near primordial germ cells (PGCs) as they migrate to the developing gonad. Through in vivo and in vitro assays, we determined that JHs are both necessary and sufficient for PGC migration. Analysis into the mechanisms of this newly uncovered paracrine JH function revealed that PGC migration was compromised when JHs were decreased or increased, suggesting that specific titers or spatiotemporal JH dynamics are required for robust PGC colonization of the gonad. Compromised PGC migration can impair fertility and cause germ cell tumors in many species, including humans. In mammals, retinoids have many roles in development and reproduction. We found that like JHs in Drosophila, RA was sufficient to impact mouse PGC migration in vitro. Together, our study reveals a previously unanticipated role of isoprenoids as local effectors of pre-gonadal PGC development and suggests a broadly shared mechanism in PGC migration.

• Keywords
• Hmgcr, cell movement, embryonic development, gametogenesis, germ cells, gonad, juvenile hormones, ovary, retinoids, testis,
• MeSH
• Drosophila melanogaster * MeSH
• Drosophila MeSH
• Gonads MeSH
• Juvenile Hormones * MeSH
• Humans MeSH
• Mice MeSH
• Cell Movement MeSH
• Mammals MeSH
• Terpenes MeSH
• Germ Cells MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Juvenile Hormones * MeSH
• Terpenes 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.

• Keywords
• hormone receptor, juvenile hormone, ligand-binding pocket, metamorphosis, oogenesis,
• MeSH
• Insecta metabolism   MeSH
• Juvenile Hormones * metabolism   MeSH
• Larva MeSH
• Ligands MeSH
• Methoprene * metabolism   MeSH
• Peptides pharmacology   MeSH
• Reproduction MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Juvenile Hormones * MeSH
• Ligands MeSH
• Methoprene * MeSH
• Peptides MeSH

Juvenile hormone (JH) signalling, via its receptor Methoprene-tolerant (Met), controls metamorphosis and reproduction in insects. Met belongs to a superfamily of transcription factors containing the basic Helix Loop Helix (bHLH) and Per Arnt Sim (PAS) domains. Since its discovery in 1986, Met has been characterized in several insect species. However, in spite of the importance as vectors of Chagas disease, our knowledge on the role of Met in JH signalling in Triatominae is limited. In this study, we cloned and sequenced the Dipetalogaster maxima Met transcript (DmaxMet). Molecular modelling was used to build the structure of Met and identify the JH binding site. To further understand the role of the JH receptor during oogenesis, transcript levels were evaluated in two main target organs of JH, fat body and ovary. Functional studies using Met RNAi revealed significant decreases of transcripts for vitellogenin (Vg) and lipophorin (Lp), as well as their receptors. Lp and Vg protein amounts in fat body, as well as Vg in hemolymph were also decreased, and ovarian development was impaired. Overall, these studies provide additional molecular insights on the roles of JH signalling in oogenesis in Triatominae; and therefore are relevant for the epidemiology of Chagas´ disease.

• MeSH
• Juvenile Hormones metabolism   MeSH
• Methoprene * metabolism   MeSH
• Oogenesis genetics   MeSH
• Triatominae * MeSH
• Vitellogenins MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Juvenile Hormones MeSH
• Methoprene * MeSH
• Vitellogenins 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.

• Keywords
• 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 genetics   metabolism   MeSH
• Phosphorylation MeSH
• Insect Proteins genetics   metabolism   MeSH
• Juvenile Hormones metabolism   MeSH
• Protein Processing, Post-Translational * MeSH
• Receptors, Cell Surface genetics   metabolism   MeSH
• Sf9 Cells MeSH
• Spodoptera MeSH
• Tribolium genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Insect Proteins MeSH
• Juvenile Hormones MeSH
• Receptors, Cell Surface 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.

• Keywords
• Aedes aegypti, corpora allata, juvenile hormone, methyl farnesoate, reproduction,
• MeSH
• Aedes enzymology   genetics   MeSH
• Metamorphosis, Biological MeSH
• Genetic Fitness * MeSH
• Juvenile Hormones biosynthesis   MeSH
• Evolution, Molecular * MeSH
• Fatty Acids, Unsaturated metabolism   MeSH
• Reproduction MeSH
• Sesquiterpenes metabolism   MeSH
• Sexual Behavior, Animal MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Juvenile Hormones MeSH
• methyl farnesoate MeSH Browser
• Fatty Acids, Unsaturated MeSH
• Sesquiterpenes 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.

• Keywords
• JH signaling, JH titer, juvenile hormone,
• Publication type
• Journal Article MeSH
• Review 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.

• Keywords
• Homologs, Juvenile hormone, Liquid chromatography, MRM, Quantification,
• MeSH
• Chromatography, Liquid * MeSH
• Diptera chemistry   MeSH
• Heteroptera chemistry   MeSH
• Hymenoptera chemistry   MeSH
• Juvenile Hormones analysis   chemistry   MeSH
• Lepidoptera chemistry   MeSH
• Tandem Mass Spectrometry * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Juvenile Hormones 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'.

• Keywords
• evolution, hormone receptor, juvenile hormone, metamorphosis, signal transduction, transcription factor,
• MeSH
• Metamorphosis, Biological * MeSH
• Insecta classification   growth & development   MeSH
• Juvenile Hormones metabolism   MeSH
• Pupa growth & development   MeSH
• Signal Transduction * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Juvenile Hormones MeSH

The sesquiterpenoid juvenile hormone (JH) is vital to insect development and reproduction. Intracellular JH receptors have recently been established as basic helix-loop-helix transcription factor (bHLH)/PAS proteins in Drosophila melanogaster known as germ cell-expressed (Gce) and its duplicate paralog, methoprene-tolerant (Met). Upon binding JH, Gce/Met activates its target genes. Insects possess multiple native JH homologs whose molecular activities remain unexplored, and diverse synthetic compounds including insecticides exert JH-like effects. How the JH receptor recognizes its ligands is unknown. To determine which structural features define an active JH receptor agonist, we tested several native JHs and their nonnative geometric and optical isomers for the ability to bind the Drosophila JH receptor Gce, to induce Gce-dependent transcription, and to affect the development of the fly. Our results revealed high ligand stereoselectivity of the receptor. The geometry of the JH skeleton, dictated by two stereogenic double bonds, was the most critical feature followed by the presence of an epoxide moiety at a terminal position. The optical isomerism at carbon C11 proved less important even though Gce preferentially bound a natural JH enantiomer. The results of receptor-ligand-binding and cell-based gene activation assays tightly correlated with the ability of different geometric JH isomers to induce gene expression and morphogenetic effects in the developing insects. Molecular modeling supported the requirement for the proper double-bond geometry of JH, which appears to be its major selective mechanism. The strict stereoselectivity of Gce toward the natural hormone contrasts with the high potency of synthetic Gce agonists of disparate chemistries.

• Keywords
• Drosophila, basic helix-loop-helix transcription factor (bHLH), development, hormone receptor, insect, juvenile hormone (JH), ligand-binding protein, reproduction, stereoselectivity,
• MeSH
• Drosophila melanogaster chemistry   genetics   metabolism   MeSH
• Juvenile Hormones chemistry   metabolism   MeSH
• Models, Molecular MeSH
• Drosophila Proteins metabolism   MeSH
• Receptors, Cell Surface metabolism   MeSH
• Stereoisomerism MeSH
• Basic Helix-Loop-Helix Transcription Factors metabolism   MeSH
• Transcription Factors metabolism   MeSH
• Protein Binding MeSH
• Gene Expression Regulation, Developmental MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• gce protein, Drosophila MeSH Browser
• Juvenile Hormones MeSH
• MET protein, Drosophila MeSH Browser
• Drosophila Proteins MeSH
• Receptors, Cell Surface MeSH
• Basic Helix-Loop-Helix Transcription Factors MeSH
• Transcription Factors MeSH