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.     

Hemolymph juvenile hormone titers in pupal and adult stages of southwestern corn borer [Diatraea grandiosella (pyralidae)] and relationship with egg development

Juvenile hormones I, II and III were monitored in hemolymph of pupal and adult stages of various ages of Diatraea grandiosella females. JH III was the predominant homologue followed by JH II, and JH I was rarely detectable. At day 5 after pupation, no JH was detectable. JH titers increased from 7.5days after pupation to a peak of 24.8ngml(-1) JH II and 26ngml(-1) JH III at adult emergence and then declined to low levels by 24h after emergence. Ovarian development in D. grandiosella parallels changes in hemolymph JH titers, but the role of JH in vitellogenesis is unclear since the time of vitellogenesis initiation has yet to be determined. No apparent vitellogenin deposition was observed in eggs 5days after pupation. Some oocytes were partially vitellogenic by 7.5days after pupation and oocytes continued to grow afterwards, but no oocytes were chorionated during the pupal stage. Chorionated oocytes were observed in 24-h-old female moths. Juvenile hormone is essential for chorion formation in this species, because decapitated pupae treated with 10&mgr;g JH III in corn oil developed chorionated oocytes while decapitated pupae treated with corn oil did not.     

Regulation of vitellogenesis in the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae)

Studies were undertaken to investigate vitellogenesis and its regulation in female adults of the fall armyworm, Spodoptera frugiperda. A single female-specific protein, likely to be the S. frugiperda vitellogenin (Vg), appeared approximately 5 h after adult eclosion in the hemolymph of virgin females. The concentration of the protein increased with age as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed. A protein with the same relative molecular mass was also present in egg extracts, but absent from hemolymph samples from male moths. The relative molecular mass of the designated S. frugiperda Vg was determined as 164.5+/-2.5 kDa. Vitellogenic oocytes became visible 36-48 h after emergence and egg deposition began on day 3 of adult life. Vg could not be detected in the hemolymph of females decapitated directly after eclosion. When decapitated virgin females were injected with the JH-mimic methoprene (MP), the level of Vg was comparable to that in non-decapitated moths, indicating that vitellogenesis in S. frugiperda depends on juvenile hormone (JH). However, the number of vitellogenic oocytes was somewhat lower than in non-decapitated virgin females. Injection of 20-hydroxyecdysone (20E) promoted Vg production to a similar extent in decapitated female moths, but in contrast to methoprene injection, treatment with 20E never resulted in the production of vitellogenic oocytes. In vitro cultivated ovaries of adult females dissected directly after eclosion produced lower amounts of ecdysteroids than those isolated on day 1 after emergence. Our results suggest a crucial role for 20E in the induction of vitellogenesis in the noctuid S. frugiperda, while JH seems to be essential for the continued uptake of Vg by developing oocytes and may trigger 20E biosynthesis in the ovary.     

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.     

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.     

Influence of juvenile hormone and mating on oogenesis and oviposition in the codling moth,Cydia pomonella

Oogenesis in the codling moth, Cydia pomonella, and the role of juvenile hormones (JHs) were addressed. Rudimentary ovarian structures were recognisable in day 3–4 pupae, when haemolymph JH was still undetectable by coupled gas chromatography‐mass spectrometry in the selected ion mode (GC‐MS/SIM). The presence of developing oocytes was observed by light microscopy on day 8, coincident with very low JH titres (0.74 ± 0.05 ng/ml JH II). Chorionation was only evident upon emergence, following an increase in JH in the pharate adult (0h old: 4.71 ± 0.34 ng/ml JH II). Analysis of haemolymph from virgin and mated females indicated that JH II was predominant, with approximately equal and lower quantities of JHs I and III (3.3‐ to 5.0‐fold less). When pupae or newly emerged adults were treated with JH homologues, no alteration in ovarian protein content was apparent, but the JH mimetic, fenoxycarb, depressed the number of oocytes filling ≥ 50% follicular volume. Chorion deposition was stimulated by JHs I, II, or III (10 μg), but not by fenoxycarb (0.05 μg, 10 μg). Mating provided correct stimuli for enhanced choriogenesis and egg laying, and, since haemolymph JH titres were concomitantly elevated (approximately 2‐fold), it was postulated that the rise in JH elicited both these events. Application of JHs to virgin females, however, could not mimic mating; only increases in choriogenesis were induced: JH‐treatment of virgins (or mated insects) significantly decreased oviposition rates over 24 and 48 h and markedly reduced the life‐time total number of eggs. Arch. Insect Biochem. Physiol. 41:186–200, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

Endocrine aspects of ovary growth and gestation in the Argentinian cockroach,Blaptica dubia (Dictyoptera,Blaberidae)

Titers of juvenile hormone (JH) III and free ecdysteroids were studied in the hemolymph of the ovoviviparous Argentinian cockroach, Blaptica dubia, related to fat body depletion and reproduction. Adult females were analyzed during the first (days 5–25) and second vitellogenic cycle (days 80–100) and during the periods of gestation. Body weight changes of adult females were closely related to ovarian growth, ootheca formation, ootheca deposition, and hatching of the nymphs. Biochemical analysis of the fat body revealed lipids as the main storage compounds, followed by glycogen, proteins, and free carbohydrates. Changes in the fat body weight and in the chemical constituents of the fat body correlated with the processes of vitellogenesis and gestation. Concentrations of JH and free ecdysteroids in the hemolymph were measured by high pressure liquid chromatography-mass spectrometry. JH III was the only JH homolog found. JH III titers were high during vitellogenesis as well as toward the end of the gestation period. Changes in the concentrations of ecdysone and 20-hydroxyecdysone were less clear. The results reveal JH III as the major gonadotropic hormone in adult females of B. dubia.     

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.     

Haemolymph vitellogenin,juvenile hormone,and oöcyte growth in the adult cockroach Nauphoeta cinerea during first pre-oviposition period

In the cockroach Nauphoeta cinerea the incorporation of a protein of low solubility into the oöcytes begins at day 5 of its adult life. An immunologically identical protein appears in the haemolymph two days earlier. The concentration of this protein, i.e. ‘vitellogenin’ in the haemolymph increases up to the onset of yolk incorporation into the oöcytes. During ovarian development no correlation could be detected between vitellogenin titre and several other parameters (ovary dry weight, length of the basal oöcytes, haemolymph protein concentration, body weight and age when ovulation occurred). In young females vitellogenin titre depends on the age, i.e. the volume of the corpora allata and hence on the presence and the titre of JH. During the period of egg maturation the total haemolymph protein concentration generally tends to drop while materials not precipitable by trichloracetic acid circulate at higher concentration after ecdysis and before ovulation.Early decapitation prevents vitellogenin synthesis and oöcyte growth, but when JH is applied to decapitated females, the normal vitellogenin titre is re-established, ovarian development, however, cannot be fully resumed. A dose-response curve shows that serial application of the hormone is much more effective than single large doses. Farnesylmethylester, a JH mimic, is about a hundred times less active, but more persistent than JH. Copulation seems to enhance the synthesis and release of endogenous JH, while food and water uptake are necessary to guarantee and optimal ovarian development. JH and high vitellogenin titre never restore ovarian development in females deprived of food and/or water or in those decapitated shortly after ecdysis.     

Juvenile hormone regulation of structural changes and DNA synthesis in the follicular epithelium of Leucophaea maderae

Juvenile hormone (JH I) stimulates specific morphological and biochemical changes in the follicular epithelium surrounding the terminal oöcytes in Leucophaea maderae. These include extracellular and intracellular structural changes, increased rates of follicle cell DNA synthesis, and elevated follicle cell DNA concentrations.Using females decapitated 24 hr after ecdysis, we have shown that JH I injections stimulate the following structural changes in the follicular epithelium: the appearance of channels between adjacent follicle cells and of spaces between the follicular epithelium and the maturing oöcyte; an increase in follicle cell size; the development of an extensive rough endoplasmic reticulum system; and an enlarged nucleus within each follicle cell. No increase in the number of follicle cells surrounding the developing terminal follicles is found in 7-day JH I-treated females, although the terminal follicles are almost twice as long as those in untreated females.In addition, we have demonstrated that JH stimulates the following biochemical events in the ovary: a 3.5 fold increase in thymidine incorporation into follicle cell DNA, with no subsequent transfer of such DNA to the developing oöcyte, and a 1.4 fold increase in ovarian DNA in 7-day JH-treated females. These data indicated that JH stimulates follicle cell DNA synthesis. The absence of any corresponding division of follicle cells suggests that JH I may induce polyploidy in follicle cells.Extended exposure of decapitated females to JH I does not result in complete ovarian maturation. Although fat bodies in the treated insects continue to display an increasing rate of vitellogenin synthesis, DNA synthesis in the terminal follicles declines rapidly after day 9, and the terminal follicles ultimately degenerate.     

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.     

Ovarian maturation in Leucophaea maderae: Juvenile hormone regulation of thymidine uptake into follicle cell DNA

Our research demonstrates that juvenile hormone (JH I) stimulates thymidine incorporation into ovarian follicle cell DNA in the ovoviviparous cockroach, Leucophaea maderae.A rapid, quantitative method for monitoring ³H-thymidine incorporation into ovarian DNA, in vitro, is described. Cultured ovarian tissue from L. maderae incorporates ³H-thymidine into DNA at a linear rate between 16 and 120 min; analysis of the incorporated label revealed at least 98% of it to be in DNA.Using L. maderae females that had been mated 7 days after adult emergence, we monitored the following biochemical phenomena during the 18–22 day period of terminal oöcyte growth: (1) ³H-thymidine incorporation into ovarian DNA: (2) general protein synthesis in fat body; and (3) specific fat body vitellogenin synthesis.Decapitation of mated females with maturing oöcytes arrested both ovarian DNA synthesis and fat body vitellogenin synthesis. Substantial restoration of both types of synthesis was induced by injection of JH I. The resumption of thymidine incorporation into DNA was localized in the follicular epithelium of the terminal oöcyte.In decapitated virgin females, injection of JH I stimulated oöcyte growth and ³H-thymidine incorporation into ovarian DNA. Dose and time response curves indicate that peak stimulation of ovarian DNA synthesis occurred between 72 and 96 hr after administration of a single optimal dose of 25 μg JH I. The concurrent manifestation of ³H-thymidine uptake into ovarian DNA and activity within the fat body indicates that a similar hormonal mode of action may be operative with respect to both tissue types in virgin females.     

Effects of juvenile hormone analogue treatment in adult females of the ovoviviparous cockroach,Blaptica dubia (Dictyoptera,Blaberidae)

Adult females of the ovoviviparous Argentinian cockroach, Blaptica dubia, were repeatedly treated with either 100?μg methoprene or 100?μg pyriproxyfen in 5?μL acetone either during the first vitellogenic cycle or during the period of gestation. Treatment during the first vitellogenic cycle (days 2–20 of adult life) did not inhibit vitellogenesis and oocyte growth, but prevented the formation of an ootheca. This was accompanied with a significant reduction of the titer of juvenile hormone (JH) III and an increased amount of ecdysone (E) and 20-hydroxyecdysone (20E) in the haemolymph of the animals. Treatment of adult females during the period of gestation (days 30–70) resulted in a complete degradation and resorption of the ootheca and induced another vitellogenic cycle. Again, this was associated with a decrease in haemolymph JH III titer, but an increase in the concentrations of free ecdysteroids.     

Juvenile hormone counteracts the action of ecdysterone on silk glands of Galleria mellonella L. (Lepidoptera : Pyralidae)

Morphological and physiological evidence is presented to show that ecdysterone (20E) and juvenile hormone (JH) directly affect the silk glands of Galleria mellonella (Lepidoptera : Pyralidae). Within 1 hr in a culture medium, 20E at 5 or 50 ng/ml stimulates, and at 5 μg/ml inhibits, RNA synthesis. Both these effects are obliterated with physiological (1 ng/ml) and higher doses of JH II or a juvenoid. Dipping of isolated larval abdomens in 0.32% 20E suppresses the rate of RNA synthesis in freshly dissected silk glands incubated in a hormone-free medium. The ultrastructure of silk glands shows functional regression, followed by histolysis within 72 hr after dipping. Both the reduction of RNA synthesis and the cytological changes are prevented when the abdomens receive JH II or a juvenoid simultaneously with 20E. Presence of JH II in the culture medium also enhances RNA synthesis in silk glands dissected from abdomens that had been treated with 20E. The results reveal that the effect of 20E is dose-dependent and may be prevented, and up to a certain point reversed, with JH.     

Temporal profiles of juvenile hormone titers and egg production in virgin and mated females of Heliothis virescens (Noctuidae)

Juvenile hormones (JH) I, II, and III were monitored in hemolymph of virgin and mated females of various ages in Heliothis virescens. JH I was the predominant homologue followed by JH II, but JH II was present at a higher level in young virgin females. JH III was detectable only at a low level. In virgin females, hemolymph JH titers were low at emergence (2.2ng/ml-total amount of JH I and JH II), but increased thereafter and reached a maximum at 24h of age (53.5ng/ml). At 30h and 36h of age, JH titers dropped to a low level, but increased again in older virgin females. After mating, JH titers increased significantly. JH titers at 0h after uncoupling (137.4ng/ml) were nearly 3 times as high as those in 24-h-old virgin females. Within 6h after uncoupling, JH titers decreased slightly, but titers increased with age of mated females and reached a level of 320.2ng/ml hemolymph at 72h after uncoupling. The titer of JH I and JH II was correlated highly with total number of eggs produced (r(2)=0.70, P<0.001). Mating stimulated JH production, resulting in an increase in egg production.     

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.     

Mechanisms of age-specific regulation of dopamine metabolism by juvenile hormone and 20-hydroxyecdysone in <Emphasis Type="Italic">Drosophila</Emphasis> females

20-hydroxyecdysone (20E) and the juvenile hormone (JH) have an age-specific effect on total dopamine (DA) content in Drosophila (Gruntenko and Rauschenbach 2008). Earlier we studied the mechanism of influence of 20E and JH on DA metabolism in young females (Rauschenbach et al. in J Insect Physiol 53:587–591, 2007a: Arch Insect Biochem Physiol 65:95–102, 2008a; Gruntenko et al. in Arch Insect Biochem Physiol 72:263–269, 2009). Here we investigate the effects of 20E and JH on the activities of the alkaline phosphatase (ALP), tyrosine hydroxylase (TH) and DA-dependent arylalkylamine N-acetyltransferase (AANAT) in mature females of wild type D. virilis under normal conditions and under heat stress (38°C). 20E feeding of the flies led to a substantial decrease in ALP and TH activities and to an increase in AANAT activity in mature females. JH application resulted in an increasing of ALP and TH activities, but did not influence AANAT activity in mature females. A rise in JH and 20E levels was found to change ALP and TH stress reactivities. Mechanisms of age-specific regulation of DA level by 20E and JH in Drosophila females are discussed.