In insects, juvenile hormone (JH) is essential for orchestrating reproductive events. For example, in the male moth Agrotis ipsilon, the behavioral response to female sex pheromone is linked to neuronal sensitivity in the primary olfactory centers (antennal lobes, ALs), and the maturation of accessory sex glands (ASGs) are known to be age- and JH-dependent. The molecular basis of this regulatory action of JH is not fully deciphered, and we show here that the heterodimerizing partner of Methoprene-tolerant called Taiman (Tai) is essential for the sexual maturation of male A. ipsilon. Tai expression in ALs and ASGs is elevated from the third day of adult life and is responsible for the acquisition of behavioral responsiveness to the sex pheromone and ASG maturation. Tai-deficient old males exhibited altered sexual behavior and delayed ASG maturation. Moreover, the expression levels of Tai and Krüppel homolog 1 (Kr-h1), an early JH-induced transcription factor, were reduced in ALs and ASGs of JH-deprived and Tai-deficient old males, respectively. Exogenous JH injection into young males resulted in precocious sexual maturation and this JH induction was suppressed by Tai silencing. Our results demonstrate that Tai is an actor of the JH signaling pathway that operates in ALs and ASGs to promote pheromone information processing and consequently the display of sexual behavior in synchrony with ASG maturation, ultimately optimizing male reproductive success. Thus, this study provides additional insights into the molecular mechanisms underlying hormonal regulation of sexual maturation in insects.

Juvenile hormone (JH) and nutrients are both essential for insect ovarian development. However, the molecular mechanisms of synergistic JH-nutrient regulation in ovarian development remain unclear. Here, trehalase 1 (Treh1) was the focus of the crosstalk between JH and nutrients in the ovarian development of Helicoverpa armigera. The data indicated that a sugar-free diet and a casein-free diet both arrested ovarian development and reduced JH levels in females. The activity of HaTreh1 was up-regulated by sugar starvation or JH treatments, but was down-regulated by casein-free diet treatment. Importantly, knockdown of HaTreh1 severely hindered ovary growth, including decreases in tubal length, egg number, ovary/total weight ratio, and egg-laying number. Interestingly, the transcription factor Methoprene-tolerant 1 (Met1) directly activated HaTreh1 transcription by binding to the E-box (GTTGAC) in its promoter. Furthermore, HaTreh1 directly bound to the methionine-rich storage protein1 (HaSP1) physically and under JH induction, and knockdown of HaSP1 delayed ovarian growth due to methionine deficiency. Overall, JH orchestrates ovarian development by modulating HaTreh1 transcription and protein activity, which in turn controls trehalose and methionine levels. As a key metabolic executor downstream of JH and nutrient signaling, HaTreh1 integrates sugar and amino acid availability to regulate ovarian development.

Trehalase (Treh) is crucial for ovarian development as it directly regulates the energy supply by hydrolyzing trehalose into glucose. Juvenile hormone (JH) is also essential for ovarian development, but how it affects Treh2 activity remains unclear. This study, which employed Helicoverpa armigera as a model, showed that HaTreh2 transcription and enzymatic activity peaks coincided with the peak of JH titers (the 2 and 3 days after emergence). Compared to the dsGFP control, knockdown of HaTreh2 transcription severely impaired ovarian development. LC-MS/MS and site mutation experiments demonstrated that JH triggered the serine 345 phosphorylation of HaTreh2 via the GPCR-cAMP-PKA pathway, thereby activating its enzymatic activity. Additionally, HaTreh2 is directly bound with trehalose transporter (HaTreT) under JH induction, thus controlling intracellular trehalose and glucose contents as well as the transcription of HaTreT. TreT controls the amount of trehalose, which serves as a substrate for Treh1, entering the cell. Treh2, on the other hand, uses extracellular trehalose as substrate, and the hydrolysis product glucose is further transported into the cell. Here, HaTreh2 regulated the substrate that HaTreh1 can act upon in the cell by directly binding with HaTreT during ovarian development when JH is induced. Therefore, JH systematically regulated trehalose metabolism during ovarian development through regulating the activity of HaTreh2. This study sheds light on the coordinated interplay between JH pathway and sugar metabolism in ovarian development.

Krüppel-homolog 1 (Kr-h1), a zinc-finger transcription factor, inhibits larval metamorphosis and promotes adult reproduction by transducing juvenile hormone (JH). Although the transcriptional regulation of Kr-h1 has been extensively studied, little is known about its regulation at the post-transcriptional level. Using the migratory locust Locusta migratoria as a model system, we report here that the microRNAs let-7 and miR-278 bound to the Kr-h1 coding sequence and downregulated its expression. Application of let-7 and miR-278 mimics (agomiRs) significantly reduced the level of Kr-h1 transcripts, resulting in partially precocious metamorphosis in nymphs as well as markedly decreased yolk protein precursors, arrested ovarian development and blocked oocyte maturation in adults. Moreover, the expression of let-7 and miR-278 was repressed by JH, constituting a regulatory loop of JH signaling. This study thus reveals a previously unknown regulatory mechanism whereby JH suppresses the expression of let-7 and miR-278, which, together with JH induction of Kr-h1 transcription, prevents the precocious metamorphosis of nymphs and stimulates the reproduction of adult females. These results advance our understanding of the coordination of JH and miRNA regulation in insect development.

Acp70A regulates Drosophila pheromones through juvenile hormone induction

Juvenile hormone regulates Aedes aegypti Krüppel homolog 1 through a conserved E box motif

Neuroparsins (NPs) are small proteins that were originally discovered in the pars intercerebralis-corpus cardiacum neurosecretory complex of the migratory locust brain. From the desert locust, Schistocerca gregaria, we recently cloned four different transcripts, each coding for a distinct NP-related peptide. In addition to the brain, some NP-like precursor (Scg-NPP) transcripts also occur in a number of peripheral tissues, and their expression levels are controlled in a gender- and stage-dependent manner. Previous studies revealed a close correlation between Scg-NPP transcript levels and the gonotrophic cycle. In the present report, we demonstrate that certain Scg-NPP transcript levels are significantly altered upon injection of juvenile hormone (JH) or 20-hydroxyecdysone (20E) in adult gregarious desert locusts (five days after final ecdysis). While Scg-NPP1 transcript levels did not significantly change as a result of hormone treatment (animals were analyzed 24 h after injection), Scg-NPP2, Scg-NPP3, and Scg-NPP4 displayed hormone-dependent regulation in various tissues. Scg-NPP2 and Scg-NPP3 transcript levels significantly increased in the brain of JH-treated locusts. In addition, JH induction of Scg-NPP3 and Scg-NPP4 transcripts was observed in male fat body and in male and female gonads. Furthermore, 20E injection also induced Scg-NPP2, Scg-NPP3, and Scg-NPP4 transcripts in desert locust gonads. This is the first report showing NP-like precursor gene expression in insect ovaries. Our study indicates that the expression levels of some Scg-NPP transcripts are regulated by developmental hormones, suggesting a close correlation between NP expression and the endocrine control of the reproductive cycle.

Using a differential display of mRNA technique we discovered that the juvenile hormone (JH) esterase gene (Cfjhe) from Choristoneura fumiferana is directly induced by juvenile hormone I (JH I), and the JH I induction is suppressed by 20-hydroxyecdysone (20E). To study the mechanism of action of these two hormones in the regulation of expression of this gene, we cloned the 1270-bp promoter region of the Cfjhe gene and identified a 30-bp region that is located between -604 and -574 and is sufficient to support both JH I induction and 20E suppression. This 30-bp region contains two conserved hormone response element half-sites separated by a 4-nucleotide spacer similar to the direct repeat 4 element and is designated as a putative juvenile hormone response element (JHRE). In CF-203 cells, a luciferase reporter placed under the control of JHRE and a minimal promoter was induced by JH I in a dose- and time-dependent manner. Moreover, 20E suppressed this JH I-induced luciferase activity in a dose- and time-dependent manner. Nuclear proteins isolated from JH I-treated CF-203 cells bound to JHRE and the binding was competed by a 100-fold excess of the cold probe but not by 100-fold excess of double-stranded oligonucleotides of unrelated sequence. JH I induced/modified nuclear proteins prior to their binding to JHRE and 20E suppressed this JH I induction/modification. These results suggest that the 30-bp JHRE identified in the Cfjhe gene promoter is sufficient to support JH induction and 20E suppression of the Cfjhe gene.

Juvenile hormone induction of pheromone gland PBAN-responsiveness in Helicoverpa armigera females

Juvenile hormone (JH) is a major hormone regulating insect development. We have obtained a cDNA and a genomic clone for juvenile hormone esterase (JHE), the enzyme that is involved in the degradation of juvenile hormone and which is critical for insect development. Analysis of the regulation of JHE during the final larval stadium in the cabbage looper, Trichoplusia ni, showed that the JHE mRNA levels are maximal on days 2 and 4 of the final stadium. Nuclear run-on analyses demonstrated that changes in JHE mRNA levels are primarily due to changes in the transcription rate of the gene, which may be a single copy in the genome. Treatment with a JH analog resulted in induction of JHE gene transcription, which could be detected within three hours after treatment. Salient features present in the 5' flanking region of this JH-sensitive gene are presented, including the presence of sequences closely resembling binding sites for members of the family of nuclear receptors. This report is the first direct demonstration, by nuclear run-on analysis, of JH induction of gene transcription.

Abstract Larvae of the Pyrochroid beetle Dendroides canadensis produce hemolymph antifreeze proteins (AFPs) as an adaptation to subzero overwintering temperatures. The AFPs are produced in early autumn in response to various environmental cues (short photoperiod, low temperature, short thermoperiod). The study presented here reinforces and extends the initial finding (Horwath and Duman, '83b) that juvenile hormone is involved in stimulating production of AFPs. Dose response curves showed seasonal variation in the sensitivity of the larvae to topical application of Juvenile Hormone (JH), with late summer larvae being more sensitive than early summer ones. The time response curve for induction of AFP activity in late summer larvae demonstrated that ∼14 days are required after JH treatment for the appearance of a significant increase in AFP activity. The timing of this response to JH induction is similar to that seen previously when larvae were acclimated to a short photoperiod.Studies with cultured fat bodies showed that the fat body is a probable site of production of the AFP, and further emphasized a role for JH in AFP induction. However, stimulation of AFP production by fat body only occurred when the fat bodies were removed from larvae that had been pretreated with JH. The presence of JH in the culture medium did not stimulate fat bodies taken from larvae that had not been pre‐treated. This suggests that an additional hormone(s), or other factor, may be required to induce fat body production of AFPs.

Purification of two distinct oocyte vitellins and identification of their corresponding vitellogenins in fat body and hemolymph of Blaberus discoidalis

An in vitro system for studying juvenile hormone induction of juvenile hormone esterase from the fat body of Trichoplusia ni (Hübner)

Juvenile hormone regulation of the second biting cycle in Culex pipiens

Juvenile hormone induction of ‘queenliness’ on female honey bee (Apis mellifera L.) larvae reared on worker jelly and on stored royal jelly

Juvenile hormone deprivation caused by surgical removal of corpora allata shortly after adult emergence blocked the initiation of biting behavior in Culex. pipiens and Culex quinquefasciatus. Reimplantation of corpora allata or injection of a synthetic juvenile hormone (JH-I) corrected the juvenile hormone deficiency and restored biting behavior. Ovariectomy experiments demonstrated that this behavioral effect of juvenile hormone was independent of ovarian involvement.

Juvenile hormone-induced biosynthesis of vitellogenin in Leucophaea maderae

In Leucophaea maderae the female specific protein, or vitellogenin, is being synthesized and secreted exclusively by the adult female fat bodies. This specific protein, which is induced by thejuvenile hormones makes up approximately 90% of the total yolk proteins. It is a lipophosphoprotein of low phosphorus (0.14‰) content. The female specific protein is synthesized on polysomes bound to membranes of the ergastoplasmic reticulum (ER). Microsome vesicles obtained from active tissues are heavily studded with ribosomes and are considerably more dense than those from “inactive” tissues. The nascent polypeptide chains of the vitellogenin are secreted into the cisternae of the ER and can be released by Na deoxycholate digestion of the membranes. Similarly, the non-specific serum proteins are also secreted into the cisternae of the ER. All evidence points to the fact that microsomes may carry mixed populations of polysomes, those associated with specific and those associated with non-sex-specific protein synthesis. The significance of the polysomal association with membranes for the synthesis of exportable sex-specific protein is discussed.

Within a few hours after injection of juvenile hormone into Hyalophora gloveri pupae, several fast-migrating carboxylesterases (EC 3.1.1.1) that are sensitive to diisopropylfluorophosphate appear in the hemolymph. Treatment of the pupae with puromycin or actinomycin D prevents the appearance of these hemolymph enzymes, suggesting de novo synthesis of the carboxylesterases. Of the several other compounds investigated, only a potent mimic of the juvenile hormone is able to induce these enzymes. When the induced enzymes are incubated in vitro with (14)C-labeled juvenile hormone, the hormone is rapidly and efficiently degraded. It is suggested that these induced carboxylesterases play an important role in the regulation of juvenile hormone titer.