A neurosecretory hormone-releasing factor from ovaries of mosquitoes fed blood

In mosquitoes, a hormone (egg development neurosecretory hormone or EDNH), produced by the medial neurosecretory cells and stored in the corpus cardiacum soon after eclosion, is released after a blood meal, and vitellogenesis begins a few hours later. When either the ovaries or the neurosecretory cells and corpus cardiacum are removed before the blood meal, vitellogenin is not synthesized. Therefore, we tested the hypothesis that the release of EDNH from the corpus cardiacum is dependent on the secretion of a releasing factor from the ovaries.Using a bioassay for EDNH in the corpus cardiacum, we found that the gland of an ovariectomized female remained active after blood feeding, and therefore, has not released EDNH. When an ovary was implanted before the blood meal, the corpus cardiacum was inactive, and therefore, had released EDNH. We concluded that the ovaries secrete an EDNH-releasing factor, and that this factor and EDNH must both be in circulation before vitellogenesis can begin. Although releasing factor alone did not stimulate vitellogenesis, it was the rate limiting process that controlled the onset of vitellogenesis.Using a bioassay for the EDNH-releasing factor from the ovaries and using rocket-immuno-electrophoresis, we showed that a Culex ovary, but not an Anopheles ovary, could replaces Aedes ovaries as a source of the releasing factor.In Ae. aegypti, EDNH-releasing factor was required again after oviposition in order to reinitiate the vitellogenic process in females that took a second blood meal. Thus, the releasing factor is part of the mechanism regulating cyclic egg maturation in mosquitoes.     

Influence of the endocrine system on tryptic activity in female Aedes aegypti

In MNC-ablated or ovariectomized mosquitoes, blood-fed by enema, the tryptic activity of midgut homogenates was reduced by half. By sealing the anus after the blood meal, we showed that the reduction in enzyme activity was not due to premature or increased excretion of active enzyme in operated females. The enzyme activity was restored to the level of unoperated controls by either reimplantation of an ovary or injection of 20-hydroxy-ecdysone in a large, single dose shortly before the blood meal.The reduction in tryptic activity in MNC-ablated or ovariectomized females did not alter the rate of protein digestion as measured by the decrease of protein in midgut homogenates. We conclude that the neurosecretory system and the ovaries are required for maximal tryptic activity in normal females, and that the enzyme apparently is secreted in excess of that required for complete digestion of the blood meal.     

The role of the male accessory gland fluid in stimulating vitellogenesis in Aedes taeniorhynchus

Injection of 20-hydroxyecdysone (20-OH-ecdysone) at high concentrations (5.0 μg) into intact or decapitated female Aedes taeniorhynchus induced vitellogenin synthesis, whereas low concentrations (5.0 ng) were ineffective. Injections of male accessory gland fluid (MAGF), however, at a concentration that was equivalent to 0.25 of the content of a pair of accessory glands, into intact or decapitated A. taeniorhynchus induced viteliogenin synthesis only in intact females. Ovariectomized mosquitoes did not synthesize vitellogenin after MAGF injection or blood feeding. Females that were first injected with MAGF and decapitated 12 h later synthesized viteliogenin at a rate that was 80% of intact controls. Egg development neurosecretory hormone (EDNH) activity in the heads of ovariectomized or intact females injected with MAGF was 9.0 pmol/min/head and 2.5 pmol/min/head, respectively, indicating that MAGF does not stimulate the corpus cardiacum (CC) to release EDNH. Incubation of MAGF and EDNH with fat bodies failed to induce vitellogenin synthesis. These results indicate that in A. taeniorhynchus the MAGF induces the ovary to release corpus cardiacum stimulating factor, which then signals CC to release stored EDNH.     

Activation of vitellogenin synthesis in the mosquito Aedes aegypti by ecdysone

Injected β-ecdysone was found to induce the synthesis of yolk protein (vitellogenin) in adult female Aedes aegypti without a blood meal. After injection of 5 μg ecdysone per mosquito, vitellogenin constituted 80 per cent of the total protein secreted by explanted fat body, a proportion comparable to that produced by fat body from blood-fed females. Moreover, the time course of induction of vitellogenin synthesis in ecdysone-injected mosquitoes was similar to that triggered by a blood meal. Response to ecdysone is dosedependent: 0·5 μg per female was required to stimulate synthesis to 50 per cent of the level found 18 hr after a blood meal. Ecdysone was effective in decapitated or ovariectomized mosquitoes, and also when applied directly to fat body preparations in vitro. Thus it appears that ecdysone acts directly on the fat body to induce specific protein synthesis, as does the vitellogenin stimulating hormone (VSH) from the ovary of blood-fed mosquitoes. These results suggest that ecdysone can replace VSH in inducing vitellogenin synthesis in the unfed mosquito.     

20-Hydroxy-ecdysone as a modulator of electrical activity in neurosecretory cells of Rhodnius prolixus

Ovariectomy of the adult female Rhodnius results in a reduction of the electrical activity of the corpus cardiacum. Injection of 20-hydroxy-ecdysone into ovariectomized animals increases the activity to control values. Incubation of heads isolated from ovariectomized females in 1.0 × 10^?7 M 20-hydroxy-ecdysone elicits a recruitment of large-amplitude spikes and the appearance of bursting activity in the corpus cardiacum of mated females which is characteristic of neurohormone release. Simultaneous recordings from the median neurosecretory cells and the corpus cardiacum demonstrate that the action neurosecretory cells.     

Vitellogenin synthesis in blood-fed Aedes aegypti in the absence of the head,thorax and ovaries

A blood meal initiates oöcyte maturation in Aedes aegypti, and we have used rocket immunoelectrophoresis to investigate the function of midgut, ovaries, and head in the onset of vitellogenin synthesis. Non-blood-fed females and those fed blood (by enema) containing soybean trypsin inhibitor never contained vitellogenin. This demonstrates that the pressure of an undigested blood meal on stretch receptors of the midgut plays no role in the induction of vitellogenin synthesis, rather the stimulus is a digestion product of blood.When females were ovariectomized or decapitated and then fed blood, the haemolymph contained newly synthesized vitellogenin 24 h later. This was also demonstrated in isolated ovariectomized abdomens. Apparently, induction of vitellogenin synthesis does not require factors from either the head, thorax, or ovaries. When ovariectomy or decapitation was postponed after a blood meal, the level of vitellogenin in the haemolymph rose. Therefore, interaction of factors from the head and ovaries maintain the synthesis needed for oöcyte maturation.     

The role of factors from the head in the regulation of egg development in the mosquito Aedes aegypti

The relationships between the release of factors from the head after blood-feeding, subsequent levels of ecdysteroids and vitellin, and the ultimate maturation of eggs in Aedes aegypti were investigated. Females were decapitated at various times after a blood meal, at 20 or 48 h after feeding the animals were dissected and divided into two groups, those with arrested oöcytes (yolk length < 100 μm) and those with maturing oöcytes (yolk length > 100 μm). These yolk lengths correspond with the levels of oöcyte growth believed to accompany the proposed initiation and promotion phases of egg development. Animals dissected at 20 h were assayed for ecdysteroid by radioimmunoassay; those dissected at 48 h were assayed for vitellin by rocket immunoelectrophoresis.Non-blood-fed unoperated females contained 8% as much ecdysteroid as blood-fed controls and no measurable vitellin. Females with arrested oöcytes (< 100 μm) were obtained only if decapitations were performed before 8 h; these females had about 20% of the ecdysteroids and 8% of the vitellogenin normally found in blood-fed animals. Females decapitated between 2 and 8 h with maturing oöcytes contained 50–60% as much ecdysteroid and vitellin as blood-fed unoperated controls. Normal ecdysteroid and vitellin levels were reached only when decapitations were delayed for 12 and 24 h, respectively. The number of developing oöcytes was also decreased by early decapitation and was closely correlated with vitellin levels.We conclude that the egg development neurosecretory hormone is released twice, once before 8 h and once after 8 h, to control ecdysteroid levels. We also suggest the presence of other factors from the head that control vitellin levels, the number of developing oöcytes, and the early growth of the oöcyte (initiation).     

Initiation and termination of host-seeking inhibition in Aedes aegypti during oöcyte maturation

The inhibition of host-seeking behaviour that accompanies vitellogenesis in the mosquito, Aedes aegypti, was examined by the removal and implantation of ovaries. Mosquitoes ovariectomized before a blood meal and between 1 and 6 hr after a blood meal responded to a host at 48 hr after a blood meal. However, when ovariectomy was delayed until 10 hr after the meal or later, most mosquitoes did not respond to the host. When a partial ovary was present for only the first 12 hr after a meal, there was no host-seeking inhibition at 48 hr, and only 58% of females with one complete ovary present during this time interval responded. Howver, these same amounts of ovarian tissue inhibited host-seeking when they remained for 48 hr after a meal. Vitellogenic ovaries from donors blood-fed 8–24 hr before, implanted into sugar-fed recipients, did not affect the host-seeking behaviour of these recipients. Ovaries removed and reimplanted before the blood meal inhibited host-seeking at 72 hr after the blood meal only in the absence of oviposition from intact ovaries. It is concluded that 2 humoral factors are involved in the promotion of host-seeking inhibition: the first factor is produced by the ovaries, and after reaching a critical threshold in the haemolymph, stimulates the release of a second factor that acts directly to inhibit mosquito behaviour. An ovary which retains 2 or fewer eggs after oviposition terminates the inhibition via nervous pathways. The role of 20-hydroxyecdysone in the behavioural inhibition is discussed.     

Artifactual stimulation of vitellogenesis in Aedes aegypti by 20-hydroxyecdysone

Single or repeated, non-physiological, high doses (0.5–5.0 μg/female) of 20-hydroxyecdysone or ecdysone injected into sugar-fed female Aedes aegypti stimulated follicular growth and deposition of yolk, but suppressed accumulation of protein yolk to approximately one-third, and lipid yolk to one-half that in an equal number of follicles with equivalent yolk length taken from blood-fed controls. Physiological doses (500 pg/female) of ecdysone or 20-hydroxyecdysone or the implantation of ecdysone-secreting ovaries (verified by bioassay), into sugar-fed females failed to induce any yolk deposition. In these experiments, yolk precursors were not the limiting factor, because in decapitated females, digesting a blood meal, the injection of a physiological dose of 20-hydroxyecdysone or the implantation of ecdysone-secreting ovaries still did not stimulate vitellogenesis. Finally, continuous infusion of 500 pg or even 50 ng 20-hydroxyecdysone/hr for 22 hr was as ineffective as single or multiple injections of equivalent doses of hormone. Consequently, rapid excretion or catabolism of 20-hydroxyecdysone by the sugar-fed female does not explain the need for high doses to induce vitellogenesis, or the failure of oöcytes to mature with normal protein and lipid content. Apparently, ovarian ecdysone is not the factor by which normal vitellogenesis is initiated and maintained in this mosquito.     

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.     

Egg development in the mosquito Anopheles albimanus

Summary

In the mosquito, Anopheles albimanus, previtellogenic egg development was completed by 48 h after emergence, and vitellogenic growth was completed by 36 h after a blood meal. Ecdysteroid levels reached a peak of 800 pg/female by 18 h, while vitellin levels rose to their maximum 36–48 h after a blood meal. Most of the ecdysteroids present in the female before 36 h behaved as ‘free’ hormone, while after 42 h the ecdysteroids were ‘conjugated’. Injection of 20-hydroxyecdysone into non-blood-fed females induced degeneration of the resting stage oocytes, but vitellogenin synthesis was detectable by autoradiography. Injection of 5 μ of 20-hy-hroxyecdysone into blood-fed decapitated females induced almost precisely normal levels of vitellin. Detailed analysis of the effect of decapitating blood-fed females suggested that the release of factors from the head (e.g., egg development neurosecretory hormone) occurs as an all-or-none phenomenon, and probably occurs twice.     

Effects of decapitation and ovariectomy on the regulation of juvenile hormone synthesis in the cockroach, Diploptera punctata

Corpora allata from Diploptera punctata females at adult ecdysis or at the end of the last-larval stadium, when implanted into decapitated females, underwent a cycle of juvenile hormone synthesis similar in timing and magnitude to that of glands implanted into control animals which had been starved and allatectomized. Starvation did not alter the cycle in rates of juvenile hormone synthesis of sham-operated animals.Decapitation of ovariectomized animals resulted in no cycle in rates of juvenile hormone synthesis by implanted adult corpora allata; however, implantation of an ovary along with the corpora allata into decapitated, ovariectomized hosts resulted in a cycle of juvenile hormone synthesis. In control animals, which retained their heads but were starved and allatectomized as well as ovariectomized, the implanted corpora allata showed a cycle of juvenile hormone synthesis only when implanted with an ovary. The maximal rates of juvenile hormone synthesis by the corpora allata in both experimental and control conditions were lower than normal, likely due to the repeated trauma of surgery. However, at no time from eclosion to the end of the first gonotrophic period was the brain necessary for the cyclic response of the corpora allata to the presence of the ovary.     

Does ovarian ecdysone stimulate mosquitoes to synthesize vitellogenin?

Physiological amounts of 20-hydroxyecdysone do not initiate vitellogenin synthesis in unfed, non-vitellogenic mosquitoes. Injecting more than 10,000 times the physiological amount induced synthesis, but considerably less than was induced by a blood meal. A dose of 20-hydroxyecdysone which exceeded the physiological level only several hundred times, did not sustain vitellogenin synthesis, when blood-fed mosquitoes were ovariectomized just prior to injection. Transplanting ovaries from vitellogenic to non-vitellogenic females did not initiate synthesis of vitellogenin in the recipient. In vitro, neither 20-hydroxyecdysone nor the ovaries of vitellogenic females were able to induce synthesis of vitellogenin in non-vitellogenic fat bodies. These experiments suggest that ecdysteroid, released by the ovaries, does not initiate ovarian development in mosquitoes.     

The roles of juvenile hormone and 20-hydroxy-ecdysone during vitellogenesis in isolated abdomens of Drosophila melanogaster

Both juvenile hormone and 20-hydroxy-ecdysone seem to be involved in the regulation of vitellogenesis in Drosophila melanogaster. It is the purpose of this paper to begin to define the functions of these two hormones. Although vitellogenin synthesis does not occur at a high rate in 1-day-old female abdomens isolated from the head and thorax before 0.75 hr after eclosion, both ZR515 (a juvenile hormone analogue) and 20-hydroxy-ecdysone can cause in these preparations vitellogenin synthesis and secretion into the haemolymph. The synthesis and secretion into the haemolymph of all three vitellogenins which are detectable by electrophoresis in sodium dodecyl sulphate-containing gels of polyacrylamide is promoted by both hormones. That result excludes the hypothesis that these two hormones regulate the synthesis of different vitellogenins. A dose-response curve showed that an injection of 0.2 μl of a 10^?6 M 20-hydroxy-ecdysone solution was sufficient to promote vitellogenin synthesis and secretion in isolated abdomens. Ovaries from isolated female abdomens treated with juvenile hormone analogue showed nearly normal amounts of all three vitellogenins and morphologically normal advanced vitellogenic follicles, whereas ovaries from isolated abdomens treated with 20-hydroxy-ecdysone contained little vitellogenin and no vitellogenic follicles. We conclude that under the conditions used, juvenile hormone permits vitellogenin uptake into the oöcyte much more readily than does 20-hydroxy-ecdysone.     

The influence of the brain hormone on retention of blood in the mid-gut of the mosquito Aedes aegypti (L.). III. The involvement of the ovaries and ecdysone.

Most female mosquitoes require a blood-meal in order to produce mature o?cytes. An egg development neurosecretory hormone (EDNH), which is produced in the medial neurosecretory cells (m.n.c.) of the brain and stored in the corpus cardiacum, is released into the haemolymph following the ingestion of blood and is essential for the promotion of ovarian development to maturity. It has been shown that a factor from the m.n.c., presumably EDNH, is necessary if the blood-meal is to be retained in the mid-gut until the o?cytes approach maturity. The present paper shows that retention is not a direct result of the action of EDNH, but is dependent on the ovaries and may well involve ecdysone. Removal of the ovaries before a blood-meal leads to early haem-defaecation, but delay can be restored by injection of ecdysterone. Sub-threshold feeders and mosquitoes decapitated immediately after the intake of blood, each of which would be expected to eliminate the blood-meal early, also show a delay in the onset of haem-defaecation when injected with ecdysterone. Further, both in ovariectomized insects and sub-threshold feeders the time of onset of haem-defaecation is associated with the dose of ecdysterone given.     

Biosynthesis and distribution of ecdysone and 20-OH-ecdysone in Aedes aegypti

The distribution and biosynthesis of ecdysone and 20-hydroxyecdysone (20-OH-ecdysone) was followed in sugar- and blood-fed female Aedes aegypti. In both sugar- and early blood-fed animals most of the ecdysteroid determined by radioimmunoassay was found outside the ovary. Twenty-four to 40 h after blood feeding, however, ecdysteroid was distributed between ovary and carcass in the ratio of 1:1.5. Ecdysteroid titer reached a plateau between 18 to 40 h after the blood meal and decreased thereafter. Analysis of the ecdysteroid titer using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) revealed that both 20-OH-ecdysone and ecdysone were synthesized after the blood meal. The ratio of 20-OH-ecdysone to ecdysone remained essentially constant and fluctuated in parallel throughout egg development. Chromatography of the early ecdysteroid peak (8 h after feeding) using TLC and HPLC indicated that although it cross-reacted with ecdysteroid antibodies, it did not have the same elution times as ecdysone and 20-OH-ecdysone and is, therefore, probably a precursor of these ecdysteroids. Injections of egg development neurosecretory hormone (EDNH) preparation purified to near homogeneity, into ligated abdomens, induced ecdysteroid synthesis only if the abdomens were first treated with methoprene (12.5 pg). Methoprene at this concentration did not stimulate ecdysteroid synthesis in these abdomens. When blood-fed females were treated with [4-¹⁴C] cholesterol and analyzed using TLC and HPLC procedures, both [¹⁴C]labeled ecdysone and [¹⁴C]labeled 20-OH-ecdysone were synthesized in the ratio of 1:1.5. This report is the first to show that both ecdysone and 20-OH-ecdysone are synthesized in vivo in female A. aegypti.     

Isolation and characterization of highly purified mosquito oostatic hormone

Oostatic hormone, the hormone that inhibits vitellogenesis in mosquitoes, was purified 7,000-fold with a recovery of 70% from the ovaries of the mosquito Aedes aegypti. The purification procedure included heat treatment and chromatography on ion exchange and gel filtration columns. The hormone is a small peptidelike molecule of molecular weight 2,200 at pH 4.5, which aggregates into larger molecular species of trimer and octamer at pH 7.0 as determined by gel filtration. The hormone is positively charged at pH 7.8 and has a low Rf at pH 9.4 on disc gel electrophoresis. Injection of purified oostatic hormone (9 ng) into female mosquitoes inhibited yolk deposition and vitellogenin synthesis. Activity of the oostatic hormone in the mosquito ovary increased rapidly following blood feeding and reached a maximum after 48 h. Oostatic hormone of A. aegypti injected into autogenous Aedes taeniorhynchus inhibited egg development. Repeated injections of dilute oostatic hormone at 24 h intervals partially arrested egg development, resulting in 60% reduction in the number of eggs laid. This hormone does not block release of egg development neurosecretory hormone (EDNH) from the mosquito brain but rather appears to act on the ovary.     

Stimulation de l'ovogenèse par la copulation chez les femelles de Drosophila melanogaster privées de leur complexe endocrine rétrocérébral

The extirpation of the corpus allatum-corpus cardiacum complex in Drosophila melanogaster females strongly reduced egg production but did not completely stop it. When the females were inseminated after the operation the increase of fertility which was observed is proportionally as important as in control females. It was concluded that the stimuli occurring from insemination were mainly acting on the neurosecretory cells of the brain. In the control of oögenesis, it is probable that the brain hormones are more important than the corpus allatum hormone.     

Ovarian control of blood meal retention the the mosquito Anopheles freeborni

Female Anopheles freeborni begin to defaecate the haem containing residue of a blood meal about the time their ova mature, 40–45 hr after feeding. Ovariectomized females begin to defaecate 15 hr prematurely, while ovariectomized females treated with 20-hydroxyecdysone retain blood meals longer, proportional to dose, than do those untreated. It is concluded that the production of ecdysteroids by the ovary initiates a mechanism for retention of the blood meal in the mosquito.     

Juvenile hormone and 20-hydroxyecdysone as primary and secondary stimuli of vitellogenesis in Aedes aegypti

Female Aedes aegypti that were fed blood and immediately abdominally ligated did not deposit yolk. Injection of 20-hydroxyecdysone (1.5–5.0 ng) or topical application of juvenile hormone (JH) analogue methoprene (25 pg) did not induce vitellogenesis in these abdomens. When blood-gorged ligated abdomens were treated with both hormones, however, vitellogenesis was stimulated in 60% of treated animals. Rocket immunoelectrophoresis indicated that vitellin concentration per follicle in treated animals was similar to that in intact controls. When ligated abdomens were first treated with methoprene and immediately injected with a crude head extract of egg development neurosecretory hormone, vitellogenin synthesis was induced at a rate similar to that in blood-fed controls. Methoprene at this concentration (25 pg), did not cause an increase in whole-body ecdysteroid titers. Larger amounts of methoprene (1.65 ng) were needed to stimulate egg development and ecdysteroid production. Implantation of ecdysone-secreting ovaries into ligated abdomens did not stimulate vitellogenesis in the recipients. However, in recipients that were first treated with methoprene (25 pg), implantation of ecdysone-secreting ovaries resulted in normal egg development. These experiments indicate that the appearance of JH precedes 20-hydroxyecdysone in stimulating vitellogenesis following blood feeding in Ae. aegypti.