- Email: [email protected]
Ecdysteroid-dependent development of the oviduct in last-instar larvae of Oncopeltus fasciatus
J. Insect fhysiol. Vol. 32, No. 7, pp. 643-647, 1986 Printed in Great Britain.
0022-1910/86$3.00+ 0.00 Pergamon Journals Ltd
ECDYSTEROID-DEPENDENT DEVELOPMENT OF THE OVIDUCT IN LAST-INSTAR LARVAE OF ONCOPELTUS FASCIATUS A. DORN, J. M. RADEMACHER and E. SEHN Institute of Zoology, University of Mainz, Saarstr. 21, D-6500 Mainz, F.R.G. (Received 3 September 1985; revised 12 November 1985) Abstract-During the last-larval instar, the oviducts undergo a drastic morphological transformation from larval to adult form. From day 3 to day 5, the long and thin larval oviducts shorten and become very wide. The transformation correlates with high ecdysteroid levels. When the ecdysteroid peak is postponed experimentally in the last-larval instars by azadirachtin treatment, oviductal transformation is also postponed and again correlates exactly with increasing hormone titre. Injections of makisterofle A into young, last-instar larvae before the build up of an endogenous ecydsteroid peak results in premature oviductal transformation. The effect on the oviducts is dose-dependent and the nearer the treatment is performed to endogenous ecdysteroid production the stronger the effect. Thus, larval-adult oviduct transformation in last-instar larvae of Oncopeltus fusciatus is ecdysteroid dependent. The possible involvement of additional factors is discussed. Key Word Index: Oviduct,
larval-adult
transformation,
INTRODUCTION
Under favourable rearing conditions, egg maturation in Oncopeltus fasciatus commences 3-4 days after the adult moult and provokes a sudden and drastic enlargment of the ovaries (Wick and Bonhag, 1955; Johansson, 1958; Dom and Paprodtka, unpublished). Also tb: oviducts, which consists of rostral paired parts I(lateral oviducts) and a caudal unpaired part (median oviduct) [Johansson, 19581 undergo morphological changes. These are, however, by no means as striking as the morphological alterations. which we observed in last (5th)-instar larvae. Prior to emergence the lateral oviducts shrink to about 4 of the length they were on day 1 of the last-larval stage. Concomitantly, the width increases more than lo-fold. These changes do not occur gradually during the development of the last-instar larvae but mainly between day 3 and day 5. This is the period when the ecdysteroid titre is high (Kelly et al., 1981; Dorn e! al., 1986). The present study analyzes whether the profound morphological changes in the lateral oviducts during the last-larval instar, which represent a transformation from larval to adult image, are ecdysteroid dependent or merely coincide with the build up of the ecdysteroid peak.
MATERIALS
AND METHODS
Oncopeftus fasciutus Dallas (Heteroptera: Lygaeidae) was reared on peeled sunflower seeds at 27.5”C, and under conditions reported earlier (Darn, 1972). Timing of Sth-instar larvae: from colonies containing primarily larvae approaching the last larval moult, all Sth-instar larvae were removed in the evening. newly hatched female Next morning, Sth-instar larvae were separated and designated as day O-larvae.
ecdysteroids,
Oncopeltus
For morphometric studies on the female genital tract, larvae were dissected in Grace’s Medium (Gibco, Karlsruhe), fixed and stored in Bouin’s fluid. Outlines of ovaries, lateral oviducts and part of the median oviduct were drawn with the help of a drawing mirror and dissecting microscope from Wild (Heersbrugg). Ecdysteroid extracts were made from normal and azadirachtin-treated whole female larvae. At least 5 larvae were pooled for each extract. They were homogenized in absolute methanol, centrifuged and filtered. The residue was washed with 60% methanol, centrifuged and filtered. The methanolic extract and wash were combined, the solvent evaporated and the residue taken in by 1 ml methanol. To remove lipids, 1 ml water and 2 ml dichloromethane were added, shaken, and the dichloromethane phase was discarded. This lipid extraction was repeated 3 times. The aqueous-methanolic phase was evaporated and the residue taken up by 500 ~1 methanol and used for radioimmunoassay (Borst and O’Connor, 1974; Spindler ef al., 1978). Ecdysone and makisterone A (Simes, Milan) served as standards. The antiserum IB2 (final concentration 0.013%) was a gift from L. I. Gilbert (University of North Carolina at Chapel Hill). The affinity of the antibody to makisterone A was about i of that to ecdysone. [23, 24 -‘HI Ecdysone (68 Ci/mmol) was a gift of P. Karlson (University of Marburg). Highly purified azadirachtin A (for detail see Rembold et al., 1984) was a gift from H. Rembold (MPI, Martinsried). Azadirachtin A is an insectgrowth inhibitor from the neem tree. It blocks ecdysis (at the applied dose) and causes “permanent” larvae (Dorn et al., 1986). It was dissolved in 10% ethanol and administered in a concentration of 0.125 pg/pl. Last-instar female larvae were injected with 1 ~1 of the solution within 18 h after previous moult. The
A. DORN et al.
644
63ng
injection site was ventro-lateral between the 1st and 2nd abdominal segment. Aqueous makisterone A solutions of 0.15 pg/pl and 3 pg/pl were injected (1 pi/larvae ventro-lateral between the 1st and 2nd abdominal segment) to induce premature oviduct transformation. Test animals obtained either a single injection or 2 injections on 2 following days. The treated larvae were either sacrificed 24 h after the single injection, or-in case of 2 injections-24 h after the last injection.
1
‘r
RESULTS
Normal development of female genital tract in lastinstar larvae The morphological changes of the ovaries and lateral oviducts taking place in the last-larval instar are pictured in Fig. 1. (The median oviduct has not been considered, since it proved too difficult to dissect in young stages.) Most conspicuous are the alterations of the lateral oviducts between day 3, day 4 and day 5. Figure 2a demonstrates that shortening is indeed most prominent from day 3 to day 4. Thickening of the tubules, however, is most drastic between day 4 and day 5 (Fig. 2b). Ovaries on the other hand exhibit an almost linear growth during larval development (Fig. 2~). The ecdysteroid titre rises in female last-instar larvae between day 3 and day 4 and declines shortly before adult ecdysis, which occurs on day 7 or day 8. An absolute peak is measured on day 5: 6.3 ng per larva-whole animal extract (Fig. 2). Thus, the transformation of lateral oviducts from the larval to adult form parallels closely the rise in the ecdysteroid titre. If this morphogenetic process is triggered by the hormone, as expected, it must be initiated within hours after the activation of the prothoracic glands. Transformation of lateral oviducts in azadirachtininduced permanent larvae Injection of 0.125 pg azadirachtin into newly moulted (O-24 h old) last-instar larvae induces long-living (permanent) larvae, which die without an adult moult. although apolysis is observed in 50% of the test animals and secretion of adult cuticle is initiated (Dorn et al., 1986). In these larvae the ecdysteroid titre increases too, but considerably later than in dl
Fig. I. Normal
d3
.4
,..r’ &_@-~ I I 0123456
I
I
I
I
I
(Days1
Fig. 2. Oviduct transformation in normal last-instar
larvae: (a) 0-0 length of oviduct; (b) 0 - - - 0 thickness of oviduct; (c) H . . . n growth of ovary; the bar indicates the days of elevated ecdysteroid titre; its peak and absolute amount of ecdysteroids (ng/larva) is indicated by the arrow head. Each datum point is derived from 5 to 10 animals (&SEM).
normal larvae (Fig. 3). From day 9 to day 10 it rises above basal level and has a maximum on day 12. On the 16h day when the last titre determination was carried out, it is still elevated, above 2 ng per larva. Thus, in comparison to normal larvae the ecdysteroid peak stretches over an extended period of time-at least 7 days versus 4 days, but has a reduced peak levelA. ng per permanent larva vs 6.3 ng per normal larva. This is a reduction of 33% in permanent larvae. Shortening of the lateral oviducts is distinct on day 10 in permanent larvae (Fig. 3a). Minimal length is attained on day 12. Thickening of the oviducts occurs between day 11 and day 12 (Fig. 3b). It is noteworthy that the thickening, as in normal transformation (see Fig. 2), slightly lags behind shortening. Whereas the transformed lateral oviducts have about the same length in permanent larvae as in normal larvae, the width does not reach the same values in the former. The diameter is about 26% less in permanent larvae. The growth of the ovaries (Fig. 3c) is more or less linear like the controls, but increases more slowly. d4
development of oviducts and ovaries in last-instar larvae. Note the drastic thickening of oviducts between day 3 (d3) and day 4 (d4).
d6
shortening
and
Ecdysteroid-dependent
oviduct development
645
4r
-lo.4
:3 II
III
III
0123456769
I
III 10
11
12
13
14
15
(Days)
Fig. 3. Oviduct transformation in azadirachtin-induced permanent larvae of the last instar: (a) 0-0 length of oviduct; (b) 0 - - - 0 thickness of oviduct; (c) n n growth of ovaries; the bar indicates the days of elevated ecdysteroid titre; its peak and absolute amount of ecdysteroids (ng/larva) is indicated by the arrow head. Note, that like in normal larvae, oviduct transformation is correlated with the rise of ecdysteroid titre. Each datum point is derived from 5 to 10 animals (f SEM).
The length of the ovaries on day 15 has not yet reached the length of the ovaries of normal larvae on day 6 (Fig. 2~).
The experiment shows clearly that the delay of the ecdysteroid peak in azadirachtin-induced permanent larvae is accompanied by a delay in oviduct transformation. The correlation between ecdysteroid peak and transformation is practically the same as in normal larvae. Induction of premature lateral oviduct transformation by injection of makisterone A info young last-instar larvae
Shortening and thickening of the lateral oviducts in normal larvae are Iconspicuous after day 3 (Fig. 2).
Table
We intended to induce premature transformation by injection of makisterone A, which is the authentic moulting hormone in Oncopeltus fasciatus (Kelly et al., 1984), at day 0, 1 and 2. Effects of low and high doses and single versus double injections on two following days were compared. They were monitored 24 h after treatment (Table 1). Shortening of lateral oviducts (Table 1). Injections of makisterone A at day 0 do not reduce the length significantly within 24 h. Single injections at day 1 or day 2, however, do so. Injection on day 2 is more effective than injection on day 1, and the high dose is more effective than the low one. In the case of the low dose, double injections on day 0 and 1 and day 1 and 2 are no more effective than single injections on
1. Premature induction of oviduct transformation by single injections (at 2 following days) of makisterone A Makisterone A dose @g/larva) resp. control*
Day(s) of injection and day of sacrifice ( )
and
double
Length of oviduct (mm)
Thickness of oviduct (rm)
Length of ovaries (mm)
I
3.03 + 0.16 2.95 f 0.05 3.17+0.11
43 * 3 35 + 2
0.63 ? 0.02 0.55 f 0.02
(day 2)
0.15 3.00 Control
day 2
2.97 k 0.08 2.08 f 0.14 3.39 ? 0.06
56 i 3 86 f 6 36 i 2
0.88 i 0.03 1.14+0.08 0.86 + 0.03
Day 0+ day I (day 2)
0.15 3.00 Control
day 2
2.96 + 0.07 1.14&0.05 3.39 + 0.06
52 f 2 131*7 36 f 2
0.67 i 0.05 I .oof 0.04 0.86 + 0.03
0.15 3.00 Control
2.10 + 0.09 I .72 k 0.07 3.18 & 0.07
121+7 103 *9 57 & 2
I .50 f
day 3
0.15 3.00 Control
day 3
2.15~0.11 1.25 +_0.08 3.18 ? 0.07
117*9 184k8 57 +2
I .38 f 0.05 I.41 * 0.05 1.16~0.04
Control Control Control
day 4 day 5 day 6
1.51 io.15 0.90 f 0.10 0.96 i 0.03
l18+ 10 426 f 19 490 * 21
Day 0
(day 1) Day
I
Day 2
_ Control
{
{ {
(day 3) Day I+ day 2 (day 3)
{
day
0.04 1.50 + 0.06 1.16+0.04
1.74*
0.03 1.89 ? 0.04 2.10 f 0.04
Two different doses were applied: 0.15 pg/larva and 3 pg/larva. Each ddlum point is derived from 5 to IO animals ( f SEM). *The data of normal larvae (controls) at the day of sacrifice are. given for comparison.
A. DORN et al.
646
day 1 and day 2 respectively. This is in contrast to the high dose, where double injections are more effective than single ones. Thickening of lateral oviducts (Table 1). Injections of makisterone A induce a premature thickening in every experiment (treated larvae have always thicker oviducts than controls). When we consider, however, that during normal larval development the thickness of the oviducts increases more than IO-fold, the experimental thickening is not as strong as could have been expected. Only a double injection of the high dose at day 1 and day 2 provokes a thickening which is comparable to that in normal larvae seen on day 4-the first day of high endogenous ecdysteroid titre. Double injections of low doses at day 0 and 1 and at day 1 and 2 are no more effective than single injections at day 1 and day 2. Double injections of high doses, however, are more effective than single injections of high doses. Length of ovaries (Table 1). The data on the length of ovaries suggest, that treatment with makisterone A slightly stimulates growth. However, the response is rather weak and will be evaluated in a separate study.
DISCUSSION
In their careful study on post-embryonic development of the ovaries of Oncopeltus fasciatus, Wick and Bonhag (1955) did not consider the oviducts. Brunt (1971) mentions, that in the Pyrrhocoridae Dysdercus fusciatus “towards the end of the fifth instar. . the oviduct appears shorter and wider. . . “. We show here, that the morphogenetic alterations (shortening and thickening) of the lateral oviducts during the last-larval stage represent a transformation from larval to adult image. Transformation occurs suddenly and coincides with high levels of ecdysteroids, the peak of which initiates adult moulting. Beyond a correlation of ecdysteroid titre and adult oviduct transformation in normal larvae, we could demonstrate that when the ecdysteroid peak is postponed by azadirachtin treatment, transformation is also postponed and again correlates precisely with the rising titre. The ecdysteroid peak in azadirachtininduced permanent larvae is about i lower than in controls. It is interesting, that the thickness of oviducts in these larvae is only about 70% of controls. The reduction of length, however, is about the same as in controls. A premature oviduct transformation can be produced by injection of makisterone A prior to endogenous ecdysteroid synthesis. The time of treatment (day 0, 1 or 2) proved important: the reaction is strongest after injections at day 2 and weakest after injections at day 0 (graded response). High doses are more effective than low doses. Double injections of high doses accelerate transformation more than single injections of high doses. This is not the case with double injections of low doses. Maybe in this instance, metabolization and/or excretion prevents accumulation of exogenous ecdysteroids. In conclusion, oviduct transformation is obviously ecdysteroid-dependent in a dose-related manner. Oviducts are largely refractory to exogenous
makisterone A at day 0, but their susceptibility is enhanced on the following days. This may be due to a gradual expression of receptor molecules or the provision of some sort of a trophic factor which increases approaching prothoracic gland activation. It is noteworthy, that shortening and thickening of oviducts do not occur completely simultaneously. Thickening lags somewhat behind shortening. In experiments where high ecdysteroid levels are delayed (azadirachtin treatment) or created prematurely (ecdysteroid injections) thickening is never as strong as in normal day-6 larvae, whereas shortening is. This may indicate, that oviduct transformation is a rather complex process. Clearly, oviducts of last-instar larvae are a target organ of ecdysteroids and must be added to the list of target tissues other than epidermis: imaginal discs, salivary glands (Diptera), larval and imaginal fat body (Diptera), larval brain (CaNiphora vicina), testicular germinal cysts (Hyalophora gloveri), pupal prothoracic glands and pupal corpora allata (Philosamia cecropia and Hyalophora cecropia) and K, cell line (Drosophila melanogaster) [for review see Koolman and Spindler, 19831. It is known from studies on insect cell lines that ecdysteroids are able to change cell shape, for instance provoke elongation (Courgeon, 1972; Berger et al., 1978; Marks and Holman, 1979). In an embryonic cell line from Manduca sexta, ecdysteroids apparently have an effect on expression and arrangement of microtubules (Lynn and Oberlander. 1981). Further studies must show how the morphological alterations of oviduct cells are realized at the subcellular level. The effect of the steroid hormone makisterone A on the oviduct during larval-adult transition is reminiscent of the effects of steroid hormones on the female genital ducts of vertebrates. Oestrogen, for instance, is responsible for full differentiation of the oviduct cells during puberty in mammals and progesterone may stimulate oviduct secretion in a number of vertebrates (cf. Reinboth, 1980). In this context, further comparative studies seem of interest. Acknowledgements-We thank Dr H. Rembold for his generous gift of purified azadirachtin A, and Dr L. I. Gilbert for supplying us with ecdysteroid antibody. The study was supported by a grant from the Deutsche Forschungsgemeinschaft. REFERENCES
Berger E., Ringler R., Alahiotis S. and Frank M. (1978) Ecdysone-induced changes in morphology and protein synthesis in Drosophila cell cultures. Deu. Biol. 62, 498-5 11. Borst D. W. and O’Connor J. D. (1974) Trace analysis of ecdysones by gas-liquid chromatography, radioimmunoassay and bioassay. Steroids 24, 637656. Brunt A. M. (1971) The histoiogy of the first batch of eggs and their associated tissues in the ovariole of Dysdercus fasciatus Signoret (Heteroptera: Pyrrhocoridae) as seen with the light microscope. J. Morph. 134, 105~130. Courgeon A. M. (1972) Effects of a- and /I-ecdysone on in vitro diploid cell multiplication in Drosophila melunogaster. Nature 238, 2.50-251. Dorn A. (1972) Die endokrinen Driisen im Embryo von Oncopeltus fasciatus Dallas (Insecta, Heteroptera) Morphogenese, Funktionsaufnahme, Beeinflussung des Gewe-
Ecdysteroid-dependent bewachstums und 13eziehungen zu den embryonalen Hautungen. Z. Morph. Tiere 71, 52-104. Dom A., Rademacher J. M. and Sehn E. (1986) Effects of azadirachtin on moulting cycle, endocrine system, and ovaries in last instar larvae of the milkweed bug, Oncopeltus fascia&s.
J. Insect Physiol.
32. 231-238.
Johansson A. S. (1958) Relation of nutrition to endocrinereproductive functions in the milkweed bug Oncopeltus fasciutus (Dallas) (Heteroptera:Lygaeidae). Nytt Mug. Zool. 7, l-132. Kelly T. J., Aldrich J. R., Woods C. W. and Borkovec A. B. (1984) Makisterone A: its distribution and physiological role as the molting hormone of true bugs. Experientia 40, 996-991.
Kelly T. J., Woods C. W., Redfern R. E. and Borkovec A. B. (1981) Makisterone A: the molting hormone of larval Oncopeltus? J exp. Zool. 218, 127-132. Koolman J. and Spindler K.-D. (1983) Mechanism of action of ecdysteroids. In Endocrinology of Insects (Ed. by Downer R. G. H. and Laufer H.). Alan R. Liss, New York. Lynn D. E. and Oberlander H. (1981) The effect of cyto-
oviduct development
647
skeletal disrupting agents on the morphological response of a Manduca line to cloned sexfa cell 20-hydroxyecdysone. Wilhelm Roux Arch. EntwMech. Org. 190, 150-155.
Marks E. P. and Holman M. (1979) Ecdysone action on insect cell lines. In Vitro IS, 30&307. Reinboth R. (1980) Vergleichende Endokrinologie.. pp. 178-183. Georg Thieme, Stuttgart. Rembold H., Forster H., Czoppelt C. and Sieber K.-P. (1984) Azadirachtins, a group of insect growth regulators from the neem tree. In Natural Pesticides from the Neem Tree. Proc. 2nd Int. Neem Conf., Rauisch Holzhausen, 1983 (Ed. by Schmutterer H. and Ascher K. R. S.), pp. 153-162. GTZ, Eschborn. Spindler K.-D., Beckers C., Groschel-Stewart U. and Emmerich H. (1978) A radioimmunoassay for arthropod molting hormones, introducing a novel method of immunogen coupling. Hoppe Seyler’s Z. physiol. Chem. 359, 1269-1275. Wick J. R. and Bonhag P. F. (1955) Postembryonic development of the ovaries of Oncopeltus fusciutus (Dallas). J. Morph. 96, 31-59.
0022-1910/86$3.00+ 0.00 Pergamon Journals Ltd
ECDYSTEROID-DEPENDENT DEVELOPMENT OF THE OVIDUCT IN LAST-INSTAR LARVAE OF ONCOPELTUS FASCIATUS A. DORN, J. M. RADEMACHER and E. SEHN Institute of Zoology, University of Mainz, Saarstr. 21, D-6500 Mainz, F.R.G. (Received 3 September 1985; revised 12 November 1985) Abstract-During the last-larval instar, the oviducts undergo a drastic morphological transformation from larval to adult form. From day 3 to day 5, the long and thin larval oviducts shorten and become very wide. The transformation correlates with high ecdysteroid levels. When the ecdysteroid peak is postponed experimentally in the last-larval instars by azadirachtin treatment, oviductal transformation is also postponed and again correlates exactly with increasing hormone titre. Injections of makisterofle A into young, last-instar larvae before the build up of an endogenous ecydsteroid peak results in premature oviductal transformation. The effect on the oviducts is dose-dependent and the nearer the treatment is performed to endogenous ecdysteroid production the stronger the effect. Thus, larval-adult oviduct transformation in last-instar larvae of Oncopeltus fusciatus is ecdysteroid dependent. The possible involvement of additional factors is discussed. Key Word Index: Oviduct,
larval-adult
transformation,
INTRODUCTION
Under favourable rearing conditions, egg maturation in Oncopeltus fasciatus commences 3-4 days after the adult moult and provokes a sudden and drastic enlargment of the ovaries (Wick and Bonhag, 1955; Johansson, 1958; Dom and Paprodtka, unpublished). Also tb: oviducts, which consists of rostral paired parts I(lateral oviducts) and a caudal unpaired part (median oviduct) [Johansson, 19581 undergo morphological changes. These are, however, by no means as striking as the morphological alterations. which we observed in last (5th)-instar larvae. Prior to emergence the lateral oviducts shrink to about 4 of the length they were on day 1 of the last-larval stage. Concomitantly, the width increases more than lo-fold. These changes do not occur gradually during the development of the last-instar larvae but mainly between day 3 and day 5. This is the period when the ecdysteroid titre is high (Kelly et al., 1981; Dorn e! al., 1986). The present study analyzes whether the profound morphological changes in the lateral oviducts during the last-larval instar, which represent a transformation from larval to adult image, are ecdysteroid dependent or merely coincide with the build up of the ecdysteroid peak.
MATERIALS
AND METHODS
Oncopeftus fasciutus Dallas (Heteroptera: Lygaeidae) was reared on peeled sunflower seeds at 27.5”C, and under conditions reported earlier (Darn, 1972). Timing of Sth-instar larvae: from colonies containing primarily larvae approaching the last larval moult, all Sth-instar larvae were removed in the evening. newly hatched female Next morning, Sth-instar larvae were separated and designated as day O-larvae.
ecdysteroids,
Oncopeltus
For morphometric studies on the female genital tract, larvae were dissected in Grace’s Medium (Gibco, Karlsruhe), fixed and stored in Bouin’s fluid. Outlines of ovaries, lateral oviducts and part of the median oviduct were drawn with the help of a drawing mirror and dissecting microscope from Wild (Heersbrugg). Ecdysteroid extracts were made from normal and azadirachtin-treated whole female larvae. At least 5 larvae were pooled for each extract. They were homogenized in absolute methanol, centrifuged and filtered. The residue was washed with 60% methanol, centrifuged and filtered. The methanolic extract and wash were combined, the solvent evaporated and the residue taken in by 1 ml methanol. To remove lipids, 1 ml water and 2 ml dichloromethane were added, shaken, and the dichloromethane phase was discarded. This lipid extraction was repeated 3 times. The aqueous-methanolic phase was evaporated and the residue taken up by 500 ~1 methanol and used for radioimmunoassay (Borst and O’Connor, 1974; Spindler ef al., 1978). Ecdysone and makisterone A (Simes, Milan) served as standards. The antiserum IB2 (final concentration 0.013%) was a gift from L. I. Gilbert (University of North Carolina at Chapel Hill). The affinity of the antibody to makisterone A was about i of that to ecdysone. [23, 24 -‘HI Ecdysone (68 Ci/mmol) was a gift of P. Karlson (University of Marburg). Highly purified azadirachtin A (for detail see Rembold et al., 1984) was a gift from H. Rembold (MPI, Martinsried). Azadirachtin A is an insectgrowth inhibitor from the neem tree. It blocks ecdysis (at the applied dose) and causes “permanent” larvae (Dorn et al., 1986). It was dissolved in 10% ethanol and administered in a concentration of 0.125 pg/pl. Last-instar female larvae were injected with 1 ~1 of the solution within 18 h after previous moult. The
A. DORN et al.
644
63ng
injection site was ventro-lateral between the 1st and 2nd abdominal segment. Aqueous makisterone A solutions of 0.15 pg/pl and 3 pg/pl were injected (1 pi/larvae ventro-lateral between the 1st and 2nd abdominal segment) to induce premature oviduct transformation. Test animals obtained either a single injection or 2 injections on 2 following days. The treated larvae were either sacrificed 24 h after the single injection, or-in case of 2 injections-24 h after the last injection.
1
‘r
RESULTS
Normal development of female genital tract in lastinstar larvae The morphological changes of the ovaries and lateral oviducts taking place in the last-larval instar are pictured in Fig. 1. (The median oviduct has not been considered, since it proved too difficult to dissect in young stages.) Most conspicuous are the alterations of the lateral oviducts between day 3, day 4 and day 5. Figure 2a demonstrates that shortening is indeed most prominent from day 3 to day 4. Thickening of the tubules, however, is most drastic between day 4 and day 5 (Fig. 2b). Ovaries on the other hand exhibit an almost linear growth during larval development (Fig. 2~). The ecdysteroid titre rises in female last-instar larvae between day 3 and day 4 and declines shortly before adult ecdysis, which occurs on day 7 or day 8. An absolute peak is measured on day 5: 6.3 ng per larva-whole animal extract (Fig. 2). Thus, the transformation of lateral oviducts from the larval to adult form parallels closely the rise in the ecdysteroid titre. If this morphogenetic process is triggered by the hormone, as expected, it must be initiated within hours after the activation of the prothoracic glands. Transformation of lateral oviducts in azadirachtininduced permanent larvae Injection of 0.125 pg azadirachtin into newly moulted (O-24 h old) last-instar larvae induces long-living (permanent) larvae, which die without an adult moult. although apolysis is observed in 50% of the test animals and secretion of adult cuticle is initiated (Dorn et al., 1986). In these larvae the ecdysteroid titre increases too, but considerably later than in dl
Fig. I. Normal
d3
.4
,..r’ &_@-~ I I 0123456
I
I
I
I
I
(Days1
Fig. 2. Oviduct transformation in normal last-instar
larvae: (a) 0-0 length of oviduct; (b) 0 - - - 0 thickness of oviduct; (c) H . . . n growth of ovary; the bar indicates the days of elevated ecdysteroid titre; its peak and absolute amount of ecdysteroids (ng/larva) is indicated by the arrow head. Each datum point is derived from 5 to 10 animals (&SEM).
normal larvae (Fig. 3). From day 9 to day 10 it rises above basal level and has a maximum on day 12. On the 16h day when the last titre determination was carried out, it is still elevated, above 2 ng per larva. Thus, in comparison to normal larvae the ecdysteroid peak stretches over an extended period of time-at least 7 days versus 4 days, but has a reduced peak levelA. ng per permanent larva vs 6.3 ng per normal larva. This is a reduction of 33% in permanent larvae. Shortening of the lateral oviducts is distinct on day 10 in permanent larvae (Fig. 3a). Minimal length is attained on day 12. Thickening of the oviducts occurs between day 11 and day 12 (Fig. 3b). It is noteworthy that the thickening, as in normal transformation (see Fig. 2), slightly lags behind shortening. Whereas the transformed lateral oviducts have about the same length in permanent larvae as in normal larvae, the width does not reach the same values in the former. The diameter is about 26% less in permanent larvae. The growth of the ovaries (Fig. 3c) is more or less linear like the controls, but increases more slowly. d4
development of oviducts and ovaries in last-instar larvae. Note the drastic thickening of oviducts between day 3 (d3) and day 4 (d4).
d6
shortening
and
Ecdysteroid-dependent
oviduct development
645
4r
-lo.4
:3 II
III
III
0123456769
I
III 10
11
12
13
14
15
(Days)
Fig. 3. Oviduct transformation in azadirachtin-induced permanent larvae of the last instar: (a) 0-0 length of oviduct; (b) 0 - - - 0 thickness of oviduct; (c) n n growth of ovaries; the bar indicates the days of elevated ecdysteroid titre; its peak and absolute amount of ecdysteroids (ng/larva) is indicated by the arrow head. Note, that like in normal larvae, oviduct transformation is correlated with the rise of ecdysteroid titre. Each datum point is derived from 5 to 10 animals (f SEM).
The length of the ovaries on day 15 has not yet reached the length of the ovaries of normal larvae on day 6 (Fig. 2~).
The experiment shows clearly that the delay of the ecdysteroid peak in azadirachtin-induced permanent larvae is accompanied by a delay in oviduct transformation. The correlation between ecdysteroid peak and transformation is practically the same as in normal larvae. Induction of premature lateral oviduct transformation by injection of makisterone A info young last-instar larvae
Shortening and thickening of the lateral oviducts in normal larvae are Iconspicuous after day 3 (Fig. 2).
Table
We intended to induce premature transformation by injection of makisterone A, which is the authentic moulting hormone in Oncopeltus fasciatus (Kelly et al., 1984), at day 0, 1 and 2. Effects of low and high doses and single versus double injections on two following days were compared. They were monitored 24 h after treatment (Table 1). Shortening of lateral oviducts (Table 1). Injections of makisterone A at day 0 do not reduce the length significantly within 24 h. Single injections at day 1 or day 2, however, do so. Injection on day 2 is more effective than injection on day 1, and the high dose is more effective than the low one. In the case of the low dose, double injections on day 0 and 1 and day 1 and 2 are no more effective than single injections on
1. Premature induction of oviduct transformation by single injections (at 2 following days) of makisterone A Makisterone A dose @g/larva) resp. control*
Day(s) of injection and day of sacrifice ( )
and
double
Length of oviduct (mm)
Thickness of oviduct (rm)
Length of ovaries (mm)
I
3.03 + 0.16 2.95 f 0.05 3.17+0.11
43 * 3 35 + 2
0.63 ? 0.02 0.55 f 0.02
(day 2)
0.15 3.00 Control
day 2
2.97 k 0.08 2.08 f 0.14 3.39 ? 0.06
56 i 3 86 f 6 36 i 2
0.88 i 0.03 1.14+0.08 0.86 + 0.03
Day 0+ day I (day 2)
0.15 3.00 Control
day 2
2.96 + 0.07 1.14&0.05 3.39 + 0.06
52 f 2 131*7 36 f 2
0.67 i 0.05 I .oof 0.04 0.86 + 0.03
0.15 3.00 Control
2.10 + 0.09 I .72 k 0.07 3.18 & 0.07
121+7 103 *9 57 & 2
I .50 f
day 3
0.15 3.00 Control
day 3
2.15~0.11 1.25 +_0.08 3.18 ? 0.07
117*9 184k8 57 +2
I .38 f 0.05 I.41 * 0.05 1.16~0.04
Control Control Control
day 4 day 5 day 6
1.51 io.15 0.90 f 0.10 0.96 i 0.03
l18+ 10 426 f 19 490 * 21
Day 0
(day 1) Day
I
Day 2
_ Control
{
{ {
(day 3) Day I+ day 2 (day 3)
{
day
0.04 1.50 + 0.06 1.16+0.04
1.74*
0.03 1.89 ? 0.04 2.10 f 0.04
Two different doses were applied: 0.15 pg/larva and 3 pg/larva. Each ddlum point is derived from 5 to IO animals ( f SEM). *The data of normal larvae (controls) at the day of sacrifice are. given for comparison.
A. DORN et al.
646
day 1 and day 2 respectively. This is in contrast to the high dose, where double injections are more effective than single ones. Thickening of lateral oviducts (Table 1). Injections of makisterone A induce a premature thickening in every experiment (treated larvae have always thicker oviducts than controls). When we consider, however, that during normal larval development the thickness of the oviducts increases more than IO-fold, the experimental thickening is not as strong as could have been expected. Only a double injection of the high dose at day 1 and day 2 provokes a thickening which is comparable to that in normal larvae seen on day 4-the first day of high endogenous ecdysteroid titre. Double injections of low doses at day 0 and 1 and at day 1 and 2 are no more effective than single injections at day 1 and day 2. Double injections of high doses, however, are more effective than single injections of high doses. Length of ovaries (Table 1). The data on the length of ovaries suggest, that treatment with makisterone A slightly stimulates growth. However, the response is rather weak and will be evaluated in a separate study.
DISCUSSION
In their careful study on post-embryonic development of the ovaries of Oncopeltus fasciatus, Wick and Bonhag (1955) did not consider the oviducts. Brunt (1971) mentions, that in the Pyrrhocoridae Dysdercus fusciatus “towards the end of the fifth instar. . the oviduct appears shorter and wider. . . “. We show here, that the morphogenetic alterations (shortening and thickening) of the lateral oviducts during the last-larval stage represent a transformation from larval to adult image. Transformation occurs suddenly and coincides with high levels of ecdysteroids, the peak of which initiates adult moulting. Beyond a correlation of ecdysteroid titre and adult oviduct transformation in normal larvae, we could demonstrate that when the ecdysteroid peak is postponed by azadirachtin treatment, transformation is also postponed and again correlates precisely with the rising titre. The ecdysteroid peak in azadirachtininduced permanent larvae is about i lower than in controls. It is interesting, that the thickness of oviducts in these larvae is only about 70% of controls. The reduction of length, however, is about the same as in controls. A premature oviduct transformation can be produced by injection of makisterone A prior to endogenous ecdysteroid synthesis. The time of treatment (day 0, 1 or 2) proved important: the reaction is strongest after injections at day 2 and weakest after injections at day 0 (graded response). High doses are more effective than low doses. Double injections of high doses accelerate transformation more than single injections of high doses. This is not the case with double injections of low doses. Maybe in this instance, metabolization and/or excretion prevents accumulation of exogenous ecdysteroids. In conclusion, oviduct transformation is obviously ecdysteroid-dependent in a dose-related manner. Oviducts are largely refractory to exogenous
makisterone A at day 0, but their susceptibility is enhanced on the following days. This may be due to a gradual expression of receptor molecules or the provision of some sort of a trophic factor which increases approaching prothoracic gland activation. It is noteworthy, that shortening and thickening of oviducts do not occur completely simultaneously. Thickening lags somewhat behind shortening. In experiments where high ecdysteroid levels are delayed (azadirachtin treatment) or created prematurely (ecdysteroid injections) thickening is never as strong as in normal day-6 larvae, whereas shortening is. This may indicate, that oviduct transformation is a rather complex process. Clearly, oviducts of last-instar larvae are a target organ of ecdysteroids and must be added to the list of target tissues other than epidermis: imaginal discs, salivary glands (Diptera), larval and imaginal fat body (Diptera), larval brain (CaNiphora vicina), testicular germinal cysts (Hyalophora gloveri), pupal prothoracic glands and pupal corpora allata (Philosamia cecropia and Hyalophora cecropia) and K, cell line (Drosophila melanogaster) [for review see Koolman and Spindler, 19831. It is known from studies on insect cell lines that ecdysteroids are able to change cell shape, for instance provoke elongation (Courgeon, 1972; Berger et al., 1978; Marks and Holman, 1979). In an embryonic cell line from Manduca sexta, ecdysteroids apparently have an effect on expression and arrangement of microtubules (Lynn and Oberlander. 1981). Further studies must show how the morphological alterations of oviduct cells are realized at the subcellular level. The effect of the steroid hormone makisterone A on the oviduct during larval-adult transition is reminiscent of the effects of steroid hormones on the female genital ducts of vertebrates. Oestrogen, for instance, is responsible for full differentiation of the oviduct cells during puberty in mammals and progesterone may stimulate oviduct secretion in a number of vertebrates (cf. Reinboth, 1980). In this context, further comparative studies seem of interest. Acknowledgements-We thank Dr H. Rembold for his generous gift of purified azadirachtin A, and Dr L. I. Gilbert for supplying us with ecdysteroid antibody. The study was supported by a grant from the Deutsche Forschungsgemeinschaft. REFERENCES
Berger E., Ringler R., Alahiotis S. and Frank M. (1978) Ecdysone-induced changes in morphology and protein synthesis in Drosophila cell cultures. Deu. Biol. 62, 498-5 11. Borst D. W. and O’Connor J. D. (1974) Trace analysis of ecdysones by gas-liquid chromatography, radioimmunoassay and bioassay. Steroids 24, 637656. Brunt A. M. (1971) The histoiogy of the first batch of eggs and their associated tissues in the ovariole of Dysdercus fasciatus Signoret (Heteroptera: Pyrrhocoridae) as seen with the light microscope. J. Morph. 134, 105~130. Courgeon A. M. (1972) Effects of a- and /I-ecdysone on in vitro diploid cell multiplication in Drosophila melunogaster. Nature 238, 2.50-251. Dorn A. (1972) Die endokrinen Driisen im Embryo von Oncopeltus fasciatus Dallas (Insecta, Heteroptera) Morphogenese, Funktionsaufnahme, Beeinflussung des Gewe-
Ecdysteroid-dependent bewachstums und 13eziehungen zu den embryonalen Hautungen. Z. Morph. Tiere 71, 52-104. Dom A., Rademacher J. M. and Sehn E. (1986) Effects of azadirachtin on moulting cycle, endocrine system, and ovaries in last instar larvae of the milkweed bug, Oncopeltus fascia&s.
J. Insect Physiol.
32. 231-238.
Johansson A. S. (1958) Relation of nutrition to endocrinereproductive functions in the milkweed bug Oncopeltus fasciutus (Dallas) (Heteroptera:Lygaeidae). Nytt Mug. Zool. 7, l-132. Kelly T. J., Aldrich J. R., Woods C. W. and Borkovec A. B. (1984) Makisterone A: its distribution and physiological role as the molting hormone of true bugs. Experientia 40, 996-991.
Kelly T. J., Woods C. W., Redfern R. E. and Borkovec A. B. (1981) Makisterone A: the molting hormone of larval Oncopeltus? J exp. Zool. 218, 127-132. Koolman J. and Spindler K.-D. (1983) Mechanism of action of ecdysteroids. In Endocrinology of Insects (Ed. by Downer R. G. H. and Laufer H.). Alan R. Liss, New York. Lynn D. E. and Oberlander H. (1981) The effect of cyto-
oviduct development
647
skeletal disrupting agents on the morphological response of a Manduca line to cloned sexfa cell 20-hydroxyecdysone. Wilhelm Roux Arch. EntwMech. Org. 190, 150-155.
Marks E. P. and Holman M. (1979) Ecdysone action on insect cell lines. In Vitro IS, 30&307. Reinboth R. (1980) Vergleichende Endokrinologie.. pp. 178-183. Georg Thieme, Stuttgart. Rembold H., Forster H., Czoppelt C. and Sieber K.-P. (1984) Azadirachtins, a group of insect growth regulators from the neem tree. In Natural Pesticides from the Neem Tree. Proc. 2nd Int. Neem Conf., Rauisch Holzhausen, 1983 (Ed. by Schmutterer H. and Ascher K. R. S.), pp. 153-162. GTZ, Eschborn. Spindler K.-D., Beckers C., Groschel-Stewart U. and Emmerich H. (1978) A radioimmunoassay for arthropod molting hormones, introducing a novel method of immunogen coupling. Hoppe Seyler’s Z. physiol. Chem. 359, 1269-1275. Wick J. R. and Bonhag P. F. (1955) Postembryonic development of the ovaries of Oncopeltus fusciutus (Dallas). J. Morph. 96, 31-59.