Ecdysis-triggering hormone (ETH) was originally discovered and characterized as a molt termination signal in insects through its regulation of the ecdysis sequence. Here we report that ETH persists in adult Drosophila melanogaster, where it functions as an obligatory allatotropin to promote juvenile hormone (JH) production and reproduction. ETH signaling deficits lead to sharply reduced JH levels and consequent reductions of ovary size, egg production, and yolk deposition in mature oocytes. Expression of ETH and ETH receptor genes is in turn dependent on ecdysone (20E). Furthermore, 20E receptor knockdown specifically in Inka cells reduces fecundity. Our findings indicate that the canonical developmental roles of 20E, ETH, and JH during juvenile stages are repurposed to function as an endocrine network essential for reproductive success.ecdysis triggering hormone | ecdysone | juvenile hormone | fecundity | oogenesis T he life history of insects is characterized by radical morphogenetic transformations, whereby tissues are reorganized and hormones are repurposed for roles associated with stage-specific functions. During development, larvae complete each molt by shedding the cuticle under control of ecdysis triggering hormones (ETHs) targeting central peptidergic ensembles to orchestrate an innate behavioral sequence (1-3). Previous observations that Inka cells, the sole source of ETHs, persist into the adult stage (4) suggest possible reproductive functions for these peptides.We hypothesized that ETHs regulate juvenile hormone (JH) levels, based on the report of ETH receptor (ETHR) expression in the corpora allata (CA) of the silkworm, Bombyx mori (5). Evidence that ETH functions as an allatotropin in the yellow fever mosquito Aedes aegypti came from a recent study showing its activation of JH acid methyltransferase (6).JH is a sesquiterpenoid hormone with well-known morphogenetic and gonadotropic functions. In Drosophila, adult phenotypes resulting from reduction of JH levels have been characterized through induction of cell death in the CA or through enhancement of its degradation (7,8). Based on evidence from studies on Bombyx and Aedes, we investigated whether ETH functions as an allatotropin in adult Drosophila and the extent to which it may be necessary for reproductive functions.Previous studies showed that ecdysone (20E) regulates synthesis and release of ETH and expression of ETHR during larval stages of moths and mosquitoes (9-12). More recently, selftranscribing active regulatory region sequencing (STARR-Seq) data confirm that 20E induces 20E receptor (EcR) enhancer activity in promoters of both ETH and ETHR genes (13). Because circulating 20E levels are of major physiological and reproductive relevance (14), we also asked whether 20E influences ETH gene expression during the adult stage.Here we describe functional roles for 20E, ETH, and JH as a hormonal triad essential for reproductive success in Drosophila.In particular, we confirm persistence of ETH signaling throughout adulthood and demonstrate it...

Male-derived sex-peptide (SP) induces profound changes in the behaviour of Drosophila females, resulting in decreased receptivity to further mating and increased egg laying. SP can mediate the switch in female reproductive behaviours via a G protein-coupled receptor, SPR, in neurons expressing fruitless, doublesex and pickpocket. Whether SPR is the sole receptor and whether SP induces the postmating switch in a single pathway has not, to our knowledge been tested. Here we report that the SP response can be induced in the absence of SPR when SP is ectopically expressed in neurons or when SP, transferred by mating, can access neurons through a leaky blood brain barrier. Membrane-tethered SP can induce oviposition via doublesex, but not fruitless and pickpocket neurons in SPR mutant females. Although pickpocket and doublesex neurons rely on G(o) signalling to reduce receptivity and induce oviposition, G(o) signalling in fruitless neurons is required only to induce oviposition, but not to reduce receptivity. Our results show that SP's action in reducing receptivity and inducing oviposition can be separated in fruitless and doublesex neurons. Hence, the SP-induced postmating switch incorporates shared, but also distinct circuitry of fruitless, doublesex and pickpocket neurons and additional receptors.

Drosophila melanogaster has become a significant model organism for alcohol research. In flies, a rich variety of behaviors can be leveraged for identifying genes affecting alcohol responses and adaptations. Furthermore, almost all genes can be easily genetically manipulated. Despite the great evolutionary distance between flies and mammals, many of the same genes have been implicated in strikingly similar alcohol-induced behaviors. A major problem in medical research today is that it is difficult to extrapolate from any single model system to humans. Strong evolutionary conservation of a mechanistic response between distantly related organisms, such as flies and mammals, is a powerful predictor that conservation will continue all the way to humans. This review describes the state of the Drosophila alcohol research field. It describes common alcohol behavioral assays, the independent origins of resistance and tolerance, the results of classical genetic screens and candidate gene analysis, and the outcomes of recent genomics studies employing GWAS, transcriptome, miRNA, and genome-wide histone acetylation surveys.

Juvenile Hormone (JH) has a prominent role in the regulation of insect development. Much less is known about its roles in adults, although functions in reproductive maturation have been described. In adult females, JH has been shown to regulate egg maturation and mating. To examine a role for JH in male reproductive behavior we created males with reduced levels of Juvenile Hormone Acid O-Methyl Transferase (JHAMT) and tested them for courtship. JHAMT regulates the last step of JH biosynthesis in the Corpora Allata (CA), the organ of JH synthesis. Males with reduced levels of JHAMT showed a reduction in courtship that could be rescued by application of Methoprene, a JH analog, shortly before the courtship assays were performed. In agreement with this, reducing JHAMT conditionally in mature flies led to courtship defects that were rescuable by Methoprene. The same result was also observed when the CA were conditionally ablated by the expression of a cellular toxin. Our findings demonstrate that JH plays an important physiological role in the regulation of male mating behavior.

Homeostatic neural adaptations to alcohol underlie the production of alcohol tolerance and the associated symptoms of withdrawal. These adaptations have been shown to persist for relatively long periods of time and are believed to be of central importance in promoting the addictive state. In Drosophila, a single exposure to alcohol results in long-lasting alcohol tolerance and symptoms of withdrawal following alcohol clearance. These persistent adaptations involve mechanisms such as long-lasting changes in gene expression and perhaps epigenetic restructuring of chromosomal regions. Histone modifications have emerged as important modulators of gene expression and are thought to orchestrate and maintain the expression of multi-gene networks. Previously genes that contribute to tolerance were identified as those that show alcohol-induced changes in histone H4 acetylation following a single alcohol exposure. However, the molecular mediator of the acetylation process that orchestrates their expression remains unknown. Here we show that the Drosophila ortholog of mammalian CBP, nejire, is the histone acetyltransferase involved in regulatory changes producing tolerance—alcohol induces nejire expression, nejire mutations suppress tolerance, and transgenic nejire induction mimics tolerance in alcohol-naive animals. Moreover, we observed that a loss-of-function mutation in the alcohol tolerance gene slo epistatically suppresses the effects of CBP induction on alcohol resistance, linking nejire to a well-established alcohol tolerance gene network. We propose that CBP is a central regulator of the network of genes underlying an alcohol adaptation.

NF-jB proteins are well known as transcription factors important in immune system activation. In this highly conserved role, they contribute to changes in behavior in response to infection and in response to a variety of other insults and experiences. In some mammalian neurons, NF-jBs can be found at the synapse and translocate to the nucleus to alter gene expression when activated by synaptic activity. Here, we demonstrate that, in Drosophila melanogaster, NF-jB action is important both inside and outside the nucleus and that the Dif gene has segregated nuclear and non-nuclear NF-jB action into different protein isoforms. The DifA isoform is a canonical nuclear-acting NF-jB protein that enters the nucleus and is important for combating infection. The DifB variant, but not the DifA variant, is found in the central nervous system (mushroom bodies and antennal lobes). DifB does not enter the nucleus and co-localizes with a synaptic protein. In males and females, a DifB mutant alters alcohol behavioral sensitivity without an obvious effect on combating infection, whereas a DifA mutant does not affect alcohol sensitivity but compromises the immune response. These data are evidence that the non-nuclear DifB variant contributes to alcohol behavioral sensitivity by a nongenomic mechanism that diverges from the NF-jB transcriptional effects used in the peripheral immune system. Enrichment of DifB in brain regions rich in synapses and biochemical enrichment of DifB in the synaptoneurosome fraction indicates that the protein may act locally at the synapse.

Consistent callus induction and regeneration of material cultured in vitro is a prerequisite for somatic cell genetic improvement of plants, through methods such as genetic engineering. This study aims to induce rapid in vitro callusing and regeneration on three local rice varieties by exposure to different culture media. When mature embryos were exposed to three levels of 2,4-dichlorophenoxyacetic acid (2,4-D) and one level of thidiazuron (TDZ) (with a short hormone free period in between), all varieties showed rapid regeneration in the absence of a visible callus phase. When the seeds were cultured in 2,4-D for three weeks, at a decreasing concentration of 2,4-D every 7 days, and subsequently treated with TDZ, non-embryogenic calli were obtained that failed to regenerate on hormone free medium. When exposed to 2,4-D for 5 days and subculturcd in hormone free medium for two weeks, embryogenic calli and greening were observed within that period. The concentration and period of exposure to 2,4-D are critical factors for callus induction and regeneration in the genotypes tested.

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