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

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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 [4].
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 [13]. 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 [19].
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 [23].
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) [13]. 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 [14]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 [28] 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
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-
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0
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-
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-
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.
3
N-
m
0
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8w
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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 [14],
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 [9],
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 [9]) 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 [24] 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 [29] 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 [5] 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 [5]. 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 [30]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).
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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).
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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).
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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).
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brain hormone in an adult mosquito. Nature 282, 92-94 (1979).
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