Responsiveness of the adult cricket (Gryllus bimaculatus andAcheta domesticus) retrocerebral complex to allatostatin-1 from a cockroach,Diploptera punctata

Brain-retrocerebral complexes of female crickets,Gryllus bimaculatus andAcheta domesticus, treated with antibody to allatostatin-1 from a cockroach,Diploptera punctata, show extensive immunoreactivity. The results suggest that allatostatins or allatostatin-like molecules are produced in neurosecretory cells of the brain and are delivered to the corpora allata through nervous connections and/or via haemolymph. Radiochemical measurements of juvenile hormone III biosynthesis by isolated corpora cardiaca-corpora allata complexes from adultG. bimaculatus have been used to demonstrate an in vitro sensitivity of these glands to allatostatin-1 fromD. punctata. Allatostatin-1 is a relatively potent inhibitor of juvenile hormone III biosynthesis in corpora allata of both young adult females and males. In glands taken from 3-day virgin females, 50% inhibition of hormone biosynthesis is reached at ca. 3 nmol·l^-1 allatostatin-1. The inhibitory action of allatostatin-1 is rapid, dose-dependent and reversible. Addition of 200 mol·l^-1 farnesol to the incubation medium prevents inhibition of juvenile hormone III biosynthesis by allatostatin-1. Juvenile hormone III biosynthesis by isolated corpora allata of 3-day female house crickets,A. domesticus, is also susceptible to inhibition by 1 mol·l^-1 allatostatin-1.Abbreviations ASB2 Diploptera punctata allatostatin-5 - CA corpora allata - CC corpora cardiaca - Dip A-1 Diploptera punctata allatostatin-1 - HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid - JH juvenile hormone(s) - Mas-AS Manduca sexta allatostatin - MF methyl farnesoate - NCA nervus corporis allati - NCC nervus corporis cardiaci - SEM standard error of mean - TRIS Tris(hydroxymethyl)aminomethane     

Apparent inhibition of in vitro juvenile hormone biosynthesis by the corpus cardiacum of adult crickets (Gryllus bimaculatus and Acheta domesticus) is due to juvenile hormone esterase

When measuring the in vitro JH III-biosynthesis by corpora allata (CA) from adult female crickets in the presence of corpora cardiaca (CC), the amount of JH III in the medium decreased in a dose dependent manner. The CC of a 4-day-old female Gryllus bimaculatus contain 42 pmol.pair CC⁻¹ Grb-AKH, 0.62 pmol.pair CC⁻¹ octopamine, and a JH-esterase activity of 9.8 pmol JH.h⁻¹.pair CC⁻¹. Comparable values for Acheta domesticus are 21 pmol.pair CC⁻¹ Grb-AKH, 0.53 pmol.pair CC⁻¹ octopamine, and 6.5 pmolJH.h⁻¹.pair CC⁻¹ of JH-esterase activity. Even if the entire octopamine content of the CC were released into the medium, the concentration would be below the 10⁻⁵ M threshold for octopamine inhibition of JH synthesis. An in vitro AKH inhibition of JH III synthesis was observed, but only at a relatively high concentration (10⁻⁵ M). If the entire AKH content (10⁻⁶ M) of the CC were released into the medium, the AKH concentration would approach JH synthesis inhibiting levels. However, the rate of release of AKH in vitro was very low, and, therefore, AKH from the CC could not affect JH synthesis. In contrast, a specific JH-esterase, released by isolated CC into the medium, was sufficiently high in both cricket species to account for the observed decrease in JH III present. OTFP-sulfone (10⁻⁵ M) restored apparent JH synthesis of the CA to the control level. There was no reduction in the amount of JH released when CA were incubated with heat treated CC. The CA themselves contained almost no JH-esterase activity. © 1997 Wiley-Liss, Inc.     

Biosynthesis and regulation of juvenile hormone III,juvenile hormone III bisepoxide,and methyl farnesoate during the reproductive cycle of the grey fleshfly,Neobellieria (Sarcophaga) bullata

To study the effect of brain signals on the biosynthesis of juvenile hormone by the corpora allata of the grey fleshfly Neobellieria bullata, exposed corpora allata connected to the brain were surgically removed from sugar-fed flies and incubated in vitro with L -[³H-methyl]methionine. After incubation, the media together with the tissues were analyzed by HPLC. [³H]Juvenile hormone III (JH III), [³H]JH III bisepoxide (BE), [³H]methyl farnesoate (MF) and an unknown [³H]labeled metabolite (Un) were identified as the primary products. The rate of synthesis of [³H]JH III bisepoxide was higher than that of [³H]JH III, [³H]MF and [³H]Un. Two days after a liver meal, female flies synthesized more JH III, MF, BE, and the Un than did males. Synthesis of JH III, BE, and MF in females was lower during the previtellogenic, sugar-feeding period than during the vitellogenic liver-feeding period. Isolated corpus cardiacum–corpus allatum (CC-CA) complexes that were incubated in vitro synthesized less JH III, MF, and BE, as compared to complexes that were attached to the brain, indicating that the brain probably modulates the biosynthesis of JH III, MF, and BE in the corpora allata. Upon incubation of brain–CC–CA complexes with Neb-TMOF (10^–8 M), Neb-colloostatin (10^–8 M), ovarian, or brain extracts resulted in significant inhibition of JH III and BE biosynthesis in the presence of ovarian extracts. These results indicate that allatostatin-like factors are present in the ovary of the flesh fly. Arch. Insect Biochem. Physiol. 37:248–256, 1998. © 1998 Wiley–Liss, Inc.     

The occurrence of a juvenile hormone binding protein and in vitro synthesis of juvenile hormone by the serosa of Locusta migratoria embryos

Summary At the end of blastokinesis, serosal epitheliae of 4- to 5-day-old embryos of Locusta migratoria contain an immunohistologically detectable cytosolic protein (M_r 240 kDa) which is related to the juvenile hormone carrier-protein in the haemolymph of the same species and which binds tritiated juvenile hormone 3 (JH3) (K_d10^–8 M). At this early stage of development the corpora allata of the embryo are not yet fully differentiated and do not synthesize JH3 in organ cultures. The earliest detectable JH3 production by corpora allata in isolated heads is on day 6. On the other hand, serosal epitheliae of 4- to 5-day-old embryos produce JH3 in organ cultures, as has been shown by methylation of (10-³H)-JH3-acid to (10-³H)-JH3, and by incorporation of tritiated CH₃ from l-(methyl-³H)-methionine into JH3. Isolated heads and abdomens of the embryos used as donors for the serosal preparations did not show methyl transferase activity responsible for JH3 biosynthesis. The serosal cells represent a hitherto unrecognized source of methyl transferase activity and of JH3 production. Degradation of JH3 to JH3-acid was also observed.Dedicated to Professor Herbert Röller on the occasion of his 60th birthday     

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.     

Correlation among corpus allatum volume,cell size,and juvenile hormone biosynthesis in ovariectomized adult Blattella germanica

The corpora allata (CA) of both intact and ovariectomized Blattella germanica adult females exhibited a high degree of bilateral symmetry in the rate of juvenile hormone (JH) biosynthesis, the mean size of CA cells, and gland volume (81.3%, 98.3%, and 100% respectively with less than a twofold difference between the two glands in CA pairs). This permitted us to split each CA pair randomly, measure JH biosynthesis in one gland, and dissociate the other gland into a cell suspension in which the size of CA cells was measured. In ovariectomized females, changes in CA volume and the spontaneous and farnesoic acid (FA)-stimulated rates of JH biosynthesis, measured from the same glands, were well correlated (r = 0.78, for both correlations). Similarly, the mean volume of CA cells in one gland increased in relation to increases in both the spontaneous and FA-stimulated rates of JH biosynthesis by the contralateral member of the pair (r = 0.83 and r = 0.91, respectively). Concurrent changes in CA cell size and activity suggest that in the CA of B. germanica cellular growth and degradation are involved in the regulation of JH biosynthesis.     

Action of a juvenile hormone analogue on the activity of Periplaneta americana corpora allata in vitro and on juvenile hormone III levels in vivo

The effects of the juvenile hormone (JH) analogue fenoxycarb (ethyl[2-(4-phenoxyphenoxy)-ethyl]carbamate) on the activity of corpora allata (CA) from adult female Periplaneta americana have been investigated. The in vitro biosynthesis of JH III by isolated CA was inhibited by about 85% in the presence of a high concentration (1 × 10⁻⁴ M) of fenoxycarb. However, at lower concentrations (1 × 10⁻⁶ M and 1 × 10⁻⁸ M) no inhibition of JH biosynthesis was apparent. Topical treatment of adult female cockroaches with fenoxycarb (100 μg/insect) did not reduce the subsequent rate of JH III biosynthesis by CA in vitro. By contrast, the same treatment markedly reduced the titre of endogenous JH III in intact cockroaches. These results suggest that CA activity in adult female P. americana may be controlled by negative feedback, and that this system of control is dependent on the maintenance of contact between the CA and nervous or humoral factors in the intact insect. Alternatively, it is possible that treatment with fenoxycarb increases the rate at which endogenous JH is metabolized.     

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.     

Control of C-16 juvenile hormone biosynthesis in active corpora allata of female African locusts

Summary

Corpora allata from 8-day-old female Locusta migratoria, during the phase of yolk deposition, exhibit high rates of C-16 juvenile hormone (JH) biosynthesis. The effect of different potential factors which may be involved in the regulation of corpora allata activity is reported. The biosynthetic activity of corpora allata was determined by radiochemical assay.

In maturing females, no changes in corpora allata activity are detected during one daily cycle. Starvation reduces JH biosynthesis only 3 days after the beginning of the food deprivation. Suppression of the median neurosecretory material by electrocoagulation of the internal cardiaca tract (TCC-I) does not disturb JH biosynthesis whereas the transection of the allata I nerve fibres (NCA-I) or the electrocoagulation of the lateral neurosecretory pericarya results in a rapid decline of JH biosynthesis. These data indicate that the median and lateral allatotropins are different, and that only the lateral neurosecretory material exerts an allatostimulating action on corpora allata at the time of vitellogenesis. The corpora allata response to the median allatotropin changes during oocyte growth. C-16 JH and/or 20-hydroxyecdysone treatments in vitro (addition in the culture medium) and in vivo (injection in female) do not influence JH production in our experimental conditions.     

Erratum

Allatotropin (AT) is a 13 amino acid neuropeptide isolated from the tobacco hornworm, Manduca sexta, which stimulates the biosynthesis of juvenile hormone (JH) in adult females of this insect. In this paper we report that AT stimulates inositol phosphate formation in both female and male corpora allata (CA) of adult Manduca sexta. Furthermore, thapsigargin, a potent Ca²⁺-ATPase inhibitor, staurosporine, a potent inhibitor of protein kinases A and C, and 2,5-di-(tert-butyl)-1,4-hydroquinone (DBHQ), a potent inhibitor of liver microsomal ATP-dependent Ca²⁺ sequestration, all stimulate JH biosynthesis without changing the basal level of inositol phosphates. These data suggest that AT activates an inositol 1,4,5-trisphosphate (IP₃) pathway. Furthermore, increases in the free intracellular Ca²⁺ concentration in the CA stimulate JH biosynthesis. © 1992 Wiley-Liss, Inc.     

The synthetic activity and glandular volume of the corpus allatum during ovarian maturation in the desert locust Schistocdrca gregaria.

We have compared, on an individual basis, the volume of the corpora allata with their ability to synthesize and release juvenile hormone (JH) using glands taken at daily intervals throughout the period of sexual maturation and the first two ovarian cycles in $\text{Schistocerca gregaria}$. A standard $\text{in vitro}$ radiochemical assay was used to measure the rates of both spontaneous JH biosynthesis from [methyl-¹⁴C]-methionine, and of JH biosynthesis stimulated by optimal concentrations of [C-2 ³H]-farnesenic acid. Computation of results showed that there are, during this period, changes of up to 250-fold in the rate of spontaneous JH biosynthesis per unit volume corpora allata. It is concluded that the volume of the corpora allata is of no value as an indicator of the spontaneous synthetic activity of the glands in this species, and that the overall rate of JH synthesis is regulated by mechanisms that do not involve large changes in the volume of the gland cells. However, in the presence of farnesenic acid, there is a corelation between stimulated JH synthesis and glandular volume, suggesting that the volume of the gland reflects the maximum activity of the final two stages in JH biosynthesis.     

Ultrastructural changes induced by precocene II and 3,4-dihydroprecocene II in the corpora allata of Blattella germanica

Summary Ultrastructural studies on corpora allata (CA) from different stages during the first gonadotropic cycle of the cockroach Blattella germanica have shown well defined changes which have a correspondence with oocyte length, CA volume and juvenile hormone (JH) biosynthesis. The most significant variations concern the mitochondria and the endoplasmic reticulum. Topically applied precocene II (P II) at a dose of 200 g induced a transient arrest of CA function, although cytotoxic effects were occasionally observed. When CA were maintained in vitro with 10^-3 M of P II, a relationship between the time of treatment (3, 6 or 9 h) and the intensity of the effects was apparent. The 9-h treatment led to an irreversible inhibition of JH production which parallels the severe damages observed in the CA (membrane lysis, nuclear pyknosis, vacuolization). Equivalent studies performed with the chroman derivative 3,4-dihydroprecocene II (DHP II) showed that it is less active than P II. Only treatments as severe as 12 h of incubation with a 10^-3 M concentration elicited cytotoxic effects which could be due to radical species involved in the in situ oxidative bioactivation of DHP II. Thus, this compound could be regarded as a new type of pro-allatocidin.     

A pharmacological and endocrinological study of female insemination inPhormia regina (Diptera: Calliphoridae)

Injections of octopamine, dopamine, and the octopaminergic agonists, clonidine and naphazoline, into the thoracic hemocoel enhanced female insemination in sugar-fed (sexually unreceptive)Phormia regina. Topical applications of the juvenile hormone (JH) analogue, methoprene, also enhanced female insemination in sugar-fed (sexually unreceptive)P. regina. Since JH plays a role in receptivity in protein-fed females, it was originally hypothesized that one agonist, clonidine, enhanced female insemination by acting on the corpus allatum (CA) to increase JH biosynthesis. Two or three doses of the antiallatal agent, precocene II, prior to administration of clonidine, did not inhibit clonidine-enhanced female insemination. Removal of the corpus allatum also did not have a significant effect on clonidine-enhanced female insemination. Measurement of juvenile hormone (JH) biosynthesis/release in corpora allata, which were removed at 1, 3, 5, and 7 h postinjection, revealed that clonidine does not affect JH biosynthesis/release. Our study reveals a possible role for the biogenic amines in female insemination in insects. We suggest that the octopaminergic agonist, clonidine, acts downstream from the corpus allatum on the regulatory mechanisms involved in female insemination.     

Suppression of JH biosynthesis by JH analog treatment: Mechanism of suppression and roles of allatostatins and nervous connections in the cockroach Diploptera punctata

Juvenile hormone analogs are known to inhibit the production of juvenile hormone (JH) by the corpora allata (CA). However, the mechanism of this inhibition remains undefined. We have used two JH mimics, fenoxycarb and pyriproxyfen, to examine the mechanism of suppression in the cockroach, Diploptera punctata. Denervation experiments demonstrated the importance of nervous connections between the brain and CA for the inhibition of JH biosynthesis by fenoxycarb. Fenoxycarb treatment alters the sensitivity of CA to allatostatin treatment in vitro. Suppression of JH biosynthesis by fenoxycarb following denervation of the CA showed that innervation was in part responsible for the inhibition. Similarly, maximal inhibition by Dippu-AST7 requires intact nervous connections between the brain and CA, particularly during rapid vitellogenesis. qPCR analysis of brain, CA, ovary and midgut extracts revealed that both allatostatin and its receptor Dippu-ASTR2 show increased levels of expression following topical fenoxycarb treatment, particularly in brain tissue on days 4 and 5 of the first gonadotrophic cycle and in CA on day 4. The correlation between inhibition of JH biosynthesis and increased expression of AST and ASTR2 in brains and CA, together with increased sensitivity of CA to allatostatin in vitro, suggests that allatostatin may be one of the effectors by which fenoxycarb inhibits JH biosynthesis.     

Inhibition of juvenile hormone III biosynthesis in cockroach corpora allata by interference with the S-adenosylmethionine-dependent transmethylation

The synthesis of juvenile hormone-III by corpora allata of the cockroach Diploptera punctata is dependent under in vitro conditions upon a supply of exogenous methionine. Radiolabelled S-adenosylmethionine was identified by HPLC in extracts of corpora allata incubated with either [methyl-³H]methionine or [³⁵S]methionine. Juvenile hormone (JH) synthesis by intact glands in vitro was inhibited by cycloleucine and selenomethionine, but this inhibition could be relieved by increasing the concentration of methionine. S-adenosylhomocysteine or sinefungin had little or no inhibitory effect on JH synthesis by intact glands, but 5′-deoxy-5′-methylthioadenosine was inhibitory. Adenosine and homocysteine synergistically inhibited JH synthesis. These results show that JH-III synthesis by intact glands can be inhibited by interfering with the S-adenosylmethionine-dependent transmethylation, and suggest that the product and inhibitor of that reaction, S-adenosyl-homocysteine, is rapidly hydrolyzed to adenosine and homocysteine in the corpora allata.     

Hormonal control of egg maturation in the corn earworm, Heliothis zea

At eclosion, the ovaries of female Corn earworm Heliothis zea do not contain mature eggs. Virgin-unfed females produced approximately 400 mature eggs in 8 days; mating or feeding doubled this number, and mating plus feeding more than tripled it. Females allatectomized or decapitated at day O matured few eggs. Egg production was restored by implantation of active corpora allata (CA) or by treatment with the juvenile hormone (JH) analogue methoprene at day 0. 20-Hydroxyecdysone, on the other hand, had no effect. Females in which the CA had been denervated or in which the median neurosecretory cells of the brain had been ablated at day O produced fewer eggs than sham-operated animals. These results indicate that egg maturation is controlled by JH and that continuous input from the brain is required for sustained CA activity for maintaining a high rates of egg maturation.The rate of JH biosynthesis by CA in vitro was determined with a radiochemical assay. The major hormones produced were JH-II and JH-III with small quantities of JH-I. The rates of JH synthesis were similar in all experimental groups which may indicate that the in vitro rate of JH synthesis does not reflect the actual state of CA activity in the female.     

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.     

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.     

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.