The mechanism of compensation in juvenile hormone synthesis following unilateral allatectomy in Diploptera punctata

Following unilateral allatectomy in the viviparous cockroach, Diploptera punctata, the remaining corpus allatum (CA) can synthesize juvenile hormone (JH) at rates equal to that of a pair of control CA. A single CA undergoes changes in volume and cell number similar in magnitude to those occurring during the normal cycle of JH synthesis. There is no hyptertrophy of the CA associated with compensation in JH synthesis. Therefore, the rate of JH synthesis per cell or per unit volume of single CA following unilateral allatectomy is twice that of a single control CA.     

The effect of juvenile hormone on protein synthesis in the transparent accessory gland of male Rhodnius prolixus

Feeding stimulates the accumulation of protein by the transparent accessory reproductive glands (TARG), allatectomy abolishes the increase, and severing the nervous connections between the corpus allatum (CA) and the brain raises the level of accumulation after feeding. TARG removed from allatectomised females exhibit a markedly decreased level of incorporation of [³H]-leucine into protein in vitro, and topical application of juvenile hormone (JH) to allatectomised males restores the ability of the glands to incorporate [³H]-leucine in vitro. Severing the nerves between the CA and the brain increases the rate of incorporation in vitro, but in both operated and normal males, the rate of incorporation decreases with time after feeding. JH applied topically to intact animals during the falling phase of incorporation, or applied directly to TARG in vitro from such animals, stimulates incorporation. These data indicate that JH from the CA controls protein synthesis in the TARG and that the CA in the male of Rhodnius may be subject to different controls than in the female.     

Control of the Corpora Allata During a Reproductive Cycle in a Viviparous Cockroach

SYNOPSIS. This paper describes factors which regulate the corporaallata (CA) during the reproductive cycle in the viviparouscockroach Diploptera punctata. Experiments, designed to alterthe rate of juvenile hormone (JH) synthesis by the CA, are performedon whole animals and their effects are demonstrated by an invitro radiochemical assay for JH synthesis by the CA. The CAis both inhibited and stimulated. Axonal pathways from the brainto the CA are required for inhibition of the CA in virgin femalesbut not in young pregnant females. Humorally transmitted inhibitionof the CA is caused in vivo by JH (in high titers) and ecdysteroidhormone. JH in low titers also stimulates the CA. Since thesehormones do not affect the CA in vitro we suggest that theyexert their effect via the central nervous system. The ovary,a possible source of ecdysteroids in the hemolymph, is necessaryboth for maintenance of JH synthesis and for inhibition in therate of JH synthesis at the end of egg growth.     

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.     

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.     

Characterization of an isopentenyl diphosphate isomerase involved in the juvenile hormone pathway in Aedes aegypti

Isopentenyl diphosphate isomerase (IPPI) is an enzyme involved in the synthesis of juvenile hormone (JH) in the corpora allata (CA) of insects. IPPI catalyzes the conversion of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP); afterward IPP and DMAPP condense in a head-to-tail manner to produce geranyl diphosphate (GPP), this head-to-tail condensation can be repeated, by the further reaction of GPP with IPP, yielding the JH precursor farnesyl diphosphate. An IPPI expressed sequence tag (EST) was obtained from an Aedes aegypti corpora-allata + corpora cardiaca library. Its full-length cDNA encodes a 244-aa protein that shows a high degree of similarity with type I IPPIs from other organisms, particularly for those residues that have important roles in catalysis, metal coordination and interaction with the diphosphate moiety of the IPP. Heterologous expression produced a recombinant protein that metabolized IPP into DMAPP; treatment of DMAPP with phosphoric acid produced isoprene, a volatile compound that was measured with an assay based on a solid-phase micro extraction protocol and direct analysis by gas chromatography. A. aegypti IPPI (AaIPPI) required Mg²⁺ or Mn²⁺ but not Zn²⁺ for full activity and it was entirely inhibited by iodoacetamide. Real time PCR experiments showed that AaIPPI is highly expressed in the CA. Changes in AaIPPI mRNA levels in the CA in the pupal and adult female mosquito corresponded well with changes in JH synthesis (Li et al., 2003). This is the first molecular and functional characterization of an isopentenyl diphosphate isomerase involved in the production of juvenile hormone in the CA of an insect.     

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.     

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.     

Morph-specific diurnal variation in allatostatin immunostaining in the corpora allata of Gryllus firmus: Implications for the regulation of a morph-specific circadian rhythm for JH biosynthetic rate

Previous studies have documented a circadian cycle in juvenile hormone (JH) biosynthesis in the long-winged, flight-capable morph, but not in the short-winged flightless morph of the cricket Gryllus firmus. One rapid and reversible inhibitor of in vitro JH biosynthesis by the corpora allata (CA) in crickets is the neuropeptide Phe-Gly-Leu/Ile-amide type of allatostatins (ASTs). To investigate the possible role of allatostatin regulation of the morph-specific circadian cycle of JH production, the quantity of this type of AST in the nerves within the CA was determined by the density of anti-AST-immunostaining in confocal images using the Image J program. The density of immunostaining was inversely related to the rate of JH biosynthesis: Immunostaining in the CA was high and did not differ between morphs early in the photophase when the in vitro rate of JH biosynthesis is low and equivalent in the morphs. However, during the end of the photophase, when the rate of JH biosynthesis rises dramatically in the flight-capable morph, but not in the flightless morph, immunostaining was significantly lower in the flight-capable compared to the flightless morph. These results indicate that morph-specific differences in delivery of AST to the CA and its probable release likely regulate the morph-specific circadian pattern of JH biosynthesis. Also, the negative correlation between AST density and JH production provides evidence for predicting the periods of altered release of these rapid-acting paracine regulators of JH biosynthesis.     

Changes in the size of corpora allata,in the juvenile hormone III titer in the hemolymph,and in the protein content of terminal oocytes throughout the first gonadotropic cycle in Aiolopus thalassinus (Insecta: Orthoptera: Acrididae)

In Aiolopus thalassinus (Fabr.), the changes in the volume of the corpora allata (CA), in the concentration of juvenile hormone III (JH III) in the hemolymph, and in protein content in the terminal (t) oocytes were studied during the first gonadotropic cycle. These parameters could be better related to the volume of t-oocytes than to age after emergence. The JH III titer curve was maximum (2.9 pmol/10 μl) at an oocyte volume of 1.2 mm³. Before oviposition (days 10–16) the JH III titer decreased to 0.65 pmol/10 μl hemolyph. The increase in JH III titer reflected a period of high protein storage in the t-oocytes. The largest volume of the CA was reached at the beginning of yolk storage in the t-oocytes. The highest JH III titer did not correspond with the largest volume of CA, which occurred much earlier. © 1993 Wiley-Liss, Inc.     

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.     

Aldehyde dehydrogenase 3 converts farnesal into farnesoic acid in the corpora allata of mosquitoes

The juvenile hormones (JHs) play a central role in insect reproduction, development and behavior. Interrupting JH biosynthesis has long been considered a promising strategy for the development of target-specific insecticides. Using a combination of RNAi, in vivo and in vitro studies we characterized the last unknown biosynthetic enzyme of the JH pathway, a fatty aldehyde dehydrogenase (AaALDH3) that oxidizes farnesal into farnesoic acid (FA) in the corpora allata (CA) of mosquitoes. The AaALDH3 is structurally and functionally a NAD⁺-dependent class 3 ALDH showing tissue- and developmental-stage-specific splice variants. Members of the ALDH3 family play critical roles in the development of cancer and Sjögren–Larsson syndrome in humans, but have not been studies in groups other than mammals. Using a newly developed assay utilizing fluorescent tags, we demonstrated that AaALDH3 activity, as well as the concentrations of farnesol, farnesal and FA were different in CA of sugar and blood-fed females. In CA of blood-fed females the low catalytic activity of AaALDH3 limited the flux of precursors and caused a remarkable increase in the pool of farnesal with a decrease in FA and JH synthesis. The accumulation of the potentially toxic farnesal stimulated the activity of a reductase that converted farnesal back into farnesol, resulting in farnesol leaking out of the CA. Our studies indicated AaALDH3 plays a key role in the regulation of JH synthesis in blood-fed females and mosquitoes seem to have developed a “trade-off” system to balance the key role of farnesal as a JH precursor with its potential toxicity.     

Relation between cell number and pupal development of wing disks in Bombyx mori

The effects of JH and ecdysone on pupal differentiation of the wing disk of Bombyx mori were studied in vivo and in vitro. JH prevents pupal differentiation during larval life by stopping a particular stage of the cell cycle. Immediately after allatectomy, a cell cycle sets in without ecdysone, but afterwards wing disks obtain the competence to differentiate to the pupal type in response to ecdysterone. Disks older than 2 days after allatectomy can develop to the pupal type with an abrupt increase of mitosis in a certain concentration of ecdysterone in vitro. Once the disks have gained such competence and begun pupal development, JH no longer exhibits the effect that prevents DNA synthesis, which is enhanced by ecdysterone. It is therefore suggested that there are two phases in DNA synthesis: one which is important for achieving competence and is inhibited by JH and relatively independent of ecdysone; and another which is important for morphogenetic development and depends on ecdysone but is not inhibited by JH.     

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.     

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.     

Decrease in Methoprene tolerant and Taiman expression reduces juvenile hormone effects and enhances the levels of juvenile hormone circulating in males of the linden bug Pyrrhocoris apterus

Juvenile hormone (JH) produced by the corpus allatum (CA) stimulates vitellogenesis and reduces the synthesis of hexamerin proteins in adult females of Pyrrhocoris apterus. At present it is unknown whether the signaling pathway involving the JH receptor gene Methoprene tolerant (Met) and its binding partner Taiman (Tai), regulates the synthesis of accessory gland proteins (ACPs) and hexamerin proteins or effects male survival. Knockdown of genes by injecting Met dsRNA or Tai dsRNA, reduced the amount of ACPs whilst enhancing the amount of hexamerin mRNA in the fat body and the release of hexamerin proteins into haemolymph, as occurs after the ablation of CA. Lifespan was enhanced by injecting Met but not Tai dsRNA. Diapause associated with the natural absence of JH had a stronger effect on all these parameters than the ablation of CA or the knockdown of genes. This indicates there is an additional regulating agent. Both Met and Tai dsRNA induced a several fold increase in JH (JH III skiped bisepoxide) but a concurrent loss of Met or Tai disabled its function. This supports the view that the Met/Tai complex functions as a JH receptor in the regulation of ACPs and hexamerins.     

Juvenile hormone biosynthesis by corpus cardiacum-corpus allatum complexes of larval Lymantria dispar

• 1.1. A radiochemical assay was used to examine juvenile hormone (JH) synthesis and secretion in vitro by incubating two pairs of larval corpus cardiacum-corpus allatum complexes (CC-CA) from, Lymantria dispar, in 50 μl of osmotically balanced Grace's medium containing 1 μC1 [³H-methyl]-methionine for 6 hr.
• 2.2. For CC-CA of fourth instar female larvae, maximal incorporation of ³H-methyl was 0.15 pmol/pr/hr between days 2 and 3. High pressure liquid chromatographic (HPLC) analysis suggested that the biosynthetic products are mainly JH III with a little JH II at times.
• 3.3. For CC-CA of last instar female larvae, incorporation of ³H-methyl was 0.48 pmol/pr/hr at the beginning of the stadium and decreased to negligible levels by day 10. HPLC analysis suggested that CC-CA of last instar larvae produced only JH III. Volume increases in CA during the last instar were associated with declining activities of JH secretion.
• 4.4. Comparisons of maximal rates of ³ H-methyl incorporation by each unit volume of CA revealed that in the last instar each unit volume (μm³) of glandular tissue secreted 50% more JH than in the fourth instar.