Ecdysone-initiated ovarian development in mosquitoes

J. Insect Physiol.,

1971,Vol.

17, pp.

1807to

1814. Pergamon Press. Printed in Great Britain

ECDYSONE-INITIATED OVARIAN DEVELOPMENT MOSQUITOES*

IN

ANDREW SPIELMAN,l ROBERT W. GWADZ,] and WINSTON A. ANDERSON27 IDepartment of Tropical Public Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, Mass. 02115 and 2Department of Anatomy and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, 25 Shattuck Street, Boston, Mass. 02115 (Received 16 April 1971) Abstract-Each of two analogues of insect growth hormone (22-isoecdysone and ecdysterone) stimulates vitellogenesis in Aedes aegypti, whereas two synthetic juvenile hormone preparations (a terpenoid mixture and Cecropia hormone) do not. Development of oiicytes is not affected by three diverse steroids (cholesterol, hydrocortisone, and estriole) or by phagostimulants normally present in vertebrate blood (ATP, serum, and haemoglobin). Two observations indicate that ecdysterone affects the ovary directly. Yolk is deposited when abdomens are ligated immediately after injection of hormone. Oocytes in excised ovaries pinocytose peroxidase from hormone-containing incubation media. Eggs mature after female A. aegypti, Culex pi&ens, or Anopheles quadrimacuZutus imbibe ecdysterone ad lib. However, relatively few eggs are deposited; most ovarian follicles degenerate. Such mature eggs are viable and give rise to normal anautogenous adult mosquitoes.

INTRODUCTION

MOSQUITOESgenerally begin vitellogenesis after taking a meal of vertebrate blood, and hormones initiate this response (DETINOVA, 1954). Although significant quantities of yolk appear about a day after feeding, other ovarian changes are detectable within a few hours. The most dramatic change is a proliferation of the micropinocytotic elements of each basal oijcyte (ROTH and PORTER,1964; ANDERSON and SPIELMAN,1971). Increased pinocytosis also occurs when certain somatic cells of moths are maintained in a medium containing insect growth hormone (JUDY, 1969). This suggests that growth hormone may likewise stimulate pinocytosis in oiicytes, and that ova may subsequently develop. Indeed, various analogues of growth hormone affect the ovaries of adult beetles (EARLE et al., 1969) and flies (WRIGHT and KAPLANIS, 1970), but the changes are degenerative. Furthermore, * This study was supported in part by Public Health Service Grant No. AI-00046, and by Research Contract No. NIH-69-2107. t Present address: Department of Anatomy, The University of Chicago, Chicago, Illinois. 1807

1808

ANDREW SPIELMAN, ROBERT W.

GWADZ,

ANDWINSTONA. ANJXR~ON

certain pharate adult moths appear to require ecdysone for normal development of eggs (SAHOTA,1969). We therefore designed experiments to determine whether growth hormone stimulates pinocytosis and vitellogenesis in the oocytes of mosquitoes and whether viable eggs result. MATERIALS AND METHODS Mosquitoes were derived from laboratory colonies of Aedes aegypti, Culex pipiens quinquefasciatus, and Anopheles quadrimaculatus; all maintained at 24°C and exposed to 16.hr of light each day. Except as otherwise noted, we used virgin female A. aegypti, 4 to 7 days after pupal-adult ecdysis. Only dry sucrose was provided as food for adult mosquitoes. For injection, unanaesthetized mosquitoes were transferred onto a screen and immobilized by air suction. Needles were fabricated from glass capillary tubing and calibrated; pressure was applied through an attached polyethylene tube and approx. 1~1 was injected into the abdomen. This was accomplished by inserting the needle between the thoracic pleurae and passing the tip posteriorly. Ecdysterone (85% pure, supplied by Dr. C. M. Williams, Harvard University) was dissolved in ethanol and diluted to a final concentration of 10, 2, 1, 0.1, and 0.01 mg/ml with saline (saline: LUM, 1961). In each solution, the final concentration of ethanol was 2 per cent. 2ZIsoecdysone (Dr. J. B. Sidall, Zoecon Corp., Palo Alto, California) was similarly prepared at a final concentration of 2 mg/ml 2% ethanol in .saline. Cholesterol (Sigma, St. Louis, MO.) was suspended at a concentration of 10 mg/ml 10% ethanol in saline. Hydracortisone sodium succinate (Upjohn, Kalamazoo, Mich.) was dissolved at a concentration of 10 mg/ml saline solution. Follicle stimulating hormone (N.I.H., Bethesda, Md.), was used undiluted. Haemoglobin (Sigma) was dissolved at a concentration of 10 mg/ml saline solution. Adenosine-5-triphosphate (Nutritional Biochemicals Co., Cleveland, Ohio) was dissolved at a concentration of 10 mg/ml saline solution. Chicken serum was used undiluted. In addition, minute amounts of crystalline ecdysterone, cholesterol, or estriole (Nutritional Biochemicals Co.) were implanted in the thorax of each of a series of mosquitoes. A crude hormonomimetic terpenoid mixture (Synthetic Juvenile Hormone, Grade B, Lot 860023, Calbiochem., Los Angeles, Calif.) was administered topically (O-002 ml of 10 mg/ml ethanol). Synthetic Cecropia juvenile hormone (Dr. J. B. Staal, Zoecon Co.) was similarly applied. Mosquitoes were dissected in Lum’s saline. Where indicated, individual ovarioles were suspended in drops of saline, and follicles measured with an ocular micrometer. Ovaries to be used in studies in sitro or in electron microscopy were excised without being disrupted. For investigations in oitro, ovaries from bloodstarved mosquitoes were maintained in KAHRI’S (1970) tissue culture medium at 24°C. In studies employing ligation, abdomens were tightly constricted in perforated elastic bands (Dr. J. B. Staal, personal communication) and thoraces cut away.

1809

ECDYSONE-INITIATED OVARIAN DEVELOPMENT IN MOSQUITOES

Horseradish peroxidase (Type IV; Sigma) was employed as a tracer compound in the tissue culture medium and in some solutions that were injected. Cultured ovaries were fixed after 2 hr of incubation, and those of injected mosquitoes were fixed 4 hr after injection. Ovaries were fixed for 30 min in 2% glutaraldehyde in 0-l M cacodylate buffer (pH 7.4) containing 5% sucrose and prepared for electron microscopy as described by GRAHAM and KARNOVSKY(1966). RESULTS

Oiicytes of A. aegypti undergo limited development during the first 3 days after ecdysis of the adult but thereafter development is arrested unless the adult obtains a blood meal. Within each arrested ovariole, basal follicles contain traces of yolk and are more than twice as long as distal follicles. Follicles of A. quadrimaculatus and C. p. quinquefasciatus are arrested in an earlier stage of development; basal follicles are approximately l-5 times as long as distal follicles, and oiiplasm is generally free of coarse yolk grains.

ETects of injected ecdysone anahgues Unfed mosquitoes were injected with ecdysterone and held for 3 days in order to determine whether follicles had developed normally. A few follicles developed in mosquitoes that received small doses (less than 1-O pg) of ecdysterone. Half of the mosquitoes receiving 1.0 pg contained well-developed ova (Table l), although TABLE ~-STAGE

OF DEVELOPMENT OF THE OVARIES OF VIRGIN

A. aegypti 3

days

AFTER INJECTION OF ECDYSTERONE

No. Ecdysterone/female

No.

with ovaries

of

females

Resting

0

22

22

0

0.01

16

14

1

1

0.1

17

7

4

6

kg)

Degenerate

Maturing 0

1-O

16

0

8

8

10-O

19

0

0

19

the mean length of these ova ( it 250 p) was approximately half that of mature ova of blood-fed mosquitoes. In other injected females, the primary oiicytes became acellular and degenerated. Ten pg of ecdysone stimulated vitellogenesis in all treated females. Follicles of control mosquitoes injected with saline were approximately 80 p long. A stereoisomer of ecdysone, 22-isoecdysone, was similarly tested. In contrast to results with ecdysterone, 10 pg doses of this material were highly toxic to female A. aegypti. Since most mosquitoes survived after 2 cl& of isoecdysone were inoculated, this dose was adopted and compared to the same dose of ecdysterone.

1810

ANDREWSPIELMAN,ROBERTW. GWADZ,ANDWINSTONA.

ANDERSON

Development of ovarian follicles proceeded after isoecdysone was injected into A. aegypti (Table 2). Some females commenced maturation of eggs while the ovaries of other mosquitoes degenerated. Thus isoecdysone and ecdysterone were equally stimulatory. TABLE 2-COMPARISON AFTRR

OF DEVELOPMENT INJECTION

OF THE

OF %ISOECDYSONE

OVARIES AND

OF A.

Uegyfiti

3

days

ECDYSTRRONE

No. with ovaries Inoculum Isoecdysone 2 pg/female Ecdysterone 2 pg/female Solvent control

No. of females

Resting

Degenerating

Maturing

31

0

20

11

9 16

0 16

4 0

5 0

To explore the range of activity of other compounds, mosquitoes 4 days after ecdysis that had never imbibed blood were treated with various nutrients and hormonally active substances. Groups of 20 A. aegypti were each injected with one of the following substances prepared as outlined above: cholesterol, hydrocortisone, follicle stimulating hormone, hemoglobin, adenosine-S-triphosphate, or chicken serum. In addition, crystalline cholesterol, estriole, and ecdysterone were implanted in groups of mosquitoes. Terpenoid mixture and synthetic Cenopia juvenile hormone were administered topically. Four days after treatment, ovaries were excised and the follicles measured. Only ecdysterone stimulated vitellogenesis. Changes in pinocytotic activity of oijcytes were studied using peroxidase, an electron-dense tracer. A. aegypti were inoculated with 1 pg of peroxidase combined with 1 pg of one of the following compounds: ecdysterone, cholesterol, hydrocortisone, or follicle stimulating hormone. In addition, 1 pg of peroxidase was injected into mosquitoes immediately after topical treatment with the terpenoid mixture. Mosquitoes were killed 4 and 7 hr after treatment (3 females/time period per treatment) and ovaries prepared for electron microscopy. Peroxidase was observed only in the oiicytes of 6 females treated with ecdysterone. Effect of ecdysterone on ovaries inoculated in vitro or after abdomenal ligation Ovaries were excised from 6 unfed mosquitoes and incubated for 4 hr in tissue culture medium containing 10 mg ecdysterone and 5 mg peroxidase/ml. They were then fixed and sectioned. Pinocytosis was a prominent feature of all oijcytes incubated with ecdysterone while it was virtually absent from 6 pairs of ovaries that were similarly handled but not exposed to ecdysterone. Peroxidase was present in pinocytotic vesicles of oiicytes incubated with ecdysterone, although these vesicles did not appear to fuse into yolk plaques. Peroxidase was present in control ovaries only in the narrow channels between cells.

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ECDYSONE-INITIATED OVARIANDEVELOPMENT IN MOSQUITOES

Each of 10 mosquitoes was injected with 10 pg of ecdysterone intra-abdominally as previously described and a ligature immediately placed between the thorax and abdomen. Four days later, ovaries were found to contain nearly mature eggs while those from 10 solvent-injected abdomens had not deposited yolk. EJfect of ecdysterone on mosquitoes of various ages

The stage of development at which ovaries of A. aegypti could be stimulated by ecdysterone was investigated. Time of adult ecdysis was recorded and mosquitoes were segregated into three age categories: O-l hr, 24 f 1 hr, and 72 I 1 hr. Ten pg of ecdysterone were injected into each female and ovaries were excised 3 days later. Newly ecdysed females were incapable of ecdysterone-induced ovarian development (Table 3). Degenerate follicles were common in l-day-old females but rare in 3-day-old females. Ovaries apparently became capable of maturing eggs during the second day after ecdysis. TABLE 3-RELATIONSHIP BETWEENAGEOF A. aegypti AT THE TIME OF INOCULATION WITH ECDYSTERONB AND RESULTINGOVARIANDEVELOPMENT

Age of female at time of inoculation 1 hr

1 day

3 days

No. of females with ovaries

No. of females

Resting

Degenerating

1Opg ecdysterone Solvent

20

16

4

0

10

10

0

0

1ocLg ecdysterone Solvent

74

0

40

34

15

15

0

0

1OcLg ecdysterone Solvent

10

0

0

10

10

10

0

0

Inoculum

Maturity

E#ect of injested ecdysterone

Male and female A. aegypti, C. p. quinquefasciatus, and A. quadrimaculatus were allowed to feed on cubes of sucrose impregnated with ecdysterone. Although eggs had never been previously observed unless females of these populations had fed upon blood, eggs were produced by mosquitoes fed an exclusive diet of ecdysterone and sucrose (Table 4). About 8 mg of ecdysterone/g of sucrose seemed optimal and most females responded in some measure to this dose. Relatively few eggs were deposited, but these were viable and gave rise to fertile adults. As a measure of the viability of eggs produced in this experiment, we studied 20 female A. aegypti that were reared from ecdysterone-stimulated eggs. Females were provided with dry sucrose but blood was withheld. All matured normally and were anautogenous; ovaries ceased development in the stage of arrest.

1812

ANDREWSPIELMAN, ROBERT W. GWADZ,ANDWINSTONA. ANDERSON TABLE ~-PRODUCTION OFEGGSBY MOSQUITOES THATIMBIBED ECDYSTERONE FROMIMPREGNATED SUGAR CUBES o/0 with eggs

Species

mg Ecdysterone/g sucrose

A. aegypti

c. p. qui?lqtqfasciatlLs A. quadrimaculatus

No. of females

Partially developed

Mature

Mean No. mature eggs/ clutch

0

61

8

0

-

2 4

29 10

30 80

3 0

-

8 16 20

97 19 28

52 84 89

14 26 0

-

0

0 100 63

0

-

8 20

14 13 57

0 4 8 16 20

6 6 6 6 10

0 33 66 100 70

23 0

2 9 3

17 -

0

-

16 0

-

16 0

-

3 4

DISCUSSION

Even when ingested, analogues of ecdysone stimulate vitellogenesis in mosquitoes. This suggests that ovarian development may be stimulated by exogenous ecdysone-like compounds in nature. Growth hormone is present in immature insects, and in various plants (review by OHTAKI et al., 1967), and mosquitoes may imbibe juices from both sources (HARRISand COOK, 1969; PATTERSONet al., 1969). The significance of exogenous steroids in the life cycle of mosquitoes remains to be determined. While ecdysone-analogues increase fecundity of blood-starved mosquitoes, these compounds may have a negative effect when blood-meals are abundant. Mosquitoes treated with ecdysterone produce far fewer eggs than do blood-fed females; follicles that fail to develop fully become degenerate. Furthermore, only arrested follicles respond to ecdysone-treatment. OScytes of teneral females are not affected while those of l-day-old A. aegypti tend to degenerate. Thus, small doses of natural steroids might serve to depress mosquito populations. Our studies indicate that ecdysterone affects the ovary directly. This steroid initiates oiigenesis after abdominal ligation and removal of the thorax. Moreover, ecdysterone stimulates excised cultured ovaries, as indicated by increased pinocytosis. Such an increase in pinocytotic activity is characteristic of ovaries of mosquitoes 4 hr after blood-feeding (ROTH and PORTER, 1964; ANDERSON and SPIELMAN, 1971).

Comparable changes have been reported for insect fat cells

(JUDY, 1969) and the excised ovaries of Drosophila take up phenol red only after in o&o exposure to ecdysone analogues (BURDETTEet al., 1968).

ECDYSONE-INITIATU) OVARIAN DEVELOPMENT IN MOsQUITOEs

1813

Although an isomer of ecdysone (22-isoecdysone) appears inactive when assayed with other insect tissues (FURLFXMEIERet al., 1967; JUDY, 1969), we found that the effect of this preparation upon the ovary of mosquitoes was similar to that Inokosterone too, is stimulatory (unpublished). However, of ecdysterone. cholesterol, which is structurally related to ecdysone, was without effect. Another steroid, hydrocortisone, induces maturation of eggs in certain fleas (ROTHSCHILDand FORD, 1964), but this compound did not stimulate ovarian activity in A. aegypti. Estriole was also tried because it is said to serve as a host attractant for mosquitoes (ROESSLERand BROWN,1964); we reasoned that a feeding stimulant might stimulate consequent ovarian function. However, estriole did not appear to influence vitellogenesis. Similar reasoning led us to try several compounds not related to ecdysone. Adenosine triphosphate is a constituent of vertebrate blood that is an essential feeding stimulant (HOSOI, 1959). S erum, too, is required for successful feeding (MASON et al., 1965). However, neither of these compounds, nor various combinations of them, nor haemoglobin, were effective in our studies. Finally, follicle stimulating hormone was tried because it induces an increased rate of pinocytosis in the oiicytes of vertebrates (SCHJEIDEet al., 1963). However, this polypeptide did not influence vitellogenesis in A. aegypti. Juvenile hormone stimulates the ovaries of diverse insects to commence vitellogenesis (WIGGLESWORTH,1961). Even such a weakly hormonomimetic compound as farnesol is effective. However, mosquitoes may not depend upon juvenile hormone as the sole initiator of oiigenesis (LEA, 1969) and our observations support this concept. Neither the crude terpenoid mixture nor pure synthetic Cecropia hormone were effective, although both compounds derange metamorphoses of mosquitoes (SPIELMANand SKAFF, 1967; and unpublished observations). The recognized source of growth hormone in insects lies in the prothoracic glands and these disappear during metamorphosis. Although CHADWICK(1956) suggests that growth hormone may normally play a role in adult insects, such has not been conclusively demonstrated. Thus, it is difficult to interpret the biological significance of the ability of ecdysone-analogues to initiate vitellogenesis. Our findings suggest a potential oiigenic function for ecdysone and indicate that this function cannot be fulfilled until 2 days after adult ecdysis. REFERENCES ANDERSON W. A. and SPISLMANA. (1971) Permeability of the ovarian follicle of Aedes aegypti mosquitoes. g. Cell Biol. In press. BURDETTEW. J., HANLBYE. W., and GROSCHJ. (1968) The effect of ecdysones on the maintenance and development of ocular imaginal discs in vitro. Texas Rept biol. Med. 26, 173-180.

CHADWICK L. E. (1956) Removal of prothoracic glands from the nymphal cockroach. 2. exp. 2001. 131, 291-305. DETINOVA T. S. (1945) On the in%ence of glands of internal secretion upon the ripening of the gonads and the imaginal diapause in Anopheles maculipemis. 298 (in Russian).

Zool. Zh. 24, 291-

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ANDR~Y SPIELMAN,ROBERTW. GWADZ, AND WINSTON A. ANDERSON

EARLE N. W., PADOVANII., THOMPSONM. J., and ROBBINSW. E. (1969) Inhibition of larval development and egg production in the boll weevil following ingestion of ecdysone analogues. J. econ. Ent. 63, 1064-1069. FURLENMEIERA., FOR~T A., LANGEMANNA., WALDVOGELG., Hoc= P., KERFI V., and WIECIIERT R. (1967) Zur Synthese des Ecdysons. Helv. chim. Acta 50, 2387-2996. GRAHAMR. C. and KARNOVSKYM. J. (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. r. Histochem. Cytochem. 14, 291-302. HARRISP. and COOKED. (1969) Survival and fecundity of mosquitoes fed on insect haemolymph. Nature, Lond. 222, 1264-1268. HOSOIT. (1959) Identification of blood components which induce gorging of the mosquito. J. Insect Physiol. 3, 191-218. JUDY K. J. (1969) Cellular response to ecdysterone in vitro. Science, Wush. 165, 1374-1375. KAHRI A. I. (1970) Selective inhibition by chloramphenicol of ACTH-induced reorganization of inner mitochondrial membranes in fetal adrenal cortical cells in tissue cultures. Am.J. Anat. 127, 103-130. LEA A. 0. (1969) Egg maturation in mosquitoes not regulated by the corpora allata. J. Insect Physiol. 15, 537-541. LUM P. T. M. (1961) The reproductive system of some Florida mosquitoes-I. The male reproductive tract. Ann. ent. Sot. Am. 54, 397-401. MASONJ. O., FROTHINGHAM T. E., SPIELMANA., and WELLER T. H. (1965) Transmission of sindbis virus by Aedes aegypti to a chick embryo cell culture system. Proc. Sot. exp. Biol. Med. 118, 736-741. OHTAKIT., MILKMANR. D., and WILLIAMSC. M. (1967) Ecdysone and ecdysone analogues: their assay on the fleshfly Sarcophaga peregrine. Proc. nut. Acad. Sci. U.S.A. 58, 981984. PATTERSON R. S., SMITTLEB. J., and DENEVE R. T. (1969) Feeding habits of male southern house mosquitoes on *3P-labeled and unlabeled plants. g. econ. Ent. 62, 1455-1458. ROESSLERP. and BROWN A. W. A. (1964) Studies on the responses of the female Aedes mosquito-x. Comparison of oestrogens and amino acids as attractants. Bull. e-nnt.Res. 55, 395-403. ROTH T. F. and PORTERK. R. (1964) Yolk protein uptake in the oijcyte of the mosquito Aedes aegypti. J. Cell Biol. 20, 313-332. ROTHSCHILDM. and FORD B. (1964) Maturation and egglaying of the rabbit flea (Spilopsyllus cuniculi Dale) induced by the external application of hydrocortisone. Nature, Lond. 203, 210-211. SAHOTAT. S. (1969) Hormonal control of ovarian development and metamorphosis in Malacosoma pluviale. Can. J. Zool. 47, 917-920. SCHJEIDE0. A., WILKINS M., MCCANDLESSR. G., MUNN R., PETERSONM., and CARLSEN E. (1963) Liver synthesis, plasma transport and structural alterations accompanying passage of yolk proteins. Am. 2001. 3, 167-184. SPIELMANA. and SKAFF V. (1967) Inhibition of metamorphosis and of ecdysis in mosquitoes. J. Insect Physiol. 13, 1087-1095. WIGGLE~W~RTHV. B. (1961) Some observations on the juvenile hormone effect of farnesol in Rhodnius prolixus SW (Hemiptera). r. Insect Physiol. 7, 73-78. WRIGHT J. E. and KAPLANISJ. N. (1970) Ecdysones and ecdysone analogues: effects on fecundity of the stable fly, Stomoxys calcitrans. Ann. ent. Sot. Am. 63, 622-623.