- Email: [email protected]
Relationship between the corpus cardiacum-allatum complex and ovaries with the haemolymph ecdysteroid profile in the housefly, Musca domestica
J. hsecr Physiol.Vol. 34, No. 12, PP. 1105-l 109. 1988 Printed in Great Britain
0022-1910188 $3.00 + 0.00 Pergamon Press plc
RELATIONSHIP BETWEEN THE CORPUS CARDIACUM-ALLATUM COMPLEX AND OVARIES WITH THE. HAEMOLYMPH ECDYSTEROID PROFILE IN THE HOUSEFLY, MUSCA DOMESTICA T. S. *Metabolism Agriculture,
ADAMS*, THOMAS J. KBLLVt
and CHARLES W. WOODSt
and Radiation Research Laboratory, Agricultural Research Service, U.S. Department of Fargo. ND 58105 and tInsect Reproduction Laboratory, Agricultural Research Service. U.S. Department of Agriculture, Beltsville, MD 20705, U.S.A. (Received
1 March 1988; revised 28 April 1988)
Abstract-Haemolymph from female houseflies at 4,48 and 96 h after emergence contained approximately equivalent amounts of 20-hydroxyecdysone that varied from 2.9 to 4. I pg/pl. Polar ecdysteroids decreased with the insect age from 12.9 pg/pl at 4 h to 5. I pg/pl at 96 h. At 48 h the haemolymph contained ecdysone and 20,26-dihydroxyecdysone in addition to 20-hydroxyecdysone and the polar materials. When ovaries were cultured in vitro, the culture medium contained ecdysone as the major ecdysteroid (70%) with lesser concentrations of 26-hydroxyecdysone. 20-hydroxyecdysone, and 20,26-dihydroxyecdysone. Haemolymph from ovariectomized flies contained 20-hydroxyecdysone. some polar materials, and very little ecdysone. Thus, it appears that the ovaries are the source of ecdysone. Since all samples contained 20-hydroxyecdysone, its source is not known. Removal of the corpus allatum-cardiacum complex decreased the amounts of all ecdysteroids in the haemolymph sample. Ecdysone and 20-hydroxyecdysone were both present in amounts of 1pg/pl and 20,26-dihydroxyecdysone was barely detectable. Key HPLC,
Word index: Ecdysone, 20-hydroxyecdysone, RIA, ovary, tissue culture
INTRODUCTION
Adams et al. (1985) have determined previously the levels of ecdysteroid by radioimmunoassay (RIA) for adult houseflies at diiferent ages and after removal of the corpus allatum-cardiacum complex or ovaries, but no attempt was made to determine the specific ecdysteroids responsible for the RIA activity. High pressure liquid chromatography (HPLC) coupled with RIA have been used with a variety of insects to identify the presence of specific ecdysteroids (Kelly et al., 1981. 1984; Loeb er al., 1982; Rubenstein et al., 1982; Bownes et al., 1984; Borovsky et al., 1986; Richard et al.. 198711.This technique was utilized to characterize specific ecdysteroids present in flies of different ages, in houseflies without the corpus cardiacurn-allatum ‘complex and/or ovaries and in cultured ovaries and tissue culture medium after in vitro incubation. MATERIALS
AND METHODS
Insects Houseflies of the Orlando regular strain were reared by methods described previously (Adams and Nelson, 1969). Flies were held at 27°C 3&40% r.h. and a photoregime of 12 h light and 12 h dark. Ovarian maturation was scored on a IO-stage scheme where stage 4 is the beginning of vitellogenesis and
Mention of a commercial or proprietary constitute a recommendation for Department of Agriculture.
product use by
does not the U.S.
20.26-dihydroxyecdysone,
stage 10 is a mature chorionated follicle (Adams, 1974). Ovaries and the corpus cardiacurn-allatum complex were removed from the flies by the technique of Adams and Hintz (1969). Insects were anaesthetized with carbon dioxide and placed on ice before surgery. The corpus cardiacum-allatum complex or the ovaries were removed from insects at 4-6 h after emergence. In some cases flies were placed in a cooler at 5°C at the above ages and held overnight before surgery. When both organs were extirpated, the corpus cardiacurn-allatum complex was removed at 4-6 h after emergence and the ovaries were removed the following morning. Haemolymph collection
Haemolymph was collected from insects in a 1~1 Dade Volupette as described by Adams et al. (1985) and placed in a borosilicate test tube that contained methanol. The sample was kept chilled on ice during the collection period. This methanol mixture was stored at - 20°C until 0.3-l .Oml haemolymph had been collected. Tissue culture
were surface Flies, 48 h after emergence, sterilized for 7min in 70% ethanol in water that contained 5% Triton X-100, rinsed successively in Schneider’s modified Drosophila medium (GIBCO) and then in Schneider’s medium with Gentomycin (50 pg/ml). Ovaries were removed, rinsed in the medium with the antibiotic and then placed in Schneider’s medium that contained 1% foetal calf serum. A single ovary was incubated for 24 h at 21°C
1105
I P 34,12--c
26-hydroxyecdysone,
T. 8. ADAMS et al.
1106
in 50~1 of the medium. Following incubation the media from 20 cultures was pooled and lyophilized. The ovaries from these cultures were placed in methanol and stored at -20°C. This experiment was repeated 4 times, Ecdysteroid extraction
Haemolymph samples in methanol for ecdysteroid extraction were placed on a water bath at 40°C and taken to dryness under nitrogen gas. Samples were diluted into 75% methanol, vortexed vigorously, centrifuged at 3000g for 10 min and the supernatant was removed and then stored until fractionation by HPLC. Lyophilized tissue culture media was extracted in the same manner as the dried haemolymph samples. Cultured ovaries were stored in methanol, ground in a Brinkmann Polytron homogenizer and dried down on a water bath under nitrogen. The resulting residue was extracted as before. HPLC fractionation
Samples containing the extracted ecdysteroids were blown down under nitrogen on a water bath at 40°C. These samples were then diluted into 40% methanol in water and fractionated on a reverse-phase Cl8 p Bondapak column (Water’s Associates; 3.9 mm id. x 37cm, 10~ particle size). The flow rate was 2ml/min and 0.6ml fractions were collected. RIA was subsequently conducted on each 0.6 ml fraction and the ecdysteroids were identified by retention times relative to known standards. At least 2 aliquots from the same haemolymph sample were analyzed by HPLC and a representative analysis was used for preparing the graphs. Radioimmunoassay
The RIA technique has been described previously (Borst and O’Conner, 1972; Bollenbacher et al., 1975). The antibody was prepared from a 22-OH-hemisuccinate derivative of ecdysone and was a gift from W. E. Bollenbacher. This antibody had a high affinity for ecdysone and 20-hydroxyecdysone (Gilbert et al., 1977). The ratio of the mass of 20-hydroxyecdysone required to displace 50% of the labelled ecdysone compared to the mass of the ecdysone is 2.8. The 23,24-3H(N)ecdysone (63.5 Ci/mmol) was purchased from New England Nuclear Corporation (Boston, MA). The 20-hydroxyecdysone used as a standard for RIA was obtained from Sigma (St Louis, MO) and the results are expressed as pg 20-hydroxyecdysone equivalents. RESULTS
Age study
The pattern of ecdysteroids found in the haemolymph of flies varied both quantitatively and qualitatively with fly age from eclosion (Fig. 1). Newly emerged flies within 4 h of eclosion had previtellogenic ovaries at stage 2 and the haemolymph contained 19.9pg/kl of total RIA positive ecdysteroid of which 64.8%, 12.9 pg/pI, was found in the fraction corresponding to the polar materials and another 19.1% of the activity, 3.8 pg/pl, co-chromatographed with 20-hydroxyecdysone (Fig. 1A). The remaining 16% of the RIA activity was associ-
NPLC FRACTION
NUMBER
Fig. 1. Ecdysteroid profiles from haemolymph removed from female Musca domestica at different ages. Samples were fractionated on an HPLC with a Waters Cl8 I( Bondapak column and fractions were then analyzed by RIA. (A) Profile from flies 4 h after adult emergence based on a 75 ~1 aliquot from a 1002 ~1 sample from 2000 flies. (B) 48 h after adult emergence based on a 100 ~1 aliquot from a 1000 ~1 sample from 3400 flies and (C) 96 h after adult emergence based on a 150 ~1 aliquot from a 943 ~1 sample from 2418 flies of haemolymph respectively.
ated with unidentified ecdysteroids. Flies 48 h after eclosion contained vitellogenic ovaries at stages 5-7 and their haemolymph had 17.7 pg/pl of RIA positive ecdysteroids that cochromatographed with 20,26-dihydroxyecdysone (11.9% of the activity, or 2.1 pg/pl) and ecdysone (20.3% of the activity, or 3.6pg/pl) [Fig. lB]. The amount of 20-hydroxyecdysone remained unchanged from the 4 h sample at 4.1 pg/pl and accounted for 23.1% of the total activity but the polar materials decreased to 7.4 pg/pl and accounted for 41.8% of the total RIA positive activity. Further changes in ecdysteroids were found in the sample of haemolymph from 96 h old flies after emergence (Fig. 1C) that had postvitellogenic ovaries at stage 10. The total ecdysteroid as well as the specific ecdysteroids decreased from that found in the 48 h sample. The total ecdysteroid level was 8.3 pg/pl and the polar materials decreased to 5.1 pg/pl (61.4% of the total), 20-hydroxyecdysone decreased to 2.9 pg/pl, ecdysone decreased to 0.1 pg/pl and 20,26-dihydroxyecdysone decreased to 0.1 pg/p 1. Removal of the corpus cardiacum-allatum and/or ovaries
complex
The haemolymph ecdysteroid pattern of these operated flies at the time of surgery is comparable to that of Fig. 1A and the unoperated control pattern at 96 h after emergence is represented by Fig. IC. Haemolymph from ovariectomized flies (Fig. 2A) contained 8.4 pg/pl of total RIA positive ecdysteroid of which 1.3 pg/pl consisted of polar material (15.5%), 4.6 pg/pl of 20-hydroxyecdysone (54.8%), and 0.6 pg/,ul of ecdysone. When the corpus allatumcardiacurn complex was removed ovarian maturation stopped at stage 4 and the amount of total ecdysteroid was only 3.5 pg/pl and consisted of polar
Ecdysteroid profile in M. domestica
HPLC FRACTION
NUMBER
Fig. 2. Haemolymph ecdysteroid profiles from ovariectomized flies based on a 300 ~1 aliquot from a 941 ~1 sample from 1809 flies (A) or flies without the corpus allatum-cardiacum complex based on a 176 ~1 aliquot from a 435 pl sample from 72.5 flies of haemolymph respectively (B). Flies were 46 h old after emergence at the time of surgery and haemolymph was collected from insects that were 46 days old. Samples were fractionated on an HPLC with a Waters C18~ Bondapak column and the fractions were then analyzed by RIA.
materialsA. pg/pl(22.8%), 20-hydroxyecdysone1.2pg/pl (34.3%), and ecdysone-1.1 pg/pl (31.4%) (Fig. 2B). Removal of both the corpus allatumcardiacum complex and the ovaries resulted in a total ecdysteroid level of 3.7 pg/pl and no HPLC fractions had RIA activity that corresponded with any of the known ecdysteroid standards. Ovaries in vitro
When ovaries at rnidvitellogenesis from 48-h old flies were cultured in vitro for 24 h, ecdysteroids were found in both the ovary (Fig. 3A) and in the media (Fig. 3B). The cultured ovaries contained 81 pg/ovary of total RIA ecdysteroid of which 35 pg, 72%, was found in the HPLC fraction that corresponded with
Fig. 3. Ecdysteroid profiles from cultured ovaries (A) and culture medium (B) based on 20 ovary equivalents. Ovaries were removed from flies 48 h after emergence and then cultured for 24 h at 21°C. Samples were fractionated on an HPLC with a Waters Cl8~ Bondapak column and the fractions were analyzed by RIA.
1107
the polar materials. Lesser amounts of 20-hydroxyecdysone, 3.5 pg (7%) and ecdysone, 9.4 pg (19%), were found. The media contained much smaller amounts of the polar material, 7.1 pg/ovary and it accounted for only 9% of the total RIA activity. The media, however, contained the following RIA active fractions that co-eluted with known ecdysteroids: 20,26-dihydroxyecdysone, 3.3. pg (4%); 20-hydroxyecdysone, 9.2 pg (11%); 26-hydroxyecdysone, 2.1 pg (3%); and ecdysone 56.6pg (70%). Thus, the majority of the ecdysteroid found in the ovary was associated with the fraction containing the polar materials but in the media it was associated with the ecdysone fraction. DISCUSSION
It is not possible to make direct comparisons between the previous work on ecdysteroid levels in Musca domesrica (Adams ef al., 1985) and the present work because a different antisera was used and the haemolymph samples were not separated by HPLC. The housefly, Musca domestica, showed both qualitative and quantitative changes in ecdysteroid patterns during the first gonotrophic cycle. Newly emerged flies contained the majority of their ecdysteroid as polar materials and material that co-eluted on HPLC with 20-hydroxyecdysone. The major ecdysteroids in the pupae of Sarcophaga argyrostoma were also 20-hydroxyecdysone and polar ecdysteroids (Richard et al., 1987). If this were the case in Musca, the 20-hydroxyecdysone and polar products found in the newly emerged fly could be holdovers from the pupal stage as suggested by Kelly et al. (1986). Flies at mid-vitellogenesis that were 48 h after emergence contained RIA active material that co-eluted with ecdysone, 20.hydroxyecdysone, and 20,26-dihydroxyecdysone. Haemolymph samples from vitellogenic female Calliphora vomitoria contained both ecdysone and 20-hydroxyecdysone with the latter being predominant (Campan et al., 1985). Aedes atropalpus (Kelly et al., 1984) and Aedes aegypti females also contained both ecdysone and 20-hydroxyecdysone 24 h after adult emergence or a blood meal respectively. Thus, it appears that vitellogenic female Diptera contain both ecdysone and 20-hydroxyecdysone. Samples from 96-h-old flies with postvitellogenic ovaries contained RIA active materials that co-eluted with 20-hydroxyecdysone and barely detectable amounts of ecdysone. Decreases in the amount of ecdysteroid found in postvitellogenic flies has been reported previously for Musca (Adams et al., 1985) and Calliphora vomitoria (Campan et al., 1985). The source of ecdysteroid has been examined for several different species of Diptera. Hagedorn et al. (1975) were the first to show that the ovaries of Aedes aegypti produced ecdysone. Subsequent studies showed that A. aegypti ovaries also produced 20-hydroxyecdysone in vitro (Smith and Mitchell, 1986). A. atropalpus (Birnbaum et al., 1984) and Drosophila melanogaster (Rubenstein et al., 1982; Schwartz ef al., 1985) ovaries produced both ecdysone and 20-hydroxyecdysone in vitro and were the major source of ecdysteroid production in the adult female. Ecdysone 20-monooxygenase hydroxylates
T. S. ADAMS et al.
1108
ecdysone to 20-hydroxyecdysone and this enzyme has been found in the body wall/fatbody, gut and Malphigian tubules, and ovaries of A. aegypti (Smith and Mitchell, 1986). Pharate pupae of Surcophugu peregrina metabolized ecdysone through 20-hydroxyecdysone to 26-hydroxyecdysone to 3-epi-26-hydroxyecdysone and 3-epi-20,26-dihydroxyecdysone (Moribayashi et al., 1985). The ovaries of Musca have
been shown to secrete and synthesize ecdysteroids (Adams et al., 1985). and the tissue culture media from Muscu ovaries at midvitellogenesis contained ecdysone as the major co-eluting. RIA active ecdysteroid (70%), with 20-hydroxyecdysone (11%). 20,26-dihydroxyecdysone (4%), 26-hydroxyecdysone (3%). These same ecdysteroids were present in the haemolymph of 48-h old flies. Ovariectomized flies contained very little ecdysone and the major ecdysteroid was 20-hydroxyecdysone. Ovariectomized Aedes uegypti also contained decreased amounts of ecdysone (Hagedorn et ai., 1975). The source of the 20-hydroxyecdysone in ovariectomized flies is not known at this time, but it may be a holdover from before emergence as suggested by Kelly et al. (1986). Thus, it is possible
that the vitellogenic
ovary is the
source of ecdysone in normal flies and the remaining ecdysteroids are ecdysone metabolites. Further experiments on tissue specific ecdysteroid production and metabolism in the housefly will be necessary to answer these questions. Cultured vitellogenic ovaries from Muscu had most of the RIA-positive ecdysteroid activity in the polar fraction with lesser amounts in the ecdysone and 20-hydroxyecdysone fractions. Ovaries of Culliphora uomitoria (Campan et al., 1985) and Sarcophagu bullatu (Briers et al., 1983) had an ecdysteroid pattern that was similar to that found in Musca. Surcophuga bullutu ovaries at midvitellogenesis were able to absorb ecdysone from the haemolymph and this was converted to 20-hydroxyecdysone and then to polar products in the oijplasm (Briers et al., 1982). Such a scheme would explain the ecdysteroid pattern found in vitellogenic Musca ovaries. The presence of the same approximate level of 20-hydroxyecdysone in haemolymph samples from houseflies at different ages and in ovariectomized insects has physiological implications in the regulation of vitellogenin production and in pheromone biosynthesis. Vitellogenin was not detected in newly emerged females but was present when the flies had vitellogenic ovaries (Adams and Filipi, 1983). Since 20-hydroxyecdysone was present at vitellogenic levels in newly emerged flies but vitellogenin was not. this would indicate that 20-hydroxyecdysone alone could not induce vitellogenin synthesis. Vitellogenin synthesis in Muscu requires both juvenile hormone and 20-hydroxyecdysone (Adams and Filipi, 1988). Other Diptera also require both hormones for vitellogenin synthesis (Borovsky et al., 1985; Kelly et al., 1987). When female flies were injected with 1.Ong of methoprene at 4 h after emergence, 50% of the samples contained vitellogenin 24 h later but none of the controls did (Adams and Filipi, unpublished). This indicates that increasing levels of juvenile hormone are probably responsible for the appearance of vitellogenin in the haemolymph of adult houseflies.
The biosynthesis of the housefly sex pheromone, (Z)-9-tricosene, did not occur in ovariectomized flies but was induced by injections of 20-hydroxyecdysone at physiological levels (0.1-10.0 pg) [Adams et al., 19841. Endogenous levels of 20-hydroxyecdysone in 48-h old flies and in ovariectomized flies was 4.1 pg/pl and 4.6 pg/pl respectively, yet the ovariectomized flies produced no (Z)-9-tricosene but the 48-h old flies did. The ovariectomized fly is missing both ecdysone and 20,26-dihydroxyecdysone, both of which are present in the haemolymph of 48-h old flies. This suggests that another ecdysteroid might be responsible for pheromone biosynthesis and that 20-hydroxy-ecdysone is acting as a mimic when it is administered at a physiological dose. Thus, these findings require a reevaluation of the role of 20-hydroxyecdysone in housefly sex pheromone biosynthesis.
REFERENCES
Adams T. S. (1974) The role of juvenile hormone in housefly
ovarian follicle morphogenesis. J. Insecr Phvsiol. 20,
263-276.
Adams T. S. and Filipi P. A. (I 983) Vitellin and vitellogenin concentrations during oogenesis in the first gonotrophic cycle of housefly, Musca domestica. J. Insect Physioi. 29, 723-733.
Adams T. S. and Filipi P. A. (1988) Interaction between juvenile hormone. 20-hydroxyecdysone. the corpuss cardiacum allatum complex, and the ovaries in regulating vitellogenin levels in the housefly, Musca domestica. J. Insect Physiol. 34,
I l-19.
Adams T. S. and Hintz A. M. (1969) Relationship of age, ovarian development. and the corpus allatum to mating in the housefly, Musca domestica. J. Insect Physiol. 15, 201-215.
Adams T. S. and Nelson D. R. (1969) Effect of corpus allatum and ovaries on the amount of pupal and adult fatbody in the housefly, Musca domestica. J. Insecr Physiol. 15, 1729-1747.
Adams T. S., Dillwith J. W. and Blomquist G. J. (1984) The role of 20-hydroxyecdysone in housefly sex pheromone biosynthesis. J. Insect Phvsiol. 30, 287-294. Adams T. S., Hagedorn H. H. and Wheelock G. D. (1985) Haemolymph ecdysteroids in the housefly, Musca domesrica, during oogenesis and its relationship with vitellogenin lev&. JT Insect Physiol. 31, 91-97.. Birnbaum M. J.. Kellv T. J.. Woods C. W. and Imberski R. B. (1984) Hormonal regulation of ovarian ecdysteroid production in the autogenous mosquito, Aedes atropalpus. Gen. camp. Endocr. 56, 9-18.
Bollenbacher W. E., Vedeckis W. V., Gilbert L. I. and O’Conner J. D. (1975) Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta. Dev. Biol. 44, 46-53.
Borovsky D., Thomas B. R.. Carlson D. A., Whisenton L. R. and Fuchs M. S. (1985) Juvenile hormone and 20-hydroxyecdysone as primary’and secondary stimuli to vitellogenesis in Aedes aegypfi. Arch. Insect Biochem. Physiol. 2, 75-96.
Borovsky D., Whisenton L. R., Thomas B. R. and Fuchs M. S. (1986) Biosynthesis and distribution of ecdysone and 20-OH-ecdysone in Aedes aegypti. Arch. Insect Biochem. Physiol. 3, 19-30.
Borst D. W. and O’Conner J. D. (1972) Arthropod molting hormone: Radioimmune assay. Science 178, 418-419. Bownes M., Dubendorfer A. and Smith T. (1984) Ecdysteroids in adult males and females of Drosophila melanogasfer. J. Insect Physiol. 30, 823-830.
Ecdysteroid profile in M. domesrica Briers T., Stoppie P. and De Loof A. (1982) In vitro uptake and metabolism of [:H]ecdysone in the vitellogenic follicles of Sarcophag,a bullato and its location by autoradiography. &t. 1. Invert. Reprod. 4, 343-347, Briers T., Van Beek E. and De Loof A. (1983) Metabolism of injected ecdysone in female Sarcophaga bullata. Comp. Biochem. Physiol. 75B, 9914. Campan M., Strambi A. and Strambi C. (1985) Variations of ecdysteroids in the ovaries and haemolymph during the first gonadotropic cycle of female Calliphora vomitoria. Comp. Biochem. Physiol. 82A, 225-23 1, Gilbert L. I., Goodman W. and Bollenbacher W. E. (1977) Biochemistry of regulatory lipids and sterols in insects. Biochemistry of lipids. Int. Rev. Biochem. 14, l-50.
Hagedorn H. H., O’Conner J. D., Fuchs M. S., Sage B., Schlaeger D. A. and Bohm M. K. (1975). The ovary as a source of ecdysone in the adult mosquito. Proc. natn Acad. Sci. U.S.A. 72, 3255-3259.
Kelly T. J., Adams T. S. and Woods C. W. (1986) Ecdysteroids and dipteran vitallogenesis. In Host Regulated Developmental Mechanisms in Vector Arthropods (Ed. by Borovsky D. and Spielman A.). pp. 6672. University of Florida Press, Vero Beach. Kelly T. J., Birnbaum M. J., Woods C. W. and Borkovec A. B. (1984) Effect of housefly oostatic hormone on egg development neurosecretory hormone action in Aedes atropalpus. J. exp. Zool. 229, 491496.
Kelly T. J.. Woods C. W., Redfern R. E. and Borkovec
1109
A. B. (1981) Makisterone A: The molting hormone of larval Oncopelius? J. exp. Zool. 218, 127-132. Kelly T. J., Adams T. S., Schwartz M. B., Birnbaum M. J.. Rubenstein E. C. and Imberski R. B. (1987) Juvenile hormone and ovarian maturation in the diptera: A review of recent results. Insect Biochem. Insect Biochem. 17, 1089-1093.
Loeb M. J., Woods C. W., Brandt E. P. and Borkovec A. B. (1982) Larval testes of the tobacco budworm: a new source of insect ecdysteroids. Science 218, 896898. Moribayashi A., Kurahashi H. and Ohtaki T. (1985) Comparative studies on ecdysone metabolism between mature larvae and pharate pupae in the fleshfly, Sarcophaga peregrina. Arch. Insect Biochem. Physiol. 2, 237-250.
Richard D. S., Warren J. T., Saunders D. S. and Gilbert L. I. (1987) Haemolymph ecdysteroid titers in diapause and non-diapause destined larvae and pupae of Sarcophaga argyrostoma. J. Insect Physiol. 33, 115-122. Rubenstein E. C., Kelly T. J., Schwartz M. B. and Woods C. W. (1982) In vitro synthesis and secretion of ecdysteroids by Drosophila melanogaster ovaries. J. exp. Zool. 223, 305-308. Schwartz M. B., Kelly T. J., Imberski R. B. and Rubenstein E. C. (1985) The effects of nutrition and methoprene treatment on ovarian ecdysteroid synthesis in Drosophila melanogaster. J. Insect Physiol. 31. 9417957.
Smith ST L. and Mitchell M. .J. (1986) Ecdysone 20.monooxygenase systems in a larval and an adult dipteran. Insect Biochem. 16, 49-55.
0022-1910188 $3.00 + 0.00 Pergamon Press plc
RELATIONSHIP BETWEEN THE CORPUS CARDIACUM-ALLATUM COMPLEX AND OVARIES WITH THE. HAEMOLYMPH ECDYSTEROID PROFILE IN THE HOUSEFLY, MUSCA DOMESTICA T. S. *Metabolism Agriculture,
ADAMS*, THOMAS J. KBLLVt
and CHARLES W. WOODSt
and Radiation Research Laboratory, Agricultural Research Service, U.S. Department of Fargo. ND 58105 and tInsect Reproduction Laboratory, Agricultural Research Service. U.S. Department of Agriculture, Beltsville, MD 20705, U.S.A. (Received
1 March 1988; revised 28 April 1988)
Abstract-Haemolymph from female houseflies at 4,48 and 96 h after emergence contained approximately equivalent amounts of 20-hydroxyecdysone that varied from 2.9 to 4. I pg/pl. Polar ecdysteroids decreased with the insect age from 12.9 pg/pl at 4 h to 5. I pg/pl at 96 h. At 48 h the haemolymph contained ecdysone and 20,26-dihydroxyecdysone in addition to 20-hydroxyecdysone and the polar materials. When ovaries were cultured in vitro, the culture medium contained ecdysone as the major ecdysteroid (70%) with lesser concentrations of 26-hydroxyecdysone. 20-hydroxyecdysone, and 20,26-dihydroxyecdysone. Haemolymph from ovariectomized flies contained 20-hydroxyecdysone. some polar materials, and very little ecdysone. Thus, it appears that the ovaries are the source of ecdysone. Since all samples contained 20-hydroxyecdysone, its source is not known. Removal of the corpus allatum-cardiacum complex decreased the amounts of all ecdysteroids in the haemolymph sample. Ecdysone and 20-hydroxyecdysone were both present in amounts of 1pg/pl and 20,26-dihydroxyecdysone was barely detectable. Key HPLC,
Word index: Ecdysone, 20-hydroxyecdysone, RIA, ovary, tissue culture
INTRODUCTION
Adams et al. (1985) have determined previously the levels of ecdysteroid by radioimmunoassay (RIA) for adult houseflies at diiferent ages and after removal of the corpus allatum-cardiacum complex or ovaries, but no attempt was made to determine the specific ecdysteroids responsible for the RIA activity. High pressure liquid chromatography (HPLC) coupled with RIA have been used with a variety of insects to identify the presence of specific ecdysteroids (Kelly et al., 1981. 1984; Loeb er al., 1982; Rubenstein et al., 1982; Bownes et al., 1984; Borovsky et al., 1986; Richard et al.. 198711.This technique was utilized to characterize specific ecdysteroids present in flies of different ages, in houseflies without the corpus cardiacurn-allatum ‘complex and/or ovaries and in cultured ovaries and tissue culture medium after in vitro incubation. MATERIALS
AND METHODS
Insects Houseflies of the Orlando regular strain were reared by methods described previously (Adams and Nelson, 1969). Flies were held at 27°C 3&40% r.h. and a photoregime of 12 h light and 12 h dark. Ovarian maturation was scored on a IO-stage scheme where stage 4 is the beginning of vitellogenesis and
Mention of a commercial or proprietary constitute a recommendation for Department of Agriculture.
product use by
does not the U.S.
20.26-dihydroxyecdysone,
stage 10 is a mature chorionated follicle (Adams, 1974). Ovaries and the corpus cardiacurn-allatum complex were removed from the flies by the technique of Adams and Hintz (1969). Insects were anaesthetized with carbon dioxide and placed on ice before surgery. The corpus cardiacum-allatum complex or the ovaries were removed from insects at 4-6 h after emergence. In some cases flies were placed in a cooler at 5°C at the above ages and held overnight before surgery. When both organs were extirpated, the corpus cardiacurn-allatum complex was removed at 4-6 h after emergence and the ovaries were removed the following morning. Haemolymph collection
Haemolymph was collected from insects in a 1~1 Dade Volupette as described by Adams et al. (1985) and placed in a borosilicate test tube that contained methanol. The sample was kept chilled on ice during the collection period. This methanol mixture was stored at - 20°C until 0.3-l .Oml haemolymph had been collected. Tissue culture
were surface Flies, 48 h after emergence, sterilized for 7min in 70% ethanol in water that contained 5% Triton X-100, rinsed successively in Schneider’s modified Drosophila medium (GIBCO) and then in Schneider’s medium with Gentomycin (50 pg/ml). Ovaries were removed, rinsed in the medium with the antibiotic and then placed in Schneider’s medium that contained 1% foetal calf serum. A single ovary was incubated for 24 h at 21°C
1105
I P 34,12--c
26-hydroxyecdysone,
T. 8. ADAMS et al.
1106
in 50~1 of the medium. Following incubation the media from 20 cultures was pooled and lyophilized. The ovaries from these cultures were placed in methanol and stored at -20°C. This experiment was repeated 4 times, Ecdysteroid extraction
Haemolymph samples in methanol for ecdysteroid extraction were placed on a water bath at 40°C and taken to dryness under nitrogen gas. Samples were diluted into 75% methanol, vortexed vigorously, centrifuged at 3000g for 10 min and the supernatant was removed and then stored until fractionation by HPLC. Lyophilized tissue culture media was extracted in the same manner as the dried haemolymph samples. Cultured ovaries were stored in methanol, ground in a Brinkmann Polytron homogenizer and dried down on a water bath under nitrogen. The resulting residue was extracted as before. HPLC fractionation
Samples containing the extracted ecdysteroids were blown down under nitrogen on a water bath at 40°C. These samples were then diluted into 40% methanol in water and fractionated on a reverse-phase Cl8 p Bondapak column (Water’s Associates; 3.9 mm id. x 37cm, 10~ particle size). The flow rate was 2ml/min and 0.6ml fractions were collected. RIA was subsequently conducted on each 0.6 ml fraction and the ecdysteroids were identified by retention times relative to known standards. At least 2 aliquots from the same haemolymph sample were analyzed by HPLC and a representative analysis was used for preparing the graphs. Radioimmunoassay
The RIA technique has been described previously (Borst and O’Conner, 1972; Bollenbacher et al., 1975). The antibody was prepared from a 22-OH-hemisuccinate derivative of ecdysone and was a gift from W. E. Bollenbacher. This antibody had a high affinity for ecdysone and 20-hydroxyecdysone (Gilbert et al., 1977). The ratio of the mass of 20-hydroxyecdysone required to displace 50% of the labelled ecdysone compared to the mass of the ecdysone is 2.8. The 23,24-3H(N)ecdysone (63.5 Ci/mmol) was purchased from New England Nuclear Corporation (Boston, MA). The 20-hydroxyecdysone used as a standard for RIA was obtained from Sigma (St Louis, MO) and the results are expressed as pg 20-hydroxyecdysone equivalents. RESULTS
Age study
The pattern of ecdysteroids found in the haemolymph of flies varied both quantitatively and qualitatively with fly age from eclosion (Fig. 1). Newly emerged flies within 4 h of eclosion had previtellogenic ovaries at stage 2 and the haemolymph contained 19.9pg/kl of total RIA positive ecdysteroid of which 64.8%, 12.9 pg/pI, was found in the fraction corresponding to the polar materials and another 19.1% of the activity, 3.8 pg/pl, co-chromatographed with 20-hydroxyecdysone (Fig. 1A). The remaining 16% of the RIA activity was associ-
NPLC FRACTION
NUMBER
Fig. 1. Ecdysteroid profiles from haemolymph removed from female Musca domestica at different ages. Samples were fractionated on an HPLC with a Waters Cl8 I( Bondapak column and fractions were then analyzed by RIA. (A) Profile from flies 4 h after adult emergence based on a 75 ~1 aliquot from a 1002 ~1 sample from 2000 flies. (B) 48 h after adult emergence based on a 100 ~1 aliquot from a 1000 ~1 sample from 3400 flies and (C) 96 h after adult emergence based on a 150 ~1 aliquot from a 943 ~1 sample from 2418 flies of haemolymph respectively.
ated with unidentified ecdysteroids. Flies 48 h after eclosion contained vitellogenic ovaries at stages 5-7 and their haemolymph had 17.7 pg/pl of RIA positive ecdysteroids that cochromatographed with 20,26-dihydroxyecdysone (11.9% of the activity, or 2.1 pg/pl) and ecdysone (20.3% of the activity, or 3.6pg/pl) [Fig. lB]. The amount of 20-hydroxyecdysone remained unchanged from the 4 h sample at 4.1 pg/pl and accounted for 23.1% of the total activity but the polar materials decreased to 7.4 pg/pl and accounted for 41.8% of the total RIA positive activity. Further changes in ecdysteroids were found in the sample of haemolymph from 96 h old flies after emergence (Fig. 1C) that had postvitellogenic ovaries at stage 10. The total ecdysteroid as well as the specific ecdysteroids decreased from that found in the 48 h sample. The total ecdysteroid level was 8.3 pg/pl and the polar materials decreased to 5.1 pg/pl (61.4% of the total), 20-hydroxyecdysone decreased to 2.9 pg/pl, ecdysone decreased to 0.1 pg/pl and 20,26-dihydroxyecdysone decreased to 0.1 pg/p 1. Removal of the corpus cardiacum-allatum and/or ovaries
complex
The haemolymph ecdysteroid pattern of these operated flies at the time of surgery is comparable to that of Fig. 1A and the unoperated control pattern at 96 h after emergence is represented by Fig. IC. Haemolymph from ovariectomized flies (Fig. 2A) contained 8.4 pg/pl of total RIA positive ecdysteroid of which 1.3 pg/pl consisted of polar material (15.5%), 4.6 pg/pl of 20-hydroxyecdysone (54.8%), and 0.6 pg/,ul of ecdysone. When the corpus allatumcardiacurn complex was removed ovarian maturation stopped at stage 4 and the amount of total ecdysteroid was only 3.5 pg/pl and consisted of polar
Ecdysteroid profile in M. domestica
HPLC FRACTION
NUMBER
Fig. 2. Haemolymph ecdysteroid profiles from ovariectomized flies based on a 300 ~1 aliquot from a 941 ~1 sample from 1809 flies (A) or flies without the corpus allatum-cardiacum complex based on a 176 ~1 aliquot from a 435 pl sample from 72.5 flies of haemolymph respectively (B). Flies were 46 h old after emergence at the time of surgery and haemolymph was collected from insects that were 46 days old. Samples were fractionated on an HPLC with a Waters C18~ Bondapak column and the fractions were then analyzed by RIA.
materialsA. pg/pl(22.8%), 20-hydroxyecdysone1.2pg/pl (34.3%), and ecdysone-1.1 pg/pl (31.4%) (Fig. 2B). Removal of both the corpus allatumcardiacum complex and the ovaries resulted in a total ecdysteroid level of 3.7 pg/pl and no HPLC fractions had RIA activity that corresponded with any of the known ecdysteroid standards. Ovaries in vitro
When ovaries at rnidvitellogenesis from 48-h old flies were cultured in vitro for 24 h, ecdysteroids were found in both the ovary (Fig. 3A) and in the media (Fig. 3B). The cultured ovaries contained 81 pg/ovary of total RIA ecdysteroid of which 35 pg, 72%, was found in the HPLC fraction that corresponded with
Fig. 3. Ecdysteroid profiles from cultured ovaries (A) and culture medium (B) based on 20 ovary equivalents. Ovaries were removed from flies 48 h after emergence and then cultured for 24 h at 21°C. Samples were fractionated on an HPLC with a Waters Cl8~ Bondapak column and the fractions were analyzed by RIA.
1107
the polar materials. Lesser amounts of 20-hydroxyecdysone, 3.5 pg (7%) and ecdysone, 9.4 pg (19%), were found. The media contained much smaller amounts of the polar material, 7.1 pg/ovary and it accounted for only 9% of the total RIA activity. The media, however, contained the following RIA active fractions that co-eluted with known ecdysteroids: 20,26-dihydroxyecdysone, 3.3. pg (4%); 20-hydroxyecdysone, 9.2 pg (11%); 26-hydroxyecdysone, 2.1 pg (3%); and ecdysone 56.6pg (70%). Thus, the majority of the ecdysteroid found in the ovary was associated with the fraction containing the polar materials but in the media it was associated with the ecdysone fraction. DISCUSSION
It is not possible to make direct comparisons between the previous work on ecdysteroid levels in Musca domesrica (Adams ef al., 1985) and the present work because a different antisera was used and the haemolymph samples were not separated by HPLC. The housefly, Musca domestica, showed both qualitative and quantitative changes in ecdysteroid patterns during the first gonotrophic cycle. Newly emerged flies contained the majority of their ecdysteroid as polar materials and material that co-eluted on HPLC with 20-hydroxyecdysone. The major ecdysteroids in the pupae of Sarcophaga argyrostoma were also 20-hydroxyecdysone and polar ecdysteroids (Richard et al., 1987). If this were the case in Musca, the 20-hydroxyecdysone and polar products found in the newly emerged fly could be holdovers from the pupal stage as suggested by Kelly et al. (1986). Flies at mid-vitellogenesis that were 48 h after emergence contained RIA active material that co-eluted with ecdysone, 20.hydroxyecdysone, and 20,26-dihydroxyecdysone. Haemolymph samples from vitellogenic female Calliphora vomitoria contained both ecdysone and 20-hydroxyecdysone with the latter being predominant (Campan et al., 1985). Aedes atropalpus (Kelly et al., 1984) and Aedes aegypti females also contained both ecdysone and 20-hydroxyecdysone 24 h after adult emergence or a blood meal respectively. Thus, it appears that vitellogenic female Diptera contain both ecdysone and 20-hydroxyecdysone. Samples from 96-h-old flies with postvitellogenic ovaries contained RIA active materials that co-eluted with 20-hydroxyecdysone and barely detectable amounts of ecdysone. Decreases in the amount of ecdysteroid found in postvitellogenic flies has been reported previously for Musca (Adams et al., 1985) and Calliphora vomitoria (Campan et al., 1985). The source of ecdysteroid has been examined for several different species of Diptera. Hagedorn et al. (1975) were the first to show that the ovaries of Aedes aegypti produced ecdysone. Subsequent studies showed that A. aegypti ovaries also produced 20-hydroxyecdysone in vitro (Smith and Mitchell, 1986). A. atropalpus (Birnbaum et al., 1984) and Drosophila melanogaster (Rubenstein et al., 1982; Schwartz ef al., 1985) ovaries produced both ecdysone and 20-hydroxyecdysone in vitro and were the major source of ecdysteroid production in the adult female. Ecdysone 20-monooxygenase hydroxylates
T. S. ADAMS et al.
1108
ecdysone to 20-hydroxyecdysone and this enzyme has been found in the body wall/fatbody, gut and Malphigian tubules, and ovaries of A. aegypti (Smith and Mitchell, 1986). Pharate pupae of Surcophugu peregrina metabolized ecdysone through 20-hydroxyecdysone to 26-hydroxyecdysone to 3-epi-26-hydroxyecdysone and 3-epi-20,26-dihydroxyecdysone (Moribayashi et al., 1985). The ovaries of Musca have
been shown to secrete and synthesize ecdysteroids (Adams et al., 1985). and the tissue culture media from Muscu ovaries at midvitellogenesis contained ecdysone as the major co-eluting. RIA active ecdysteroid (70%), with 20-hydroxyecdysone (11%). 20,26-dihydroxyecdysone (4%), 26-hydroxyecdysone (3%). These same ecdysteroids were present in the haemolymph of 48-h old flies. Ovariectomized flies contained very little ecdysone and the major ecdysteroid was 20-hydroxyecdysone. Ovariectomized Aedes uegypti also contained decreased amounts of ecdysone (Hagedorn et ai., 1975). The source of the 20-hydroxyecdysone in ovariectomized flies is not known at this time, but it may be a holdover from before emergence as suggested by Kelly et al. (1986). Thus, it is possible
that the vitellogenic
ovary is the
source of ecdysone in normal flies and the remaining ecdysteroids are ecdysone metabolites. Further experiments on tissue specific ecdysteroid production and metabolism in the housefly will be necessary to answer these questions. Cultured vitellogenic ovaries from Muscu had most of the RIA-positive ecdysteroid activity in the polar fraction with lesser amounts in the ecdysone and 20-hydroxyecdysone fractions. Ovaries of Culliphora uomitoria (Campan et al., 1985) and Sarcophagu bullatu (Briers et al., 1983) had an ecdysteroid pattern that was similar to that found in Musca. Surcophuga bullutu ovaries at midvitellogenesis were able to absorb ecdysone from the haemolymph and this was converted to 20-hydroxyecdysone and then to polar products in the oijplasm (Briers et al., 1982). Such a scheme would explain the ecdysteroid pattern found in vitellogenic Musca ovaries. The presence of the same approximate level of 20-hydroxyecdysone in haemolymph samples from houseflies at different ages and in ovariectomized insects has physiological implications in the regulation of vitellogenin production and in pheromone biosynthesis. Vitellogenin was not detected in newly emerged females but was present when the flies had vitellogenic ovaries (Adams and Filipi, 1983). Since 20-hydroxyecdysone was present at vitellogenic levels in newly emerged flies but vitellogenin was not. this would indicate that 20-hydroxyecdysone alone could not induce vitellogenin synthesis. Vitellogenin synthesis in Muscu requires both juvenile hormone and 20-hydroxyecdysone (Adams and Filipi, 1988). Other Diptera also require both hormones for vitellogenin synthesis (Borovsky et al., 1985; Kelly et al., 1987). When female flies were injected with 1.Ong of methoprene at 4 h after emergence, 50% of the samples contained vitellogenin 24 h later but none of the controls did (Adams and Filipi, unpublished). This indicates that increasing levels of juvenile hormone are probably responsible for the appearance of vitellogenin in the haemolymph of adult houseflies.
The biosynthesis of the housefly sex pheromone, (Z)-9-tricosene, did not occur in ovariectomized flies but was induced by injections of 20-hydroxyecdysone at physiological levels (0.1-10.0 pg) [Adams et al., 19841. Endogenous levels of 20-hydroxyecdysone in 48-h old flies and in ovariectomized flies was 4.1 pg/pl and 4.6 pg/pl respectively, yet the ovariectomized flies produced no (Z)-9-tricosene but the 48-h old flies did. The ovariectomized fly is missing both ecdysone and 20,26-dihydroxyecdysone, both of which are present in the haemolymph of 48-h old flies. This suggests that another ecdysteroid might be responsible for pheromone biosynthesis and that 20-hydroxy-ecdysone is acting as a mimic when it is administered at a physiological dose. Thus, these findings require a reevaluation of the role of 20-hydroxyecdysone in housefly sex pheromone biosynthesis.
REFERENCES
Adams T. S. (1974) The role of juvenile hormone in housefly
ovarian follicle morphogenesis. J. Insecr Phvsiol. 20,
263-276.
Adams T. S. and Filipi P. A. (I 983) Vitellin and vitellogenin concentrations during oogenesis in the first gonotrophic cycle of housefly, Musca domestica. J. Insect Physioi. 29, 723-733.
Adams T. S. and Filipi P. A. (1988) Interaction between juvenile hormone. 20-hydroxyecdysone. the corpuss cardiacum allatum complex, and the ovaries in regulating vitellogenin levels in the housefly, Musca domestica. J. Insect Physiol. 34,
I l-19.
Adams T. S. and Hintz A. M. (1969) Relationship of age, ovarian development. and the corpus allatum to mating in the housefly, Musca domestica. J. Insect Physiol. 15, 201-215.
Adams T. S. and Nelson D. R. (1969) Effect of corpus allatum and ovaries on the amount of pupal and adult fatbody in the housefly, Musca domestica. J. Insecr Physiol. 15, 1729-1747.
Adams T. S., Dillwith J. W. and Blomquist G. J. (1984) The role of 20-hydroxyecdysone in housefly sex pheromone biosynthesis. J. Insect Phvsiol. 30, 287-294. Adams T. S., Hagedorn H. H. and Wheelock G. D. (1985) Haemolymph ecdysteroids in the housefly, Musca domesrica, during oogenesis and its relationship with vitellogenin lev&. JT Insect Physiol. 31, 91-97.. Birnbaum M. J.. Kellv T. J.. Woods C. W. and Imberski R. B. (1984) Hormonal regulation of ovarian ecdysteroid production in the autogenous mosquito, Aedes atropalpus. Gen. camp. Endocr. 56, 9-18.
Bollenbacher W. E., Vedeckis W. V., Gilbert L. I. and O’Conner J. D. (1975) Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta. Dev. Biol. 44, 46-53.
Borovsky D., Thomas B. R.. Carlson D. A., Whisenton L. R. and Fuchs M. S. (1985) Juvenile hormone and 20-hydroxyecdysone as primary’and secondary stimuli to vitellogenesis in Aedes aegypfi. Arch. Insect Biochem. Physiol. 2, 75-96.
Borovsky D., Whisenton L. R., Thomas B. R. and Fuchs M. S. (1986) Biosynthesis and distribution of ecdysone and 20-OH-ecdysone in Aedes aegypti. Arch. Insect Biochem. Physiol. 3, 19-30.
Borst D. W. and O’Conner J. D. (1972) Arthropod molting hormone: Radioimmune assay. Science 178, 418-419. Bownes M., Dubendorfer A. and Smith T. (1984) Ecdysteroids in adult males and females of Drosophila melanogasfer. J. Insect Physiol. 30, 823-830.
Ecdysteroid profile in M. domesrica Briers T., Stoppie P. and De Loof A. (1982) In vitro uptake and metabolism of [:H]ecdysone in the vitellogenic follicles of Sarcophag,a bullato and its location by autoradiography. &t. 1. Invert. Reprod. 4, 343-347, Briers T., Van Beek E. and De Loof A. (1983) Metabolism of injected ecdysone in female Sarcophaga bullata. Comp. Biochem. Physiol. 75B, 9914. Campan M., Strambi A. and Strambi C. (1985) Variations of ecdysteroids in the ovaries and haemolymph during the first gonadotropic cycle of female Calliphora vomitoria. Comp. Biochem. Physiol. 82A, 225-23 1, Gilbert L. I., Goodman W. and Bollenbacher W. E. (1977) Biochemistry of regulatory lipids and sterols in insects. Biochemistry of lipids. Int. Rev. Biochem. 14, l-50.
Hagedorn H. H., O’Conner J. D., Fuchs M. S., Sage B., Schlaeger D. A. and Bohm M. K. (1975). The ovary as a source of ecdysone in the adult mosquito. Proc. natn Acad. Sci. U.S.A. 72, 3255-3259.
Kelly T. J., Adams T. S. and Woods C. W. (1986) Ecdysteroids and dipteran vitallogenesis. In Host Regulated Developmental Mechanisms in Vector Arthropods (Ed. by Borovsky D. and Spielman A.). pp. 6672. University of Florida Press, Vero Beach. Kelly T. J., Birnbaum M. J., Woods C. W. and Borkovec A. B. (1984) Effect of housefly oostatic hormone on egg development neurosecretory hormone action in Aedes atropalpus. J. exp. Zool. 229, 491496.
Kelly T. J.. Woods C. W., Redfern R. E. and Borkovec
1109
A. B. (1981) Makisterone A: The molting hormone of larval Oncopelius? J. exp. Zool. 218, 127-132. Kelly T. J., Adams T. S., Schwartz M. B., Birnbaum M. J.. Rubenstein E. C. and Imberski R. B. (1987) Juvenile hormone and ovarian maturation in the diptera: A review of recent results. Insect Biochem. Insect Biochem. 17, 1089-1093.
Loeb M. J., Woods C. W., Brandt E. P. and Borkovec A. B. (1982) Larval testes of the tobacco budworm: a new source of insect ecdysteroids. Science 218, 896898. Moribayashi A., Kurahashi H. and Ohtaki T. (1985) Comparative studies on ecdysone metabolism between mature larvae and pharate pupae in the fleshfly, Sarcophaga peregrina. Arch. Insect Biochem. Physiol. 2, 237-250.
Richard D. S., Warren J. T., Saunders D. S. and Gilbert L. I. (1987) Haemolymph ecdysteroid titers in diapause and non-diapause destined larvae and pupae of Sarcophaga argyrostoma. J. Insect Physiol. 33, 115-122. Rubenstein E. C., Kelly T. J., Schwartz M. B. and Woods C. W. (1982) In vitro synthesis and secretion of ecdysteroids by Drosophila melanogaster ovaries. J. exp. Zool. 223, 305-308. Schwartz M. B., Kelly T. J., Imberski R. B. and Rubenstein E. C. (1985) The effects of nutrition and methoprene treatment on ovarian ecdysteroid synthesis in Drosophila melanogaster. J. Insect Physiol. 31. 9417957.
Smith ST L. and Mitchell M. .J. (1986) Ecdysone 20.monooxygenase systems in a larval and an adult dipteran. Insect Biochem. 16, 49-55.