DrosophilaCG10527 mutants are resistant to juvenile hormone and its analog methoprene

Juvenile hormone (JH) is critical for development, metamorphosis, and reproduction in insects. While the physiological importance of JH has been appreciated for decades, its biosynthetic pathway and molecular action remain poorly understood. DrosophilaCG10527 encodes a protein with high homology to crustacean farnesoic acid methyltransferase (FAMeT) that converts farnesoic acid to methyl farnesoate (MF), a precursor of JH, but its in vivo functions remain unclear. Here we report that CG10527 is expressed widely in secondary cells in the male accessory glands, in ovarian follicle cells, and in glial cells in the nervous system. Furthermore, CG10527 is expressed abundantly in the corpora allata where JH is synthesized. To understand the physiological functions of CG10527, we generated specific CG10527 deletions. Phenotypic analysis showed that CG10527 null mutants are fully viable and fertile in both sexes, indicating that CG10527 is not essential for survival and fertility. Surprisingly, CG10527 mutants showed no defects in the biosynthesis of MF and JH. However, CG10527 mutants were 3-5 times more resistant than wild-type flies to topically applied MF and JH as well as the JH analog methoprene at both sub-lethal and lethal doses. Taken together, our data indicate that DrosophilaCG10527 plays little, if any, role in JH biosynthesis but may participate in the JH signaling pathway.     

1,5-Disubstituted imidazoles inhibit juvenile hormone biosynthesis by the corpora allata of the mosquito Aedes aegypti

We investigated the effect of fifteen 1,5-disubstituted imidazoles (1,5-dis) on juvenile hormone III (JH III) and methyl farnesoate (MF) biosynthesis by the corpora allata (CA) of the mosquito Aedes aegypti in vitro. Four compounds (TH-35, TH-83, TH-62 and TH-28) significantly decreased JH biosynthesis in the CA dissected from 3-day old sugar-fed females. The decrease of JH synthesis was not always associated with increased MF. TH-30 and TH-83 increased MF levels, while TH-85 and TH-61 significantly decreased MF levels. Five compounds (TH-26, TH-60, TH-83, TH-35 and TH-30) significantly inhibited JH biosynthesis in the CA dissected from females 15 h after a blood meal. Four 1,5-dis (TH-30, TH-26, TH-28 and TH-66) caused MF increases in CA from blood-fed females. 1,5-Disubstituted imidazoles had higher inhibitory activity on JH synthesis when substituted at position 5 by a 3-benzyloxyphenyl group and at position 1 by a benzyl group (such as TH-35). Inhibition of JH and MF biosynthesis by TH-35 was age-dependent and influenced by nutritional status; inhibition differed when evaluated in the CA dissected from sugar-fed females at different days after emergence and in the CA dissected from females at different hours after a blood meal. Inhibition was always higher when the CA was more active. The addition of TH-35 significantly reduced the stimulatory effect of Aedes-allatotropin and farnesoic acid on JH synthesis. This is the first report of an inhibitory effect of 1,5-disubstituted imidazoles on JH synthesis in Diptera.     

The circadian clock genes affect reproductive capacity in the desert locust Schistocerca gregaria

The circadian clocks govern many metabolic and behavioral processes in an organism. In insects, these clocks and their molecular machinery have been found to influence reproduction in many different ways. Reproductive behavior including courtship, copulation and egg deposition, is under strong influence of the daily rhythm. At the molecular level, the individual clock components also have their role in normal progress of oogenesis and spermatogenesis. In this study on the desert locust Schistocerca gregaria, three circadian clock genes were identified and their expression profiles were determined. High expression was predominantly found in reproductive tissues. Similar daily expression profiles were found for period (per) and timeless (tim), while the clock (clk) mRNA level is higher 12 h before the first per and tim peak. A knockdown of either per or tim resulted in a significant decrease in the progeny produced by dsRNA treated females confirming the role of clock genes in reproduction and providing evidence that both PER and TIM are needed in the ovaries for egg development. Since the knockdown of clk is lethal for the desert locust, its function remains yet to be elucidated.     

Is juvenile hormone involved in the maternal regulation of egg size and progeny characteristics in the desert locust?

The role of juvenile hormone (JH) in the maternal regulation of progeny characteristics was examined in the desert locust, Schistocerca gregaria. Female adults of this species are known to produce smaller but more eggs when reared in isolation than do those reared in a group. Eggs laid by isolated females develop green hatchlings typical of solitarious forms, whereas those laid by the latter produce black hatchlings typical of gregarious forms. Topical application of a juvenile hormone analog (JHA), fenoxycarb, or implantation of corpora allata (CA) taken from the migratory locust, Locusta migratoria, caused crowded S. gregaria females to deposit smaller eggs, but did not have a significant effect on the number of eggs per egg pod except at high doses of JHA. The production of smaller eggs by treated and untreated crowded females was closely associated with earlier deposition of the egg pods and shorter oviposition intervals. However, neither JHA application nor CA implantation influenced the progeny characteristics in actively reproducing aged females under crowded conditions, while untreated control females started producing smaller and more eggs upon transfer to isolated conditions. These results may suggest that JH is not directly involved in the maternal regulation of phase-dependent progeny characteristics.     

Terminal stages in juvenile hormone biosynthesis in corpora allata of Diploptera punctata: Developmental changes in enzyme activity and regulation by allatostatins

The O-methyltransferase, which is responsible for the methylation of farnesoic acid in the corpora allata of Diploptera punctata, is a cytosolic enzyme. The activity of O-methyltransferase closely parallels JH biosynthesis in last instars and adult females. Because allatostatin 4 (AST 4) from D. punctata and callatostatin 5 (CAST 5) from Calliphora vomitoria can inhibit juvenile hormone biosynthesis, their effects on the activity of O-methyltransferase and epoxidase, the enzymes involved in the final two steps of juvenile hormone biosynthesis, were investigated in vitro. AST 4 can inhibit methyltransferase activity whereas CAST 5 stimulates it. AST 4 inhibits epoxidase activity slightly whereas CAST 5 inhibits it significantly (36%). Treatment of corpora allata with farnesoic acid (40 μM) can reverse the inhibitory effect of AST 4 and CAST 5 on JH release by corpora allata. Thus, allatostatins appear to exert their inhibitory effect on JH biosynthesis at least partially through inhibition of the activity of terminal enzymes. Two biosynthetic pathways for the conversion of farnesoic acid to JH may exist in corpora allata of D. punctata: the predominant pathway is farnesoic acid to methyl farnesoate, then to JH whereas the other, representing about 5–10% of total JH production, is farnesoic acid to JH III acid, then to JH.     

Non-swarming grasshoppers exhibit density-dependent phenotypic plasticity reminiscent of swarming locusts

Locusts are well known for exhibiting an extreme form of density-dependent phenotypic plasticity known as locust phase polyphenism. At low density, locust nymphs are cryptically colored and shy, but at high density they transform into conspicuously colored and gregarious individuals. Most of what we know about locust phase polyphenism come from the study of the desert locust Schistocerca gregaria (Forskål), which is a devastating pest species affecting many countries in North Africa and the Middle East. The desert locust belongs to the grasshopper genus Schistocerca Stål, which includes mostly non-swarming, sedentary species. Recent phylogenetic studies suggest that the desert locust is the earliest branching lineage within Schistocerca, which raises a possibility that the presence of density-dependent phenotypic plasticity may be a plesiomorphic trait for the whole genus. In order to test this idea, we have quantified the effect of rearing density in terms of the resulting behavior, color, and morphology in two non-swarming Schistocerca species native to Florida. When reared in both isolated and crowded conditions, the two non-swarming species, Schistocerca americana (Drury) and Schistocerca serialis cubense (Saussure) clearly exhibited plastic reaction norms in all traits measured, which were reminiscent of the desert locust. Specifically, we found that both species were more active and more attracted to each other when reared in a crowded condition than in isolation. They were mainly bright green in color when isolated, but developed strong black patterns and conspicuous background colors when crowded. We found a strong effect of rearing density in terms of size. There were also more mechanoreceptor hairs on the outer face of the hind femora in the crowded nymphs in both species. Although both species responded similarly, there were some clear species-specific differences in terms of color and behavior. Furthermore, we compare and contrast our findings with those on the desert locust and other relevant studies. We attribute the presence of density-dependent phenotypic plasticity in the non-swarming Schistocerca species to phylogenetic conservatism, but there may be a possible role of local adaptation in further shaping the ultimate expressions of plasticity.     

JH biosynthesis by reproductive tissues and corpora allata in adult longhorned beetles, Apriona germari

We report on juvenile hormone (JH) biosynthesis from long‐chain intermediates by specific reproductive tissues and the corpora allata (CA) prepared from adult longhorned beetles, Apriona germari. The testes, male accessory glands (MAGs), ovaries, and CA contained the long‐chain intermediates in the JH biosynthetic pathway, farnesoic acid (FA), methyl farnesoate (MF), and JH III. The testes and ovaries, but not CA, produced radioactive JH III after the addition of ³H‐methionine and, separately, unlabeled methionine, to the incubation medium. We inferred that endogenous FA is methylated to MF in the testes and ovaries. Addition of farnesol led to increased amounts of FA in the testes, MAGs, ovaries, and CA, indicating oxidation of farnesol to FA. Addition of FA to incubation medium yielded increased JH III, again indicating methylation of FA to MF in the testes, MAGs, ovaries, but not CA. Addition of MF to incubation medium also led to JH III, from which we inferred the epoxidation of MF to JH III. JH biosynthesis from farnesol in the testes, MAGs, and ovaries of A. germari proceeds via oxidation to FA, methylation to MF, and epoxidation to JH III. This is a well‐known pathway to JH III, described here for the first time in reproductive tissues of longhorned beetles. © 2010 Wiley Periodicals, Inc.     

Methyl farnesoate synthesis is necessary for the environmental sex determination in the water flea Daphnia pulex

Sex-determination systems can be divided into two groups: genotypic sex determination (GSD) and environmental sex determination (ESD). ESD is an adaptive life-history strategy that allows control of sex in response to environmental cues in order to optimize fitness. However, the molecular basis of ESD remains largely unknown. The micro crustacean Daphnia pulex exhibits ESD in response to various external stimuli. Although methyl farnesoate (MF: putative juvenile hormone, JH, in daphnids) has been reported to induce male production in daphnids, the role of MF as a sex-determining factor remains elusive due to the lack of a suitable model system for its study. Here, we establish such a system for ESD studies in D. pulex. The WTN6 strain switches from producing females to producing males in response to the shortened day condition, while the MFP strain only produces females, irrespective of day-length. To clarify whether MF has a novel physiological role as a sex-determining factor in D. pulex, we demonstrate that a MF/JH biosynthesis inhibitor suppressed male production in WTN6 strain reared under the male-inducible condition, shortened day-length. Moreover, we show that juvenile hormone acid O-methyltransferase (JHAMT), a critical enzyme of MF/JH biosynthesis, displays MF-generating activity by catalyzing farnesoic acid. Expression of the JHAMT gene increased significantly just before the MF-sensitive period for male production in the WTN6 strain, but not in the MFP strain, when maintained under male-inducible conditions. These results suggest that MF synthesis regulated by JHAMT is necessary for male offspring production in D. pulex. Our findings provide novel insights into the genetic underpinnings of ESD and they begin to shed light on the physiological function of MF as a male-fate determiner in D. pulex.     

Behavioural phase change in the Australian plague locust, Chortoicetes terminifera, is triggered by tactile stimulation of the antennae

Density-dependent phase polyphenism is a defining characteristic of the paraphyletic group of acridid grasshoppers known as locusts. The cues and mechanisms associated with crowding that induce behavioural gregarization are best understood in the desert locust, Schistocerca gregaria, and involve a combination of sensory inputs from the head (visual and olfactory) and mechanostimulation of the hind legs, acting via a transient increase in serotonin in the thoracic ganglia. Since behavioural gregarization has apparently arisen independently multiple times within the Acrididae, the important question arises as to whether the same mechanisms have been recruited each time. Here we explored the roles of visual, olfactory and tactile stimulation in the induction of behavioural gregarization in the Australian plague locust, Chortoicetes terminifera. We show that the primary gregarizing input is tactile stimulation of the antennae, with no evidence for an effect of visual and olfactory stimulation or tactile stimulation of the hind legs. Our results show that convergent behavioural responses to crowding have evolved employing different sites of sensory input in the Australian plague locust and the desert locust.     

Activities of natural methyl farnesoids on pupariation and metamorphosis of Drosophila melanogaster

Methyl farnesoate (MF) and juvenile hormone (JH III), which bind with high affinity to the receptors USP and MET, respectively, and bisepoxy JH III (bisJH III) were assessed for several activities during Drosophila larval development, and during prepupal development to eclosed adults. Dietary MF and JH III were similarly active, and more active than bisJH III, in lengthening larval development prior to pupariation. However, the order of activity was changed (JH III > bisJH III > MF) with respect to preventing prepupae from eclosing as normal adults, whether administered in the larval diet or as topically applied at the white puparium stage. If endogenous production of all three larval methyl farnesoids was suppressed by a strongly driven RNAi against HMGCR in the corpora allata cells, most larvae did not attain pupariation. Farnesol (which has no demonstrated life-necessary function in larval life except in corpora allata cells as a precursor to methyl farnesoid biosynthesis) when incorporated into the diet rescued attainment of pupariation in a dose-dependent manner, presumably by rescuing endogenous production of all three hormones. A more mild suppression of endogenous methyl farnesoid production enabled larval attainment of pupariation. However, in this background dietary MF had increased activity in preventing puparia from attaining normal adult eclosion. The physiological relevance of using exogenous methyl farnesoids to block prepupal development to normally eclosed adults was tested by, instead, protecting in prepupae the endogenous titer of methyl farnesoids. JH esterase normally increases during the mid-late prepupal stage, presumably to clear endogenous methyl farnesoids. When JH esterase was inhibited with an RNAi, it prevented attainment of adult eclosion. Cultured adult corpora allata from male and female Aedes aegypti released both MF and JH III, and the A. aegypti nuclear receptor USP bound MF with nanomolar affinity. These A. aegypti data support the use of Drosophila as a model for mosquitoes of the binding of secreted MF to USP.     

Enhancement by farnesol and farnesoic acid of juvenile-hormone biosynthesis in induced low-activity locust corpora allata

Exogenous farnesol or farnesoic acid (FA) stimulates juvenile hormone III (JH III) biosynthesis by isolated corpora allata from Locusta migratoria in a dose-dependent manner. Farnesol and FA also stimulate a dose-dependent accumulation of substantial amounts of methyl farnesoate (MF), identified by gas chromatography-mass spectroscopy (GCMS) analysis, in the corpora allata. Lower quantities of MF were found in the incubation medium. Corpora allata, denervated 2 days prior to assay, showed low spontaneous rates of JH biosynthesis which were stimulated by farnesol and FA. The dose-response curves for control and denervated corpora allata were similar. During oocyte maturation the rate of farnesol and FA stimulation of JH biosynthesis increased gradually. However, after transection of nervus corporis allati 1 (NCA-1), the rate of stimulated JH synthesis was maintained at preoperative levels. Although the spontaneous rate of JH biosynthesis decreased rapidly after NCA-1 transection, denervated glands could still be stimulated by farnesol or FA to produce large amounts of JH. These results suggest that the low spontaneous rate of JH biosynthesis in denervated corpora allata is not caused by inhibition of the final steps of JH biosynthesis.     

Biosynthetic maturation of the corpus allatum of the female adult medfly,Ceratitis capitata,and its putative control

The major juvenile hormone (JH) homolog synthesized in vitro by the adult female Medfly (Ceratitis capitata) corpus allatum (CA) is JHB(3), with JH-III the minor homolog. Methyl-incorporation in vitro in post-eclosion virgin females is age-dependent. Basal activity occurs during the first four days post-eclosion and increases significantly thereafter, peaking at five days. Biosynthetic maturation of the mated female CA is delayed by one day and reduced considerably. The delayed response may be due to direct cerebral or neural inhibition. Synthetic Drosophila melanogaster sex peptide depresses JH biosynthesis by the Medfly female CA in vitro. Male-derived accessory gland peptides of the Medfly are transferred to the female during mating and a Medfly SP-analog may be responsible for down-regulation of JH synthesis by the CA in mated Medfly females. Mevinolin, an inhibitor of the mevalonate pathway, significantly reduces the biosynthesis of JHB(3), while farnesoic acid, a proximate precursor of JHIII, significantly stimulates the biosynthesis of both JHB(3) and JHIII in vitro.     

Methyl Farnesoate Plays a Dual Role in Regulating Drosophila Metamorphosis

Corpus allatum (CA) ablation results in juvenile hormone (JH) deficiency and pupal lethality in Drosophila. The fly CA produces and releases three sesquiterpenoid hormones: JH III bisepoxide (JHB3), JH III, and methyl farnesoate (MF). In the whole body extracts, MF is the most abundant sesquiterpenoid, followed by JHB3 and JH III. Knockout of JH acid methyl transferase (jhamt) did not result in lethality; it decreased biosynthesis of JHB3, but MF biosynthesis was not affected. RNAi-mediated reduction of 3-hydroxy-3-methylglutaryl CoA reductase (hmgcr) expression in the CA decreased biosynthesis and titers of the three sesquiterpenoids, resulting in partial lethality. Reducing hmgcr expression in the CA of the jhamt mutant further decreased MF titer to a very low level, and caused complete lethality. JH III, JHB3, and MF function through Met and Gce, the two JH receptors, and induce expression of Kr-h1, a JH primary-response gene. As well, a portion of MF is converted to JHB3 in the hemolymph or peripheral tissues. Topical application of JHB3, JH III, or MF precluded lethality in JH-deficient animals, but not in the Met gce double mutant. Taken together, these experiments show that MF is produced by the larval CA and released into the hemolymph, from where it exerts its anti-metamorphic effects indirectly after conversion to JHB3, as well as acting as a hormone itself through the two JH receptors, Met and Gce.     

Biosynthesis and release of juvenile hormone and its precursors in insects and crustaceans: The search for a unifying arthropod endocrinology

It now appears that arthropods produce and release a wider variety of juvenile hormones (JH) and related compounds than previously thought. For instance, in the adult crayfish, Procambarus clarkii, the mandibular organs, the homologous structure to insect corpora allata (CA), release both farnesoic acid (FA) and methyl farnesoate (MF), the immediate precursors of JH III, but not JH III itself. In larvae of the cockroach Diploptera punctata, JH III production ceases during the last half of the 4th stadium, but the CA continue to produce and release FA throughout this period. The embryos of the same species also release JH III and a product that coelutes with MF on HPLC. In adult blowfly, Calliphora vomitoria, the CA release JH III bisepoxide and possibly the 6,7-epoxide, in addition to JH III. In the lepidopteran species Pseudaletia unipuncta, male CA produce and release JH acids I, II, and III as well as a product which we have tentatively identified as homo-(and/or) dihomo-FA. In the females, CA produce and release the three common JH homologues and a product that we believe is the esterified version of the male compound, homo/dihomo-MF. Although the release of JH precursors from their sites of synthesis might result in their conversion to the active hormone in peripheral tissues, there is only limited evidence for such a process. Studies on biological activities of these compounds and on the developmental changes in biosynthesis and its regulation should provide information necessary for the defining of these compounds as hormones or otherwise and should improve our understanding of the evolution of the JH biosynthetic pathway in the phylum Arthropoda.     

Spatial expression of the mevalonate enzymes involved in juvenile hormone biosynthesis in the corpora allata in Bombyx mori

The developmental expressions of the mRNA of JH synthetic enzymes have been studied using homogenates of the corpora cardiaca-corpora allata (CC-CA) complexes in Bombyx mori [Kinjoh, T., Kaneko, Y., Itoyama, K., Mita, K., Hiruma, K., Shinoda, T., 2007. Control of juvenile hormone biosynthesis in Bombyx mori: cloning of the enzymes in the mevalonate pathway and assessment of their developmental expression in the corpora allata. Insect Biochemistry and Molecular Biology 37, 808-818]. The in situ hybridization analyses in the CC-CA complex showed that the distribution of the mRNAs of all the mevalonate enzymes and juvenile hormone (JH) acid O-methyltransferase occurred only in the CA cells, indicating that the fluctuations of the enzyme mRNA amounts in the CC-CA complexes were derived solely from the CA. In addition, the size of the CA and their nuclei was not associated with the JH synthetic activity by the CA until the pharate adult. Only female adult CA synthesized JH in B. mori, and the CA and the nuclei were significantly larger than those of male CA which do not synthesize JH.     

Novel cross-stage solitarising effect of gregarious-phase adult desert locust (Schistocerca gregaria (Forskål)) pheromone on hoppers

Previous studies had demonstrated stage differentiation in the cohesion (aggregation) pheromone systems of the desert locust, Schistocerca gregaria. In laboratory arena, the nymphal and adult stages responded aggregatively to their own pheromone, but dispersed evenly within the arena in the presence of the other. In the present study, we explored the effects of longer-term contact of field gregarious hopper bands and laboratory crowd-reared nymphs with the major constituent of the adult pheromone. During the first few days, hoppers in treated bands became relatively hyperactive. Over the next few days, their movements became random and they stopped marching as coherent groups, they started to roost for longer periods on vegetations, and they fragmented into smaller and smaller groupings and individuals. When attacked by birds, they demonstrated subdued levels of collective defensive behaviour compared to normal hoppers, and there were clear signs of increased predation and cannibalism at the roosting sites. In cage experiments, crowd-reared nymphs treated with the pheromone component became hyperactive, showed abnormal diel patterns and reduced feeding on plants but increased cannibalism. Our observations show that the major adult pheromone constituent has a solitarising effect on gregarious hoppers. The mechanism underlying this effect and the potential of the agent in desert locust control are discussed.     

Candida albicans PHO81 is required for the inhibition of hyphal development by farnesoic acid

Farnesoic acid is a signaling molecule that inhibits the transition from budding yeast to filament formation in Candida albicans, but the molecular mechanism regulated by this substance is unknown. In this study, we analyzed the function of CaPHO81, which is induced by farnesoic acid. The pho81Δ mutant cells existed exclusively as filaments under favorable yeast growth conditions. Furthermore, the inhibition of hyphal growth and repression of CPH1, EFG1, HWP1, and GAP1 mRNA expression in response to farnesoic acid were defective in pho81Δ mutant cells. These data suggest a role for CaPHO81 in the inhibition of hyphal development by farnesoic acid.