ECDYSONE-MEDIATED STIMULATION OFDOPA DECARBOXYLASE ACTIVITY

AND ITS RELATIONSHIP TO

OVARIAN DEVELOPMENT IN AEDES AEGYPTI

DOROTHY A . SCHLAEGER, MORTON S . FUCHS,and SUK-HEE KANGFrom the Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556 . Dr.Schlaeger's present address is the Department of Biology, St. Francis College, Fort Wayne,Indiana 46808 .

ABSTRACT

Very little dopa decarboxylase activity is detectable in adult female mosquitoes Aedesaegypti which have not been allowed to engorge blood . However, when such females areinjected with the molting hormone ß-ecdysone a marked stimulation of this enzyme's ac-tivity is observable . No stimulation is observed in males similarly injected, nor in femalesinjected with cholesterol or a juvenile hormone mimic . In addition, ecdysone injection initi-ates ovarian development in these anautogenous non-blood-fed mosquitoes . The extent ofstimulation in both cases is dependent upon the amount of ß-ecdysone administered . Theseresults suggested that ecdysone may play a role in ovarian development in Aedes and led usto hypothesize that a normal blood meal may trigger the synthesis, activation, or release ofthis hormone endogenously . Using the radioimmune assay for ecdysone developed byBorst and O'Connor (Science [Wash . D. C.] 178 :4-18.), we found that the titer of anantigenic-positive material, presumably ecdysone or a closely related analogue, substan-tially increased 24 h after blood feeding, thereby supporting our postulation .

INTRODUCTION

The chorions of both fertilized and unfertilized essary for the synthesis of the sclerotizing agenteggs of the mosquito Aedes aegypti are soft and N-acetyldopamine (2) . However, this in turnwhite at the time oviposition . Within a short did suggest a possible correlation between thistime, in air, they gradually darken and harden enzyme and ovarian development in A. aegypti .and after 4 h the egg is completely black . This This postulation was confirmed when it was foundprocess resembles the hardening and darkening that 5-day old non-blood-fed female adults ex-of the cuticles of newly molted insects, referred hibit very little dopa decarboxylase activity untilto as sclerotization and/or tanning. Therefore, after they are given a blood meal, a necessaryfrom this point of view it was not surprising to requirement for subsequent normal ovarianfind that mature oocytes obtained from virgin A . development in this anautogenous species . Aaegypti contained high levels of dopa decarboxylase dramatic increase in enzymatic activity thenactivity (1), an enzyme which in Diptera is nec- ensued which paralleled oocyte maturation (3) .

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Removal of the resting ovaries before blood feed-ing resulted in no increase in dopa decarboxylaseactivity whatsoever, suggesting that the ovaryitself may be the site of synthesis or activation ofthis enzyme (4) .

The rise in dopa decarboxylase activity in adultfemale A . aegypti after a blood meal was rem-iniscent of an observation made by Karlson andco-workers who after injecting the molting hor-mone ecdysone into ligated Calliphora larvaefound an increase in dopa decarboxylase activity(5) . This led us to ask if injection of ß-ecdysoneinto non-blood-fed adult females would alsoelicit an increase in enzymatic activity? Ourpreliminary observations were affirmative, i .e .,injection of the molting hormone into 5-day oldnon-blood-fed females did result in a significantstimulation of the dopa decarboxylase activity(3, 6) . The first part of this report will examinethis enzymatic stimulation in more detail withreference to hormonal dosage effects and spec-ificity .

Spielman et al . (7) reported that injection ofß-ecdysone into non-blood fed, anautogenousadult female A. aegypti stimulated vitellogenesisand that prolonged feeding of the hormone re-sulted in the production of a few normal eggs.Thus, in the second part of this paper we willfurther address ourselves to the relationship be-tween ß-ecdysone, dopa decarboxylase activity,and ovarian development in these insects .

MATERIALS AND METHODS

Rearing of Mosquitoes

The wild type ROCK strain of A. aegypti (main-tained at the Vector Biology Laboratory, Universityof Notre Dame) was used throughout the study .Mosquitoes were reared according to the generalizedmethods of Craig and VandeHey (8) . All experi-ments were performed using 5-day old adult mosqui-toes maintained on apple slices which had been com-mercially blanched, packed in sugar, and frozen .Adults were given apple at the time of emergence .They received fresh apple on day 3, were starved onday 4, and then utilized for experimentation on day5. When required, a blood meal was administeredusing laboratory mice which had been anesthetizedwith sodium pentobarbital (Nembutal, Abbott La-boratories, South Pasadena, Calif.) . The time interval,post blood meal, was calculated from the midpoint ofthe feeding period . Only mosquitoes from the samerearing batch were used for any given experiment .

Injection

Adult mosquitoes were injected intrathoracically .The injection apparatus consisted of a mouthpiece,rubber tubing, a Leitz micromanipulator needleholder, and an injection needle made by drawing acapillary tube to a fine point . Routine injections in-volved a sample dose of 1 .6 f 0 .1 µl . This volumedistended the abdomen to approximately the samedegree as does a normal blood meal . In the "un-gorged" abdomen, the tergites and sternites slightlyoverlap, such that no portion of the intersegmentalmembrane is visible when viewed laterally . To avoiddilution of the injected sample, care was taken to in-ject only those individuals that met the requirementof the ungorged . For this reason, individuals werestarved 1 day before injection . After injection, themosquitoes were again given fresh apple slices .

The following substances were injected into A .aegypti adult mosquitoes to determine their effect ondopa decarboxylase activity : Aedes saline (9) ; 1%Tween 80 in saline; mouse blood :Alsevers (1 :1) ;mouse serum ; cholesterol (ICN Nutritional Biochemi-cals Div., International Chemical & Nuclear Corp .,Cleveland, Ohio) ; synthetic juvenile hormone (Cal-biochem, San Diego, Calif.) ; ecdysterone (Schwarz/Mann Div., Becton, Dickinson & Co ., Orangeburg,N. Y. ; Rohto Pharmaceutical Co ., Ltd., Osaka, Ja-pan). The cholesterol (2 µg/µl) and synthetic juvenilehormone (5 µg/µl) samples were prepared in 1 %Tween 80 in saline, while saline alone was used forecdysterone .

Dopa Deearboxylase Assay

The enzyme dopa decarboxylase (E .C . 4 .1 .1 .28)converts 3,4-dihydroxyphenylalanine (dopa) to 3,4-dihydroxyphenylethylarnine (dopamine) . Its activitywas assayed by measuring the rate of conversion ofradioactive substrate [2-14C]dopa to radioactivedopamine [1-14C]dopamine. Separation of productand substrate, and subsequent monitoring of radio-activity was accomplished by using a modification ofthe radiochromatographic procedure of Lunan andMitchell (10) . Further details can be obtained else-where (3) . Protein concentration was determined bythe method of Lowry et al . (11) using a standardcurve corrected for phenylthiourea (PTU) inter-ference.

For each enzymatic assay, at least 50 but moreusually 75 individuals were homogenized in order toobtain the extract . Each value reported herein repre-sents the average of six replicates . The range of vari-ability in all cases was less than ±5% .

Photomicroscopy

Transmitted-light photomicrographs were takenwith a Zeiss Ultraphot II equipped with a Luminar

SCHLAEGER ET AL . Ecdysone-Mediated Stimulation of Dopa Decarboxylase Activity

455

head. 4- by 5-inch Kodak Ektapan Professional filmwas used with this instrument .

Radioimmune Assay for Ecdysone

The radioimmune method of Borst and O'Connor(12) was used to assay for ecdysone in adult mosqui-toes . The extraction and subsequent assay procedureswere performed in Dr. O'Connor's laboratory atU.C.L.A. under the supervision of Dr . David Borst.Six samples of mosquitoes were tested for the presenceof ecdysone : newly pupated females ; 5-day old adultmales; 5-day old pre-blood-fed adult females ; andfemales 4-, 12-, and 24-h post blood feeding . Thesample of newly emerged female pupae was includedto serve as a control, testing the effectiveness of thisassay in detecting the presence of ecdysone in mosqui-toes . Approximately 1 g of each sample of mosquitoesof known number was respectively homogenized inapproximately 5 vol (wt/vol) of 60% methanol for 2min at room temperature using an Omnimixer . Thehomogenate was centrifuged at 5,000 g for 15 min,and the resulting supernate was filtered throughpaper. The precipitate was extracted two additionaltimes in methanol as before ; the filtrates were thencombined and mixed with chloroform (for protein de-naturation) to give a final concentration of 20% . Afterthe resulting mixture was filtered into a round-bottomflask, the solvents were removed by flash evaporationat 48 °C. The residue was dissolved in 5 ml of metha-nol ; that which failed to dissolve was removed bycentrifugation . About 10-50 µl of the methanol ex-

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FIGURE 1 Dosage effect of ß-ecdysone injection on dopa decarboxylase activity in unfed adult femaleA. aegypti . Activity after saline, o, and ß-ecdysone injection : N, 0 .1 µg/µl ; •, 1 .0 µg/µ1 ; A, 2.5 µg/µl ;and S, 5 .0 µg/µl . Total volume injected in all cases was 1 .6 f 0.1 µl . Fig. I a depicts specific activityand Fig. I b activity per individual .

THE JOURNAL OF CELL BIOLOGY • VOLUME 61, 1974

tract were assayed for the presence of ecdysone . Analiquot of this extract was pipetted into a 6- by 50-mm culture tube and evaporated to dryness by N 2flushing . The residue was suspended in 100 µl of bor-ate buffer, pH 8 .4, to which was added 50 µl of anti-ecdysterone-bovine serum albumin (BSA) serum .The ecdysterone-BSA conjugate and the antiserumderived thereof has been previously prepared byBorst and O'Connor (12) . After thorough mixing, thetube was incubated for 2 h at room temperature. Itwas then cooled to 4 °C, and the haptene-antibodyconjugate was precipitated by the addition of 200 µlof saturated (NH4)2SO4 . After 30 min at 4 °C, themixture was centrifuged at 5,000 g for 20 min . Thesupernate was removed by suction, and the precipi-tate was washed with 50% (NH4)2SO4 . The finalpellet was dissolved in 25 µl of water and mixed with600 µl of Aquasol (New England Nuclear, Boston,Mass .) . This was monitored for radioactivity in aBeckman LS-233 liquid scintillation counter (Beck-man Instruments, Inc., Fullerton, Calif.) .

RESULTS

Ecdysone and Dopa Decarboxylase

An examination of Fig . I a and I b clearlyreveals that ß-ecdysone (also called 20-hydroxy-ecdysone or ecdysterone) does indeed stimulate theactivity of the enzyme dopa decarboxylase wheninjected into unfed adult A. aegypti females. Aftera lag period of approximately 24 h, the amount

HOURS

POST INJECTION

of activity increases dramatically until it beginsto level off at approximately 72 h . Furthermore,although the amount of enzymatic activity ob-served is not directly proportional to the amountof hormone injected, a dose-dependent response isevident . This positive correlation between theamount of hormone injected and the amount ofdopa decarboxylase activity observed is seenwhether the specific activity (Fig . 1 a) or the en-zymatic activity per female (Fig. 1 b) is monitored .

The increase in the specific activity of dopadecarboxylase suggests that ß-ecdysone is notcausing a general increase in protein synthesisbut does suggest some specificity in the mode ofaction of the hormone . To gain further insight intothis possibility two questions were asked : first, willß-ecdysone stimulate dopa decarboxylase activityin males and, secondly, will other materials in-jected into females mimic the hormonal effect ob-served? In order to answer the above questionscritically, the enzymatic assay procedure was

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POST INJECTION

FIGURE 2 Effect of 5 .0 pg/ftl ß-ecdysone injectionon the adult levels of dopa decarboxylase activity inmale and female A. aegypti . The volume injected inall cases was 1 .6 f 0.1 µl : -•- , specific activity offemales ; . . . ./ • • • •, specific activity of males ; --A--,specific activity of saline-injected female controls .

altered . The standard assay utilizes 10 µl of en-zyme extract in which the reaction mixtures areallowed to incubate for 10 min . These conditionsgive one the true initial velocity of the reactionand serve as a measure of the amount of activeenzyme present (3) . However, to be absolutelycertain that we would not miss small increasesin dopa decarboxylase activity, the volume of theenzymatic extracts were increased to 40 µl and thereaction mixtures were incubated for 1 h beforetermination . Thus if any dopa were converted todopamine enzymatically, the chances of it beingundetected would be considerably diminished . Thismodified assay procedure for dopa decarboxylaseactivity was utilized for the experiments depictedin Figs . 2 and 3 . Fig . 2 clearly demonstrates thatadult males injected with ß-ecdysone show noincrease in dopa decarboxylase activity . Fig. 3

FIGURE 3 Activity of dopa decarboxylase exhibitedby non-blood-fed adult female A. aegypti after in-jection of : x, ß-ecdysone ; •, mouse blood ; O, mouseserum ; L, juvenile hormone mimic ; A, uninjectedfemales; /, 1% Tween 80 in Aedes saline ; and c,cholesterol . All females were 5-day old adults at thetime of injection . In all cases 1 .6 f 0 .1 µl per femaleof sample were injected in the following concentra-tions : ß-ecdysone 5 mg/ml in Aedes physiologicalsaline, juvenile hormone mimic, 5 mg/ml in 1% Tween80 and Aedes saline and cholesterol, 2 mg/ml in thesame solvent as the juvenile hormone mimic .

SCRLAEGER ET AL . Eedysone-Mediated Stimulation of Dopa Decarboxylase Activity

457

also shows that mouse blood, mouse serum, cho-

lesterol, and a juvenile hormone mimic (Williams-Law mixture) do not by themselves stimulate dopadecarboxylase activity.

Ecdysone and Ovarian Development

Figs. 4-6 show the progression of ovarian

development at 24, 48, and 72 h, respectively,after a normal blood meal and ß-ecdysone in-jection. It appears that the 0.1 ug/ul ß-ecdysoneinjection is below the threshold concentrationrequired to elicit any significant yolk deposition .Likewise, this concentration failed to significantlyenhance dopa decarboxylase activity (Fig. 1) .

At 24-h post injection there seems to be no realdifference between the ovarian response to 2 .5

ug/ul and to 5 .0 ug/ul ß-ecdysone. Much greatervariation is observed among females injected with1 .0 ug/ul ß-ecdysone. Some females show con-siderable development, others very little, whilethe majority exhibit a moderate degree of yolkdeposition as seen in Fig . 4 d. The difference inovarian development due to differences in

ecdysone concentration become much morepronounced by 48-h post injection. While it ap-

peared that ovarian development had beeninitiated in approximately equal numbers offollicles by 24-h post injection, we observe strikingdifferences in the amount of yolk deposited andin the number of oocytes continuing to developafter 48-h post injection . There appears to be acorrelation between the concentration of the in-jected ecdysone and the number of oocytes con-

tinuing to develop . The lower the ecdysone con-

centration, the fewer the oocytes that continue todevelop, but the greater the yolk deposition ineach oocyte. These same observations apply tothe 72-h post injection stage. The fact that higherconcentrations of injected ecdysone result inprogressively greater amounts of dopa decar-boxylase activity correlates exceedingly well withthe morphological observations illustrated in

Figs. 4-6 .

The Radioimmune Assay for Detection ofEndogenous Levels of Ecdysone

The data reported herein by us clearly confirmand extend the original observation of Spielman

et al. (7) that ß-ecdysone initiates yolk deposition,and, they are consistent with Fallon andHagedorn's report (13) that ß-ecdysone activates

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the synthesis of vitellogenin by mosquito fat bodies.Moreover, the ineffectiveness of ß-ecdysone tostimulate dopa decarboxylase activity in males,and the unique effectiveness (of the substancestested) of ß-ecdysone to stimulate dopa decar-boxylase activity suggest that the responses elic-ited by this hormone in adult females are notartifactual . As a working hypothesis we propose

that in anautogenous adult A. aegypti females,the engorgement of blood triggers the endogenousrelease, activation, or synthesis of ecdysone,which now assumes a gonadotropic role by ac-tivating or inducing the components necessaryfor normal ovarian development, including theenzyme dopa decarboxylase . Note that we are

not suggesting that ecdysone is the only gonado-tropic hormone, but only that it is a necessary one.A consequence of this postulation is the predictionthat blood-fed females would exhibit a highertiter of ecdysone than non-fed individuals . Utiliz-ing the Borst-O'Connor radioimmune assay forecdysone (12), hormone levels were determined innewly emerged pupae (used as a guide), 5-day oldadult non-blood fed males and females, and 5-dayold females assayed at 4, 12, and 24 h after bloodfeeding.

Fig . 7 shows the levels of "ecdysone" detected inA. aegypti pupae (obtained within 10 min after thelarval-pupal ecdysis) and adults . The data clearlyestablish the presence of ecdysone in the pupalcontrol and also illustrate an increase in ecdysonelevels in blood-fed females over the non-blood-fedcontrol. Although some variation in the valuesobtained is indicated, there is a very obviouschange in ecdysone titer by 4 h after a blood meal,and at 24-h post blood meal the ecdysone titer iscomparable to that of newly emerged pupae. No

other female samples were tested at this time .The 5-day old males show ecdysone levels com-parable to that of 4-12-h blood-fed females . Nodetectable ecdysone-like antigenic activity wasfound in mouse blood .

DISCUSSION

The activation of egg development by ecdysonewas first demonstrated by Spielman et al. (7) whofound that injection of ß-ecdysone into non-bloodfed, anautogenous female A. aegypti stimulatedvitellogenesis, and that prolonged feeding of thehormone resulted in the production of a few nor-mal eggs. Development of oocytes was not ini-tiated by three other steroids (cholesterol, hy-

FIGURE 4 Comparison of normal ovarian development at 24-h post blood meal with that initiated byinjection of ß-ecdysone : (a) normal post blood meal ovary, (b) ovaries of a saline-injected control female ;ovaries of ß-ecdysone-injected females, (c) 0.1 µg/µl, (d) 1 .0 µg/µl, (e) 2 .5 µg/µl, and (f) 5 .0 µg/µl,respectively . X 5!5 .

4 59

FIGURE 5 Comparison of normal ovarian development at 48-h post blood meal with that initiatedby injection of ß-ecdysone : (a) normal post blood meal ovary, (b) ovaries of a saline-injected controlfemale ; ovaries of ß-ecdysone-injected females, (c) 0 .1 µg/µl, (d) 1 .0 µg/µl, (e) 2 .5 µg/µl, and (f) 5 .0µg/µl, respectively. X 25 . Note that in Fig. 5 d two pairs of ovaries are shown and in Fig . 5 e two com-plete pairs and part of a third pair can be seen .

4 60

FIGURE 6 Comparison of normal ovarian development at 72-h post blood meal with that initiatedby injection of ,ß-ecdysone : (a) normal post blood meal ovary, (b) ovaries of a saline-injected controlfemale ; ovaries of ß-ecdysone-injected females, (c) 0 .1 µg/µl, (d) 1 .0 µg/µ1, (e) 2.5 µg/µI, and (f) 5 .0µg/µI, respectively . X 25 . Note that Fig. 6 d depicts two complete pairs of ovaries .

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drocortisone, and estriole), nor by phagostim-ulants normally present in vertebrate blood (ATP,serum, and hemoglobin) . In addition, a juvenilehormone mimic (a terpenoid mixture) and syn-thetic Cecropia juvenile hormone had no dis-cernible affect on ovarian development . Our workreported herein confirms their original observa-tions in all aspects with regard to ovarian develop-ment and extends them to include the regulationof dopa decarboxylase by ecdysone. It was withthis background that we were led to suggest thatecdysone is normally involved in ovarian develop-ment in A . aegypti . The results of our initial at-tempts to determine the endogenous levels ofecdysone in pre- and post-blood meal femalessupport our hypothesis .

A major implication of our hypothesis is thatactive ecdysone is produced, activated, or re-leased and subsequently functions in an adultinsect . Until recently, it was believed that theprothoracic glands synthesized the active hor-mone, and since mosquitoes (14) as well as mostother adult insects do not retain this gland (15),

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THE JOURNAL OF CELL BIOLOGY . VOLUME 61, 1974

ecdysone was thought to have little if any normalfunction in adults . The fact that significant levelsof ecdysone have been detected in several adultinsects, such as the locust (16), the silkworm (17),and the milkweed bug (18), has been referred toas "a slightly puzzling fact" by so eminent anauthority as Wigglesworth (15) . ß-Ecdysone isformed by enzymatic conversion from its pre-cursor a-ecdysone (19), and there is now ampleevidence indicating that ß-ecdysone is the activeform of the hormone (20) . Nakanishi et al. (21)have shown that cholesterol is converted into botha- and ß-ecdysone in isolated abdomens of silk-worm larvae in the absence of the prothoracicgland, and Ellis et al . (22), working with locustprothoracic glands, concluded that this glanddoes not produce ß-ecdysone. Thus, althoughthere is not unanimous agreement among insectphysiologists, more and more evidence is ac-cumulating which strongly suggests that thepresence of the prothoracic gland is not universallyrequired for ecdysone activity . Conversely, theabsence of these glands in adult insects does notnecessarily mean, as had been previously as-sumed, that ecdysone is not produced and doesnot function in the adult .

The results of the Borst-O'Connor radioimmuneassay for ecdysone are consistent with our hy-pothesis which supposes a normal gonadotropicfunction for ecdysone in adult mosquitoes, butby no means does it prove it . Our only firm con-clusion from this assay can be that the concen-tration of a substance which is antigenically sim-ilar to ß-ecdysone is present in low levels in non-blood-fed females and increases after blood feed-ing. Given that known ß-ecdysone will initiateovarian development, we suggest that the increasein antigenic specificity observed after a bloodmeal toward ß-ecdysone antibody is due to anendogenous increase in an active ecdysone-likehormone. We are not prepared, at present, tospecifically identify this material . This point isespecially pertinent because Spielman et al . (7)found that 22-isoecdysone, an isomer of ecdysonewhich is devoid of molting hormone activity inother insect tissues, will also stimulate ovariandevelopment in A . aegypti . Experiments to deter-mine the biological activity of our antigenic-positive material as well as its chemical andphysical properties in order to identify it are nowbeing performed . Concurrently, the synthesisand function of this material in ovarian develop-

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ment, whether it be bonified ß-ecdysone or an

analogue, are also being investigated .Considerable literature has accumulated deal-

ing with the relationship of the blood meal to the

hormonal control of ovarian development inmosquitoes. Larsen and Bodenstein (23) reportedthat a blood meal is not obligatory for ovariandevelopment in anautogenous mosquitoes . Theysealed the anus with wax in a group of mosquitoes

and fed them fruit juice . This resulted in a marked,prolonged distension of the gut, and by the 4thday, ovarian development had been initiated in a

number of the mosquitoes . They concluded thatdistension of the abdomen for a sufficient periodof time stimulates afferent nerve impulses leadingdirectly or indirectly to the release of gonado-

tropic hormone. In mosquitoes, stretch receptorson the midgut or in the abdominal wall whichsend nerve impulses in order to initiate ovarian

development have not been described . In fact,there is firm evidence that they do not exist (24,25). Moreover, the suggestion that sustained gutstretching initiates egg development by anauto-genous mosquitoes has not been confirmed byother workers (26) . However, if gut distensiondoes initiate a nervous impulse to the brain, it is

possible that the pathway may proceed via thestomatogastric system (24) .

Lea and co-workers (25, 27, 28), on the basis ofelectron microscopy and histological and ablationexperiments, have concluded that in mosquitoesas in other Diptera, the corpus allatum (CA)and medial neurosecretory cells (MNC) eachproduce a hormone that is required for yolk

deposition. The neurosecretory hormone (EDNH)appears to be stored in the corpus cardiacumwhich "meters" the release of EDNH for eachbatch of eggs after a blood meal . Parabiosis ex-periments indicated that a humoral factor wasnecessary for this release (25) . The chemicalnature of EDNH is unknown except that its pres-

ence in the cardiacum was found to be independ-ent of positive paraldehyde-fuchsin staining

material (29) . The suggestion that a hemolymph-

borne humoral factor is needed for ovarian

development which is regulated by the blood

meal is a viable alternative to the stretch receptor-

nerve impulse idea (25) . Other workers (30)

have shown that the ovary is the source of a

humoral factor which activates the fat body to

synthesize vitellogenin in mosquitoes. They fur-

ther state that injected ecdysone mimics the effect

of the ovary. Obviously then, the ovary is a primesuspect as the likely anatomical site responsiblefor the increase of the ecdysone-like material wehave observed after the blood meal . Experimentsdesigned to determine the ecdysone titer of ovari-

ectomized blood-fed females are now underway .There are at least three major difficulties with

our hypothesis: (a) Why do we observe so long atime lag between the ecdysone injections and thefirst indication of dopa decarboxylase activity?(b) Why is so high a concentration of ecdysonerequired to elicit a response? And, as corollaries

to (a) and (b) above, (c) How can the injectedecdysone remain biologically active over a periodof days? These are difficult questions and, frankly,require much more data than is presently availablebefore definitive answers can be offered. However,we can offer what we think are reasonable spec-ulations . Objections (b) and (c) are related in that

exogenous ecdysone may be degraded in adultmosquitoes as has been shown in other adultinsects (31) . This fact may lend credibility to thelarge doses of ecdysone required to elicit a positiveresponse. Even if ecdysone is not degraded, it ispresumed that the injected material must go tosome target tissue(s) in order to elicit a givenresponse; therefore, it is possible that the large

amount required for such a response is a reflectionof random diffusion of the hormone in the insect .In reality, it may well be that only a very smallamount of ecdysone actually reaches the ap-propriate site . The time lag question may not bedebatable at all, since a similar lag is observedafter a blood meal (3) . The question should moreprobably focus on the action of ecdysone at the

molecular level, and our work is proceeding alongthese lines .

It has been shown in other insects that highdoses of exogenously introduced ecdysones resultin abnormalities (32), and more specifically, itwas found that high doses of ecdysone, when fedto adults, severely inhibited ovarian develop-

ment in houseflies (33) . Wright et al . (34) reportedthat ß-ecdysone, when ingested by the stable fly,

prevented vitellogenesis in developing oocytes .

The opposite occurs in A . aegypti and, it is difficultto conceive that the positive biological responses

we and others have observed after ecdysone treat-ment (i .e . initiation of ovarian development,

stimulation of dopa decarboxylase activity, andvitellogenin synthesis by the fat body) are merely

artifactual .

SCHLAEGER ET AL . Ecdysone-Mediated Stimulation of Dopa Deearboxylase Activity 463

A most intriguing result was our recent findingthat when cyclic AMP and ß-ecdysone are in-jected simultaneously into non-blood-fed females,the dopa decarboxylase activity after 72 h is in-creased by approximately 50% (6) . Cyclic AMPinjected alone did not elicit any enzymatic ac-

tivity above that of the saline control. Thus, thenucleotide is not a second messenger for ecdysonein this system . Further investigations to deter-mine the endogenous levels of cyclic AMP (aswell as other cyclic nucleotides) and the ap-propriate cyclases and their relationship to ec-dysone are underway .

The authors thank Dr. D. W. Borst and Dr . J . D.O'Connor of U.C.L.A. for graciously providing uswith their time, laboratory and facilities, materials,and most important their knowledge which allowedone of us (S . H. Kang) to perform the radioimmuneassay under their supervision . We also thank Dr . A .Spielman of Harvard University for helpful discus-sions and gratefully acknowledge our indebtness toDr. J. J . McGrath of the University of Notre Damefor his help with the photography .

This work was supported by National Institutes ofHealth Research Grant no. At 10707, and TrainingGrant no . At 00378, both from the National Instituteof Allergy and Infectious Diseases.

Received for publication 17 September 1973, and in revisedform 21 January 1974 .

REFERENCES

1 . SCHLAEGER, D . A., and M . S . FucHs. 1974 . Effectof dopa decarboxylase inhibition on Aedesaegypti eggs. Evidence for sclerotization . J .Insect Physiol . 20 :349.

2. KARLSON, P., and C. E . SEKERIS . 1962 . N-acetyl-dopamine as sclerotizing agent of the insectcuticle . Nature (Lond.) . 195 :183 .

3. SCHLAEGER, D . A., and M . S . Fucus . 1974 . Dopadecarboxylase activity in Aedes aegypti . A pre-adult profile and its subsequent correlationwith ovarian development . Dev . Biol . In Press .

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