Insect Biochem. Vol. 17, No. 7, pp. 1095-1098, 1987 0020-1790/87 $3.00+0.00 Printed in Great Britain Pergamon Journals Ltd

DEVELOPMENT OF RESPONSIVENESS TO HORMONES AFTER A BLOOD MEAL IN THE MOSQUITO AEDES AEGYPTI

TERESA MARTI~rEZ* and HENRY H. HAGEDORNt Department of Entomology, Cornell University, Ithaca, NY 14853, U.S.A.

Abstract--The effects of exogenous hormones on oocyte development in isolated abdomens from blood-fed female Aedes aegypti were examined. Abdomens were prepared immediately after a blood meal. Single applications of hormones were administered immediately after tigation or 18 hr after the blood meal. Double applications were done at both times. Oocyte development was assayed by measuring the amount of yolk in oocytes 66 hr after the blood meal. Topical application of maximum doses of methoprene immediately after ligation caused oocytes to mature in 60% of the abdomens; a half- maximum response was obtained with 300 pg. Injection of 700 ng of 20-hydroxyecdysone (20-HE) was necessary to cause an equivalent response. Delaying the injection of 20-HE until 18 hr after feeding reduced the amount necessary to obtain a half-maximum response to 150 ng. Treating the abdomens twice dramatically reduced the amount of 20-HE needed for the second dose: pretreatment of abdomens immediately after ligation with 50 pg of 20-HE reduced the amount of 20-HE needed in the second injection to 30 ng. Pretreatment with a topical application of 50 pg of methoprene had a similar effect. These data indicate that the sensitivity of the mosquito to exogenous hormones changes after a blood meal, and that either 20-HE or methoprene can promote a further increase in sensitivity.

INTRODUCTION

Early experiments on egg development in mosquitoes implicated either juvenile hormone (JH) (Larsen and Bodenstein, 1959), a hormone from the medial neurosecretory cells of the brain (Lea, 1967), or 20-hydroxyecdysone (20-HE) (Spielman et ai., 1971; Hagedorn et al., 1975) as the controlling factor involved in stimulating growth of the eggs after a blood meal. We now know that the blood meal in Aedes aegypti causes a decrease in the titer of JH (Shapiro et al., 1986), the release of the egg devel- opment neurosecretory hormone, EDNH (Lea, 1972; Hanaoka and I-Iagedom, 1980; Greenplate et al., 1985) and an increase in the titer of20-HE (Hagedorn et al., 1975; Greenplate et al., 1985). These events somehow result in the synthesis of vitellogenin and the development of a batch of eggs. Egg development is the result of a complex interaction between these known hormones and others about which we know very little. It has become increasingly clear that no one factor is in control of the process.

The most intensively studied part of the process of egg development is the production of the yolk pro- tein, vitellogenin. At the peak of vitellogenin syn- thesis the female A. aegypti makes about 1O#g of vitellogenin per hour for a total of about 160 #g. We have presented evidence that 20-HE stimulates the synthesis of vitellogenin by the fat body [for review see Hagedorn (1985)]. However, it now appears that JH is also involved. Experiments with decapitated, blood-fed animals and with isolated abdomens from blood-fed animals have shown that JH somehow potentiates the action of 20-HE (Borovsky, 1981;

*Present address: Instituto de Quimica Bio-Organica, CSIC, Barcelona, Spain.

tTo whom correspondence should be addressed.

Borovsky et al., 1985). We have recently shown that an effect of JH can also be demonstrated in vitro (Racioppi et al., 1986); however, it is only effective in concert with 20-HE and has no apparent effect by itself.

To gain a better understanding of the effects of these two hormones, we examined the effect of single and double doses of methoprene and 20-HE on oocyte development. We show here that the isolated abdomen becomes increasingly sensitive to injected 20-HE after a blood meal. We also demonstrate that both 20-HE and methoprene can promote an even greater change in sensitivity.

MATERIALS AND METHODS

Animals

Mosquitoes were derived from the NIH-Rockefeller strain and were reared as described by Shapiro and Hagedorn (1982). Four-day-old females were used in all experiments. Animals were starved for about 2 hr before a blood meal and were then fed on warmed cow blood (37-40°C) through a Parafilm membrane. Ligation of the abdomen was performed within I hr of the blood meal, as described by Baker et al. (1983). Ligated abdomens were held at 27°C in a humidified chamber. Ovaries were re- moved 66 hr after the blood meal and the length of the oocytes was measured at 30 x using a binocular microscope. Under these conditions, oocyte length is a measure of the amount of yolk deposited. Oocytes shorter than I00 #m were considered undeveloped. Maturing oocytes were longer than 250 #m. Oocytes of intermediate length were rarely seen, except after treatment with a single dose of meth- oprene as noted.

Hormones

(RS) Methoprene was obtained from Zoecon Corpor- ation (Palo Alto, CA). Stock solutions were made in hexane and stored at -20°C. Fresh dilutions were prepared for each experiment. Hexane was evaporated under nitrogen, and the residue was dissolved in spectrograde acetone.

1095

1096 TERESA MARTINEZ and HENRY H. HAGEDORN

Concentrations were checked spectrophotometrically. A 0.25-#1 volume of the appropriate dilution was topically applied on the abdomen immediately after ligature. Con- trols were treated with 0.25 #1 of acetone.

20-Hydroxyecdysone was obtained from Rhoto Pharma- ceutical Co. (Japan). Stock solutions were prepared in Aedes saline (Hagedorn et al., 1977) with added penicillin (150 rag/L) and streptomycin (250mg/L). Dilutions were prepared in the same saline before each experiment. For injection the abdomens were placed on a paraffin surface under netting and 0.25/~1 of the appropriate dilution was injected between the 4th and 5th abdominal tergites with a needle drawn from a glass capillary tube. 20-HE injections were administered immediately after ligature (between 1 and 2 hr after feeding) and/or at 18 hr (18-19 hr after feeding). Controls were injected with 0.25 #1 saline.

RESULTS

Effect o f methoprene

Topical application of methoprene to isolated ab- domens immediately after ligation resulted in oocyte maturation (Fig. 1). In response to the maximum dose, 60% of the abdomens had developed oocytes. A dose of 250-300 pg resulted in a half-maximal response (i.e. 30% of the abdomens responding). Borovsky (1981) obtained a half-maximal response with about 100 pg of methoprene, and 80% of the abdomens responded to the maximum dose.

Effect of methoprene and 20-hydroxyecdysone

Borovsky et al. (1985) found that a small dose of methoprene (25 pg) followed by an injection of 5 ng of 20-HE 18 hr later caused oocytes to mature in 60%

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z~ 5'o ,c;o s6o ,8o0 rnethoprene pg / obdomen

Fig. 1. Effect of methoprene and 20-hydroxyecdysone on oocyte development in isolated abdomens of ,4. aegypti. Females were fed blood and immediately ligated. &, Meth- oprene topically applied immediately after ligation; O, variable amounts of methoprene applied immediately after ligation followed by an injection of 5 ng of 20-hydroxy- ecdysone 18 hr after the blood meal; II, 100 pg of meth- oprene applied immediately after ligation followed by an injection of 50ng of 20-hydroxyecdysone 18 hr after the blood meal. Abscissa, the amount of methoprene topically applied in 0.25/~1 of acetone. Ordinate, the percentage of abdomens that contained maturing oocytes 66 hr after the blood meal. Each point represents the mean percentage of abdomens responding. The number of abdomens used to calculate the per cent maturing is indicated next to each

point.

of the abdomens. Using this protocol, our animals showed very little response to 25 pg of methoprene, so we varied the dose of methoprene while keeping the amount of 20-HE injected constant at 5 ng. As shown in Fig. 1, increasing the amount of meth- oprene caused an increasing response: the amount needed for a half-maximal response was 50 pg. A maximum response required over 100pg of meth- oprene. Sixty per cent of the abdomens responded to the maximum dose, but if the amount of 20-HE injected at 18 hr was increased from 5 to 50 ng, then 90% of the abdomens developed oocytes (Fig. 1).

Thus, we have been able to repeat the observations of Borovsky (1981) and Borovsky et al. (1985), but our animals required increased amounts of meth- oprene to show the same response. The differences between our results are possibly due either to the strain of animals used, since recently published re- suits show that our strains also differ in the timing of the ecdysteroid peaks and in the ratio of ecdysone vs 20-HE present (Borovsky et al., 1986), or to slight differences in the timing of ligation.

Effect of 20-hydroxyecdysone injections Abdomens were injected with 20-HE shortly after

ligation or 18 hr after feeding (Fig. 2). If the hormone was injected immediately after ligation, ~ 700 ng was needed to obtain a half-maximal response (i.e. 45% of the abdomens responding). Injection at 18 hr reduced the amount needed for a half-maximal re- sponse to 150 ng/abdomen. The highest doses used caused oocytes to mature in 90% of the abdomens.

Abdomens were injected with 20-HE twice. The first injection of 50 pg was done immediately after ligation. The second injection of a variable amount followed at 18 hr after feeding. The effect of two injections was to lower the dose needed for a half- maximal response to about 30 ng (Fig. 2). At the highest doses used oocytes matured in 90% of the abdomens. Lowering the amount of hormone in the first dose to 25 pg reduced the response; only 50% of the abdomens contained mature oocytes and the number of developing oocytes was consistently half of the normal number.

A greater number of large oocytes (oocyte lengths 300-450 # m) developed in those abdomens given two doses of 20-HE, or a single dose at 18 hr. Abdomens injected with 20-HE immediately after ligation had fewer developing oocytes. Abdomens treated with a single dose of methoprene had smaller oocytes (oo- cyte lengths 200--350 #m) than those that received both methoprene and 20-HE.

DISCUSSION

The data reported here reveal two new aspects of egg development in A. aegypti. First, it is clear that the ability of the mosquito to respond to injected 20-HE changes after a blood meal. The isolated abdomen requires large amounts of hormone (700 ng) if the injection is given immediately after ligation; however, if the injection is delayed for the amount of time needed for 20-HE titers to rise in the normal animal [i.e. 18hr--Hagedorn et al. (1975), Greenplate et al. (1985)], then the female is over 4½ times more sensitive to injected hormone (Fig. 2).

Responsiveness to hormones in Aedes aegypti 1097

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I i I S i i i i 5 2 5 5 0 I 0 0 2 5 0 5 0 0 IOOO

2 0 - h y d r o x y e c d y s o n e n(] / a b d o m e n

Fig. 2. Effect of single and double injections of 20-hydroxyecdysone on oocyte developm¢nt in isolatecl abdomens of .4. aegypti. Females were fed blood and immediately ligated. &, 20-Hydroxyecdysone injected immediately after ligation; O, 20-hydroxyecdysone injected 18 hr after the blood meal; i , 50 pg of 20-hydroxyecdysone injected immediately after ligation followed by a second injection of variable amounts of 20-hydroxyecdysone 18 hr after the blood meal. Abscissa, the amount of 20-hydroxyecdyson¢ injected in 0.25 #l of saline. Ordinate, the percentage of abdomens with maturing oocytes 66 hr after the blood meal. Each point indicates the mean percentage of abdomens responding. The number of abdomens

used to calculate the per cent maturing is indicated next to each point.

Thus, the abdomen became more sensitive to 20-HE in response to the blood meal alone, independent of exogenous treatments.

Several earlier experiments had suggested that the female changes in its response to other hormones after a blood meal. For example, the egg development neurosecretory hormone (EDNH) has no effect when injected into a female that has been fed blood and immediately decapitated (Fuchs et al., 1981; Wheelock and Hagedorn, 1985). However, if the animal is not decapitated until 2 hr after the blood meal, then an injection of EDNH causes egg devel- opment OVheelock and Hagedorn, 1985). Thus, the blood meal initiates changes in the female that result in greater sensitivity to hormones that appear shortly afterward.

The second major conclusion is that two doses of 20-HE, spaced 18 hr apart, reduce the amount of 20-HE needed to stimulate egg develoment over 20-fold. We also confirmed the results of Borovsky et al. (1985) showing that methoprene also had this effect. Thus, we conclude that both 20-HE and methoprene render the abdomen more sensitive to an injection of 20-HE at 18 hr. This raises two questions: how does the increased sensitivity occur, and which hormone is normally involved? With regard to the first question, the priming effect of hormones, in which a target tissue becomes more sensitive, is a well-known phenomenon, and often the effect is on receptor levels. In some cases, two hormones are involved. For example, in the case of the hamster uterus, estrogen stimulates the appearance of recep- tors for progesterone (Leavitt et aL, 1974; Clark and Peek, 1979). However, a hormone can also stimulate synthesis of its own receptor. For example, in the chicken liver estrogen stimulates the appearance of estrogen receptor (Snow et al., 1978). An increase in the population of 20-HE receptors is a possible

reason for the observed change in sensitivity in the mosquito to 20-HE.

Another explanation for the effect of JH is its known effect of stimulating an increase in eedysteroid titers. This stimulation has been seen when large doses (ng) of either JH I (Fuchs et al., 1981) or methoprene (Borovsky et aL, 1985) are used. It is not clear whether the small doses used in the experiments described here would also elevate ecdysteroid titers. Borovsky et aL (1985) reported a small increase (50 pg) in eedysteroid levels in response to the appli- cation of 25 pg of methoprene, but the abdomens were assayed 24 hr after application. If we assume that the methoprene is stimulating a small increase in levels of 20-HE that mimics the early increase after a blood meal, then the treated abdomens should be assayed soon after application. In preliminary experi- ments we have not been able to detect a response 6 hr after application of 100 pg of methoprene. However, since the expected effect is small and transitory it may be difficult to observe. This possibility needs further investigation.

To answer the second question, which hormone is normally involved, our results suggest that either juvenile hormone or 20-HE could act as a primer for vitellogenin synthesis. A case can be built for either of these two hormones acting as a primer. Three facts suggest that in the normal animal 20-HE is both the primary and secondary stimulator of vitellogenin synthesis. First, a small peak of 20-HE occurs in whole body extracts 4-6hr after a blood meal (Hagedorn et aL, 1975; Greenplate et aL, 1985; Borovsky et aL, 1986; Wheelock, unpublished obser- vations). This small peak could represent the primer. Second, the titer of JH falls rapidly after a blood meal (Shapiro et aL, 1986). Third, Lea (1969) showed that females allatectomized 3 days after emergence devel- oped eggs normally following a blood meal.

1098 TERESA MARTINEZ and HENRY H. HAGEDORN

It is also possible that JH is the primer, and two facts can be advanced to support this hypothesis. First, since it takes 12 hr for the JH titer to fall to its lowest level (Shapiro et al., 1986) there may be sufficient hormone over a long enough period to act as a primer. Second, Racioppi et al. (1986), using incubated fat body, showed an effect of methoprene on the expression of the vitellogenin gene. Fat bodies from non-blood-fed females responded to 20-HE by synthesizing vitellogenin messenger RNA. However, they made twice as much message in the presence of both methoprene and 20-HE. Methoprene by itself had no effect. This experiment is crucial to the interpretation of the function of JH. Clearly, it cannot turn on the vitellogenin gene by itself, whereas 20-HE can. Yet it somehow interacts with 20-HE, potentiating the effect of 20-HE on the expression of the vitellogenin gene. An effect of methoprene on 20-HE receptors is consistent with these results. However, methoprene may be merely stimulating ecdysone production by some cells in the fat body preparation (which consists of the abdominal wall plus the attached fat body, oenocytes and epidermis). If so, the methoprene is merely causing an increase in the titers of 20-HE in the culture medium. This could cause the increase in vitellogenin messenger RNA observed by Racioppi et al. (1986).

Since oocyte development was more normal when a primer of either methoprene or 20-HE was given, it seems very likely that two increases in hormone titer occur in the mosquito after a blood meal and that this is necessary for normal oocyte development. Whether JH or 20-HE constitute the first peak is not clear. It is possible that both are present and have the same effect. Such redundancy is known to occur in endocrine systems (Bahr et al., 1974).

Acknowledgements--We thank Gerald Chu and Kimberly Wade for their assistance with some of these experiments. This work was supported by a Fulbright Fellowship to TM and the New York State Experiment Station at Cornell (project 420).

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