Juvenile hormone and 20-hydroxyecdysone as primary and secondary stimuli of vitellogenesis in Aedes aegypti.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 2:75-90 (1985) juvenile Hormone and 20-Hydroxyecdysone as Primary and Secondary Stimuli of Vitellogenesis in Aedes aegypti Dov Borovsky, Billy R. Thomas, David A. Carlson, Lavern R. Whisenton, and Morton S . Fuchs lnstitute of Food and Agricultural Sciences, University of Florida, Florida Medical Entomology Laboratoy, Vero Beach (DB); Department of Microbiology, Galvin Life Science Center, Notre Dame University, Notre Dame, Indiana (B.R.T.); USDA. Insects Affecting Man and Animals Research Laboratory, Gainesville, Florida (D.A.C.); Department of Biology, Notre Dame University, Notre Dame, Indiana (L.R. W., M.S.F.) 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:Ianalogue methoprene (25 pg) did not induce vitellogenesis in these abdomens. When blood-gorged ligated abdomens were treated with both hormones, however, vitellogenesis was stimulated i n 60% of treated animals. Rocket immunoelectrophoresis indicated that vitellin concentration per follicle in treated animals was similar t o 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 t o that in blood-fed controls. blethoprene at this concentration (25 pg), did not cause an increase i n 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 20hydroxyecdysone in stimulating vitellogenesis following blood feeding in Ae. aegypti. Mention of a commercial or proprietary product in this report does not constitute an endorsement of this product by the U.S. Department of Agriculture. Acknowledgments: This work was partially supported by NIH Research Grant A l 77347 and Career Development Awards Al 00429 to D.B. and Al ‘10707 to M.S.F. L.R.W. was supported by NIH Training Grant A l 07030, and B.R.T. by a Universily of Florida postdoctoral award. Received March 19,1984; accepted July 2,1984. Address reprint requests to Dr. Dov Borovsky, Florida Medical Entomology Laboratory, 200 9th Street SE, Vero Beach, FL 32962. 0 1985 Alan R. Liss, Inc. 76 Borovsky et al Using high-pressure liquid chromatography, gas chromatography, and gas chromatography/mass spectrometry with chemical ionization, we isolated and identified JH Ill i n a group of 2- t o 10-h and 8-h blood-fed animals. Our data provide evidence that JH Ill (6.0 pg per female), which has been recently identified in larval and adult Ae. aegypti, is also present 8 h after a blood meal. Key words: Aedes aegypti, JH, 20-hydroxyecdysone, mass spectrometry, HPLC, gas chromatography, vitellogenesis, fat body, rocket immunoelectrophoresis, tissue culture INTRODUCTION Egg development in anautogenous mosquitoes is regulated by a complex series of hormonal interactions. Although a general consensus has not yet been reached (see Fuchs and Kang [l], for a recent review) the following sequence of events, as pertains to Aedes aegypti, has much experimental evidence supporting it. Juvenile hormone from the corpus allatum is released shortly after adult emergence. Its function here is to permit the further development and differentiation of the previtellogenic primary follicles and allow these follicles to attain their so-called “resting stage” [2,3,5,27]. Vitellogenesis is initiated by blood feeding. The first step in this process is the secretion of an ovarian factor, the corpus cardiacum stimulating factor [6,g, which causes the CC* to release another factor, the egg development neurosecretory hormone [B]. EDNH stimulates the previtellogenic ovary to secrete ecdysone [9,10,31].Ecdysone is then presumably converted to 20hydroxyecdysone which induces the fat body to synthesize vitellogenin [lo]. The newly synthesized yolk proteins are then subsequently sequestered by the oocytes . A major point of contention and disagreement in the above scheme is the proposed role of 20-hydroxyecdysone. Three major inconsistencies have emerged: 1) vitellogenin synthesis in vivo in non-blood-fed females is induced only by injecting amounts of 20-hydroxyecdysone far in excess of known physiological levels; 2) in vitro incubations of fat bodies from unfed females with physiological amounts of 20-hydroxyecdysone failed to induce significant vitellogenin synthesis (Borovsky and Hagedorn, unpublished observations, cited in Borovsky [ll]); 3) continuous infusion of sugar-fed females with physiological concentrations of 20-hydroxyecdysone or implantation of ovaries that actively secrete ecdysone failed to stimulate vitellogenin synthesis in unfed or blood-fed recipients [12-141. The experiments cited above tend to negate the direct involvement of 20hydroxyecdysone with reference to fat-body vitellogenin synthesis in anau*Abbreviations: absorbance units full scale = a.u.t.s; CA; corpus cardiacum = CC; chemical ionization = CI; dimethylchlorosilane = DMCS; data system = DS; egg development neurosecretory hormone = EDNH; gas chromatograph = GC; high-performance liquid chromatography = HPLC; juvenile hormone = JH; mass spectrometry = MS; radioimmunoassay = RIA; trichloroacetic acid = TCA; tris (hydroxymethyl) aminoethane = TRIS. JHA Primary Stimulus of Vitellogenesis 77 togenous mosquitoes. Further complicating the picture was the observation that injection of physiological amounts of 20-hydroxyecdysone into isolated abdomens of autogenous Ae. afropulpus will restore ovarian development provided they have first been exposed to low levels (0.5 ng) of JH [15-17]. Interestingly, topical application of 500 ng of JH I onto Ae. afropalpus abdomens without subsequent injection of 20-hydroxyecdysone will also induce ovarian development in these animals. In this case it was found that excess JH I (500 ng) increased the endogenous ecdysteroid titer; thus the animals had in effect ”seen” both hormones and ovarian development was able to ensue [lq. Borovsky [ll] independently observed that injection of low levels of 20hydroxyecdysone, into either decapitated females or isolated abdomens, failed to restore significant ovarian development. However, topical application of either JH I or methoprene in amounts which should (based on previous results with Ae. afuopalpus) induce the ovaries to release ecdysone was successful in inducing ovarian development. Therefore the results obtained using excess JH on abdomens or decapitated autogenous Ae. aegypfi appear consistent with one major exception. It is clear in Ae. afropulpus that vitellogenesis will not occur in isolated abdomens unless both JH I and 20hydroxyecdysone are exogenously applied. Aedes aegypfi females whose ovaries have reached the ”resting stage” have already been exposed to an increase in JH titer prior to blood feeding. Therefore, it would seem logical to assume that once blood-fed (in order to provide protein which can be resynthesized into vitellogenin), all that should be required for vitellogenesis to proceed in Ae. aegypfi would be ecdysone . Moreover, this would also seem to explain why females decapitated shortly after blood feeding do not undergo vitellogenesis, viz, because they are deprived of EDNH (stored in the CC and removed by decapitation)which is responsible for ecdysone release from the ovaries. However, injection of physiological amounts of 20-hydroxyecdysone into blood-fed decapitated Ae. aegypfi does not induce ovarian development whereas excess JH alone (which also presumably increased the ecdysone titer as well) does [ll]. This observation suggests that even after blood feeding both JH and ecdysone are required for successful oocyte maturation in this species. An extrapolation of all of these considerations leads to the prediction that JH is synthesized after blood feeding. The results reported herein support this hypothesis. MATERIALS AND METHODS Experimental Animals Larvae of Ae. aegypti were reared at 26°C in the laboratory on a diet of brewer’s yeast, lactalbumin, and lab chow (l:l:l), with 16:8 h 1ight:dark cycle. Adults were fed on 10% sucrose and on chicken blood. Females were used 3-5 days after emergence. surgery Mosquitoes were ligated with a fine thread between the thorax and abdomen, thereby isolating the ovaries from the medial neurosecretory cells, the 78 Borovsky et al corpus cardiacum, and corpora data. Ovaries in saline were implanted through a dorsal slit made between the fourth and fifth abdominal segment via a finely drawn capillary tube. The slit was then sealed with paraffin wax. Postoperated mosquitoes were held at 26°C in cages lined with moist paper. Injection of 20-hydroxyecdysone (0.25 p1) in saline (Rhoto Pharmaceutical Co, Japan) or [3H]valine(0.5 pl) 200 pcilpmole (New England Nuclear, USA) were administered dorsally between the fourth and fifth abdominal segments via a finely drawn capillary tube. Methoprene (ZR 515 isopropyl ll-methoxy3,7,1l-trimethyl-trans-2,trans-4-docecadienoate) (Zoecon Co, Palo Alto, CA) was dissolved in acetone and 0.25 pI was applied topically to the abdomens with a finely drawn capillary tube. Ligated abdomens were held at 26°C in a humidified chamber and sprayed daily with a fine mist of water. Oocyte growth was measured using an ocular micrometer. We compared length of the yolk in several oocytes of each ovary and the data are given as the mean f standard error of the mean. Yolk length at resting stage was approximately 50 pm. Assay for Vitellogenin Synthesis Vitello enin synthesis was measured 2 h after injection (0.5 pl) of 0.1 pCi (0.1 nM)53 [ Hlvaline. Groups of five mosquitoes were homogenized in a glass homogenizer, centrifuged at 4°C at 4,OOOg and the supernatants assayed for labeled vitellogenin and total protein synthesis as described by Borovsky and Van Handel [12,18]. Double diffusion analysis was done on microscope slides coated with 1%agar . Rocket immunoelectrophoresis was performed on microscope slides layered with 1%agarose containing 4% vitellogenin antiserum in 50 mM TRIS-citrate pH 8.5, substituted for TRIS-glycine buffer . Ecdysteroid Extraction and Radioimmunoassay Ecdysteroid was extracted from adult mosquitoes (30-40) in methanol/ water, followed with chloroform [17,20]. Final supernatants were dried under air at 50°C. The dried products were kept frozen until assayed. Ecdysteroids were analyzed by a radioimmunoassay [21,22] using [3H]ecdysone (specific activity; 68 Cilmmol; New England Nuclear, Boston) as the radioisotope. Calibration curves were constructed with 20-hydroxyecdysone as the unlabeled ligand. The antibody had equal affinity to ecdysone and 20-hydroxyecdysone and the results are expressed as 20-hydroxyecdysone equivalents. Data of ecdysteroid titer is expressed as the mean standard error of the mean. * Extraction and Purification of JH Aedes aegypti females (2,000-3,000) were extracted with acentonitrile (50 ml) in a glass homogenizer at 4°Cin the presence of [3H]JH-III(300,000 cpm, 11CilmM) which served as an internal standard for isotope dilution calculations of recoveries. The extract was centrifuged at 30,OOOg for 30 min at 4"C, the supernatant removed and transferred to a separatory funnel containing 50 ml of 4% aqueous sodium chloride and extracted three times in pentane (30 ml). The pentane extract (90 ml) was reduced to 20 ml in a rotary JHA Primary Stimulus of Vitellogenesis 79 evaporator under reduced pressure and was layered on a column (10 x 2 cm) packed with 6.0 cm aluminum oxide (activity 2-3, Merck). The bottom and top 2.0 cm of the column were packed with anhydrous sodium sulfate. The column was then eluted with benzene (100 ml) (JH acid and diol forms did not elute in benzene but in more polar solvents). The benzene fraction was evaporated to dryness in a rotary evaporator under reduced pressure. The dried extract was redissolved in hexane (2.0 ml) and chromatographed with 1%tetrahydrofuranlhexane on an Acisorbosphere silica column 5 pm particle size (100 x 4.6 mm) at a flow rate of 1ml/min using high-performance liquid chromatography (Laboratory Data Control, Rivera Beach, FL). The HPLC system included a dual minipump, a variable wavelength UV detector, and a computing integrator. JH peaks were detected by UV at 214 nm and 0.1 a.u.f.s. Under these conditions elution times for JH I, 11, and I11 standards were 17, 19, and 22 min. Areas or eluate corresponding to the same elution time of JH standards were collected, evaporated, redissolved in acetonitrile (200 pl), and rechromatographed on a Cl8 reverse-phase column (100 x 4.6 mm; Adsorbosphere, 5 pm particles). The column was eluted with acetonitrilelwater (1:l) at 1.5 mllmin, and monitored at 214 nm and 0.1 a.u.f.s. Under these conditions JH 111, 11, and I eluted at 15, 24, and 38 min. Areas corresponding to elution times of standards were collected, 4% sodium chloride added, and eluate was partitioned against pentane five times. The pentane layer was removed and evaporated under N2. Recoveries of internal standard at the end of the purification procedure were between 30% and 50%. These values were then used to calculate the JHs found in our samples using isotope dilution calculations . Gas Chromatography and Mass-Spectroscopy Analysis Aliquots from HPLC-purified eluates were injected into a Varian model 2100 gas chromatograph equipped with a flame ionization detector, an integrator, and a 1.8 x 2 mm ID glass column packed with 3% OV-1 on 120-140 mesh Chromosorb W DMCS. Samples were analyzed with the column oven temperature programmed from 150-300°C at 6°C per minute. Other parameters were: injector port, 315°C; detector, 320°C.The carrier gas was helium, 20 mllmin. Under these conditions JH 111, 11, and I were eluted at 5.8, 7.0, and 8.0 min. Samples that ran with JH standards on GC were further analyzed on a Finnigan model 1015 SIL GC mass spectrometer data system in the chemical ionization mode using isobutane ionization gas (400 pm pressure). Samples were introduced (splitless) via a fused silica into the MS via the column (15 m x 0.3 mm ID, DB-1, helium carrier gas) that was temperature-programmed from 80°C to 220°C at 20°C per min after a 3-min delay. The data system was used in the normal scanning mode. RESULTS Synergistic Effects of Physiological Doses of Methoprene and 20-Hydroxyecdysone on Vitellogenesis Since pharmacological doses of 20-hytlroxyecdysone (1.25-5.0 pg) are needed to elicit egg development in Ae. aegypti that have not imbibed blood, 80 Borovsky et al we explored the possibility that females may be exposed to JH soon after a blood meal and that this stimulus potentiates vitellogenesis following treatment with physiological amounts of 20-hydroxyecdysone.Ae. aegypti females were fed on chicken blood and ligated immediately thereafter. Females were then divided into two groups, one treated with methoprene (25 pg) and the other with acetone. Eighteen hours after treatment, 20-hydroxyecdysonewas titrated against each group of mosquitoes, and 66 h after blood feeding ovaries were examined for egg development. When treated with methoprene (25 pg) 20-hydroxyecdysone (5.0 ng) induced 60% of the ovaries to deposit yolk and mature eggs. In acetone-treated abdomens this treatment induced only 5% of the ovaries to mature eggs, and 20 ng 20-hydroxyecdysone was needed for 18% response (Fig. 1).The yolk length 66 h after blood feeding and subsequent treatment with methoprene and 20-hydroxyecdysone was between 300 & 30 pm and 486 9 pm in those ovaries that deposited yolk. Ovaries with mature eggs were then extracted in buffered saline (0.15 M sodium chloride, 50 mM sodium phosphate buffer, pH 7.0). The extract tested for cross reactivity with vitellin antibodies using double-diffusion analysis and rocket immunoelectrophoresis. Both techniques showed that the antibodies recognized the yolk extract by forming a single precipitin line or a rocket. When the same number of eggs (1,OOO; 21 ovaries) were extracted from 20-hydroxyecdysone- and methoprene-treated animals and compared with similar numbers of ovaries and eggs extracted from blood-fed controls on rocket immunoelectrophoresis, peak heights and vitellogenin content in the eggs were similar whereas in controls no appreciable rockets were found (Fig. 2). We concluded that methoprene pretreatment potentiates the action of 20-hydroxyecdysone. 20-HYDROXYECDYSONE (ng) Fig. 1. Ovarian maturation in ligated abdomens of Aedes aegypti after treatment with methoprene and 20-hydroxyecdysone. Females were fed on blood and their abdomens ligated immediately. Abdomens were then treated with methoprene (open circles) or acetone (solid circles). Eighteen hours later various concentrations of 20-hydroxyecdysone were injected into the abdomens. The percentage of females with yolk in their oocytes was determined by dissection of the ovaries 66 h after the blood meal. Forty animals were used for each point. Ten percent of the methoprene-smeared abdomens and 5% of acetone-treated abdomens developed eggs. These values were subtracted from each point. JHA Primary Stimulus of Vitellogenesis 81 30 I - - 20 E E I- I a W I Y U 10 - W a L&LA i1 B C Fig. 2. Rocket immunoelectrophoresis of eggs removed from ligated abdomens treated with methoprene, from ligated abdomens treated with methoprene and 20-hydroxyecdysone, and from nonligated, blood-fed controls. Three groups of females (21 per group) were fed on blood. The first group was immediately ligated and smeared with methoprene (25 pg), the second group was treated as the first and 18 h later injected with 5.0 ng 20-hydroxyecdysone. Sixty hours after the blood meal eggs (1,000) were removed from 21 pairs of ovaries from each group, extracted in 50 m M TRIS-citrate pH 8.5 (0.5 ml), and assayed for vitellogenin content (620 pg in methoprene and 20-hydroxyecdysone treated and 650 pg in blood-fed control) . Similar dilutions (1:24) of groups A, B, and C were then run for 4 h at 100 V at 4OC. A) Methoprene-treated abdomens; 6) rnethoprene- and 20-hydroxyecdysone-treated abdomens (26 pg vitellin); C) Blood-fed controls (25 pg vitellin). Each bar represents the average of three determinations & SEM. Comparison Between Ecdysteroid Titer in Methoprene-Treated and Nontreated Females Excess JH I (500 ng) significantly increases the endogenous titer of ecdysteroids in decapitated Ae. atropalpus [lq.To determine whether our pretreatment with methoprene caused egg development owing to an artifactual increase in ecdysteroid titer, females were fed chicken blood and divided into four groups. Twenty-four hours later the first group was assayed for ecdysteroid content by radioimmunoassay and for yolk deposition (Table 1, a). Females in the second and third groups were ligated immediately and smeared with methoprene (25 pg and 1.65 ng respectively). Twenty-four hours later, abdomens were assayed for ecdysteroid content and for yolk deposition (Table 1, b and c). Abdomens in the third group that were smeared with 1.65 ng methoprene underwent vitellogenesis and the ecdysteroid titer 414 rh 63 pg per female was similar to that of blood-fed controls 528 -t 17 pg per female. A fourth group consisted of females that were fed chicken blood and immediately asayed for ecdysteroid (Table 1, d) to show that there was not a sudden increase in ecdysteroid titer immediately after the blood meal. The titer per sugar-fed female was 82 k 26 pg (Table 1, e). These results indicate that mosquitoes treated with 25 pg methoprene have less than half as much ecdysteroid as do mosquitoes 1 day after blood feeding. Egg development was not observed, whereas 1.65 ng caused egg 82 Borovsky et al TABLE 1. Ecdysteroid Equivalents in Intact Females and Methoprene-Smeared Abdomens Aedes aegypti females were fed on blood and then: a) Assayed 24 h later b) Immediately ligated, smeared with methoprene (25 pg), and assayed 24 h later c) Immediately ligated, meared with methoprene (1.65 ng), and assayed 24 h later d) Immediately assayed e) Unfed control Number of females Ecdysteroida per Q pg f SEM Females with yolk length > 100 prn Yolk lengthb prn SEM 80 120 528 f 17 204 f 10 80 188 k 20 0 < 50 30 414 f 63 24 150 f 10 30 140 & 30 82 k 26 0 < 50 < 50 30 0 aExpressed in 20-hydroxyecdysone equivalents. bFernales with yolk > 100 pm developed 100 follicles. Vitellin extracted from these ovaries cross-reacted with vitellogenin antibody using double-diffusion analysis and rocket immunoelectrophoresis. development. The increase in ecdysteroid titer occurring immediately after the blood meal may be due to nonspecific binding in the RIA. Stimulation of Vitellogenesis with JH Analogue and EDNH Because EDNH stimulates the ovary to mature [S], we tested the possibility that head extract with EDNH activity synergizes with JH-stimulated vitellogenesis. Three groups of females were fed on chicken blood and ligated immediately. Abdomens in the fist group were treated with methoprene (12.5 pg) and 24 h later injected with 0.5 p1 [3H]valine and analyzed. Vitellogenin synthesis was not initiated and no yolk was deposited in the ovaries (Table 2, a). Abdomens in the second group were treated with methoprene (12.5 pg) followed by head extract containing EDNH activity (1p1; equivalent to 2 heads). Twenty-four hours later abdomens were injected with 0.5 pl [3H]valineand analyzed. Vitellogenin synthesis was stimulated and yolk was deposited in the ovaries (Table 2, b). Abdomens in a third group were injected with head extract containing EDNH activity (1 pl; equivalent to 2 heads) and 24 h later injected with 0.5 pl [3H]valine and assayed. Vitellogenin was not synthesized and yolk was not deposited (Table 2, c). These results indicate that both JH and EDNH may be required for oogenesis following blood feeding. Stimulation of Vitellogenesis With Methoprene and Developing Ovaries Borovsky [6,13] and Lea reported that developing ovaries implanted into blood-fed decapitated females failed to stimulate vitellogenesis in the recipients, and it may be that lack of JH was the source of this failure. Three groups of female Ae. aegypti were fed chicken blood and ligated immediately. Two groups were then treated with methoprene (20 pg) and 72 h later the first group was analyzed. Yolk deposition in the ovaries was found in only 20 20 410 f 9 < 50 4.40 f 10 15 0 30 23 22 30 20 13,000 f 800 20 < 50 0 20 16,000 f 400 300 f 70 500 f 50 Number of females Yolk lengtha pm k SEM Number of females VitelIogeninb synthesis cpm k SEM Females with yolk length > 100 pm 800 17,500 k 20,000 5 800 14,500 k 1,000 800 15,500 & Total protein synthesis (TCA) cpm f SEM “Yolk length was measured 72 h after blood feeding. Females in groups b and d matured 100 oocytes which formed a single precipitin line or a rocket using double-diffusion analysis and rocket immunoelectrophoresis. bTwenty-four hours after blood feeding females (5 per group) were injected with 0.5 p1 [3H]-valineand analyzed for vitellogenin 2 h later. ‘Head extract with EDNH activity was prepared as previously described by Fuchs et a1  and 1 pl (equivalent to 2 heads) injected per abdomen. Smeared with methoprene (12.5 pg) and analyzed Smeared with methoprene (12.5 pg), injected with EDNH‘ and analyzed Injected with EDNH and analyzed Control: Intact females fed on blood and 24 h later analyzed Aedes aegypti females were fed on blood, immediately ligated and then: TABLE 2. Stimulation of Vitellogenesis in Ligated Abdomens with JH Analogue and EDNH 3 23 0 41 34 Ovaries with yolk > 116 pm 30 Number of females Host + 12 < 50 432 415 f 10 Yolk lengthb pm k SEM 0 2 None Donora Ovaries with yolk length > 116pm < 50 418 k 10 None Yolk sizeb pm f SEM 'Since average length of oocytes in donor's ovary was 116 pm, females with yolk length < 116 pm were not counted. bFemaleswith yolk length > 116 p m matured 100 oocytes in a pair of ovaries. Vitellogenin synthesis was normal, and extracted vitellin crossreacted with vitellogenin antibody using double-diffusion analysis. Smeared with methoprene (20 pg) and assayed 72 h later Smeared with methoprene (20 pg) 24 h later, implanted with an ovary removed from a donor fed on blood 15 h earlier, and assayed 48 h after implantation Implanted 24 h later with an ovary from donor fed on blood 15 h earlier and assayed 48 h after implantation Aedes aegypfi females were fed on blood, immediately ligated and: Vitellogenesis TABLE 3. Implantation of Developing Ovaries Into Ligated Abdomens of Aedes aegypti Smeared With Methoprene: Effect on JHA Primary Stimulus of Vitellogenesis 85 10% of the treated animals (yolk length 415 t 10 pm; Table 3, a). The second group was implanted with ovaries removed from a donor that was fed chicken blood 15 h earlier. Forty-eight hours later, host and donor ovaries were assayed. Oocyte growth was stimulated in 56% of the recipients (Table 3, b), and 5% of the implant ovaries also grew (Table 3, b). A third group not treated with methoprene was implanted, 24 h after abdominal ligation, with an ovary removed from a donor 16 h after a blood meal and assayed 48 h later. Ovaries did not grow in recipients, and implants started to degenerate after 24 h (Table 3, c). Thus, it seems that both JH and the ecdysteroid secreted by the implants are required to stimulate vitellogenesis. Identification of JH Titer After Blood Feeding Since our results suggest that JH may participate in stimulating vitellogenesis, we explored the possibility that JH titer appears after the blood meal. Females were fed chicken blood and five groups of 1,000 each were collected at 2-h intervals for up to 10h after blood feeding. All mosquitoes in each time cohort were combined, and JH was extracted and purified using HPLC as described above. After C18 reverse-phase chromatography eluates collected with elution times of JH I, 11, and I11 standards were further analyzed on GC. The JH I11 region were resolved on GC into a major peak which ran in the JH I11 region. A fraction of that sample (20%)was further analyzed by GC-MSDS-CI as described above and compared with a JH I11 standard (10.0 ng) which is characterized by three major fragments: rnlz 235 (MH' -CH30H, base peak); 249 (MH+-H20, 40%); 267 (MH+, 75%) (Fig. 3a). The analyzed peak gave similar mlz fragments which eluted at the same time as JH 111: ml z 235 (MH+-CH30H, base peak); 249 (MH+-H20, 50%); and 267 (MH+, goo/,) (Fig. 3b). The total amount of JH I11 was about 15 ng or 3 pg per female in the combined group of 5,000 females collected 2-10 hr after the blood meal. In a group of females collected 8 h after the blood meal the amount of JH I11 was about 6.3 pg per female. DISCUSSION The work presented herein clearly demonstrates that a physiologically relevant amount of 20-hydroxyecdysone administered to prepared isolated abdomens obtained from blood-fed Ae. aegypti females is capable of initiating and supporting vitellogenesis. By "prepared" we refer to a pretreatment with JH. This is critical and must be emphasized because administration of 20-hydroxyecdysone followed with methoprene was not effective (results not shown). Administration of physiological amounts of 20-hydroxyecdysone or ecdysone alone does not induce vitellogenesis [11,12,14]. When a low level of methoprene is previously applied to these isolated abdomens, ovarian development ensues but only after the 20-hydroxyecdysone level is increased. Since 20-hydroxyecdysone titer peaks between 16 and 20 h after the blood meal [lo], we chose to wait 18 h before injecting 20-hydroxyecdysone. Therefore, physiological amounts of 20-hydroxyecdysone and ecdysone (via EDNH and ovarian transplants) were able to induce vitellogenesis in anautogenous Ae. aegypti strongly supports the contention that this hormone Borovsky et al 86 0 N 0- N u1 0 ul . ul I 0 - CT- N 0 N- - N - 30 0 w- P0 " . 3 N- m 0 N sN m0 N 8w m 8 I . ul (u -- N N N 0 0- -_ - - --- Fig. 3. Mass spectra (GC/CI, isobutane) of JHlll (10 ng) (a), and 20% of the LC fraction with the J H Ill retention time (b). Total ions of base peak (235) are 60,000 (a) and 6,000 (recovery 33%) (b). functions biologically in this system. Objections to this idea based on previous experiences that only nonphysiological, high doses of 20-hydroxyecdysone or ecdysone resulted in ovarian development in this species are no longer valid. Our experiments indicated that 20-hydroxyecdysone at close to physiological concentrations (1.5-5.0 ng) can stimulate vitellogenesis in blood-fed ligated abdomens that were first treated with 25 pg of methoprene (Fig. 1). The appearance of yolk in the maturing oocytes under the microscope seemed normal and it cross-reacted with vitellin antiserum. Rocket immunoelectrophoresis indicated that peak heights of various concentrations of vitellin obtained from a similar number of egg extracts of blood-fed and methoprene and 20-hydroxyecdysone-treated females were the same, whereas controls did not have appreciable amounts of vitellin (Fig. 2). Our treatment with both methoprene and 20-hydroxyecdysone (1.5-5.0 ng) caused normal egg development in ligated abdomens, whereas treatment with only 20-hydrox- JHA Primary Stimulus of Vitellogenesis 87 yecdysone (1.5-5.0 ng) failed to stimulate egg development. On the other hand, nonphysiological concentration of 20-hydroxyecdysone (2 pg) caused artifactual stimulation of vitellogenesis in At.. aegypti , Table 1, c, shows that 1.65 ng of methoprene alone (ie, without injecting 20-hydroxyecdysone)is capable of initiating ovarian development in isolated abdomens of blood-fed females. However, it is significant to note that this amount of methoprene (1.65 ng) also significantly increased the endogenous ecdysteroid titer to more than 78% of the intact blood-fed control (compare Table 1, a and c). Thus again it appears that a JH-20-hydroxyecdysone combination was capable of inducing ovarian development. Conversely 25 pg of methoprene when applied alone did not increase yolk size and only slightly increased the endogenous ecdysteroid titer above the nontreated control (compare Table 1,b and d). Since EDNH stimulates the ovary to synthesize and secrete ecdysone , we tested whether head extracts with EDNH activity can replace 20-hydroxyecdysone. When blood-fed, ligated abdomens were smeared with methoprene (12.5 pg) and immediately injected with head extract containing EDNH activity, vitellogenin synthesis was induced at a rate similar to that of bloodfed controls (Table 2). Neither head extract with EDNH activity nor methoprene alone could stimulate yolk deposition or vitellogenin synthesis (Table 2). Since injection of head extracts with EDNH activity causes a normal level of 20-hydroxyecdysone in Ae. aegypti (Borovsky and Fuchs, unpublished observations), this again supports our conclusion that both 20-hydroxyecdysone and JH are required after blood feeding. Previous reports have shown that developing mosquito ovaries (which secrete ecdysone after EDNH stimulation -- see ) implanted into bloodfed decapitated females failed to stimulate vitellogenesis [6,13,14]. We repeated these experiments using ligated abdomens from blood-fed animals as hosts, with and without previous application of 20 pg of methoprene. Implantation of donor ovaries without methoprene treatment did not result in any yolk deposition (Table 3, c) in either the host or donor ovaries, confirming previous observations. However, when methoprene was topically applied before ovarian implantation, 56% of the hosts ovaries exhibited considerable yolk deposition (Table 3, b). In this same experiment only 5% of the donor ovaries had yolk, indicating that the host ovaries selectively sequestered the available yolk proteins. Nonimplanted controls (ie, abdomens treated with only 20 pg of methopreme) showed some ovarian development (10%-see Table 3, a) but considerably less than those provided with active ovaries. These results again substantiate the need for both JH and ecdysone for vitellogenesis in Ae. aegypfi. Using various physicochemical techniques (HPLC, GC, and GC-MS-DSCI), we isolated and identified JH I11 in blood-fed females. This is the first time that isolation and identification of a specific JH after the blood meal in Ae. aegypfi has been reported, although recently Basker et a1  identified JH I11 in sugar-fed Ae. aegypti. The total amount of JH 111 from GC-MS-CI estimation was about 15 ng in the combined!group of 5,000 females collected 2, 4, 6, 8, and 10 h after the blood meal, and 10 ng in a group of 1,500 females collected 8 h after blood feeding. Since our extraction was done on groups of 88 Borovsky et females that were fed on blood at different intervals and then combined into one group, we do not know the amount of JH circulated per female at peak synthesis. The use of GC-MS techniques in the identification of JH titers in insects is remarkably specific [25,26]. Using isobutane as reactant gas, we could detect and identify as little as 200 pg of each JH without resorting to more sensitive technique of specific ion detection [23,25]. Even though the appearance of JH in blood-fed females supports a vitellogenic role for this hormone, it by no means proves it. That both JH and ecdysone are required for ovarian development has been previously documented with autogenous Ae. atrupalpus [15-17,201. It is now clear that this can be extended to anautogenous Ae. aegypti with specific reference to the initiation and maintenance of vitellogenesis. This is especially highlighted if one assumes that the availability of yolk precursors in isolated abdomens of autogenous Ae. atrupalpus and anautogenous blood-fed Ae. aegypti is equivalent. Then in both of these cases ovarian development only proceeds after application of both JH and ecdysone or 20-hydroxyecdysone. This in turn suggests that the fundamental hormonal regulation of vitellogenesis in autogenous and anautogenous mosquitoes may be very similar if not identical. The only differences would appear to be in the signals which account for the release of the various hormones involved. Recently Guilvard et a1  have shown using RIA that both titers of ecdysteroids and juvenile hormones were present during egg development after the oocytes of the autogenous Ae. caspius and Ae. detritus have already reached the previtellogenic stage IIlb. Although both titers overlapped, the ecdysteroids titer peaked 4-8 h before the JH titer. The postulation of a vitellogenic role for JH in Ae. aegypti after blood feeding does not in any way cast doubt upon the previously suggested function for this hormone in previtellogenic follicular growth and development [5,27]. We do, though, now hypothesize that JH may be secreted more than once during one female gonadotrophic cycle; first, as mentioned above, prior to blood feeding, and then again after blood feeding, where the JH in conjunction with ecdysone induces vitellogenesis. Our evidence for the resynthesis of JH is based on the positive effect the JH-20-hydroxyecdysone combination has on vitellogenesis and the presence of JH I11 in females 2-10 h after blood-feeding (Fig. 3). A serious possible contradiction concerning the role or nonrole of JH in vitellogenesis still remains. Lea [2,3] and Gwadz and Spielman  reported that when Ae. aegypti females were allatectomized 3-5 days after adult ecolsion (a time period sufficient for their ovaries to attain their resting stage), subsequent blood feeding still resulted in mature eggs. This observation would argue that JH is not required for vitellogenesis, and is obviously in conflict with the interpretation of our and Guilvard et al’s 1291 results. The reason(s) for this difference might lie with the criteria used for assaying vitellogenesis by both Lea [2,3] and Gwadz and Spielman . Lea [2,3] measured the length of the longest follicle and the length of its yolk mass and on this basis scored the ”number females with mature eggs.” Gwadz and Speilman 151 simply recorded whether or not yolk was present. It is difficult, however, to know from these reports how much vitellogenin was JHA Primary Stimulus of Vitellogenesis 89 synthesized and how many oocytes were matured in each allatectomized female. If these original results were indeed false positives then the role of the corpora allata as the sole source of JH in Ae. aegypfi after the blood meal is firmly established. If, on the other hand, confirmation of positive vitellogenesis in allatectomized females is obtained, then the possibility that JH is synthesized or stored outside the allata and released by the blood meal should be explored. The phenomenon in which a hormone is synthesized in one organ and is stored in several others is not without a precedence. Gilbert et a1 have shown that the prothoracicotropic hormone in manduca which is synthesized in the neurosecretory cells is stored in both the corpora cardiaca and the corpora allata. LITERATURE CITED 1. Fuchs MS, Kang S: Ecdysone and mosquito vitellogenesis: A critical appraisal. Insect Biochem 11, 627 (1981). 2. Lea AO: Some relationships between environment, corpora allata, and egg maturation in aedine mosquitoes. J Insect Physiol 9, 793 (1963). 3. Lea AO: Egg maturation in mosquitoes not regulated by the corpora allata. J Insect Physiol 15, 537 (1969). 4. Anderson WA, Spielman A: Permeability of the ovarian follicle of Aedes aegypti mosquitoes. J Cell Biol 50, 201 (1971). 5. Gwadz RW, Spielman A: Corpus allatum control of ovarian development in Aedes aegypti. J Insect Physiol 19, 1441 (1973). 6. Borovsky D: Release of egg development neurosecretory hormone in Aedes aegypti and Aedes taeniorhynchus induced by an ovarian factor. J Insect Physiol28, 311 (1982). 7. Lea AO, Van Handel E: A neurosecretory hormone-releasing factor from ovaries of mosquitoes fed blood. J Insect Physiol 28, 503 (1982). 8. Lea AO: Regulation of egg maturation in the mosquito by the neurosecretory system. The role of the corpus cardiacum. Gen Comp Endocrinol Suppl3, 602 (1972). 9. Hanaoka K, Hagedorn HH: Brain hormone control of ecdysone secretion by the ovary in mosquito. In: Progress in Ecdysone Research. Hoffman JA, ed. ElsevieriNorth-Holland, Amsterdam, pp. 467-479 (1980). 10. Hagedorn HH, O’Connor JD, Fuchs MS, Sage B, Schlaeger DA, Bohm MK: The ovary as a source of ecdysone in adult mosquitoes. Proc Natl Acad Sci USA 72, 3255 (1975). 11. Borovsky D: In vivo stimulation of vitellogenesis in Aedes aegypti with juvenile hormone, juvenile hormone analogue (ZR 515) and 20-hyclroxyecdysone. J Insect Physiol 27, 371 (1981) 12. Borovsky D, Van Handel E: Does ovarian ecdysone stimulate mosquitoes to synthesize vitellogenin? J Insect Physiol25, 861 (1979). 13. Borovsky D: Feedback regulation of vitellogenin synthesis in Aedes aegypti and Aedes atropalpus. Insect Biochem 1 2 , 207 (1981). 14. Lea AO: Artifactual stimulation of vitellogenesis -inAedes aegypti by 20-hydroxyecdysone. J Insect Physiol28, 173 (1982). 15. Kelly TJ, Fuchs MS: In vivo induction of ovarian development in decapitated Aedes atropalpus by physiological levels of 20-hydroxyectlysone. J Exp Zoo1 213, 25 (1980). 16. Kelly TJ, Fuchs MS, Kang S-H: Induction of ovarian development in autogenous Aedes atropalpus by juvenile hormone and 20-hydroxyecdysone. Int J Invetebr Reprod 3, 101 (1981). 17. Fuchs MS, Kang S-H, Kelly TJ, Masler EP, Whisenton LR: Endocrine control of ovarian development in an autogenous mosquito. In: Regulation of Insect Development and Behaviour International Conference. Sehnal F, Zabza A, Menn JJ, Cymborowski B, eds. WrocIaw Technical University Press, Wroclaw, pp 569-590 (1981). 18. Borovsky D, Van Handel E: Synthesis of ovary specific proteins in mosquitoes. Int J Invertebr Reprod 2, 153 (1980). 90 Borovsky et al 19. Kelly TJ, Hunt LM: Endocrine influence upon development of vitellogenic competency in Oncopeltus fasciutus. J Insect Physiol28, 935 (1982). 20. Masler PE, Fuchs MS, Sage B, O’Connor JD: Endocrine regulation of ovarian development in the autogenous mosquito, Aedes atropulpus. Gen Comp Endocrinol42, 250 (1980). 21. Borst DW, O’Connor JD: Arthropod molting hormone: Radioimmunoassay Science 278, 418 (1972). 22. Borst DW, O’Connor JD: Trace analysis of ecdysone by gas-liquid chromatography, radioimmunoassay and bioassay. Steroids 24, 637 (1974). 23. Rembold H, Hagenguth H, Rascher J: A sensitive method for detection and estimation of juvenile hormones from biological samples by glass capillary combined gas chromatography-selected ion monitoring mass spectrometry. Anal Biochem 101, 356 (1980). 24. Baker FC, Hagedorn HH, Schooley DA, Wheelock G: Mosquito juvenile hormone: Identification and bioassay activity. J Insect Physiol29, 465 (1983). 25. Bergot BJ, Ratcliff M, Schooley DA: Method for quantitative determination of the four known juvenile hormones in insect tissue using gas chromatography-mass spectroscopy. J Chromatogr 204, 231 (1981). 26. Lanzrien B, Hashimoto M, Parmakovich V, Nakanishi K, Wilhelm R, Luscher M: Identification and quantification of juvenile hormones from different developmental stages of the cockroach Nuuphoeta cinerea. Life Sci 16, 1271 (1975). 27. Hagedorn HH, Turner S, Hagedorn EA, Pontecorvo D, Greenbaum P, Pffiffer D, Wheelock G, Flanagan TR: Postemergence growth of the ovarian follicles of Aedes uegypti. J Insect Physiol23, 203 (1977). 28. Fuchs MS, Sundland BR, Kang S-H: In vivo induction of ovarian development in Aedes atropulpus by a head extract from Aedes aegypti. Int J Invertebr Reprod 2, 121 (1980). 29. Guilvard E, DeReggi M, Rioux J-A: Changes in ecdysteroid and juvenile hormone titers correlated to the initiation of vitellogenesis in two Aedes species (Diptera, Culicidae). Gen Comp Endocrinol 53, 218 (1984). 30. Gilbert LI, Bollenbacher WE, Agui N, Granger NA, Sedlak BJ, Gibbs D, Buys CM: The prothoracicotropes: Source of the prothoracicotropic hormone. Am Zoo1 22, 641 (1981). 31. Hagedorn HH, Shapiro JP, Hanaoka K: Ovarian ecdysone secretion is controlled by a brain hormone in an adult mosquito. Nature 282, 92-94 (1979).