Control of the ecdysteroid level plays a crucial role in density-dependent metamorphosis in the giant mealworm beetle Zophobas atratus.

Metamorphic transition in some tenebrionid beetles is dependent on population density. This phenomenon is useful for pupae that are vulnerable to cannibalism. The physiological mechanism of this adaptive developmental phenomenon remains unclear. In Zophobas atratus, which show density-dependent metamorphosis, larval isolation can induce metamorphosis. We herein demonstrated that the return of isolated larvae to a crowded condition (re-crowding) inhibited their metamorphosis. The timing of metamorphic initiation was slightly extended according to the duration of re-crowding experienced by the isolated larvae. Therefore, the re-crowding induced physiological changes needed for metamorphic inhibition. We investigated whether hormone-related genes involved in signaling of metamorphic inhibitor (juvenile hormone, JH) and molting hormone (ecdysteroid) responded to the re-crowding. An expression analysis showed that gene expression of ecdysteroid signaling was maintained at low levels under the re-crowded condition. Actually, ecdysteroid levels decreased responding to re-crowding. Ecdysteroid injections induced metamorphosis in re-crowded larvae. In contrast, the JH signaling gene showed little fluctuation in both isolated and re-crowded conditions, and knockdown of JH signaling factors did not affect inhibition of metamorphosis under the re-crowded condition. The present study suggests that regulation of ecdysteroid level rather than JH is more crucial in the density dependent metamorphosis in Z. atratus.

Ecdysteroids affect female reproductive status and outcome of contest over hosts in the parasitoid wasp Eupelmus vuilleti.

Ecdysteroids are a family of insect hormones that may play a role in modulating aggressive behavior in reproductive contexts. In Hymenoptera, the few studies investigating the link between ecdysteroid titers, reproduction and aggressiveness during contests concern solely eusocial species. Here, we explored whether ecdysteroid titers influenced female reproductive status as well as aggressiveness and resolution of conflict in a solitary ectoparasitoid, Eupelmus vuilleti (Hymenoptera: Eupelmidae). Eupelmus vuilleti females parasitize and feed upon juvenile stages of Callosobruchus maculatus (Coleoptera: Bruchidae). When two E. vuilleti females are simultaneously present on a patch, they tend to protect the host they exploit by displaying aggressive behaviors towards conspecific competitors. To our knowledge, nothing is known about the association between ecdysteroids and aggressiveness or the outcome of contests for host access in solitary insects. First, we quantified ecdysteroid titers that naturally circulate in females without fighting experience and after a contest over host access. Ecdysteroid titers measured after the contest did not correlate with female aggressiveness during the contest, but winner wasps had higher titers than both losers and females that did not fight. Then, we manipulated hormone titers via injection: ecdysone favored egg maturation (i.e., gonadotropic effect) within 24 h and increased almost immediately the females' probability of winning host access without affecting their aggressiveness. Our results represent an important step in understanding how hormones, such as ecdysteroids, mediate insect behavior during intraspecific competition.

[Exploration of Insect Growth Regulators Targeting Noppera-bo, an Enzyme Involved in Ecdysteroid Biosynthesis].

Insect growth regulators (IGRs) are chemicals that adversely affect the physiological processes associated with insect development and cause abnormalities that impair insect survival. Ecdysone, an insect steroid hormone originally identified as a molting hormone, plays an essential role in developmental transition, such as during molting and metamorphosis. Recently, a member of the epsilon class of glutathione S-transferases (GST), GSTe14, also called Noppera-bo (Nobo), has been identified as essential for regulating the biosynthesis of ecdysone. Knockout or knockdown of the nobo gene causes ecdysone deficiency, leading to either death or arrested phenotype development at the larval stage. It is therefore considered that Nobo is potentially well suited as a target for novel IGRs. In this review, we focus on the development of a high-throughput screening strategy for Nobo inhibitors using a GST fluorogenic substrate.

On gonads and gadflies: the estrus angle.

The first sex steroid to be crystallized was the vertebrate ovarian hormone, estrone - a less potent metabolite of 17ß-estradiol, which in mammals stimulates the female urge to mate (estrus). The gadfly (Greek oistros) lent its name to the process of estrus, as an insect that bites and torments in classical Greek mythology. With the purification and crystallization of a moult-inducing steroid (ecdysone) from insects, an interesting parallel emerged between mating and moulting in lower mammals and arthropods. Ecdysterone (potent ecdysone metabolite) has anabolic effects in mammalian muscle cells that can be blocked by selective estrogen receptor antagonists. Insects utilize ecdysteroids in similar ways that vertebrates use estrogens, including stimulation of oocyte growth and maturation. Ecdysteroids also modify precopulatory insect mating behaviour, further reinforcing the gonad-gadfly/mate-moult analogy.

Circadian control of prothoracicotropic hormone release in an adult insect and the induction of its rhythmicity by light cues.

The insect neuropeptide prothoracicotropic hormone (PTTH) is a critical regulator of larval development. We recently demonstrated that PTTH is also present in adult Rhodnius prolixus and is released by adult brains in vitro with a clear daily rhythm during egg development. Here, we employ a well-established in vitro bioassay, to show that the daily rhythm of PTTH release by brains in vitro is under circadian control since it persists in aperiodic conditions with a free running period of around 24h that is temperature compensated. Prolonged exposure (3weeks) of insects to continuous constant light (LL) completely eliminated PTTH release. Subsequent transfer of such insects from LL to constant darkness (DD) rapidly induced rhythmic PTTH release, indicating that the circadian rhythm of PTTH release is induced by photic cues. Western analysis identified PTTH in the adult hemolymph, suggesting that PTTH acts as a functional neurohormone in the adult insect. Dot blot analysis revealed that PTTH levels in the hemolymph also cycled with a daily rhythm that persisted in DD and was synchronous with the rhythm of PTTH release by brains in vitro. We conclude that the previously documented photosensitive clock in the brain regulates rhythmic PTTH release and thus generates the rhythm seen in the hemolymph. These results emphasize the importance of rhythmic PTTH release in the adult insect and support a role for PTTH in adult physiology and possibly within the adult circadian system.

UVB Radiation Delays Tribolium castaneum Metamorphosis by Influencing Ecdysteroid Metabolism.

Ultraviolet B (UVB) radiation is an important environmental factor. It is generally known that UVB exhibits high genotoxicity due to causing DNA damage, potentially leading to skin carcinogenesis and aging in mammals. However, little is known about the effects of UVB on the development and metamorphosis of insects, which are the most abundant terrestrial animals. In the present study, we performed dose-response analyses of the effects UVB irradiation on Tribolium castaneum metamorphosis, assessed the function of the T. castaneum prothoracicotropic hormone gene (Trcptth), and analyzed ecdysteroid pathway gene expression profile and ecdysterone titers post-UVB irradiation. The results showed that UVB not only caused death of T. castaneum larvae, but also delayed larval-pupal metamorphosis and reduced the size and emergence rate of pupae. In addition, we verified the function of Trcptth, which is responsible for regulating metamorphosis. It was also found that the expression profiles of Trcptth as well as ecdysteroidogenesis and response genes were influenced by UVB radiation. Therefore, a disturbance pulse of ecdysteroid may be involved in delaying development under exposure to irradiation. To our knowledge, this is the first report indicating that UVB can influence the metamorphosis of insects. This study will contribute to a better understanding of the impact of UVB on signaling mechanisms in insect metamorphosis.

Ecdysteroid promotes cell cycle progression in the Bombyx wing disc through activation of c-Myc.

Developmental switching from growth to metamorphosis in imaginal primordia is an essential process of adult body planning in holometabolous insects. Although it is disciplined by a sequential action of the ecdysteroid, molecular mechanisms linking to cell proliferation are poorly understood. In the present study, we investigated the expression control of cell cycle-related genes by the ecdysteroid using the wing disc of the final-instar larvae of the silkworm, Bombyx mori. We found that the expression level of c-myc was remarkably elevated in the post-feeding cell proliferation phase, which coincided with a small increase in ecdysteroid titer. An in vitro wing disc culture showed that supplementation of the moderate level of the ecdysteroid upregulated c-myc expression within an hour and subsequently increased the expression of cell cycle core regulators, including A-, B-, D-, and E-type cyclin genes, Cdc25 and E2F1. We demonstrated that c-myc upregulation by the ecdysteroid was not inhibited in the presence of a protein synthesis inhibitor, suggesting a possibility that the ecdysteroid directly stimulates c-myc expression. Finally, results from the administration of a c-Myc inhibitor demonstrated that c-Myc plays an essential role in 20E-inducible cell proliferation. These findings suggested a novel pathway for ecdysteroid-inducible cell proliferation in insects, and it is likely to be conserved between insects and mammals in terms of steroid hormone regulation.

A genome-wide survey of sexually dimorphic expression of Drosophila miRNAs identifies the steroid hormone-induced miRNA let-7 as a regulator of sexual identity.

MiRNAs bear an increasing number of functions throughout development and in the aging adult. Here we address their role in establishing sexually dimorphic traits and sexual identity in male and female Drosophila. Our survey of miRNA populations in each sex identifies sets of miRNAs differentially expressed in male and female tissues across various stages of development. The pervasive sex-biased expression of miRNAs generally increases with the complexity and sexual dimorphism of tissues, gonads revealing the most striking biases. We find that the male-specific regulation of the X chromosome is relevant to miRNA expression on two levels. First, in the male gonad, testis-biased miRNAs tend to reside on the X chromosome. Second, in the soma, X-linked miRNAs do not systematically rely on dosage compensation. We set out to address the importance of a sex-biased expression of miRNAs in establishing sexually dimorphic traits. Our study of the conserved let-7-C miRNA cluster controlled by the sex-biased hormone ecdysone places let-7 as a primary modulator of the sex-determination hierarchy. Flies with modified let-7 levels present doublesex-related phenotypes and express sex-determination genes normally restricted to the opposite sex. In testes and ovaries, alterations of the ecdysone-induced let-7 result in aberrant gonadal somatic cell behavior and non-cell-autonomous defects in early germline differentiation. Gonadal defects as well as aberrant expression of sex-determination genes persist in aging adults under hormonal control. Together, our findings place ecdysone and let-7 as modulators of a somatic systemic signal that helps establish and sustain sexual identity in males and females and differentiation in gonads. This work establishes the foundation for a role of miRNAs in sexual dimorphism and demonstrates that similar to vertebrate hormonal control of cellular sexual identity exists in Drosophila.

Circadian regulation of hemolymph and ovarian ecdysteroids during egg development in the insect Rhodnius prolixus (Hemiptera).

Ecdysteroids, classically studied as the molting hormones of insects, occur at lower levels in the adult stage, but their source and significance in adult physiology is debated. In Rhodnius prolixus, a blood meal initiates a cycle of egg development and ecdysteroids appeared in the hemolymph within 1-2h of feeding. Systematic changes in hemolymph ecdysteroid titer then occurred during egg development. There was a clear circadian rhythm in the hemolymph ecdysteroid titer throughout egg development. This is the first report of an ecdysteroid rhythm in any adult female insect. Of various tissues examined in vitro, only ovaries released ecdysteroids. The amounts released were adequate to account for observed hemolymph ecdysteroid titers. Therefore, ovaries are the primary, perhaps sole, source of hemolymph ecdysteroids. Ovaries in vitro showed a circadian rhythm of changes in ecdysteroid content and release that was in synchrony with the hemolymph ecdysteroid rhythm, indicating that ovarian ecdysteroid release drives the rhythm seen in the hemolymph. Potential regulation of ovarian ecdysteroids is discussed in relation to the known rhythms in prothoracicotropic hormone and insulin-like peptides that occur during egg development. The prospect of circadian regulation of egg development itself by hormones and/or an ovarian clock is also discussed.

Interplay between insulin signaling, juvenile hormone, and vitellogenin regulates maternal effects on polyphenism in ants.

Polyphenism is the phenomenon in which alternative phenotypes are produced by a single genotype in response to environmental cues. An extreme case is found in social insects, in which reproductive queens and sterile workers that greatly differ in morphology and behavior can arise from a single genotype. Experimental evidence for maternal effects on caste determination, the differential larval development toward the queen or worker caste, was recently documented in Pogonomyrmex seed harvester ants, in which only colonies with a hibernated queen produce new queens. However, the proximate mechanisms behind these intergenerational effects have remained elusive. We used a combination of artificial hibernation, hormonal treatments, gene expression analyses, hormone measurements, and vitellogenin quantification to investigate how the combined effect of environmental cues and hormonal signaling affects the process of caste determination in Pogonomyrmex rugosus. The results show that the interplay between insulin signaling, juvenile hormone, and vitellogenin regulates maternal effects on the production of alternative phenotypes and set vitellogenin as a likely key player in the intergenerational transmission of information. This study reveals how hibernation triggers the production of new queens in Pogonomyrmex ant colonies. More generally, it provides important information on maternal effects by showing how environmental cues experienced by one generation can translate into phenotypic variation in the next generation.

Drosophila female precopulatory behavior is modulated by ecdysteroids.

The effect of ecdysteroid signaling on Drosophila female precopulatory behavior was investigated using two types of mutants with either globally reduced ecdysteroid availability or reduced expression of ecdysone receptors in fruitless neurons, known to control sexual behavior. While being courted by males, mutant females performed significantly less full ovipositor extrusion behavior to reject male copulation attempts. Ecdysteroid depleted females (ecdysoneless(1)) performed male-like courtship behaviors, including unilateral wing extension and song production with patterns very similar to male courtship song. These results support the hypothesis that ecdysteroids modulate female sexual behavior, perhaps acting as a regulator of sexual motivation, and as a component affecting the performance of sex specific behavior patterns.

Reproduction, dominance, and caste: endocrine profiles of queens and workers of the ant Harpegnathos saltator.

The regulation of reproduction within insect societies is a key component of the evolution of eusociality. Differential patterns of hormone levels often underlie the reproductive division of labor observed among colony members, and further task partitioning among workers is also often correlated with differences in juvenile hormone (JH) and ecdysteroid content. We measured JH and ecdysteroid content of workers and queens of the ant Harpegnathos saltator. In this species, new colonies are founded by a single queen, but after she dies workers compete in an elaborate dominance tournament to decide a new group of reproductives termed "gamergates." Our comparisons revealed that queens, gamergates, and inside workers (non-reproductive) did not differ in levels of JH or ecdysteroids. However, increased JH and decreased ecdysteroid content was observed in outside workers exhibiting foraging behavior. Application of a JH analog to virgin queens of H. saltator, although effective at inducing dealation, failed to promote egg production. Together, these results support the hypothesis that JH has lost its reproductive function in H. saltator to regulate foraging among the worker caste.

Drosophila male courtship behavior is modulated by ecdysteroids.

Temperature-dependent induction of ecdysteroid deficiency in the ecdysoneless mutant ecd(1) adult Drosophila melanogaster results in altered courtship behavior in males. Ecdysteroid deficiency brings about significantly elevated male-male courtship behavior including song production resembling that directed toward females. Supplementation with dietary 20-hydroxyecdysone reduces male-male attraction, but does not change motor activity, courtship patterns or attraction to females. These observations support the hypothesis that reduced levels of ecdysteroids increase the probability that male fruit flies will display courtship behaviors to male stimuli.

Ontogenic potentialities of the worker caste in two sympatric subterranean termites in France.

In termites, the capacity of workers to differentiate into neotenic reproductives is an important characteristic that deserves particular attention. To gain insight into the differentiation pathway, the potentialities of workers and the endocrinal changes during the formation of neotenics were compared in two sympatric termites, Reticulitermes flavipes and Reticulitermes grassei. After 1 year of development, 100% of R. flavipes worker groups produced neotenics against only 63% of R. grassei groups. The average production of female neotenics was significantly higher in R. flavipes worker groups compared with R. grassei groups and R. flavipes produced a greater proportion of female neotenics. Moreover, R. flavipes produced more offspring, not only because there were more females, but also because R. flavipes females were more productive. Moreover, the offspring produced by R. flavipes grew faster than the offspring of R. grassei. Both ecdysteroid and juvenile hormone (JH) titers varied significantly during the development of neotenics. The two species showed similar ecdysteroid titer variation patterns. However, the JH titer variation patterns strongly differed: in R. grassei, the concentration of JH increased in maturing neotenics then dropped in mature neotenics, whereas in R. flavipes, the level of JH was significantly higher than in R. grassei and remained constantly high in mature neotenics. Overall, these results suggest that these two species differ strongly in many life-history traits as well as in the physiological control of their caste differentiation system. Possible origins and mechanisms of such interspecific variations are discussed, as well as their evolutionary and ecological consequences.

Surgically increased ovarian mass in the honey bee confirms link between reproductive physiology and worker behavior.

Honey bee (Apis mellifera L.) workers are essentially sterile females that are used to study how complex social behavior develops. Workers perform nest tasks, like nursing larvae, prior to field tasks, like foraging. Despite worker sterility, this behavioral progression correlates with ovary size: workers with larger ovaries (many ovary filaments) start foraging at younger ages on average. It is untested, however, whether the correlation confers a causal relationship between ovary size and behavioral development. Here, we successfully grafted supernumerary ovaries into worker bees to produce an artificial increase in the amount of ovary tissue. We next measured fat body mRNA levels for the yolk precursor gene vitellogenin, which influences honey bee behavioral development and can correlate with ovary size. Vitellogenin was equally expressed in surgical controls and bees with supernumerary ovaries, leading us to predict that these groups would be characterized by equal behavior. Contrary to our prediction, bees with supernumerary ovaries showed accelerated behavioral development compared to surgical controls, which behaved like reference bees that were not treated surgically. To explore this result we monitored fat body expression levels of a putative ecdysteroid-response gene, HR46, which is genetically linked to ovary size in workers. Our data establish that social insect worker behavior can be directly influenced by ovaries, and that HR46 expression changes with ovary size independent of vitellogenin.

Ecdysis triggering hormone signaling in the yellow fever mosquito Aedes aegypti.

At the end of each developmental stage, the yellow fever mosquito Aedes aegypti performs the ecdysis behavioral sequence, a precisely timed series of behaviors that culminates in shedding of the old exoskeleton. Here we describe ecdysis triggering hormone-immunoreactive Inka cells located at branch points of major tracheal trunks and loss of staining coincident with ecdysis. Peptides (AeaETH1, AeaETH2) purified from extracts of pharate 4th instar larvae have--PRXamide C-terminal amino acid sequence motifs similar to ETHs previously identified in moths and flies. Injection of synthetic AeaETHs induced premature ecdysis behavior in pharate larvae, pupae and adults. Two functionally distinct subtypes of ETH receptors (AeaETHR-A, AeaETHR-B) of A. aegypti are identified and show high sensitivity and selectivity to ETHs. Increased ETHR transcript levels and behavioral sensitivity to AeaETHs arising in the hours preceding the 4th instar larva-to-pupa ecdysis are correlated with rising ecdysteroid levels, suggesting steroid regulation of receptor gene expression. Our description of natural and ETH-induced ecdysis in A. aegypti should facilitate future approaches directed toward hormone-based interference strategies for control of mosquitoes as human disease vectors.

Roles of ecdysteroid and juvenile hormone in vitellogenesis in an endoparasitic wasp, Pteromalus puparum (Hymenoptera: Pteromalidae).

To elucidate the endocrine regulation of vitellogenesis in an endoparastic wasp (Pteromalus puparum), the titers of ecdysteroid and juvenile hormone (JH) from the whole bodies are measured using the method of radioimmunoassay and GC-MS, and compared with the levels of vitellogenin (Vg) mRNA in the fat bodies, hemolymph Vg and ovarian vitellin (Vt), respectively. The results show that the ecdysteroid titer and fat body Vg mRNA level have a similar dynamics tendency, and the peak titer is at adult eclosion. The titer of JH III and ovarian Vt also have a similar dynamics tendency, and the peak titer is at 48h after eclosion. The profiles of hemolymph Vg, Vg mRNA in fat bodies and ovarian Vt, are also measured in the wasps after treated with different amounts of 20-hydroxyecdysone (20HE) or JH III in female pupa and adults. The results show that 20HE stimulates Vg synthesis in the fat bodies and its release into the hemolymph, and that JH III only accelerates Vg sequestration in the oocytes. Decapitation, which is believed to terminate synthesis of JH in insects, can not inhibit vitellogenesis and oocyte maturation in P. puparum. Furthermore, Vg gene is expressed with a lower titer of JH and depressed by a higher titer of JH III. These studies suggest that ecdysteroids play a role in Vg synthesis and believed to be the dominant hormones in regulation of vitellogenesis in P. puparum, and JHs are not the essential factors to female reproduction in this wasp.

The buzz on fly neuronal remodeling.

Hormone-dependent rewiring of axons and dendrites is a conserved feature of nervous system development and plasticity. During metamorphosis in insects, steroid hormones (the ecdysteroids) and terpenoid hormones (the juvenile hormones) regulate extensive remodeling of the nervous system. These changes retool the nervous system for new behavioral and physiological functions that are required for the adult stage of the life cycle. In honey bees and other highly social insects, hormones also regulate behavioral changes and neuronal plasticity associated with transitions between social caste roles. This review focuses on recent work in fruit flies and honey bees that reveals hormonal and molecular mechanisms underlying metamorphic and caste-dependent neuronal remodeling, with specific emphasis on the role of Krüppel homolog 1.

Ecdysteroids, juvenile hormone and insect neuropeptides: Recent successes and remaining major challenges.

In the recent decade, tremendous progress has been realized in insect endocrinology as the result of the application of a variety of advanced methods in neuropeptidome- and receptor research. Hormones of which the existence had been shown by bioassays four decades ago, e.g. bursicon (a member of the glycoprotein hormone family) and pupariation factor (Neb-pyrokinin 2, a myotropin), could be identified, along with their respective receptors. In control of diurnal rhythms, clock genes got company from the neuropeptide Pigment Dispersing Factor (PDF), of which the receptor could also be identified. The discovery of Inka cells and their function in metamorphosis was a true hallmark. Analysis of the genomes of Caenorhabditis elegans, Drosophila melanogaster and Apis mellifera yielded about 75, 100 and 200 genes coding for putative signaling peptides, respectively, corresponding to approximately 57, 100 and 100 peptides of which the expression could already be proven by means of mass spectrometry. The comparative approach invertebrates-vertebrates recently yielded indications for the existence of counterparts in insects for prolactin, atrial natriuretic hormone and Growth Hormone Releasing Hormone (GRH). Substantial progress has been realized in identifying the Halloween genes, a membrane receptor(s) for ecdysteroids, a nuclear receptor for methylfarnesoate, and dozens of GPCRs for insect neuropeptides. The major remaining challenges concern the making match numerous orphan GPCRs with orphan peptidic ligands, and elucidating their functions. Furthermore, the endocrine control of growth, feeding-digestion, and of sexual differentiation, in particular of males, is still poorly understood. The finding that the prothoracic glands produce an autocrine factor with growth factor-like properties and secrete proteins necessitates a reevaluation of their role in development.

[Ecdysone 20-monooxygenase activity in Drosophila virilis strains varying in ecdysteroid response to heat stress].

The effect of various duration of heat stress (38 degrees C) on the activity of ecdysone 20-monooxygenase converting ecdysone into 20-hydroxyecdysone has been studied in D. virilis of wild type and mutant strain females, which differ by the mode of heat stress response of ecdysone and 20-hydroxyecdysone. We are the first to show that heat stress induces activity of ecdysone 20-monooxygenase in Drosophila females and enzyme activity correlates with the level of 20-hydroxyecdysone.