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Ecdysteroids in adults, ovaries and eggs of Xyleborus ferrugineus (Coleoptera:Scolytidae)
Insect Biochem., Vol. 12, No. 5, pp. 531-536, 1982. Printed in Great Britain.
0020-1790/82/050531-05503.00/0 © 1982 Pergamon Press Ltd.
ECDYSTEROIDS IN ADULTS, OVARIES AND EGGS OF X Y L E B O R U S FERRUGINEUS (COLEOPTERA: SCOLYTIDAE) K. D. P. RAO, DALE M. NORRIS and H. M. CHU 642 Russell Labs, University of Wisconsin, Madison, WI 53706, U.S.A. (Received 9 November 1981; revised 18 February 1982) Abstract--Free ecdysteroids in the extract of various aged males, females and embryos, and of ovaries of different aged females were estimated by radioimmunoassay. Such ecdysteroids were present during all stages of ovarian development and their titre peaked (534 pg/mg body wt) at day 6 in the cycle, the time of major yolk deposition and initial ovulation. Ovaries did not contain the whole body complement of free ecdysteroids; however, they showed a cyclical pattern of titre that coincided with cycles of oocyte maturation and ovulation. The observed cyclical decline in these free steroids in ovaries was approximately two egg equivalents. Such ecdysteroids were present during all phases of embryonic development and peaked at 362 pg/mg egg wt in embryos that had undergone gastrulation and germ band extension. Free ecdysteroid content in males showed no significant peak at any stage of development. Key Word Index: Xyleborus ferruoineus, radioimmunoassay, free ecdysteroids, development, ovary, egg, male, and female
criteria of BEEMANand NORRIS(1977). For the quantitative study of free ecdysteroids the eggs were aged to four stages. (l) Embryos had mostly undergone gastrulation and germ band extension (20-25 hr old), (2) embryos were between germ band extension and definitive dorsal closure (30-60 hr old), (3) embryos had undergone initial organogenesis (about 90 hr old) and (4) embryos were mature and just before eclosion (96-102 hr old).
INTRODUCTION ECDYSTEROIDS were previously thought to function mainly in the control of moulting events in the immature stages of insects (GILBERT, 1974). However, during the last decade these steroids have been found both in free and conjugated forms in the adults of several species (see HSIAO and HSIAO, 1979; HOFFMANN, 1980). The ovary was reported as the site of adult ecdysteroid synthesis and in some studies most, if not all, of the titre in the body was recoverable from ovary (LAGUEUXe t al., 1977; BOLLENBACHER et al., 1978; HSIAO and HSIAO, 1979; HAGEDORN, 1980). Consequently, new roles have been assigned to these steroidal hormones, and these include interacting with J H in the control of reproduction (HANDLER and POSTLETHWAIT, 1978). The present study was initiated to determine free (i.e., radioimmunologically detectable) ecdysteroid titres in the whole body of adult female Xyleborus ferruyineus (in an attempt to correlate such titres to ovarian development and oocyte maturation), in the ovary (to determine whether the whole body complement is present in the ovary), in adult males (to establish whether cyclic increases in the titres of such ecdysteroids are specific to female gonadal events), and in eggs (to demonstrate whether cyclic quantitative declines in titres in the ovary essentially equal the titres in eggs laid during the period).
Extraction of ecdysteroids Adult females of known age and stage of ovarian cycle were homogenized and extracted with 50~o (v/v) methanol in water (1.0 ml/fifteen adults) for 2-3 rain at 27°C using a glass homogenizer. The resultant extract was allowed to stand overnight at 4-6°C. The extract then was spun at l l00g for 30min at 0°C. The supernatant was decanted and the residue was further extracted sequentially with 50~o methanol and methanol. The supernatants were combined and concentrated under a stream of nitrogen. Free ecdysteroids were similarly extracted from males (150#1/ two-three adults), eggs (150gl/0.Smg eggs), or ovaries (150/A/ten ovaries) which had been dissected into 0.8~o saline. Each extraction was repeated twice as described above. The dried residues were either taken into known volumes of methanol or used directly for RIA estimations.
MATERIALS AND METHODS Xyleborus ferrugineus beetles were reared on a standard sawdust (SDS)-based diet (NORRIS and BAKER, 1967) at 28°C and 70~ RH. Pupae were collected from brood galleries in such stock cultures and were placed on water-agar medium in Petri plates at 28°C and 70~ RH for the subsequent collection of resultant transformed adults at 2 hr intervals. Eggs were collected from stock cultures and the stage of embryogenesis was determined using the detailed
Radioimmunoassay (RI A ) Quantification of free ecdysteroids was by radioimmunoassay (RIA) (BOLLENBACHERet al., 1975). In the initial experiments RIA estimations compared crude methanolic vs purified extracts. The purification of crude extracts involved the use of a hexane: water: methanol (40: 40: 20, by vol) solvent system according to the method detailed by BOLLENBACHERet al. (1975). Because only small differences (<4~o) were found between the absolute values of RIAactive materials in crude as compared with the purified extracts, subsequent RIA estimations used only the former type of extract. An antiserum (D-10) prepared against a carboxymethyloxime derivative of 20-hydroxyecdysone was used for the RIA. This serum exhibited an approximately equivalent competition for ecdysone and 20-hydroxyecdysone (see GILBERTet al., 1977). The radiolabelled
531
K.D.P. RAO,DALEM. NORRISAND H. M. CHU
532
ligand used in the RIA was ct-[23,24-3H(N)] ecdysone (63.5 Ci/m-mole). Authentic ecdysone was used to establish a standard curve and therefore RIA data are expressed as ecdysone equivalents. The results are stated as pg of ecdysane equivalents/mg of body wt. The standard curve is a logit-log plot with a regression coefficient of >0.99. For histological observations the ovaries were fixed in picric acid: formalin: 0.5~o(w/v) trichloroacetic acid, 112.5: 37.5:7.5 ml (by vol), respectively; dehydrated through a series of ethanol; and embedded in paraffin. Serial sections of 6/t thickness were cut and stained with haematoxylin and eosin. Ecdysone antiserum was a generous gift from Prof. L. I. GILBERT, Department of Zoology, University of North Carolina, Chapel Hill, NC U.S.A. ct-[23,24-3HiN)]-Ecdysane was purchased from New England Nuclear. Ecdysone was purchased from Eco Chemical Intermediates. Purity of ecdysone was determined using TLC and high performance liquid chromatography (RAOet al., 1981). RESULTS
Ovarian events and free ecdysteroids in whole body extracts of X yleborus X. ferruyineus has telotrophic ovaries. Each ovariole may be arbitrarily divided into terminal filament, germarium and vitellarium. The anterior portion of the germarium consists of trophocytes (nurse cells) and the posterior portion, previtellogenic and vitellogenic oocytes. The oldest oocyte is present in the proximal part of the ovariole (Fig. 1). In the newly emerged adult the primary oocytes were smaller than trophocytes and were located in the posterior part of the germarium. At this time the free ecdysteroid titre was 61 + 22 S.D. pg/mg body wt (Fig. 4). During the intitial 24 hr of development, the oocyte started moving medially in the posterior part of the germarium and became enlarged (Fig. 2) compared to the preceding stage and free ecdysteroid titre increased to 170 + 16 S.D. pg/mg body wt. The significant ovarian changes during days 2-5 inclusive were (a) the most mature oocyte became surrounded by prefollicular cells, (b) it next became separated from the germarium and moved into the vitellarium, and (c) the associated follicular cells became well developed and cuboidal (Fig. 3). During these ovarian events no significant changes in the titres of free ecdysteroids were observed (Fig. 4). At the sixth day of adult development, the ovarian events included: (a) appearance of yolk globules at the periphery of the most mature oocyte, (b) rapid enlargement of that oocyte, (c) completion of yolk deposition, and (d) the beginning of oviposition (Fig. 3). In concert with these events the free ecdysteroid titre rose rapidly to 534 _ 20 S.D. pg/mg body wt (Fig. 4). Between days 6 and 7 the ecdysteroid titre declined sharply to 125 + 35 S.D. pg/mg body wt. It then varied in a basal-titre range of about 106-150 pg/mg body wt up to the tenth day of adult development. X. ferrugineus grown for six days on standard sawdust (SDS) diet containing 0.08~o sorbic acid remain healthy but do not reproduce (CHu et al., 1982) and the ovarioles do not possess oocytes that pass the pre-vitellogenic stage. The capacity to produce and lay viable eggs however is regained by females shortly after transfer to SDS diet. When free ecdysteroids were estimated in six-day-old beetles on SDS diet plus
6o~ ~ac ~ 4oc ~, o ~' 3ac 2ac ,c~ o
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2
3
4
5 Age
6
7
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8
9
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Fig. 4. Changes in ecdysteroid titres during ovarian development in adult female X. ferrugineus. Each point represents the average of three independent estimations each duplicated at two different concentrations. Vertical bars represent + S.D. 0.08~o sorbic acid, the peak normally found in sixday-old beetles on SDS diet was absent. The free ecdysteroid content in one to seven-day-old adult females remained within the basal range of 100 pg/mg body wt or less. Having quantified the whole body titres of free ecdysteroids in various aged adult females, we next determined whether or not these titres were completely contained in the ovaries. Titres in the ovaries of different aged females in various stages of oocyte maturation were determined (Fig. 5). Ovaries from the freshly eclosed females contained 7 + 2 S.D. pg/ovary. This situation changed little up to, and including, the fourth day of adult development. The titre in ovaries subsequently increased to 22 ___4S.D. pg/ovary between the fourth and fifth day. This is the interval when each ovary contains its first maturing egg (Fig. 1). The titre in ovaries returned to basal level (e.g., 11 + 0.9 S.D. pg/ovary) by the sixth day in conjunction with oviposition of the mature oocytes. The titre peaked again in seven-day-old adult ovaries as the second oocyte matured and then fell suddenly once 28
24
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6
Age of ovory~
i B
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Fig. 5. Changes in ecdysteroid titres per paired ovaries of X.ferrugineus with the age of the insect. Each datum point represents the average of three independent estimations; and vertical bars, +S.D. Fourteen-day ovaries involved only one estimation.
533
Fig. 1. Light photograph of the paired two-ovariole ovaries of X. ferrugineus showing terminal filament (TO, germarium (G), developing oocyte (DO), mature oocyte (MO) and seminal vesicle (SV). (Scale = 200 ~). Fig. 2. Histological details of the ovary showing germarium (G), well developed cuboidal follicular cells (Fc) and oocyte (O) invested by follicular ceils. (Scale = 100/~). Fig. 3. Histological details of the ovary showing deposition of yolk (Y) into oocyte. Note the change in the morphology of the follicular cells (Fc). (Scale = 100/~).
Ecdysteroids of Xyleborus Table 1. Ecdysteroid titres during various stages of embryogenesis in X. ferrugineus as estimated by RIA Stage*
535
evidence of a cycling of the titre as had been observed in fertile females. We did not detect RIA-positive compounds in day-3 males.
pg/mg _+ S.D. pg/egg _+ S.D. DISCUSSION
Freshly laid eggs I II III IV
The data presented in this paper provide bases for correlating the morphological and physiological events in the Xyleborus ovary with free ecdysteroid titres in the whole body and ovary. Because the employed radioimmunoassay method only estimates free ecdysteroids and not conjugated ecdysteroids, * Stage of embryogenesis was determined only a portion of the total titre of ecdysteroids in the using the detailed criteria of BEEMANand NORRIS insect was detected. In contrast with the findings (1977). See text in "Materials and Methods" for regarding free ecdysteroids in the adult coleopteran the details of stages I through IV. Leptinotarsa decemlineata (HSIAO et al., 1976), X. fer** Each datum represents the average of three rugineus contained immunoreactive (i.e., free) comindependent estimations. pounds during all ovarian developmental stages. The small increase in free ecdysteroids from day 0 to day 1 more in the ovaries of ovipositing eight-day-old of adult X. ferrugineus development agrees with the females. This rise and fall of free ecdysteroid titre in findings in Aedes aegypti (HAGEDORNet al., 1975) and ovaries was repeated in concert with cyclic oocyte A. atropalpus (MASLER et al., 1981). The major inmaturation and oviposition (Fig. 5). From these data crease in titre of free ecdysteroids during day 6 of it is evident that the ovary did not contain more than adult development, combined with our yet unpub5-7% of the total free ecdysteroid activity extractable lished observations on juvenile hormone titres (i.e., an from the whole beetles. increase in JH 1, JH 2 and JH 3 titres during day 0 to The peaking of this free ecdysteroid titre in ovaries, day 1 in adult development; followed by a decline by followed by a concomitant fall during oviposition, day 3), strongly suggest that juvenile hormones are suggested that most of the free ecdysteroids detected involved in the early ovarian events and that free in ovaries may be in the eggs. Thus, the amounts of ecdysteroids are essential to the later events (i.e., yolk free ecdysteroids present in newly laid eggs and at deposition and initial ovulation). Such dual endocrine several stages of embryonic development were further control of female insect reproductive events has been determined (Table 1). Newly deposited eggs contained suggested for A. aegypti (HANOKA and HAGEDORN, 142 _ 21 S.D. pg/mg egg wt (7.9 pg/egg). During the 1980). Our findings on free ecdysteroid titres in ovarfirst 25 hr period (stage 1) of embryonic development iectomized (caused by sorbic acid treatment) females free ecdysteroid titre peaked at 362 _+ 47 S.D. pg/mg substantiate the findings of SCHL~GER et al. (1974) egg wt (15 pg/egg). By the time embryos reached the and HAGEDORN (1980) in A. aegypti (i.e., ovariectodorsal closure stage (i.e., stage 2) the ecdysteroid con- mized females still contained free ecdysteroids, and tent had dropped significantly (P < 0.05) by about ovariectomy abolished the peaking of free ecdysteroid 100pg/mg egg wt (ll.33pg/egg). This value subse- titre in females, respectively). quently remained little changed until the stage of The more or less steady state of free ecdysteroids organogenesis (i.e., stage IV) when a decline to during day 1-6 of adult male development and the 173 _+ 21 S.D. pg/mg, or 9.5 pg/egg, occurred. abolition of the peak of free ecdysteroids in the ovariectomized beetles strongly suggest that these horFree ecdysteroids of male X yleborus mones play roles in the ovarian events. The observed titres in various aged males (Table 2) The amount of free ecdysteroids present in the X. were about one-half, or less, of those observed in the ferrugineus ovary was a very small fraction of the connewly metamorphosed adult females. The titres dunng tent of the whole female beetle. Thus, X. ferrugineus the first six days of adult male development never differs in these regards from L. migratoria (LAGUEUX exceeded 3(P40 pg/mg body wt. Thus, there was no et al., 1977), Macrotermis bellicosus and M. subhyalinus (DELBECQUEet al., 1978). This is compatible with the ovaries of X.ferrugineus not being the sole organ Table 2. Ecdysteroid titres in various aged male X. ferrugiof ecdysteroid synthesis (Fig. 5). This possibility gains neus as estimated by RIA further support from the findings of our detailed Wt/adult ultrastructural studies on ovaries which revealed no Age of the (mg) pg/adult pg/mg compelling ultrastructural evidence for steroidal synmale in d a y s ±SEM _+S.D. _S.D. thesis by the ovarian tissues (CHU et al., 1982). If most of the free ecdysteroids in X. ferrugineus females are 0 0.63 _+ 0.09 21 + 8 32 ___9 synthesized in the ovary, then they must be quickly 1 0.62 _ 0.02 15 _+ 2 25 _ 2 released into the haemolymph or bound in conju2 0.65 + 0.03 20 _ 4 38 _ 11 gated forms until just before yolk deposition and in4 0.58 + 0.01 19 + 6 32 + 11 itial ovulation. 5 0.68 + 0.04 21 _ 2 31 _ 5 Ovaries showed a cyclical pattern of free ecdyster6 0.51 + 0.05 14 ___4 29 _ 7 oid titre that coincided with oocyte maturation and Each point represents the average of three independent ovulation. The observed cyclical decline in these fre~ estimations. steroids in ovaries was approximately two egg equiv. 142"* _ 21 362 + 47 254 +_ 63 238 + 58 173 ___21
7.9 _ 1.1 15.3 _ 2.9 11.3 ___3.0 ll.0 + 2.8 9.5 _ 0.4
536
K. D. P. RAO, DALE M. NORRISAND H. M. CHU
alents (Fig. 5, Table 1). This suggests a role of the ovary in transmitting maternally derived steroidal hormones to the developing eggs. The observed major increase in RIA-positive compounds (i.e., free ecdysteroids) in eggs at stage 1 of embryonic development is probably due to liberation of ecdysteroids from the conjugated state. The pattern of free ecdysteroid titres in various aged eggs of X. ferrugineus is similar to that for Oncopeltusfasciatus in that the peak occurred after the initial embryonic development (DORN, 1977). Though X.ferrugineus is the first Coleopteran egg in which free ecdysteroids were found, they were previously known in eggs of Blaberus craniifer (BULLIERE et al., 1979), Galleria mellonella (HSIAO and HSlAO, 1979), Manduca sexta (KAPLANIS et al., 1976), O. fasciatus (DORN, 1977) and Schistocerca gregaria (GANDE and MORGAN, 1979). In summary our results suggest that there is a temporal relationship between the peaking of free ecdysteroids and the ovarian events of yolk deposition and initial ovulation. The lack of such a peak in the male at any stage of development and abolition of the peak by ovariectomy support the major peaking of the free ecdysteroid titre in day 6 as being female specific. The whole body complement of free ecdysteroids was not found in the ovary. It may not be the sole organ of ecdysteroid synthesis; however, it at least acts as a vehicle to transmit maternal ecdysteroids to the eggs. The rapid increase of free ecdysteroids found in gastrulated eggs suggests their release from conjugated forms rather than synthesis by the egg genome. Acknowledgements--We sincerely thank Prof. L. I. GILBERT, Department of Zoology, University of North Carolina, Chapel Hill, NC, for his generous supply of ecdysone antiserum and for critically going through the manuscript. We also thank STEVENSCHNURRERand JANICEANN Busch for valuable technical assistance with the insect material. This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison; and in part by funds from the Wisconsin Department of Natural Resources and research grants No. RR-00779, Division of Research Resources, and No. AG-01271, Institute on Aging, The National Institutes of Health, to D.M.N.
REFERENCES BEEMANS. L. and NORRIS D. M. (1977) Embryogenesis of Xyleborus ferrugineus (Fabr.) (Coleoptera:Scolytidae) I. External morphogenesis of male and female embryos. J. Morphol. 152, 177-220. BOLLENBACHERW. E., VEDECKISW. V., GILBERT L. I. and O'CONNOR J. D. (1975) Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta. Devl. Biol. 44, 46-53. BOLLENBACHER W. E., ZVENKO H., KUMARAN A. K. and
GILBERTL. I. (1978) Changes in ecdysone content during post embryonic development of the wax moth Galleria mellonella: The role of the ovary. Gen. Comp. Endocrinol. 34, 169-179. BULLIERE D., BULLIEREF. and DEREGGI M. (1979) Ecdysteroid titers during ovarian and embryonic development in Blaberus craniifer. Wilhelm Roux Arch. DeE. Biol. 186, 103-114.
CHU H. M., NORRISD. M. and RAO K. D. P. (1982) Sorbic acid induced differences in ultrastructural development of oocytes in ectosymbotic female Xyleborus ferruoineus. J. Morphol. (in press). DELBECQUEJ. P., LANZREINB., BORDEREAUC., IMBODENH., HIRN M., O'CONNORJ. D., NOIROT C. and LUSCHERM. (1978) Ecdysone and ecdysterone in physogastric termite queens and eggs of Macrotermis bellicosus and Macrotermis subhyalinus. Gen. Comp. Endocrinol. 36, 40-47. DORN A. (1977) Hormonal control of egg maturation and embryonic development in insects. In Advances in Invertebrate Reproduction. (Ed. by ADIYODI K. G. and ADIYODI R. G.), Vol. 1, pp. 451~481. GANDE A. R. and MORGANE. D. (1979) Ecdysteroids in the developing eggs of the desert locust, Schistocerca #re#aria. J. Insect Physiol. 25, 289-293. GILBERT L. I. (1974) Endocrine action during insect growth. In Recent Progress in Hormone Research. (Ed. by GREEP R. O.), Vol. 30, pp. 347-390. Academic Press, New York. GILBERT L. I., GOODMAN W. and BOLLENBACHERW. E. (1977) Biochemistry of regulatory lipids and sterols in insects. In International Reviews of Biochemistry, Biochemistry ofLipids II, (Ed. by GOODWlNT. W.), Vol. 14, pp. 1 50. University Park Press, Baltimore. HAGEDORNH. H., O'CONNORJ. D., FUCHS M. S., SAGE B., SCHLAEGERD. A. and BOHMM. K. (1975) The ovary as a source of ecdysone in an adult mosquito. Proc. natn. Acad. Sci. U.S.A. 72, 3255-3259. HA~EDORN H. H. (1980) Ecdysone, a gonadal hormone in insects. In Advances in Invertebrate Reproduction (Ed. by CLARK W. H. and ADAMS T. S.), pp. 97-107. Elsevier/ North Holland, Amsterdam. HANDLERA. M. and POSTLETHWAITJ. H. (1978) Regulation of vitellogenin synthesis in Drosophila by ecdysterone and juvenile hormone. J. exp. Zool. 206, 267-254. HANOKA K. and HA~EDORN H. H. (1980) Brain hormone control of ecdysone secretion by the ovary in a mosquito. In Progress in Ecdysone Research (Ed. by HOFFMANN J. A.), pp. 467-480. Elsevier/North Holland Biomedical Press, Amsterdam. HOFFMANN J. A. (1980) Progress in Ecdysone Research. Elsevier/North Holland Biomedical Press, Amsterdam. HSIAO T. H., HSIAO C. and DE WILDE I. (1976) Molting hormone titre changes and their significance during development of the Colorado beetle, Leptinotarsa decemlineata. J. Insect. Physiol. 22, 1252-1261. HSIAOT. H. and HSIAOC. (1979) Ecdysteroids in the ovary and the eggs of the greater wax moth. d. Insect. Physiol. 25, 45-52. KAPLANISJ. N., ROBBINSW. E., THOMSONM. J. and DUTKY S. R. (1976) The molting hormones from the embyronated egg of the tobacco hornworm, Manduca sexta (L.). Steroids 27, 675-679. LAGUEUXM., HIRN M. and HOFFMANNJ. A. (1977) Ecdysone during ovarian development in Locusta miyratoria. J. Insect Physiol. 23, 109-119. MASLERE. P., Fvcns M. S., SAGE B. and O'CONNOR J. D. (1981) A positive correlation between oocyte production and ecdysteroid levels in adult Aedes. Physiological Entotool. 6, 45-49. NORRIS D. M. and BAKERJ. M. (1967) Symbiosis: effects of mutualistic fungus upon the growth and reproduction of X yleborus f errugineus. Science 156, 1120-1122. RAO K. D. P., NORRISD. M. and Crtu H. M. (1981) Ecdysteroids during pupal development of female Xyleborus ferrugineus. Ent. exp. & appl. 30, 151-156. SCHLAE~ER D. A., Fucns M. S. and KANG S. H. (1974) Ecdysone mediated stimulation of dopa decarboxylase activity and its relationship to ovarian development in Aedes aegypti. J. Cell Biol. 61,454-465.
0020-1790/82/050531-05503.00/0 © 1982 Pergamon Press Ltd.
ECDYSTEROIDS IN ADULTS, OVARIES AND EGGS OF X Y L E B O R U S FERRUGINEUS (COLEOPTERA: SCOLYTIDAE) K. D. P. RAO, DALE M. NORRIS and H. M. CHU 642 Russell Labs, University of Wisconsin, Madison, WI 53706, U.S.A. (Received 9 November 1981; revised 18 February 1982) Abstract--Free ecdysteroids in the extract of various aged males, females and embryos, and of ovaries of different aged females were estimated by radioimmunoassay. Such ecdysteroids were present during all stages of ovarian development and their titre peaked (534 pg/mg body wt) at day 6 in the cycle, the time of major yolk deposition and initial ovulation. Ovaries did not contain the whole body complement of free ecdysteroids; however, they showed a cyclical pattern of titre that coincided with cycles of oocyte maturation and ovulation. The observed cyclical decline in these free steroids in ovaries was approximately two egg equivalents. Such ecdysteroids were present during all phases of embryonic development and peaked at 362 pg/mg egg wt in embryos that had undergone gastrulation and germ band extension. Free ecdysteroid content in males showed no significant peak at any stage of development. Key Word Index: Xyleborus ferruoineus, radioimmunoassay, free ecdysteroids, development, ovary, egg, male, and female
criteria of BEEMANand NORRIS(1977). For the quantitative study of free ecdysteroids the eggs were aged to four stages. (l) Embryos had mostly undergone gastrulation and germ band extension (20-25 hr old), (2) embryos were between germ band extension and definitive dorsal closure (30-60 hr old), (3) embryos had undergone initial organogenesis (about 90 hr old) and (4) embryos were mature and just before eclosion (96-102 hr old).
INTRODUCTION ECDYSTEROIDS were previously thought to function mainly in the control of moulting events in the immature stages of insects (GILBERT, 1974). However, during the last decade these steroids have been found both in free and conjugated forms in the adults of several species (see HSIAO and HSIAO, 1979; HOFFMANN, 1980). The ovary was reported as the site of adult ecdysteroid synthesis and in some studies most, if not all, of the titre in the body was recoverable from ovary (LAGUEUXe t al., 1977; BOLLENBACHER et al., 1978; HSIAO and HSIAO, 1979; HAGEDORN, 1980). Consequently, new roles have been assigned to these steroidal hormones, and these include interacting with J H in the control of reproduction (HANDLER and POSTLETHWAIT, 1978). The present study was initiated to determine free (i.e., radioimmunologically detectable) ecdysteroid titres in the whole body of adult female Xyleborus ferruyineus (in an attempt to correlate such titres to ovarian development and oocyte maturation), in the ovary (to determine whether the whole body complement is present in the ovary), in adult males (to establish whether cyclic increases in the titres of such ecdysteroids are specific to female gonadal events), and in eggs (to demonstrate whether cyclic quantitative declines in titres in the ovary essentially equal the titres in eggs laid during the period).
Extraction of ecdysteroids Adult females of known age and stage of ovarian cycle were homogenized and extracted with 50~o (v/v) methanol in water (1.0 ml/fifteen adults) for 2-3 rain at 27°C using a glass homogenizer. The resultant extract was allowed to stand overnight at 4-6°C. The extract then was spun at l l00g for 30min at 0°C. The supernatant was decanted and the residue was further extracted sequentially with 50~o methanol and methanol. The supernatants were combined and concentrated under a stream of nitrogen. Free ecdysteroids were similarly extracted from males (150#1/ two-three adults), eggs (150gl/0.Smg eggs), or ovaries (150/A/ten ovaries) which had been dissected into 0.8~o saline. Each extraction was repeated twice as described above. The dried residues were either taken into known volumes of methanol or used directly for RIA estimations.
MATERIALS AND METHODS Xyleborus ferrugineus beetles were reared on a standard sawdust (SDS)-based diet (NORRIS and BAKER, 1967) at 28°C and 70~ RH. Pupae were collected from brood galleries in such stock cultures and were placed on water-agar medium in Petri plates at 28°C and 70~ RH for the subsequent collection of resultant transformed adults at 2 hr intervals. Eggs were collected from stock cultures and the stage of embryogenesis was determined using the detailed
Radioimmunoassay (RI A ) Quantification of free ecdysteroids was by radioimmunoassay (RIA) (BOLLENBACHERet al., 1975). In the initial experiments RIA estimations compared crude methanolic vs purified extracts. The purification of crude extracts involved the use of a hexane: water: methanol (40: 40: 20, by vol) solvent system according to the method detailed by BOLLENBACHERet al. (1975). Because only small differences (<4~o) were found between the absolute values of RIAactive materials in crude as compared with the purified extracts, subsequent RIA estimations used only the former type of extract. An antiserum (D-10) prepared against a carboxymethyloxime derivative of 20-hydroxyecdysone was used for the RIA. This serum exhibited an approximately equivalent competition for ecdysone and 20-hydroxyecdysone (see GILBERTet al., 1977). The radiolabelled
531
K.D.P. RAO,DALEM. NORRISAND H. M. CHU
532
ligand used in the RIA was ct-[23,24-3H(N)] ecdysone (63.5 Ci/m-mole). Authentic ecdysone was used to establish a standard curve and therefore RIA data are expressed as ecdysone equivalents. The results are stated as pg of ecdysane equivalents/mg of body wt. The standard curve is a logit-log plot with a regression coefficient of >0.99. For histological observations the ovaries were fixed in picric acid: formalin: 0.5~o(w/v) trichloroacetic acid, 112.5: 37.5:7.5 ml (by vol), respectively; dehydrated through a series of ethanol; and embedded in paraffin. Serial sections of 6/t thickness were cut and stained with haematoxylin and eosin. Ecdysone antiserum was a generous gift from Prof. L. I. GILBERT, Department of Zoology, University of North Carolina, Chapel Hill, NC U.S.A. ct-[23,24-3HiN)]-Ecdysane was purchased from New England Nuclear. Ecdysone was purchased from Eco Chemical Intermediates. Purity of ecdysone was determined using TLC and high performance liquid chromatography (RAOet al., 1981). RESULTS
Ovarian events and free ecdysteroids in whole body extracts of X yleborus X. ferruyineus has telotrophic ovaries. Each ovariole may be arbitrarily divided into terminal filament, germarium and vitellarium. The anterior portion of the germarium consists of trophocytes (nurse cells) and the posterior portion, previtellogenic and vitellogenic oocytes. The oldest oocyte is present in the proximal part of the ovariole (Fig. 1). In the newly emerged adult the primary oocytes were smaller than trophocytes and were located in the posterior part of the germarium. At this time the free ecdysteroid titre was 61 + 22 S.D. pg/mg body wt (Fig. 4). During the intitial 24 hr of development, the oocyte started moving medially in the posterior part of the germarium and became enlarged (Fig. 2) compared to the preceding stage and free ecdysteroid titre increased to 170 + 16 S.D. pg/mg body wt. The significant ovarian changes during days 2-5 inclusive were (a) the most mature oocyte became surrounded by prefollicular cells, (b) it next became separated from the germarium and moved into the vitellarium, and (c) the associated follicular cells became well developed and cuboidal (Fig. 3). During these ovarian events no significant changes in the titres of free ecdysteroids were observed (Fig. 4). At the sixth day of adult development, the ovarian events included: (a) appearance of yolk globules at the periphery of the most mature oocyte, (b) rapid enlargement of that oocyte, (c) completion of yolk deposition, and (d) the beginning of oviposition (Fig. 3). In concert with these events the free ecdysteroid titre rose rapidly to 534 _ 20 S.D. pg/mg body wt (Fig. 4). Between days 6 and 7 the ecdysteroid titre declined sharply to 125 + 35 S.D. pg/mg body wt. It then varied in a basal-titre range of about 106-150 pg/mg body wt up to the tenth day of adult development. X. ferrugineus grown for six days on standard sawdust (SDS) diet containing 0.08~o sorbic acid remain healthy but do not reproduce (CHu et al., 1982) and the ovarioles do not possess oocytes that pass the pre-vitellogenic stage. The capacity to produce and lay viable eggs however is regained by females shortly after transfer to SDS diet. When free ecdysteroids were estimated in six-day-old beetles on SDS diet plus
6o~ ~ac ~ 4oc ~, o ~' 3ac 2ac ,c~ o
i
2
3
4
5 Age
6
7
of aduLt,
8
9
io
days
Fig. 4. Changes in ecdysteroid titres during ovarian development in adult female X. ferrugineus. Each point represents the average of three independent estimations each duplicated at two different concentrations. Vertical bars represent + S.D. 0.08~o sorbic acid, the peak normally found in sixday-old beetles on SDS diet was absent. The free ecdysteroid content in one to seven-day-old adult females remained within the basal range of 100 pg/mg body wt or less. Having quantified the whole body titres of free ecdysteroids in various aged adult females, we next determined whether or not these titres were completely contained in the ovaries. Titres in the ovaries of different aged females in various stages of oocyte maturation were determined (Fig. 5). Ovaries from the freshly eclosed females contained 7 + 2 S.D. pg/ovary. This situation changed little up to, and including, the fourth day of adult development. The titre in ovaries subsequently increased to 22 ___4S.D. pg/ovary between the fourth and fifth day. This is the interval when each ovary contains its first maturing egg (Fig. 1). The titre in ovaries returned to basal level (e.g., 11 + 0.9 S.D. pg/ovary) by the sixth day in conjunction with oviposition of the mature oocytes. The titre peaked again in seven-day-old adult ovaries as the second oocyte matured and then fell suddenly once 28
24
2C
>, o c:f i~,
5
6
Age of ovory~
i B
I0
days
Fig. 5. Changes in ecdysteroid titres per paired ovaries of X.ferrugineus with the age of the insect. Each datum point represents the average of three independent estimations; and vertical bars, +S.D. Fourteen-day ovaries involved only one estimation.
533
Fig. 1. Light photograph of the paired two-ovariole ovaries of X. ferrugineus showing terminal filament (TO, germarium (G), developing oocyte (DO), mature oocyte (MO) and seminal vesicle (SV). (Scale = 200 ~). Fig. 2. Histological details of the ovary showing germarium (G), well developed cuboidal follicular cells (Fc) and oocyte (O) invested by follicular ceils. (Scale = 100/~). Fig. 3. Histological details of the ovary showing deposition of yolk (Y) into oocyte. Note the change in the morphology of the follicular cells (Fc). (Scale = 100/~).
Ecdysteroids of Xyleborus Table 1. Ecdysteroid titres during various stages of embryogenesis in X. ferrugineus as estimated by RIA Stage*
535
evidence of a cycling of the titre as had been observed in fertile females. We did not detect RIA-positive compounds in day-3 males.
pg/mg _+ S.D. pg/egg _+ S.D. DISCUSSION
Freshly laid eggs I II III IV
The data presented in this paper provide bases for correlating the morphological and physiological events in the Xyleborus ovary with free ecdysteroid titres in the whole body and ovary. Because the employed radioimmunoassay method only estimates free ecdysteroids and not conjugated ecdysteroids, * Stage of embryogenesis was determined only a portion of the total titre of ecdysteroids in the using the detailed criteria of BEEMANand NORRIS insect was detected. In contrast with the findings (1977). See text in "Materials and Methods" for regarding free ecdysteroids in the adult coleopteran the details of stages I through IV. Leptinotarsa decemlineata (HSIAO et al., 1976), X. fer** Each datum represents the average of three rugineus contained immunoreactive (i.e., free) comindependent estimations. pounds during all ovarian developmental stages. The small increase in free ecdysteroids from day 0 to day 1 more in the ovaries of ovipositing eight-day-old of adult X. ferrugineus development agrees with the females. This rise and fall of free ecdysteroid titre in findings in Aedes aegypti (HAGEDORNet al., 1975) and ovaries was repeated in concert with cyclic oocyte A. atropalpus (MASLER et al., 1981). The major inmaturation and oviposition (Fig. 5). From these data crease in titre of free ecdysteroids during day 6 of it is evident that the ovary did not contain more than adult development, combined with our yet unpub5-7% of the total free ecdysteroid activity extractable lished observations on juvenile hormone titres (i.e., an from the whole beetles. increase in JH 1, JH 2 and JH 3 titres during day 0 to The peaking of this free ecdysteroid titre in ovaries, day 1 in adult development; followed by a decline by followed by a concomitant fall during oviposition, day 3), strongly suggest that juvenile hormones are suggested that most of the free ecdysteroids detected involved in the early ovarian events and that free in ovaries may be in the eggs. Thus, the amounts of ecdysteroids are essential to the later events (i.e., yolk free ecdysteroids present in newly laid eggs and at deposition and initial ovulation). Such dual endocrine several stages of embryonic development were further control of female insect reproductive events has been determined (Table 1). Newly deposited eggs contained suggested for A. aegypti (HANOKA and HAGEDORN, 142 _ 21 S.D. pg/mg egg wt (7.9 pg/egg). During the 1980). Our findings on free ecdysteroid titres in ovarfirst 25 hr period (stage 1) of embryonic development iectomized (caused by sorbic acid treatment) females free ecdysteroid titre peaked at 362 _+ 47 S.D. pg/mg substantiate the findings of SCHL~GER et al. (1974) egg wt (15 pg/egg). By the time embryos reached the and HAGEDORN (1980) in A. aegypti (i.e., ovariectodorsal closure stage (i.e., stage 2) the ecdysteroid con- mized females still contained free ecdysteroids, and tent had dropped significantly (P < 0.05) by about ovariectomy abolished the peaking of free ecdysteroid 100pg/mg egg wt (ll.33pg/egg). This value subse- titre in females, respectively). quently remained little changed until the stage of The more or less steady state of free ecdysteroids organogenesis (i.e., stage IV) when a decline to during day 1-6 of adult male development and the 173 _+ 21 S.D. pg/mg, or 9.5 pg/egg, occurred. abolition of the peak of free ecdysteroids in the ovariectomized beetles strongly suggest that these horFree ecdysteroids of male X yleborus mones play roles in the ovarian events. The observed titres in various aged males (Table 2) The amount of free ecdysteroids present in the X. were about one-half, or less, of those observed in the ferrugineus ovary was a very small fraction of the connewly metamorphosed adult females. The titres dunng tent of the whole female beetle. Thus, X. ferrugineus the first six days of adult male development never differs in these regards from L. migratoria (LAGUEUX exceeded 3(P40 pg/mg body wt. Thus, there was no et al., 1977), Macrotermis bellicosus and M. subhyalinus (DELBECQUEet al., 1978). This is compatible with the ovaries of X.ferrugineus not being the sole organ Table 2. Ecdysteroid titres in various aged male X. ferrugiof ecdysteroid synthesis (Fig. 5). This possibility gains neus as estimated by RIA further support from the findings of our detailed Wt/adult ultrastructural studies on ovaries which revealed no Age of the (mg) pg/adult pg/mg compelling ultrastructural evidence for steroidal synmale in d a y s ±SEM _+S.D. _S.D. thesis by the ovarian tissues (CHU et al., 1982). If most of the free ecdysteroids in X. ferrugineus females are 0 0.63 _+ 0.09 21 + 8 32 ___9 synthesized in the ovary, then they must be quickly 1 0.62 _ 0.02 15 _+ 2 25 _ 2 released into the haemolymph or bound in conju2 0.65 + 0.03 20 _ 4 38 _ 11 gated forms until just before yolk deposition and in4 0.58 + 0.01 19 + 6 32 + 11 itial ovulation. 5 0.68 + 0.04 21 _ 2 31 _ 5 Ovaries showed a cyclical pattern of free ecdyster6 0.51 + 0.05 14 ___4 29 _ 7 oid titre that coincided with oocyte maturation and Each point represents the average of three independent ovulation. The observed cyclical decline in these fre~ estimations. steroids in ovaries was approximately two egg equiv. 142"* _ 21 362 + 47 254 +_ 63 238 + 58 173 ___21
7.9 _ 1.1 15.3 _ 2.9 11.3 ___3.0 ll.0 + 2.8 9.5 _ 0.4
536
K. D. P. RAO, DALE M. NORRISAND H. M. CHU
alents (Fig. 5, Table 1). This suggests a role of the ovary in transmitting maternally derived steroidal hormones to the developing eggs. The observed major increase in RIA-positive compounds (i.e., free ecdysteroids) in eggs at stage 1 of embryonic development is probably due to liberation of ecdysteroids from the conjugated state. The pattern of free ecdysteroid titres in various aged eggs of X. ferrugineus is similar to that for Oncopeltusfasciatus in that the peak occurred after the initial embryonic development (DORN, 1977). Though X.ferrugineus is the first Coleopteran egg in which free ecdysteroids were found, they were previously known in eggs of Blaberus craniifer (BULLIERE et al., 1979), Galleria mellonella (HSIAO and HSlAO, 1979), Manduca sexta (KAPLANIS et al., 1976), O. fasciatus (DORN, 1977) and Schistocerca gregaria (GANDE and MORGAN, 1979). In summary our results suggest that there is a temporal relationship between the peaking of free ecdysteroids and the ovarian events of yolk deposition and initial ovulation. The lack of such a peak in the male at any stage of development and abolition of the peak by ovariectomy support the major peaking of the free ecdysteroid titre in day 6 as being female specific. The whole body complement of free ecdysteroids was not found in the ovary. It may not be the sole organ of ecdysteroid synthesis; however, it at least acts as a vehicle to transmit maternal ecdysteroids to the eggs. The rapid increase of free ecdysteroids found in gastrulated eggs suggests their release from conjugated forms rather than synthesis by the egg genome. Acknowledgements--We sincerely thank Prof. L. I. GILBERT, Department of Zoology, University of North Carolina, Chapel Hill, NC, for his generous supply of ecdysone antiserum and for critically going through the manuscript. We also thank STEVENSCHNURRERand JANICEANN Busch for valuable technical assistance with the insect material. This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison; and in part by funds from the Wisconsin Department of Natural Resources and research grants No. RR-00779, Division of Research Resources, and No. AG-01271, Institute on Aging, The National Institutes of Health, to D.M.N.
REFERENCES BEEMANS. L. and NORRIS D. M. (1977) Embryogenesis of Xyleborus ferrugineus (Fabr.) (Coleoptera:Scolytidae) I. External morphogenesis of male and female embryos. J. Morphol. 152, 177-220. BOLLENBACHERW. E., VEDECKISW. V., GILBERT L. I. and O'CONNOR J. D. (1975) Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta. Devl. Biol. 44, 46-53. BOLLENBACHER W. E., ZVENKO H., KUMARAN A. K. and
GILBERTL. I. (1978) Changes in ecdysone content during post embryonic development of the wax moth Galleria mellonella: The role of the ovary. Gen. Comp. Endocrinol. 34, 169-179. BULLIERE D., BULLIEREF. and DEREGGI M. (1979) Ecdysteroid titers during ovarian and embryonic development in Blaberus craniifer. Wilhelm Roux Arch. DeE. Biol. 186, 103-114.
CHU H. M., NORRISD. M. and RAO K. D. P. (1982) Sorbic acid induced differences in ultrastructural development of oocytes in ectosymbotic female Xyleborus ferruoineus. J. Morphol. (in press). DELBECQUEJ. P., LANZREINB., BORDEREAUC., IMBODENH., HIRN M., O'CONNORJ. D., NOIROT C. and LUSCHERM. (1978) Ecdysone and ecdysterone in physogastric termite queens and eggs of Macrotermis bellicosus and Macrotermis subhyalinus. Gen. Comp. Endocrinol. 36, 40-47. DORN A. (1977) Hormonal control of egg maturation and embryonic development in insects. In Advances in Invertebrate Reproduction. (Ed. by ADIYODI K. G. and ADIYODI R. G.), Vol. 1, pp. 451~481. GANDE A. R. and MORGANE. D. (1979) Ecdysteroids in the developing eggs of the desert locust, Schistocerca #re#aria. J. Insect Physiol. 25, 289-293. GILBERT L. I. (1974) Endocrine action during insect growth. In Recent Progress in Hormone Research. (Ed. by GREEP R. O.), Vol. 30, pp. 347-390. Academic Press, New York. GILBERT L. I., GOODMAN W. and BOLLENBACHERW. E. (1977) Biochemistry of regulatory lipids and sterols in insects. In International Reviews of Biochemistry, Biochemistry ofLipids II, (Ed. by GOODWlNT. W.), Vol. 14, pp. 1 50. University Park Press, Baltimore. HAGEDORNH. H., O'CONNORJ. D., FUCHS M. S., SAGE B., SCHLAEGERD. A. and BOHMM. K. (1975) The ovary as a source of ecdysone in an adult mosquito. Proc. natn. Acad. Sci. U.S.A. 72, 3255-3259. HA~EDORN H. H. (1980) Ecdysone, a gonadal hormone in insects. In Advances in Invertebrate Reproduction (Ed. by CLARK W. H. and ADAMS T. S.), pp. 97-107. Elsevier/ North Holland, Amsterdam. HANDLERA. M. and POSTLETHWAITJ. H. (1978) Regulation of vitellogenin synthesis in Drosophila by ecdysterone and juvenile hormone. J. exp. Zool. 206, 267-254. HANOKA K. and HA~EDORN H. H. (1980) Brain hormone control of ecdysone secretion by the ovary in a mosquito. In Progress in Ecdysone Research (Ed. by HOFFMANN J. A.), pp. 467-480. Elsevier/North Holland Biomedical Press, Amsterdam. HOFFMANN J. A. (1980) Progress in Ecdysone Research. Elsevier/North Holland Biomedical Press, Amsterdam. HSIAO T. H., HSIAO C. and DE WILDE I. (1976) Molting hormone titre changes and their significance during development of the Colorado beetle, Leptinotarsa decemlineata. J. Insect. Physiol. 22, 1252-1261. HSIAOT. H. and HSIAOC. (1979) Ecdysteroids in the ovary and the eggs of the greater wax moth. d. Insect. Physiol. 25, 45-52. KAPLANISJ. N., ROBBINSW. E., THOMSONM. J. and DUTKY S. R. (1976) The molting hormones from the embyronated egg of the tobacco hornworm, Manduca sexta (L.). Steroids 27, 675-679. LAGUEUXM., HIRN M. and HOFFMANNJ. A. (1977) Ecdysone during ovarian development in Locusta miyratoria. J. Insect Physiol. 23, 109-119. MASLERE. P., Fvcns M. S., SAGE B. and O'CONNOR J. D. (1981) A positive correlation between oocyte production and ecdysteroid levels in adult Aedes. Physiological Entotool. 6, 45-49. NORRIS D. M. and BAKERJ. M. (1967) Symbiosis: effects of mutualistic fungus upon the growth and reproduction of X yleborus f errugineus. Science 156, 1120-1122. RAO K. D. P., NORRISD. M. and Crtu H. M. (1981) Ecdysteroids during pupal development of female Xyleborus ferrugineus. Ent. exp. & appl. 30, 151-156. SCHLAE~ER D. A., Fucns M. S. and KANG S. H. (1974) Ecdysone mediated stimulation of dopa decarboxylase activity and its relationship to ovarian development in Aedes aegypti. J. Cell Biol. 61,454-465.