Farnesyl Phosphatase,a Corpora allata Enzyme Involved in Juvenile Hormone Biosynthesis in Aedes aegypti

Background

The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The late steps of JH III biosynthesis in the mosquito Aedes aegypti involve the hydrolysis of farnesyl pyrophosphate (FPP) to farnesol (FOL), which is then successively oxidized to farnesal and farnesoic acid, methylated to form methyl farnesoate and finally transformed to JH III by a P450 epoxidase. The only recognized FPP phosphatase (FPPase) expressed in the corpora allata (CA) of an insect was recently described in Drosophila melanogaster (DmFPPase). In the present study we sought to molecularly and biochemically characterize the FPP phosphatase responsible for the transformation of FPP into FOL in the CA of A. aegypti.

Methods

A search for orthologs of the DmFPPase in Aedes aegypti led to the identification of 3 putative FPPase paralogs expressed in the CA of the mosquito (AaFPPases-1, -2, and -3). The activities of recombinant AaFPPases were tested against general phosphatase substrates and isoprenoid pyrophosphates. Using a newly developed assay utilizing fluorescent tags, we analyzed AaFPPase activities in CA of sugar and blood-fed females. Double-stranded RNA (dsRNA) was used to evaluate the effect of reduction of AaFPPase mRNAs on JH biosynthesis.

Conclusions

AaFPPase-1 and AaFPPase-2 are members of the NagD family of the Class IIA C2 cap-containing haloalkanoic acid dehalogenase (HAD) super family and efficiently hydrolyzed FPP into FOL. AaFPPase activities were different in CA of sugar and blood-fed females. Injection of dsRNAs resulted in a significant reduction of AaFPPase-1 and AaFPPase-2 mRNAs, but only reduction of AaFPPase-1 caused a significant decrease of JH biosynthesis. These results suggest that AaFPPase-1 is predominantly involved in the catalysis of FPP into FOL in the CA of A. aegypti.     

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.     

In vivo stimulation of vitellogenesis in Aedes aegypti with juvenile hormone,juvenile hormone analogue (ZR 515) and 20-hydroxyecdysone

Decapitated blood-fed Aedes aegypti mosquitoes do not undergo normal oöcyte maturation. Topical application of 1.25 ng JH analogue (ZR 515) or 250 ng JH-I restored ovarian development in 70–80% of the treated females. The rate of vitellogenin synthesis in these animals was 80% of normal blood-fed controls.When ligated abdomens were treated, 125 pg ZR 515 or 12.5 ng JH-I were sufficient to restore ovarian development in 80% of the animals. The rate of vitellogenin synthesis in these animals was 70% of normal blood-fed controls. On the other hand, injection of 1.25 μg 20-hydroxyecdysone was needed to restore ovarian development and vitellogenin synthesis in decapitated and abdominally ligated females.These experiments indicate that JH concentrations closer to the physiological norm than 20-hydroxyecdysone, can restore ovarian development and vitellogenin synthesis in vivo.     

Independently regulated juvenile hormone activity and vitellogenesis in mosquitoes

Temporally distinct, head-mediated processes regulate vitellogenic development as well as juvenile hormone (JH)-mediated development of ovarian follicles of Aedes aegypti. In blood-fed adult mosquitoes, vitellogenic development is stimulated during the first day after blood is imbibed and JH secretion is stimulated 2 days later. JH secretion in recently ecdysed adult mosquitoes is stimulated during or shortly before ecdysis. These observations suggest that vitellogenesis follows blood-ingestion, whereas JH activity may secondarily be promoted by vitellogenesis. It may be that vitellogenesis and JH activity are mediated by different brain hormones     

Ecdysis triggering hormone ensures proper timing of juvenile hormone biosynthesis in pharate adult mosquitoes

Juvenile hormones (JHs) are synthesized by the corpora allata (CA) and play a key role in insect development. A decrease of JH titer in the last instar larvae allows pupation and metamorphosis to proceed. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa (or pharate adult) becomes again “competent” to synthesize JH, which would play an essential role orchestrating reproductive maturation. In the present study, we provide evidence that ecdysis triggering hormone (ETH), a key endocrine factor involved in ecdysis control, acts as an allatotropic regulator of JH biosynthesis, controlling the exact timing of CA activation in the pharate adult mosquito. Analysis of the expression of Aedes aegypti ETH receptors (AeaETHRs) revealed that they are present in the CA and the corpora cardiaca (CC), and their expression peaks 4 h before eclosion. In vitro stimulation of the pupal CA glands with ETH resulted in an increase in JH synthesis. Consistent with this finding, silencing AeaETHRs by RNA interference (RNAi) in pupa resulted in reduced JH synthesis by the CA of one day-old adult females. Stimulation with ETH resulted in increases in the activity of juvenile hormone acid methyltransferase (JHAMT), a key JH biosynthetic enzyme. Furthermore, inhibition of IP₃R-operated mobilization of endoplasmic reticulum Ca²⁺ stores prevented the ETH-dependent increases of JH biosynthesis and JHAMT activity. All together these findings provide compelling evidence that ETH acts as a regulatory peptide that ensures proper developmental timing of JH synthesis in pharate adult mosquitoes.     

Negative Feedbacks by Isoprenoids on a Mevalonate Kinase Expressed in the Corpora Allata of Mosquitoes

Background

Juvenile hormones (JH) regulate development and reproductive maturation in insects. JHs are synthesized through the mevalonate pathway (MVAP), an ancient metabolic pathway present in the three domains of life. Mevalonate kinase (MVK) is a key enzyme in the MVAP. MVK catalyzes the synthesis of phosphomevalonate (PM) by transferring the γ-phosphoryl group from ATP to the C₅ hydroxyl oxygen of mevalonic acid (MA). Despite the importance of MVKs, these enzymes have been poorly characterized in insects.

Results

We functionally characterized an Aedes aegypti MVK (AaMVK) expressed in the corpora allata (CA) of the mosquito. AaMVK displayed its activity in the presence of metal cofactors. Different nucleotides were used by AaMVK as phosphoryl donors. In the presence of Mg²⁺, the enzyme has higher affinity for MA than ATP. The activity of AaMVK was regulated by feedback inhibition from long-chain isoprenoids, such as geranyl diphosphate (GPP) and farnesyl diphosphate (FPP).

Conclusions

AaMVK exhibited efficient inhibition by GPP and FPP (Ki less than 1 μM), and none by isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DPPM). These results suggest that GPP and FPP might act as physiological inhibitors in the synthesis of isoprenoids in the CA of mosquitoes. Changing MVK activity can alter the flux of precursors and therefore regulate juvenile hormone biosynthesis.     

Juvenile hormone biosynthesis in moths: synthesis and evaluation of farnesol homologs as alternate substrates of farnesol oxidase

The oxidation of farnesol to farnesal is an important step in insect juvenile hormone (JH) biosynthesis and is mediated by one or more alcohol oxidases located within the minute endocrine gland, the corpus allatum. Because lepidopteran insects have the capacity to produce homologous JH structures, the substrate selectivity of farnesol oxidase was examined by determining the ability of several terpenol homologs to inhibit farnesol oxidation in moths. Results utilizing corpora allata homogenates from larval, adult, and embryonic Manduca sexta indicate that increased steric bulk at the C-3 position of the sesquiterpenol chain is detrimental to inhibitory potency. Triethylhomofarnesol (1h), which is precursor to JH 0 and therefore a physiologically important metabolite of M. sexta embryos, was found to be a poor inhibitor of farnesol oxidation but was oxidized in almost same amount as farnesol. This data indicate that farnesol oxidase of the corpus allatum plays a limited role in controlling JH homolog production in moths, and suggests that another oxidative enzyme, which is present at early stages of moth development, is involved in JH homolog construction.     

Evidence of controlled corpus allatum activity in the adult Colorado potato beetle

The in vivo control of corpus allatum (CA) activity in females of Leptinotarsa decemlineata was investigated. Evidence was obtained that CA activity is adjusted by negative feedback when juvenile hormone (JH) titres are changed experimentally. Conclusions are based on determination of the rate of in vitro JH synthesis by the CA, on changes in CA volume, and on JH titres in the haemolymph. These assay methods are used alternatively in some of the experiments.After unilateral allatectomy, the remaining CA had doubled its activity 7 days later. On the other hand, the activity of CA in young adults was suppressed after the JH titre was elevated by the implantations of 2 CA taken from active females. Similarly, in beetles treated topically with exogenous JH the CA atrophied and showed a much reduced activity after 5 days. Denervation of CA in 0-day-old long-day and 7-day-old short-day females did not change CA activity when measured 1 day later.     

Coordinated changes in JH biosynthesis and JH hemolymph titers in Aedes aegypti mosquitoes

Juvenile hormone III (JH) is synthesized by the corpora allata (CA) and plays a key role in mosquito development and reproduction. A decrease in JH titer during the last instar larvae allows pupation and metamorphosis to proceed. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa once again synthesizes JH, which plays an essential role in orchestrating reproductive maturation. In spite of the importance of Aedes aegypti as a vector, a detailed study of the changes of JH hemolymph titers during the gonotrophic cycle has never been performed. In the present studies, using a high performance liquid chromatography coupled to a fluorescent detector (HPLC–FD) method, we measured changes in JH levels in the hemolymph of female mosquitoes during the pupal and adult stages. Our results revealed tightly concomitant changes in JH biosynthesis and JH hemolymph titers during the gonotrophic cycle of female mosquito. Feeding high sugar diets resulted in an increase of JH titers, and mating also modified JH titers in hemolymph. In addition these studies confirmed that JH titer in mosquitoes is fundamentally determined by the rate of biosynthesis in the CA.     

Studies on the de novo synthesis of juvenile hormone III and methyl farnesoate by isolated corpora allata of adult female Gryllus bimaculatus

Activity of the corpora allata (CA) in vitro of adult female Gryllus bimaculatus was studied following incorporation of radioactivity from [2-¹⁴C]acetate and l-[methyl-³H]methionine into juvenile hormone III (JH III) and its immediate precursor methyl farnesoate (MF). Spontaneously active glands from females reared at 27°C utilized exogenous labelled acetate extensively for synthesis of MF and JH III (incorporation 80–84% at 2 mM acetate). 10⁻⁷ to 10⁻⁵ M exogenous JH III in the incubation medium had no effect on the rate of JH biosynthesis in spontaneously active glands. At 10⁻⁴ M JH III incorporation of acetate into JH III was reduced. The amount of MF was also lowered. JH III treatment (10⁻⁸–10⁻⁶ M) of spontaneously inactive glands led to an increase in the amount of MF. This increase was due to a de novo synthesis. Exogenous farnesol (20–200 μM) increased JH III biosynthesis and the amount of MF, but suppressed [2-¹⁴C]acetate incorporation. Dilution of the endogenous precursors is probably the most important cause of this suppression. As shown by the abnormally high MF levels in farnesol treated glands, epoxidation seems to be a rate-limiting step under certain experimental conditions.     

Biosynthesis of (10R)-Juvenile hormone III from farnesoic acid by Aedes aegypti ovary

Synthesis of (10R)-juvenile hormone III (JH III) outside the corpora allata (CA) was investigated in female Aedes aegypti. Intact females or ligated abdomens of blood-fed and sugar-fed females synthesized in vivo [12-³H]JH III-like molecules from [12-³H]-methyl farnesoate, indicating that an organ(s) in the female abdomen, other than the CA, converted methyl farnesoate into JH III. To find out the organ(s) that synthesized JH III-like molecules, ovaries, fat bodies, and midguts were incubated in vitro with [12-³H]methyl farnesoate and the synthesis of JH III-like molecules was compared with JH III synthesized by CA. To identify tissue(s) having both farnesoic acid methyl transferase and farnesoate epoxidase, enzymes that convert farnesoic acid into JH III, ovaries, and fat bodies were removed from sugar and blood-fed females and incubated with [12-³H]farnesoic acid. Chemical derivatization by methoxyhydrin formation followed by esterification with (+)-α-methoxy- α-trifluoromethyl phenylacetic (MTPA) acid chloride and reversed phase liquid chromatography identified (10R)-JH III methoxyhydrin (+)-MTPA ester as the sole JH III-like molecule produced in tissue culture incubation of ovaries. Since only (10R)-JH III is produced and not racemic JH III, the oxidation of farnesoic acid must be enzymatically mediated. Ovaries and corpora allata of female A. aegypti also synthesized [³H,¹⁴C]JH III from L-[methyl-³H]methionine and [¹⁴C]acetate which was characterized by HPLC and gas chromatography. These results suggest that mosquito ovary can synthesize (10R)-JH III from farnesoic acid, and that this tissue synthesizes JH III-like molecules from L-methionine and acetate. © 1994 Wiley-Liss, Inc.     

The fate of follicles after a blood meal is dependent on previtellogenic nutrition and juvenile hormone in Aedes aegypti

Juvenile hormone (JH) mediates the relationship between fecundity and nutrition during the gonotrophic cycle of the mosquito in three ways: (1) by regulating initial previtellogenic development, (2) by mediating previtellogenic resorption of follicles and (3) by altering intrinsic previtellogenic follicle “quality”, physiology, and competitiveness thereby predetermining the fate of follicles after a blood meal. To support a role for JH in mediating the response of ovarian follicles after a blood meal, we explored three main questions: (1) Do changes in nutrition during the previtellogenic resting stage lead to relevant biochemical and molecular changes in the previtellogenic ovary? (2) Do hormonal manipulations during the previtellogenic resting stage lead to the same biochemical and molecular changes? (3) Does nutrition and hormones during the previtellogenic resting stage affect vitellogenic resorption and reproductive output? We examined the accumulation of neutral lipids in the previtellogenic ovary as well as the previtellogenic expression of genes integral to endocytosis and oocyte development such as the: vitellogenin receptor (AaVgR), lipophorin receptor (AaLpRov), heavy-chain clathrin (AaCHC), and ribosomal protein L32 (rpL32) under various previtellogenic nutritional and hormonal conditions. mRNA abundance and neutral lipid content increased within the previtellogenic ovary as previtellogenic mosquitoes were offered increasing sucrose concentrations. Methoprene application mimicked the effect of offering the highest sucrose concentrations on mRNA abundance and lipid accumulation in the previtellogenic ovary. These same nutritional and hormonal manipulations altered the extent of vitellogenic resorption. Mosquitoes offered 20% sucrose during the previtellogenic resting stage had nearly 3 times less vitellogenic resorption than mosquitoes offered 3% sucrose despite taking smaller blood meals and developed ～10% more eggs during the first gonotrophic cycle. Mosquitoes treated with JH III during the previtellogenic resting stage and then offered a blood meal had a ～40% reduction in the amount of vitellogenic resorption and developed ～12% more eggs. Taken together, these results suggest that previtellogenic nutrition alters the extent and pattern of resorption after a blood meal through the effect of JH on mRNA abundance and lipid accumulation in previtellogenic follicles.     

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.     

Juvenile hormone biosynthesis in larval and adult stick insects,Carausius morosus

Corpora allata (CA) from adult egg-carrying Indian stick insects, Carausius morosus, synthesise and release juvenile hormone (JH) III in vitro. No JH biosynthesis was observed in larvae, young adults, and old adult females that do not carry sclerotised eggs. In females, which bear sclerotised eggs, a consistent JH biosynthesis was observed. Supplementation of precursors of JH biosynthesis (farnesol, mevalonic acid lactone) greatly enhanced JH biosynthesis in a stage-, age-, and dose-dependent manner, but CA from the last larval instar retained the biosynthesised JH within the gland. Elevated calcium concentration in the incubation medium stimulated JH biosynthesis by CA from older adults but had either no or a poor effect on CA from young adults and larvae. The results obtained with farnesol, mevalonic acid lactone, and calcium indicate that the rate-limiting steps of JH biosynthesis very likely occur before the formation of mevalonic acid and that these early steps cannot be stimulated by elevated calcium concentrations in larvae and young adults. In older adults, in which spontaneous JH biosynthesis occurs, elevated calcium concentration can markedly stimulate JH biosynthesis. A pre-purified extract from brains of adult females had a stimulating effect on JH biosynthesis by CA from adult females. The results indicate that JH biosynthesis in C. morosus may require food-derived farnesol and may be regulated by allatotropic signals from the brain, possibly triggered by sclerotised oocytes in the ovary.     

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.     

Nutritional regulation of JH synthesis: a mechanism to control reproductive maturation in mosquitoes?

Juvenile hormone (JH) titers must be modulated to permit the normal progress of development and reproduction in mosquitoes. In adult female Aedes aegypti, JH levels are low at adult eclosion, elevated in sugar-fed females and low again after a blood meal. Although degradation plays a role, JH titer is fundamentally determined by the rate of biosynthesis in the corpora allata gland (CA). CA from newly eclosed females (0-1 h after emergence) exhibit a very low basal JH biosynthetic activity, Aedes-allatotropin stimulates the CA in newly emerged females to produce JH. There is a correlation between nutritional reserves at adult emergence (teneral reserves) and CA activity. JH synthesis is significantly reduced in teneral females that emerge with low nutritional reserves. Taking a blood meal results in a reduction of CA activity. The biosynthetic activity of Ae. aegypti CA is significantly inhibited by factors present in the head, as well as by Anopheles gambiae PISCF-allatostatin. Nutritional signals affect the release of allatotropin and allatostatins by the brain resulting in the activation or inhibition of JH synthesis. JH is therefore an important part of a transduction mechanism that connects changes in the nutritional status with activation of specific physiological events during reproduction.