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.     

Release of egg development neurosecretory hormone in Aedes aegypti and Aedes taeniorhynchus induced by an ovarian factor

Ovariectomized Aedes aegypti do not synthesize vitellogenin after a blood meal, unless an ovary from a blood-fed donor is implanted. Decapitation, however, prior to implantation inhibits vitellogenin synthesis. A female ovariectomized and decapitated 6 hr after a blood meal, synthesizes vitellogenin if an ovary from a blood-fed donor is implanted. On the other hand, females that are fed on blood and immediately decapitated can not be stimulated to synthesize vitellogenin with implanted ovaries removed from blood-fed donors. These experiments led to the hypothesis that the blood meal stimulates the ovary to secrete a corpus cardiacum stimulating factor, that in turn promotes release of egg development neurosecretory hormone stored in the corpus cardiacum.Injection of 20-hydroxy-ecdysone or ovarian extract prepared from ovaries removed from unfed females does not release egg development neurosecretory hormone. Thus corpus cardiacum stimulating factor is not 20-hydroxy-ecdysone, and ovaries removed from unfed females do not store it.The rate of inactivation of egg development neurosecretory hormone released from the corpus cardiacum after a blood meal was investigated by implanting an ovary into females that were blood fed for various intervals than decapitated and ovariectomized. Seventy per cent of implants grow when the operation is done 18 hr after feeding, and 30% when the operation is done between 18 and 24 hr after feeding, indicating that egg development neurosecretory hormone is stable for the first 18 hr after a blood meal.Aedes taeniorhynchus females ovariectomized 24 hr after adult emergence do not synthesize vitellogenin. When such a female is implanted with an ovary removed from a sugar-fed or blood-fed Aedes aegypti donor vitellogenin synthesis is initiated, and the implant grows. Decapitation prior to implantation inhibit vitellogenin synthesis and implants do not grow. These results indicate that corpus cardiacum stimulating factor is not species specific.     

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).     

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.     

Protein synthesis in the fat body of Leucophaea maderae during vitellogenesis

During the early stages of vitellogenesis in Leucophaea, vitellogenin accounted for most if not all of the secreted protein synthesized by the fat body. Synthesis began about 5 days after mating and continued until 24 hr or so before the formation of the oötheca. Ligation resulted in the degeneration of the oöcytes, the first evidence of which was seen within 24 hr. Ligation also curtailed the synthesis of vitellogenin at about the same time. Isolated abdomens treated with an analog of juvenile hormone commenced vitellogenesis within 12 to 24 hr and measureable oöcyte growth occurred after 5 days. Despite continued synthesis of vitellogenin, the oöcytes in isolated abdomens always degenerated.     

Corpus allatum control of ovarian development in Aedes aegypti

Ovarian follicles of Aedes aegypti normally pass through a potential stage of arrest 1 day after adult ecdysis and after blood feeding. Removal of the corpora allata or decapitation within an hour of these events interrupts normal development, and development is restored by reimplantation of active allata or by administration of synthetic analogues of juvenile hormone. Only follicles in the normal stage of arrest deposit yolk when stimulated by exogenous ecdysone or after the female takes a meal of blood. Follicles that ceased development as a result of allatectomy or decapitation respond to these oögenic stimuli, but degenerate without depositing yolk.     

Uptake of vitellogenin into developing oöcytes of Locusta migratoria

The selective incorporation of vitellogenin into developing locust oöcytes was studied using ¹²⁵I-vitellin. Vitellogenin incorporation does not start before the oöcytes are 1.5 mm in length. It increases rapidly up to a maximum at 4.7 mm oöcyte length and decreases steadily until the eggs are fully developed (6.5 mm). Concentrations of serum proteins and vitellogenin in the haemolymph show parallel changes, vitellogenin titre reaching a maximum of 7.5 mg/ml. Incorporation rates for vitellogenin increase from 1.5 μg/hr/oöcyte (2.2 mm) up to 13.8 μg/hr/oöcyte (4.7 mm). In this range incorporation per unit surface area increases 4-fold. While the vitelline and chorionic membranes are being formed, the incorporation rates as well as the protein concentrations in the haemolymph decrease steadily until the second gonotrophic cycle starts. The hormonal basis for oögenesis and the mechanism for selective uptake of locust vitellogenin are discussed.     

Vitellin and vitellogenin incorporation by isolated oöcytes of Locusta migratoria migratorioides (R.F.)

Vitellin and vitellogenin labelled in vitro with ¹²⁵I and in vivo with ³H were incorporated into yolk by locust oöcytes incubated in an in vitro system. This incorporation was specific and linear with the duration of incubation. Uptake of vitellin by oöcytes was 3–4 times higher than ¹²⁵I-bovine serum albumin in 2.1-mm oöcytes and 20 times higher than ¹²⁵I-bovine serum albumin in 4.0-mm long oöcytes. The uptake of the albumin was enhanced by the presence of vitellin in the incubation medium. ³H-labelled yolk protein was incorporated at higher rates than that labelled with ¹²⁵I. The addition of the juvenile hormone analogue ZR 515, caused the incorporation rates of vitellogenin to be increased. The amount of vitellin or vitellogenin taken up by the oöcytes increased with their length, and the rate of incorporation per unit surface area was highest in 3–4-mm long oöcytes. These results corroborate previously reported in vivo patterns of incorporation rates of developing oöcytes.     

Vitellogenin fluctuations in haemolymph and fat body and dynamics of uptake into oöcytes during the reproductive cycle of Diploptera punctata

Haemolymph and fat body soluble protein titres have been examined during the reproductive cycle of Diploptera punctata, with particular emphasis on the occurrence of vitellogenin and its uptake into the developing oöcytes. Vitellogenin was first detected in the haemolymph of mated females 2 days after adult eclosion at about the same time that vitellin deposition in basal oöcytes began. Peak haemolymph titres of vitellogenin occurred on day 6, correlated with the completion of yolk uptake. Thereafter vitellogenin levels declined and were generally undetectable throughout most of gestation, rising again shortly before parturition in association with the second gonotrophic cycle. Total haemolymph protein levels were not correlated with vitellogenesis.Soluble fat body vitellogenin titres of mated females remained low during the first oöcyte growth period but then rose several-fold at its completion and remained high throughout pregnancy and the second gonotrophic cycle. Total fat body soluble proteins decline after adult eclosion in association with oöcyte growth.Vitellin accumulation in basal oöcytes was related linearly to increase in volume until the onset of chorion formation. Thus no post-vitellogenic growth period was detected.     

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.     

Cell replication in the follicular epithelium of the adult mosquito

Females of the mosquitoes Culex pipiens molestus, Aedes togoi, Mansonia uniformis, and Aedes aegypti show active mitosis and an increase in cell number in the follicular epithelium of the ovary immediately after emergence from the pupa. Development up to stage III in the autogenous species, when mitosis in the follicular epithelium ceases, depends upon the continuity of cell proliferation over the first 48 hr after emergence (C. p. molestus, A. togoi) and on the number of cells originally in the follicle at emergence (A. togoi). The follicular epithelium in the anautogenous species ceases to divide in Mansonia, or the rate of division is considerably reduced in A. aegypti, 24–48 hr after emergence. Mitosis in the follicular epithelium of A. aegypti is renewed after the blood meal. A number of cells are released from phase G2 of the mitotic cycle into mitosis and other cells are initiated into DNA synthesis within 4 hr of the mosquito feeding on blood.     

Vitellin and vitellogenin concentrations during oögenesis in the first gonotrophic cycle of the house fly,Musca domestica

The house fly, Musca domestica, contains at least two native vitellin and two vitellogenin proteins. Both vitellins appear to have an identical vitellogenin partner. The major native vitellin has a mol. wt of 281 K Daltons, and the major native vitellogenin has a mol. wt of 283 K Daltons. These proteins are composed of three subunits with mol. wt of 48, 45 and 40 K Daltons. The relationship of the subunits to the native proteins is not known.Haemolymph vitellogenin levels are cyclical during oögenesis with no detectable amounts in previtellogenic flies and low levels in postvitellogenic flies. The highest level of vitellogenin, 10.5 μg/μl, occurred in flies with stage-7 ovaries. The vitellogenin levels during oögenesis fit a parabolic curve and the fat body vitellogenin content during oögenesis showed this same pattern.Uptake of vitellogenin into the ovary during each stage of oögenesis also fit a parabolic curve and produced a high linear correlation with haemolymph vitellogenin levels. The greatest uptake was 37 μg/stage and occurred during stage 6.     

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.     

Changes in follicle cell morphology,ovarian protein synthesis and ovarian DNA synthesis during oöcyte maturation in Leucophaea maderae: Role of juvenile hormone

Changes in follicle cell morphology were correlated with changes in rates of protein synthesis and DNA synthesis by the ovary during ovarian maturation in Leucophaea maderae. During the vitellogenic period of oöcyte development, which lasts approx, 15 days, morphological changes in the follicle cells are accompanied by moderate rates of ovarian protein synthesis and rapid rates of ovarian DNA synthesis. At approx. 15 days after mating, the shape of the follicle cells changes from cuboidal to squamous, ovarian DNA synthesis is arrested, and ovarian protein synthesis increases slightly. During the final period of oöcyte development, which lasts approx, two days, the interfollicular channels between the follicle cells have disappeared and the squamous follicle cells, which contain an extensive rough endoplasmic reticulum, deposit a chorion around the mature oöcyte. These morphological changes are accompanied by a radical increase in ovarian protein synthesis, while ovarian DNA synthesis remains arrested. Immediately before ovulation, ovarian protein synthesis starts to decline, reaching a minimal level 24 hr post-ovulation.Ovarian maturation is dependent on the presence of juvenile hormone (JH) only during the vitellogenic stage of oöcyte development. Decapitation of insects at any point during the first 10 days after mating arrests the synthesis of DNA and retards the synthesis of protein by the ovary, resulting in degeneration of the oöcyte. Subsequent injection of JH restores both events to normal levels within 72 hr. Decapitation on or after the tenth day following mating does not alter normal oöcyte development, chorion deposition, ovulation or egg case formation.Primary induction of protein synthesis in ovaries from virgin females can be achieved by either an in vivo or in vitro exposure of the tissue to JH, thus confirming a site of action for JH to be ovarian tissue. Electrophoretic analysis of the soluble proteins from JH-exposed ovaries in vivo reveals that JH stimulates general protein synthesis, rather than the synthesis of a specific major protein such as vitellogenin.     

A comparison of the morphogenetic and sterilizing activities of juvenile hormone mimics on Aedes aegypti

At 25°C, adult female aedes aegypti are most sensitive to sterilization by juvenile hormone (JH) mimics when such chemicals are applied 32 to 36 hr after the blood meal (when the ovaries are at late stage III to early stage IV). Application of JH mimics during this period reduces egg fertility and female fecundity and induces the production of large numbers of visually abnormal eggs. As the most sensitive phase for sterilization with JH mimics is well before oviposition, and as many abnormal eggs are laid following JH mimic treatment, it is likely that in this species sterilization effects are induced by some action on the developing oöcyte rather than on embryonic development.The relative activities of several JH mimics in sterilizing adult female A. aegypti are very similar to their relative activities in inhibiting metamorphosis. Thus the sterilizing action of JH mimics is likely to be a true JH effect and can be used as a test for JH activity for A. aegypti.     

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.     

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.     

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.     

Phase polymorphism in Schistocerca gregaria: Assessment of juvenile hormone synthesis in relation to vitellogenesis

Juvenile hormone (JH) synthesis by the corpora allata of gregarious and solitarious phase females of Schistocerca gregaria was determined in vitro during the penultimate and last stadia as well as during the first gonotrophic period of adults. Generally, the corpora allata of solitarious females showed higher rates of JH synthetic activity. In addition, in adult females there was a temporal difference between the corpora allata activities of gregarious and solitarious locusts, the latter exhibiting relatively higher rates of JH synthesis early in the first gonotrophic period. The corpus allatum volumes of solitarious females were also generally larger than those of their gregarious counterparts; there was no synchrony between fluctuations in JH synthetic activity and changes in corpus allatum volume in either phase.The early onset of relatively high JH synthetic rates in solitarious females was correlated with the early detection, by rocket immunoelectrophoresis, of vitellogenin in the haemolymph and vitellin in the oöcytes. Vitellogenin appeared in the haemolymph on day 4 in solitarious females and on day 6 in gregarious females and vitellin appeared in the oöcytes on days 6 and 8 respectively. Oöcyte length at which vitellogenesis was first detected was 1.8 mm for gregarious and 1.3 mm for solitarious females. However, despite the accelerated onset of both vitellogenin synthesis and uptake, oöcyte maturation time of solitarious females was longer. In both gregarious and solitarious females, vitellogenin titres increased until oöcytes reached a length of about 4 mm and declined thereafter. Vitellin content of ovaries increased proportionately to oöcyte growth until they attained a length of 5.0 mm. The subsequent increase in length of oöcytes to maturity is attributed to postvitellogenic growth, possibly by hydration.