Steroid receptor coactivator TAIMAN is a new modulator of insect circadian clock.

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.

Evolution of proteins involved in the final steps of juvenile hormone synthesis.

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.

Evolution of casein kinase 1 and functional analysis of new doubletime mutants in Drosophila.

Circadian clocks are timing devices that rhythmically adjust organism's behavior, physiology, and metabolism to the 24-h day-night cycle. Eukaryotic circadian clocks rely on several interlocked transcription-translation feedback loops, where protein stability is the key part of the delay between transcription and the appearance of the mature proteins within the feedback loops. In bilaterian animals, including mammals and insects, the circadian clock depends on a homologous set of proteins. Despite mostly conserved clock components among the fruit fly Drosophila and mammals, several lineage-specific differences exist. Here we have systematically explored the evolution and sequence variability of insect DBT proteins and their vertebrate homologs casein kinase 1 delta (CKIδ) and epsilon (CKIε), dated the origin and separation of CKIδ from CKIε, and identified at least three additional independent duplications of the CKIδ/ε gene in Petromyzon, Danio, and Xenopus. We determined conserved regions in DBT specific to Diptera, and functionally tested a subset of those in D. melanogaster. Replacement of Lysine K224 with acidic residues strongly impacts the free-running period even in heterozygous flies, whereas homozygous mutants are not viable. K224D mutants have a temperature compensation defect with longer free-running periods at higher temperatures, which is exactly the opposite trend of what was reported for corresponding mammalian mutants. All DBTs of dipteran insects contain the NKRQK motif at positions 220-224. The occurrence of this motif perfectly correlates with the presence of BRIDE OF DOUBLETIME, BDBT, in Diptera. BDBT is a non-canonical FK506-binding protein that physically interacts with Drosophila DBT. The phylogeny of FK506-binding proteins suggests that BDBT is either absent or highly modified in non-dipteran insects. In addition to in silico analysis of DBT/CKIδ/ε evolution and diversity, we have identified four novel casein kinase 1 genes specific to the Drosophila genus.

Loss of Timeless Underlies an Evolutionary Transition within the Circadian Clock.

Most organisms possess time-keeping devices called circadian clocks. At the molecular level, circadian clocks consist of transcription-translation feedback loops (TTFLs). Although some components of the negative TTFL are conserved across the animals, important differences exist between typical models, such as mouse and the fruit fly. In Drosophila, the key components are PERIOD (PER) and TIMELESS (TIM-d) proteins, whereas the mammalian clock relies on PER and CRYPTOCHROME (CRY-m). Importantly, how the clock has maintained functionality during evolutionary transitions between different states remains elusive. Therefore, we systematically described the circadian clock gene setup in major bilaterian lineages and identified marked lineage-specific differences in their clock constitution. Then we performed a thorough functional analysis of the linden bug Pyrrhocoris apterus, an insect species comprising features characteristic of both the Drosophila and the mammalian clocks. Unexpectedly, the knockout of timeless-d, a gene essential for the clock ticking in Drosophila, did not compromise rhythmicity in P. apterus, it only accelerated its pace. Furthermore, silencing timeless-m, the ancestral timeless type ubiquitously present across animals, resulted in a mild gradual loss of rhythmicity, supporting its possible participation in the linden bug clock, which is consistent with timeless-m role suggested by research on mammalian models. The dispensability of timeless-d in P. apterus allows drawing a scenario in which the clock has remained functional at each step of transition from an ancestral state to the TIM-d-independent PER + CRY-m system operating in extant vertebrates, including humans.

Complex Evolution of Insect Insulin Receptors and Homologous Decoy Receptors, and Functional Significance of Their Multiplicity.

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.

HR38, an ortholog of NR4A family nuclear receptors, mediates 20-hydroxyecdysone regulation of carbohydrate metabolism during mosquito reproduction.

The Aedes aegypti mosquito is the principal vector for many dangerous human viral diseases. Carbohydrate metabolism (CM) is essential for supplying the energy necessary for host seeking, blood digestion and rapid egg development of this vector insect. The steroid hormone 20-hydroxyecdysone (20E) and the ecdysone receptor (EcR) are important regulators of CM, coordinating it with female reproductive events. We report here that the NR4A nuclear receptor AHR38 plays a critical role in mediating these actions of 20E and EcR. AHR38 RNA interference (RNAi) depletion in female mosquitoes blocked the transcriptional activation of CM genes encoding phosphoglucomutase (PGM) and trehalose-6-phophate synthase (TPS); it caused an increase of glycogen accumulation and a decrease of the circulating sugar trehalose. This treatment also resulted in a dramatic reduction in fecundity. Considering that these phenotypes resulting from AHR38 RNAi depletion are similar to those of EcR RNAi, we investigated a possible connection between these transcription factors in CM regulation. EcR RNAi inhibits the AHR38 gene expression. Moreover, the 20E-induced EcR complex directly activates AHR38 by binding to the ecdysone response element (EcRE) in the upstream regulatory region of this gene. The present work has implicated AHR38 in the 20E-mediated control of CM and provided new insight into mechanisms of 20E regulation of metabolism during female mosquito reproduction.

Nutritional Control of Insect Reproduction.

The amino acid-Target of Rapamycin (AA/TOR) and insulin pathways play a pivotal role in reproduction of female insects, serving as regulatory checkpoints that guarantee the sufficiency of nutrients for developing eggs. Being evolutionary older, the AA/TOR pathway functions as an initial nutritional sensor that not only activates nutritional responses in a tissue-specific manner, but is also involved in the control of insect insulin-like peptides (ILPs) secretion. Insulin and AA/TOR pathways also assert their nutritionally linked influence on reproductive events by contributing to the control of biosynthesis and secretion of juvenile hormone and ecdysone. This review covers the present status of our understanding of the contributions of AA/TOR and insulin pathways in insect reproduction.

Genetic Evidence for Function of the bHLH-PAS Protein Gce/Met As a Juvenile Hormone Receptor.

Juvenile hormones (JHs) play a major role in controlling development and reproduction in insects and other arthropods. Synthetic JH-mimicking compounds such as methoprene are employed as potent insecticides against significant agricultural, household and disease vector pests. However, a receptor mediating effects of JH and its insecticidal mimics has long been the subject of controversy. The bHLH-PAS protein Methoprene-tolerant (Met), along with its Drosophila melanogaster paralog germ cell-expressed (Gce), has emerged as a prime JH receptor candidate, but critical evidence that this protein must bind JH to fulfill its role in normal insect development has been missing. Here, we show that Gce binds a native D. melanogaster JH, its precursor methyl farnesoate, and some synthetic JH mimics. Conditional on this ligand binding, Gce mediates JH-dependent gene expression and the hormone's vital role during development of the fly. Any one of three different single amino acid mutations in the ligand-binding pocket that prevent binding of JH to the protein block these functions. Only transgenic Gce capable of binding JH can restore sensitivity to JH mimics in D. melanogaster Met-null mutants and rescue viability in flies lacking both Gce and Met that would otherwise die at pupation. Similarly, the absence of Gce and Met can be compensated by expression of wild-type but not mutated transgenic D. melanogaster Met protein. This genetic evidence definitively establishes Gce/Met in a JH receptor role, thus resolving a long-standing question in arthropod biology.

Hormonal enhancement of insecticide efficacy in Tribolium castaneum: oxidative stress and metabolic aspects.

Insect anti-stress responses, including those induced by insecticides, are controlled by adipokinetic hormones (AKHs). We examined the physiological consequences of Pyrap-AKH application on Tribolium castaneum adults (AKH-normal and AKH-deficient prepared by the RNAi technique) treated by two insecticides, pirimiphos-methyl and deltamethrin. Co-application of pirimiphos-methyl and/or deltamethrin with AKH significantly increased beetle mortality compared with application of the insecticides alone. This co-treatment was accompanied by substantial stimulation of general metabolism, as monitored by carbon dioxide production. Further, the insecticide treatment alone affected some basic markers of oxidative stress: it lowered total antioxidative capacity as well as the activity of superoxide dismutase in the beetle body; in addition, it enhanced the activity of catalase and glutathione-S-transferase. However, these discrepancies in oxidative stress markers were eliminated/reduced by co-application with Pyrap-AKH. We suggest that the elevation of metabolism, which is probably accompanied with faster turnover of toxins, might be responsible for the higher mortality that results after AKH and insecticide co-application. Changes in oxidative stress markers are probably not included in the mechanisms responsible for increased mortality.

Importance of juvenile hormone signaling arises with competence of insect larvae to metamorphose.

Juvenile hormone (JH) postpones metamorphosis of insect larvae until they have attained an appropriate stage and size. Then, during the final larval instar, a drop in JH secretion permits a metamorphic molt that transforms larvae to adults either directly (hemimetaboly) or via a pupal stage (holometaboly). In both scenarios, JH precludes metamorphosis by activating the Kr-h1 gene through a JH receptor, Methoprene-tolerant (Met). Removal of Met, Kr-h1, or JH itself triggers deleterious precocious metamorphosis. Although JH is thought to maintain the juvenile status throughout larval life, various methods of depleting JH failed to induce metamorphosis in early-instar larvae. To determine when does JH signaling become important for the prevention of precocious metamorphosis, we chose the hemimetabolous bug, Pyrrhocoris apterus, and the holometabolous silkworm, Bombyx mori. Both species undergo a fixed number of five larval instars. Pyrrhocoris larvae subjected to RNAi-mediated knockdown of Met or Kr-h1 underwent precocious adult development when treated during the fourth (penultimate) instar, but younger larvae proved increasingly resistant to loss of either gene. The earliest instar developing minor signs of precocious metamorphosis was the third. Therefore, the JH-response genes may not be required to maintain the larval program during the first two larval instars. Next, we examined Bombyx mod mutants that cannot synthesize authentic, epoxidized forms of JH. Although mod larvae expressed Kr-h1 mRNA at severely reduced levels since hatching, they only entered metamorphosis by pupating after four, rarely three instars. Based on findings in Pyrrhocoris and Bombyx, we propose that insect postembryonic development is initially independent of JH. Only later, when larvae gain competence to enter metamorphosis, JH signaling becomes necessary to prevent precocious metamorphosis and to optimize growth.

Juvenile hormone signaling during reproduction and development of the linden bug, Pyrrhocoris apterus.

Juvenile hormone (JH), a sesquiterpenoid produced by the insect corpus allatum gland (CA), prevents metamorphosis in larvae and stimulates vitellogenesis in adult females. Whether the same JH signaling pathway regulates both processes is presently unknown. Here, we employ the robust JH response during reproduction and development of the linden bug, Pyrrhocoris apterus, to compare the function of key JH-signaling genes encoding the JH receptor, Methoprene-tolerant (Met), its binding partner Taiman (Tai), and a JH-inducible protein, Krüppel-homolog 1 (Kr-h1). RNA interference (RNAi) with Met or Tai, but not Kr-h1, blocked ovarian development and suppressed vitellogenin gene expression in the fat body of females raised under reproduction-inducing conditions. Loss of Met and Tai matched the effects of CA ablation or the natural absence of JH during reproductive diapause. Stimulation of vitellogenesis by treatment of diapausing females with a JH mimic methoprene also required both Met and Tai in the fat body, whereas Kr-h1 RNAi had no effect. Therefore, the Met-Tai complex likely functions as a JH receptor during vitellogenesis. In contrast to Met and Kr-h1 that are both required for JH to prevent precocious metamorphosis in P. apterus larvae, removal of Tai disrupted larval ecdysis without causing premature adult development. Our results show that while Met operates during metamorphosis in larvae and reproduction in adult females, its partner Tai is only required for the latter. The diverse functions of JH thus likely rely on a common receptor whose actions are modulated by distinct components.

Common and distinct roles of juvenile hormone signaling genes in metamorphosis of holometabolous and hemimetabolous insects.

Insect larvae metamorphose to winged and reproductive adults either directly (hemimetaboly) or through an intermediary pupal stage (holometaboly). In either case juvenile hormone (JH) prevents metamorphosis until a larva has attained an appropriate phase of development. In holometabolous insects, JH acts through its putative receptor Methoprene-tolerant (Met) to regulate Krüppel-homolog 1 (Kr-h1) and Broad-Complex (BR-C) genes. While Met and Kr-h1 prevent precocious metamorphosis in pre-final larval instars, BR-C specifies the pupal stage. How JH signaling operates in hemimetabolous insects is poorly understood. Here, we compare the function of Met, Kr-h1 and BR-C genes in the two types of insects. Using systemic RNAi in the hemimetabolous true bug, Pyrrhocoris apterus, we show that Met conveys the JH signal to prevent premature metamorphosis by maintaining high expression of Kr-h1. Knockdown of either Met or Kr-h1 (but not of BR-C) in penultimate-instar Pyrrhocoris larvae causes precocious development of adult color pattern, wings and genitalia. A natural fall of Kr-h1 expression in the last larval instar normally permits adult development, and treatment with an exogenous JH mimic methoprene at this time requires both Met and Kr-h1 to block the adult program and induce an extra larval instar. Met and Kr-h1 therefore serve as JH-dependent repressors of deleterious precocious metamorphic changes in both hemimetabolous and holometabolous juveniles, whereas BR-C has been recruited for a new role in specifying the holometabolous pupa. These results show that despite considerable evolutionary distance, insects with diverse developmental strategies employ a common-core JH signaling pathway to commit to adult morphogenesis.