Activity of the corpora allata of adult female Leucophaea maderae: effects of mating and feeding

Juvenile hormone III biosynthesis by corpora allata of adult female Leucophaea maderae was measured by an in vitro radiochemical assay. In fed females, JH III synthesis increases more than 20-fold after mating to a peak of 55 pmol/pair/h on day 9 and then rapidly declines. This increase in JH III synthesis concomitant with rapid oocyte growth in mated females is not observed in virgin females. The corpora allata from starved, virgin females appear to be inactive. The addition of 150 microM 2E,6E-farnesol (a) JH III precursor) to the incubation medium stimulates the corpora allata from starved, virgin females less than the corpora allata from starved, mated females. Both feeding and mating are necessary for the expression of a normal cycle of JH III synthesis in this cockroach.     

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.     

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 effect of enforced virginity and subsequent mating on the activity of the corpus allatum of Periplaneta americana measured in vitro, as related to changes in the rate of ovarian maturation

ABSTRACT. Female P. americana, reared with males from the time of adult emergence, mated on the 4th–5th day after metamorphosis, produced the first ootheca on the 8th or 9th day, and then produced successive oothecae at intervals of 3.0 days, whereas, only 50% of virgin females had produced their first ootheca by the 28th day after adult emergence. Examination of the ovaries indicated that oocyte development is normal in virgins until shortly after the time when they first become receptive to males. When mating was not allowed there was a dramatic reduction in the rate of vitellogenic growth of the terminal batch of oocytes which persisted until mating was allowed, and was often accompanied by resorption of a percentage of the oocytes. Short-term, in vitro, radiochemical assay of juvenile hormone (JH III) biosynthesis by corpora allata (CA) showed that, in females reared with males, the cycles of ovarian development are accompanied by regular pulses of CA activity. There is a small, possibly preparatory peak of JH III biosynthesis before vitellogenesis of the first wave of oocytes, followed by a larger peak of JH III production during vitellogenesis of this batch of eggs and one peak of CA activity between ovulation of each subsequent wave of oocytes. Activities as low as 0.25 pmol C₁₆JH/CA pair/h and as high as 48.38 pmol/CA pair/h were observed in CA from mated females after the onset of cyclic activity. Stimuli received during mating are somehow responsible for the cyclic activity of the CA, for when females were subjected to enforced virginity the first small peak was normal but the second peak was not fully realized and there was then a gradual decline in CA activity until approximately 2 weeks post-emergence. Thereafter the glands exhibited a more or less constant rate of JH biosynthesis (mean = 3.45 ± 0.32 pmol/CA pair/h.) When females were mated after 21 days of enforced virginity the activity of the CA was enhanced. By 48 h after mating the mean glandular activity was at least four times that found in virgins of the same age, and by 72 h rates as high as 40 pmol/CA pair/h were observed. This was followed by normal cyclic activity of the CA. The increase in rate of JH biosynthesis appears to result in a recommencement of oocyte development in these ‘delayed-mated’ females.     

Mating in Heliothis virescens: Transfer of juvenile hormone during copulation by male to female and stimulation of biosynthesis of endogenous juvenile hormone

Studies were undertaken to determine whether adult males of Heliothis virescens transfer juvenile hormone (JH) to females during copulation, and an in vitro radiochemical assay was used to determine whether mating causes an allatotropic effect, i.e., stimulation of JH biosynthesis by corpora allata (CA). In vitro, CA from 3-day-old mated females synthesized and released approximately 2.5 times total JH as that of CA from comparably aged virgin females. Of the homologues, JH II exhibited significant increase in mated females; JH I also increased but not significantly. JH III remained similar to that of virgin females. This is the first demonstration of an allatotropic effect of mating in moths. In contrast to the female, CA of virgin males did not produce any JH, but accessory sex glands (ASG) in 3-day-old males synthesized small amounts of JH. Immediately after adult emergence, male ASG contained approximately 1.5 ng JH I and II, which increased by 12 h after emergence and remained at this high level up to 54 h after emergence. JH III was barely detected in ASG. JH in ASG of mated male immediately after uncoupling was depleted almost completely, and 24 h later recovered to levels comparable to that of 54-h-old virgin male. Virgin female bursa copulatrix did not contain any JH, but mated female bursa, immediately after uncoupling, had JH at levels comparable to that observed in virgin male ASG. By 6 h after uncoupling, JH levels decreased dramatically in mated female bursa. These data suggest the transfer of JH to females by the male. Arch. Insect Biochem. Physiol. 38:100–107, 1998. © 1998 Wiley-Liss, Inc.     

Juvenile hormone biosynthesis,oocyte growth and vitellogenin accumulation in Choristoneura fumiferana and C. rosaceana: a comparative study

We assessed the effects of age and mating status on in vitro juvenile hormone (JH) biosynthesis, oocyte growth, egg production and vitellogenin (Vg) accumulation in the tortricid moths, Choristoneura fumiferana and C. rosaceana. To determine whether vitellogenesis is dependent on the presence of JH, we also examined the effects of decapitation and JH analog treatments on egg production. In both species, the corpora allata (CA) of adult females released fmol quantities of JH, with JH II being the major homolog produced. The CA began producing detectable quantities of JH around the time of emergence. Full activation of the CA was observed a few hours sooner in C. fumiferana than in C. rosaceana. In pharate adults and young virgin females of both species, growth of the basal oocyte reflected changes in CA activity. Decapitation of newly emerged females significantly reduced egg production, but treatment of decapitated females with the JH analog methoprene resulted in egg production that was similar to (C. fumiferana) or greater than (C. rosaceana) that of controls, indicating that JH is required for oocyte maturation. Vg was first observed in the hemolymph before the presumptive time of CA activation, suggesting that the synthesis of this protein is not dependent on JH. The presence of normal quantities of Vg in the hemolymph of pupae decapitated before CA activation confirmed this hypothesis. The Vg titer underwent a transient decline following CA activation and was significantly lower in mated than in virgin females of both species 3 and 5 days after copulation. Since CA activation at emergence and mating are both expected to cause a rise in the JH titer, we suggest that the declines in the levels of Vg result from JH-enhanced Vg uptake by the developing oocytes. Mating induced a significant increase in egg production but had no measurable impact on rates of JH biosynthesis in vitro.     

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.     

Exogenous juvenile hormone and methoprene, but not male accessory gland substances or ovariectomy, affect the blood/nectar choice of female Culex nigripalpus mosquitoes

When newly emerged females of the mosquito Culex nigripalpus Theobald (Diptera: Culicidae) take a sugar meal, they develop both a propensity to seek a host and resting-stage ovarian follicles. As follicle growth is indicative of Juvenile Hormone (JH) activity, we searched for possible roles of JH in the behavioural shift from nectar to blood-host odour preference by Cx. nigripalpus after emergence, using an olfactometer-choice assay. Topically applied or injected methoprene (a JH analogue) resulted in a behavioural shift and follicular growth in unfed females that increased with increasing dosages. Topical methoprene 500 ng and 4 microg resulted in a switch to bird-odour preference. JH III injection resulted in a lesser shift from honey responses to bird responses. Methoprene application caused no detectable changes in glycogen, total sugars or total lipid when assayed 24h after treatment. Additionally, as male accessory glands (MAG) have been shown to synthesize JH, we implanted intact paired MAGs from either conspecific or Aedes aegypti (L.) donors, or injected 1/3 gland pair equivalents of conspecific MAG homogenate into unfed newly emerged females. All MAG treatments failed to induce behavioural or ovarian modifications. Ovariectomy had no effect on the sugar-induced shift from nectar to host preference in the olfactometer. Thus JH, but not MAG, mimicked the effects of a sugar meal by causing both follicular growth and the shift to preference for a host.     

Allatostatin-C reversibly blocks the transport of citrate out of the mitochondria and inhibits juvenile hormone synthesis in mosquitoes

Aedes aegypti allatostatin-C (AeaAST-C or PISCF-AST) is a strong and fast reversible inhibitor of juvenile hormone III (JH III) synthesis by the corpora allata (CA) of mosquitoes; however, its mechanism of action remains poorly understood. AeaAST-C showed no inhibitory activity in the presence of any of the intermediate precursors of JH III indicating that the AeaAST-C target is located before the entry of acetyl-CoA in the pathway. Stimulation experiments using different sources of carbon (glucose, pyruvate, acetate and citrate) suggest that AST-C acts after pyruvate is transformed to citrate in the mitochondria. In vitro inhibition of the citrate mitochondrial carrier (CIC) mimicked the effect of AeaAST-C, and was overridden by addition of citrate or acetate. Our results provide compelling evidence that AeaAST-C inhibits JH III synthesis by blocking the CIC carrier that transports citrate from the mitochondria to the cytosol, obstructing the production of cytoplasmic acetyl-CoA that sustains JH III synthesis in the CA of mosquitoes.     

Corpus allatum function during sexual maturation of male Diploptera punctata

ABSTRACT. The effect of allatectomy on synthesis of accessory reproductive gland secretion, spermatophore production and sexual behaviour in male Diploptera punctata was investigated during the first 6 weeks of adult life. After allatectomy, synthesis of the secretion and production of spermatophores was slightly reduced relative to sham-operated animals (by 16%), but not relative to normal animals. However, sexual behaviour of the operated animals appeared normal. Thus, the corpora allata (CA) may not be necessary for the sexual functioning of male D.punctata. The synthesis of C₁₆ juvenile hormone (C₁₆ JH; JH III) by isolated pairs of CA from individual males was followed during this period and, at all times, the rate of synthesis was less than 8pmolh^-1 per pair, a rate similar to that observed in pregnant females. The significance of this continued synthesis of JH by male CA is unknown, although it may be related to the maintenance of general metabolic activities.     

Role of juvenile hormone and allatotropin on nutrient allocation, ovarian development and survivorship in mosquitoes

Teneral reserves are utilized to initiate previtellogenic ovarian development in mosquitoes. Females having emerged with low teneral reserves have reduced juvenile hormone (JH) synthesis and previtellogenic development. We investigated what role JH, allatotropin (AT) and other head-factors play in the regulation of previtellogenic ovarian development and adult survivorship. Factors from the head are essential for corpora allata (CA) activation and reproductive maturation. We have shown that decapitation of females within 9-12h after adult ecdysis prevented normal development of the previtellogenic follicles; however maximum previtellogenic ovarian development could be induced in decapitated females by topically applying a JH analog. When females were decapitated 12 or more hours after emergence nutritional resources had been committed to ovarian development and survivorship was significantly reduced. To study if allatotropin levels correlated with teneral reserves, we measured AT titers in the heads of two adult phenotypes (large and small females) generated by raising larvae under different nutritional diets. In large mosquitoes AT levels increased to a maximum of 45 fmol in day 4; in contrast, the levels of allatotropin in the heads of small mosquitoes remained below 9 fmol during the 7 days evaluated. These results suggest that only when nutrients are appropriate, factors released from the brain induce the CA to synthesize enough JH to activate reproductive maturation.     

Effects of chilling stress on allatal growth and juvenile hormone synthesis in the cockroach, Diploptera punctata

During the ovarian cycle of the cockroach, Diploptera punctata, a mitotic wave occurs in the corpora allata before an increase in gland volume and juvenile hormone (JH) synthesis. Previous studies have demonstrated that the brain inhibits mitosis and JH synthesis in corpus allatum (CA) cells until adult females have mated. Herein, we report that chilling stress effectively suppresses mating induced proliferation of CA cells. In mated females, chilling on melting ice for 0.5-3 hours caused a strong, dose-dependent decrease in mitotic activity. In insects chilled for 3 hours, although the mitotic wave in the CA was practically abolished, CA volume and JH synthesis finally reached peak levels typical of unchilled insects, despite a 2-day delay. Consequently, oocyte maturation and oviposition were also delayed by 2 days, yet in both chilled and unchilled insects, peak values of basal oocyte length were the same. By allowing virgin females to mate on different days after chilling, we found that the chilling effect could be retained in the insect body for at least 2 days. During this period, signals from mating could not effectively remove inhibition of CA cell proliferation. Unilaterally disconnecting the CA from the brain revealed that chilling stress mediated CA cell proliferation via the brain, and did not directly affect the CA.     

Structure,haemolymph titre,and regulatory effects of juvenile homone during ovarian maturation in Leucophaea maderae

Juvenile hormone (JH) synthesized and secreted in vitro by the corpora allata of mated adult Leucophaea maderae females was determined to be JH III (methyl-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate).The haemolymph titre of JH was determined during maturation of the terminal oöcytes in the first reproductive cycle of L. maderae. In virgin females, JH is not detectable in the haemolymph during the first eight days following adult emergence; however, by 10 days after emergence, trace quantities of JH are apparent. Mating stimuli induce a dramatic increase in the concentration of haemolymph JH, with a peak occurring approximately 12 days after mating; thereafter, the JH concentration declines until it has reached an undetectable level 19 days after mating, at the time of chorion deposition.During ovarian maturation, changes in the rates of synthesis of vitellogenin by the fat body and DNA by the ovary correlate closely with the haemolymph titre of JH. However, no such correlation exists between the JH titre and the extensive ovarian protein synthesis that occurs in L. maderae coincident with chorion formation.The effects of JH I and JH III on both vitellogenin synthesis and ovarain DNA synthesis are statistically similar.     

A stage-specific ovarian factor with stable stimulation of juvenile hormone synthesis in corpora allata of the cockroach Diploptera punctata

This is a study of a feedback loop from a stimulated organ to glands that produce the stimulatory hormone in the cockroach Diploptera punctata. In this insect as in many others, juvenile hormone (JH) produced by corpora allata (CA) stimulates vitellogenesis. In our previous studies, transplantations of ovaries at certain stages of development into ovariectomized mated females stimulated JH synthesis within 24h. An in vitro study by other investigators showed that all stages of ovaries release a stimulatory factor into culture medium that was not retained on a solid-phase extraction column but occurred in the aqueous flow-through. The present study is a comparison of the effect of medium conditioned with ovaries from days 1-4 and 8 of the first reproductive cycle, to the effect of the flow-through of that medium on members of a pair of CA from day 3 females. Results provide evidence for an ovarian factor that stimulates JH synthesis by CA in vitro after removal from the conditioning medium (i.e., stable stimulation). Only medium conditioned with ovaries from days 2 or 3 females significantly stimulated CA more than flow-through. Stimulation was dose dependent, sensitive to trypsin, and survived freezing. These results indicate that CA can be directly and stably stimulated by a stage-specific peptidergic ovarian factor.     

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.     

Changes in juvenile hormone synthesis in the termite Reticulitermes flavipes during development of soldiers and neotenic reproductives from groups of isolated workers

Workers of Reticulitermes flavipes were isolated in groups of increasing numbers to determine the in vitro rates of juvenile hormone (JH) synthesis by individual pairs of corpora allata (CA) as other castes differentiated. Only neotenic reproductives developed in groups of 12. Mean JH synthesis rates increased after 5 weeks but only a few individuals had significantly higher rates, about 0.4 pmol/pair/h, which occurred at about 3 weeks before neotenics developed. Soldiers and neotenics developed in groups of 50. Mean rates increased to a peak at week 6 after isolation, but only a few individuals had rates approaching 1 pmol/pair/h, which occurred at the same time after isolation as the development of pre-soldiers. JH synthesis by CA of pharate pre-soldiers and soldiers was low compared to that of pharate workers and neotenics. CA of pre-soldiers attained a peak mean rate of JH synthesis of 0.9 pmol/pair/h at 6 days of age, whereas CA of soldiers attained only a peak mean rate of 0.3 pmol/pair/h. These measurements of JH synthesis by individual pairs of CA suggest that the few workers destined to become pre-soldiers have 2.5-fold higher JH synthesis than the few that would develop into neotenic reproductives, and show that a cycle of synthesis accompanies the development of pre-soldiers into soldiers.     

Switchover to the mated state by spermathecal activation in female Anopheles gambiae mosquitoes

Both Aedes aegypti and Anopheles gambiae mosquitoes undergo physiological and behavioral changes after the females mate, but unlike Ae. aegypti, the mated state in An. gambiae is not attained through the action of male accessory gland substances. Experiments in which the spermathecae of mated females were manipulated suggest that a spermatheca filled with sperm is responsible for triggering oviposition behavior. An estimated 48% of the previously mated An. gambiae females mated again when the interval between encounters with males was less than 24h. Unlike male Ae. aegypti that can inseminate up to seven females after their testes are removed, An. gambiae do not store sperm in the vas deferens and without testes would be unable to inseminate any females.