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Different profiles of ecdysone secretion and its metabolism between diapause- and nondiapause-destined cultures of the fleshfly, Boettcherisca peregrina
Camp. Biochem. fhysiol. Vol. 9lA, No. 1, pp. 157-164, 1988 Printed in Great Britain
0300-9629/88 163.00+ 0.00 0 1988 Pergamon Press plc
DIFFERENT PROFILES OF ECDYSONE SECRETION AND ITS METABOLISM BETWEEN DIAPAUSE- AND NONDIAPAUSE-DESTINED CULTURES OF THE FLESHFLY, BOETTCHERISCA PEREGRINA ATSUKO MORIBAYASHI*, HIROMU KURAHASHI and TETSUYA OHTAKI Department of Technology and Medical Entomology, National Institute of Health, 10-35, Kamiosaki 2-chome, Shinagawa-ku, Tokyo 141 and Department of Biology, Faculty of Science, Kanazawa University, I-1, Marunouchi, Kanazawa 920, Japan. Telephone: (444) 2 181 (Received 5 January 1988) Abstract--l. Change of ecdysteroid content in whole body and ecdysteroid metabolism in larval and pupal stages were studied in diapause- and nondiapause-destined individuals of Eoettcherisca peregrina. 2. In nondiapause-destined animals, a small and large peak of ecdysteroid level were observed 16 and 116 hr after pupariation, respectively. In diapause-destined animals, however, only one peak was detected during larval-pupal transformation, and thereafter ecdysteroid content remained at a very low level over 360 hr of the observation period. 3. Injected radioactive ecdysone was metabolized into ecdysteroids more polar than 20-hydroxyecdysone and very polar degraded products in developing pupae, while in diapausing pupae ecdysone was mainly converted to epimers of ecdysteroid without hydroxylation at C26.
INTRODUCTION In Boettcherisca
peregrina (Ohtaki and Takahashi, 1972; Kurahashi and Ohtaki, 1979), as in other species of the flesh fly, exposure of embryos and larvae to a short-day light regime results in diapause at the pupal stage (Fraenkel and Hsiao, 1968; Denlinger, 1971; Saunders, 1971; Roberts and Warren, 1975; Vinogradova, 1976). In many Lepidopteran and Dipteran species, environmental stimuli, such as short day-length and/or low temperature, induce pupal diapause. During pupal diapause brain remains in an inactive state and PTTH release is stopped. Eventually, prothoracic gland does not secrete moulting hormone which is responsible for adult development (Denlinger, 1984). In sarcophagid flies, injection of ecdysone into diapausing pupae effectively induced their adult development (Fraenkel and Hsiao, 1968) and ecdysone level in whole body determined by sarcophaga-assay rose to more than 100 rig/g body weight from 2 days after the puparium formation in developing pupae, but remained at a low level during the same period in diapausing pupae (Ohtaki and Takahashi, 1972). This is confirmed by bioassay and radioimmunoassay of ecdysone in S. crassipafpis (Walker and Denlinger, 1980) and S. argyrostoma (Richard et al., 1987). Change of ecdysteroid content in the whole body is based on the balance between the secretion of ecdysone and the degradation of secreted ecdysone. Difference of the content in nondiapause-destined individuals from that in diapause-destined ones must be derived from (1) a different pattern of ecdysone
*To whom all correspondence
should be addressed
secretion and (2) a different process of ecdysone metabolism. We have studied the inactivation process of ecdysone in the nondiapause-destined larvae and pharate pupae (Moribayashi and Ohtaki, 1978,198O; Moribayashi et al., 1985). In the present paper, we describe different profiles of ecdysone metabolism between nondiapause- and diapause-destined animals of B. peregrina and discuss the biological implications of the different endocrinological events between these two systems. MATERIALS AND
METHODS
Experimental animals
The colony of the temperate race of Boettcherisca perewas routinely maintained in our laborat&y.In-this-experiment, adults were reared at 20°C on a liehtdark cvcle of 1I : 13 or 9: 15 hr. Dianausedestined larvae and puiae were obtained by rearing-larvae continuously under the same environment, whereas nondiapause-destined ones were obtained by culturing them from embryonic through whole larval stage under a longday condition (lightdark cycle of 15 : 9 or 16 : 8 hr) at 20°C. To synchronize their developmental stage, both populations of mature larvae were kept in contact with water for another 2 days, when their crops became empty, in the same condition as they were in growing stage. A portion of these full-grown larvae of both cultures in wet conditions was used immediately as “mature larvae” in the present experiment. The remainder were then transferred to dry conditions and allowed to undergo metamorphosis. The animals 20 hr after pupariation were used as “pharate pupae”. The animals were staged again at the pupariation. In nondiapausing animals, ecdysteroid content was determined by radioimmunoassay every 4 or 8 hr up to 360 hr when adult eclosion occurs. Diapause-destined animals were also sampled for ecdysteroid radioimmunoassay as above until 72 hr after the pupariation, and thereafter sampled every 12 hr until 360 hr after the pupariation. ,qrina (Sarcophaga peregrina)
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ATSUKOMORIBAYASHI et al
158 Chemicals
Radioactive [23,24--‘H(N)]ecdysone (specific activity 70.0 Ciimmol) was purchased from New England Nuclear, Boston. Unlabeled ecdysone was bought from Sigma, St. Louis. 20-Hydroxyecdysone and 25-deoxy, 20,26-dihydroxyecdysone (inokosterone) were obtained from Rhoto Pharmaceutical Chemistry, Osaka, Japan. Extraction and separation of the ecdysteroid metabolites
A certain amount of radioactive ecdysone dissolved in 1 ~1 of water was injected into each mature larva or pharate pupa of the diapause- and nondiapause-destined animals. Ecdysone metabolites were extracted with a mixture of acetone and ethanol (1: 1 v/v). and separated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) as previously described (Moribayashi et al., 1985). Two development solvents were used for TLC separation. One of them (CHCI,: CH,OH = 4: 1 v/v) (system A) was used for separation of very polar metabolites of ecdysone from free ecdysteroids with moderate polarity. The other solvent (CH,COOC2H5: C,H,OH :H,O = 2: 8: 1 v/v) (system B) was used for separation of very polar metabolites into two groups (OA and OB) (Moribayashi and Ohtaki, 1980). Free ecdysteroid metabolites were extracted from the TLC plates by methanol. The methanol solution was passed through Millipore filters (catalog No. SJHVOO4NS) and subjected to HPLC under the following conditions: column Zorbax Sil, 15 cm/4.6mm; mobile phase CHj(CH,), CH,:C,H,OH:CH,CN:CH,OH=85:15:3:3(v/v); flow rate 0.6 ml/min; column pressure 25 kg/cm*; UV detector set at 254nm. Identification of free ecdysteroid metabolites
In order to identify free ecdysteroid metabolites separated by HPLC, the compounds were acetylated by acetic anhydride and pyridine (1: 1, v/v). The acetylated derivatives of metabolites and the standard tri- and tetra-acetylated ecdysone and 20-hydroxyecdysone were run together on TLC with the solvent (CH,COOC,H,:CH,(CH,),CH, = 8: 2 v/v) (system C). Every 5 mm of silica gel of the plates was scraped into miniviais, and then 0.1 ml of methanol and 1.0 ml of Insta-Gel were added for counting radioactivity. Radioimmunoassay of ecdysteroid content in whole body
Three animals, each taken from diapause- and nondiapause-destined colonies at certain intervals, were weighed and homogenized with a hand-driven homogenizer in 0.5 ml methanol and the homogenates were centrifuged at 10,OOOrpm for 2 min at 0°C. The supernatant was transferred to another tube and 0.5 ml of petroleum ether was added to remove lipids. The delipidated samples were kept at - 20°C and graded aliquots of the samples were then transferred to small tubes. After solvent was evaporated, samples were subjected to radioimmunoassay (RIA) of ecdysteroid according to the method of Horn ef al. (1976)
(A)
0 1
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I
60
90
I
I
f
0
30
i!
Pupariatian +
I
20
40
I 60
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I I 100 120 h
Fig. 1. Time of pupariation of diapause-destined (0) and nondiapause-destined (0) cultures. Eash point represents average time of three trials using 100 individuals in each test. Bars show ranges of the standard error. and Carrow ef al. (1981). Further details were described in a previous paper (Ohtaki et al., 1986). RESULTS
Efect of different photoperiods on pupariation and pupation Almost all larvae reared in a short-day condition from embryonic stage entered pupal diapause. In these animals, 50% pupariation took place 38.5-41.5 hr after the transfer to dry conditions,
whereas that of nondiapause-destined larvae kept in a long-day condition during whole larval stage was 29.5-31.5 hr after the dry treatment (Fig. 1). There was approx. a 10 hr difference in 50% pupariation between these two cultures. Pupation time was determined by observing the head evagination of pharate pupa, on opening the anterior tip of the puparium every 2 hr. Pupation of diapause- and nondiapause-destined animals occurred 48-50 hr and 58-60 hr after the pupariation, respectively. The delay in pupariation of diapausedestined larvae was entirely recovered by a shorter duration between pupariation and pupation, so that pupation of both groups took place almost at the same time after the dry treatment (Fig. 2). Changes of ecdysteroid levels during pupal and adult development
The level of ecdysteroids from mature larvae through adult development determined by RIA was shown in Fig. 3. The ecdysteroid titre in mature larvae was approx. 20 pg ecdysone equivalent/mg fresh body weight.
390 I‘
h
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t
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Pupation
(B) I
0
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t
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390 h
I, Adult emergence
Fig. 2. Comparison of the times of 50% pupariation and pupation between diapause-destined (A) and nondiapause-destined (B) cultures. For determination of pupation time 10 animals each for every 2 hr were observed in either of the cultures by removing anterior part of pupal shell.
Ecdysteroid in diapausing and nondiapausing flies
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aa t D
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”
192
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240
264
”
266
312
”
336
360
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Fig. 3. Ecdysteroid content in whole body in diapause-inducing condition (A) and in nondiapause-inducing condition (B). See text for details.
In white puparial stage of nondiapause-destined animals, the titre was nearly 150pg/mg and this increased towards pupation. Sixteen hours after the pupariation the titre peaked at approx. 230pg/mg, and then decreased 40 hr after pupariation, showing a minimum at about lOOpg/mg. This small peak of ecdysone titre was also observed in diapause-destined pharate pupae. After that, only in nondiapausedestined pharate pupae, the titre again increased with nearly daily fluctuations and reached the second maximum of 1500 pg/mg at 116 hr after pupariation, and the titre then began to decrease toward adult eclosion in which only 80 pg/mg of ecdysteroids was found. In the later pharate adult stage ecdysone titre was determined in female and male separately, but no significant differences were observed. In diapause-destined animals, however, the peak of ecdysteroid titre was only one at the pharate pupal stage and that was approx. 200pg/mg fresh body weight. The titre then decreased to almost undetectable level several hours before pupation and remained at a low level (20-40 pg/mg) until the end of observation period (360 hr after pupariation).
Radioactive metabolites were extracted and separated into the following three groups by TLC: OA (mainly composed by conjugated ecdysteroids), OB (very polar final metabolites) and free ecdysteroid metabolites. When labeled ecdysone was given to mature larvae of both groups, almost equal amounts of OA, OB and free ecdysteroid metabolites were produced 72 hr after the injection. Amounts of OA produced in animals treated with ecdysone in larval stage was much more than that treated in pharate
cm 20000
r
Ecdysteroid metabolism
Radioactivity of labeled ecdysone injected into both diapause- and nondiapause-destined pharate pupae reduced rapidly, and only half of the radioactivity could be recovered 24 hr after the injection. Then. the labeled substance gradually reduced to about 45% of original activity in 72 hr, and there was no remarkable difference between both groups (Fig. 4).
4oooL--0
24
48
72
hr
Fig. 4. Time course of recovered radioactivity after injection of 3[H]ecdysone into diapause-destined (A) and nondiapause-destined (0) pharate pupae.
ATSUKO MORIBAYASHI et
160
pupal stage in both groups. As shown in Fig. 5, in diapause-destined pharate pupae, labeled metabolites were mainly a free ecdysteroid group, composed of approx. 80% of total radioactivity recovered, whereas OB and free ecdysteroids were produced almost equally in nondiapause-destined pharate pupae. At 96 hr, the amount of OA fractions were reduced, but no difference was observed between both cultures (Fig. 5). HPLC analysis of the free ecdysteroid metabolites Elution patterns of the free ecdysteroid metabolites recovered 48 hr and 72 hr after injection of ecdysone into both diapause- and nondiapause-destined pharate pupae are shown in Figs 6 and 7. In developing pupae, ecdysone was mainly metabolized to several compounds, which were temporarily
IO
20
30
al. OA
08
........... ................ ................................
48h
( N I pharate
pupae
72
h
I-
( D )pharate
48h 72 h
1
pupae
Free
; ............................... .... ................ : : : : : :.:.:.:.....................
u
Fig. 5. Percent radioactivity of three major groups of metabolites: OA (mainly conjugated ecdysteroids), OB (very polar final metabolites) and free ecdysteroids, in the extracts 72 and 96 hr after injection of 3[H]ecdysone into diapause-destined (D) and nondiapause-destined (N) larvae (upper), and in those 48 and 75 hr after injection of ‘[HJecdysone into diapause-destined (D) and nondiapausedestined (N) pharate pupae (lower).
40
50
60
min
50
60
min
Fig. 6. HPLC profiles of free ecdysteroid metabolites extracted 48 hr (A) and 72 hr (B) after injection of ‘[Hlecdysone into nondiapause-destined pharatae pupae. Fractions were collected every 30 set (tilled columns) from the 21st min to 40th min, or every 1 min for the rest of the time. For comparison the halves of open columns, that are equivalent to the radioactivity of 30 set collection, are filled. See text for further details.
Eedysteroid in diapausing and nondiapausing flies
161
IA)
10
CPm
1000 750
500
20
min
40
I 1
10
20
30
40
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min
Fig. 7. HPLC profiles of free ecdysteroid metabolite extracted 48 hr (A) and 72 hr (B) after injection of ‘[Hlecdysone into diapause-destined pharate pupae, Explanations are the same as in Fig. 6.
identified as 20.hydroxyecdysone, 26-hydroxyecdysone, 20,26dihydroxyecdysone and the epimers of the last two by their elution time, being compared with those of the cochromatographed standards (Fig. 6). In diapausing pupae, however, mainly 3-epiecdysone, 3-epi-20-hydroxyecdysone were found, but 26hydroxy and 20,2&dihydroxy derivatives were scarcely in the free ecdysteroid group (Fig. 7). From 48 to 72 hr after injection, in developing pupae, almost all of unchanged ecdysone disappeared, but the amounts of 20-hydroxyecdysone, 26-hydroxylated and 20,26-dihydroxylated derivatives inreased considerably. Identification
of acetylated metaboiites
The free ecdysteroid metabolites were then subjected to acetylation for more detailed identification. Seven free ecdysteroid metabolites were isolated from the extracts of developing pupae in order of increasing elution time (compounds I to VII shown in Fig. 6). Three free ecdysteroid metabolites (compounds I to III shown in Fig. 7) were isolated from
diapausing pupae. All these isolated samples were acetylated and applied on TLC separation (System C, detailed in Materials and Methods). The gel on the TLC plates was scraped in 5 mm strips and the radioactivity was determined. As shown in Fig. 8, standard samples of tri-acetyl and tetra-acetyl ecdysone migrated to the positions shown as a(I) and n(2), and those of 20-hydroxyecdysone migrated to /$(I) and p(2). The R, value of tetra-acetyl inokosterone was almost the same as that of tetra-acetyl 20-hydroxyecdysone. Acetylated product of compound I, which was temporarily identified as 3-epi-ecdysone by HPLC, migrated to the same region of acetylated ecdysone, confirming the identification by HPLC analysis. Compound II obtained from developing pupae behaved the same as ecdysone. However, compound II extracted from diapausing pupae was identified as 3-epi-20-hydroxyecdysone, but not as ecdysone, by the acetylation test. Compound III obtained from the extract of both groups was identified as 20-hydroxyecdysone. Similarly, compounds IV and VI were identified as
ATSUKO MORIBAYASHI
162
(A)
@d
(I31
O&Q
1>
cm 3-Epi-ecdysone
400
@aa
@dO +31
cpm 1500
600
et al
(I)
1250
3-Epi-ecdysone
1000 200
750. 500 250
er n
Ill
t
(II) Ecdysone
?
( 3-Epi-20-hydroxyecdysone
500 250 -
em@da
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*
(III,
4
(III)
20-Hydroxyecdysone
20-Hydroxyecdysone
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500 250
0
cpml n
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BOO 600
HI
cm, 3-Epi-26-hydroxyecdysone
600
(VI 3-Epi-20,26-dihydroxyecdysone
, “I, 26-Hydroxyecdysone
20,26-Dihydroxyecdyrone
To,
Fig. 8. Comparative radioactivity of acetylated derivatives of ecdysteroid metabolites separated by HPLC in nondiapause-destined pharate pupae (A) and diapause-destined pharate pupae (B). Shaded circles show the migrated positions of the following standard compounds co-chromatographed with tested samples (Moribayashi et al., 1985). a 0, triacetylated ecdysone; a 0, tetraacetylated ecdysone; p 0, triacetylated 20-hydroxyecdysone; b 0, tetraacetylated 20-hydroxyecdysone; I, tetraacetylated inokosterone. Numbers in the right shoulder of each chromatogram are those of fractions separated by HPLC shown in Figs 6 and 7. The substances in each fraction are temporarily identified as ecdysteroid listed under the number.
1
Ecdysteroid
in diapausing and nondiapausing flies
3-epi-26-hydroxyecdysone and 26-hydroxyecdysone, respectively. Amounts of compounds V and VII isolated from developing pupae were not enough to obtain definite data, but they could be temporarily identified as 3-epi-20,26-dihydroxyecdysone and 20,26-dihydroxyecdysone, with reference to data described in previous report (Moribayashi et al., 1985). DISCUSSION
The ecdysteroid content in the whole body determined by RIA peaked between pupariation and pupation at approx. 200 rig/g body weight in both diapause- and nondiapause-destined individuals of Boettcherisca peregrina and is well within the range of physiological level and of titre measured by sarcophaga-assay which was reported previously (Ohtaki and Takahashi, 1972). The second peak of ecdysteroid content was observed in developing pupae of several species of the fly by either bioassay or RIA (see review by Richards, 1981). But, in some cases, the content determined by RIA was represented as much higher than that measured by bioassay (Koolman, 1980). We also found a high level of ecdysteroid content during the adult development. This may be caused by both non-specificity of antisera of ecdysone used and determining ecdysteroid levels of the whole body, in which inactivated ecdysteroids were stored, but not of the haemolymph. The absence of the second peak in diapausing pupae was also reported, using bioassays and RIAs (Ohtaki and Takahashi, 1972; Walker and Denlinger, 1980; Richard, D. S. et al., 1987). In the present study we analysed and identified ecdysone metabolites by TLC, HPLC and chemical methods. Although definite identification of the compounds could not be attained by these methods, we found clear differences in ecdysteroid metabolism between diapause- and nondiapause-destined animals In nondiapause-destined animals, 20,26-dihydrovylation of ecdysone at larval stage, and in addition to that 26-hydroxylation and epimerization in pharate pupal stage, were shown to be main inactivation processes of ecdysone, confirming the results reported in the previous paper (Moribayashi et al., 1978, 1985). It is worthy to note, however, that 26-hydroxyl derivatives were rarely found in the free ecdysteroid metabolites in diapausing pupae. The main inactivated products belonging to free ecdysteroid groups in these animals were 3-epimers of ecdysone and 20-hydroxyecdysone. These results suggest that, in diapausing pupae, either 26-hydroxylation of ecdysone did not occur or 26-hydroxyecdysone produced was rapidly converted to more polar metabolites, such as ecdysonoic acid or conjugated ecdysteroids (Richard et al., 1987). However, the latter case may not be possible because both OB (very polar metabolites of ecdysone) and OA (mainly conjugates of ecdysone) were produced in diapausing pupae less than in developing pupae (Fig. 5). A short-day condition induces pupal diapause in which ecdysone release and probably foregoing
PTTH secretion are absent in adult development of the flesh flies. From an evolutionary point of view, this species of sarcophagid flies is thought to emerge
163
in some tropical region (Kurahashi and Kano, 1984; Denlinger, 1986). Among sarcophagid species, Boettcherisca peregrina has the widest distribution, covering tropical and temperate countries, including Japan, where its races with facultative diapause or continuously developing races are found (Kurahashi and Ohtaki, 1977, and in preparation). It could be suggested, therefore, that incorporation of a new set of genes, which is responsible for the change of the endocrinological mechanisms, including stoppage of the PTTH release and different metabolic pathway of ecdysone, should be involved in the expansion of their territory to the temperate zone. A sequential process between environmental stimuli and endocrinological events is still entirely unknown. Further studies should be put forward to elucidate this process together with other regulatory mechanisms of metabolisms, for instance the induction of a high content of glycerol in diapausing pupae (Kitoh, 1979; Richard, E. Lee Jr et al., 1987). Acknowledgement-This work was partly supported by a grant from the Ministry of Education, Science and Culture, Japan (No. 62540541) to T.O.
REFERENCES
Carrow G. M., Calabrese R. L. and Williams C. M. (1981) Spontaneous and evoked release of prothoracicotropin from multiple neurohumal organs of the tobacco hornworm. Proc. Natl. Acad. Sci., USA 78, 5866-5870. Denlinger D. L. (1971) Embryonic determination of pupal diapause in the flesh fly, Sarcophaga crasipalpis. J. Insect Physiol. 17, 1815-1822. Denlinger D. L. (1984) Hormonal control of diapause. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), pp. 354412. Pergamon Press, Oxford. Denlinger D. L. (1986) Dormancy in tropical insects. Ann. Rev. Entomol. 31, 239-264. Fraenkel G. and Hsiao C. (1968) Morphological and endocrinological aspects of pupal diapause in a fleshfly, Sarcophaga argyrostoma and S. bullata. J. Insect Physiol. 14, 689-705. Horn D. H. S., Wilkie J. S., Sage B. A. and O’Conner J. D. (1976) A high affinity antiserum specific for the ecdysone nucleus. J. Insect Physiol. 22, 901-905. Kitoh K. (1979) Glycerol content in diapausing and developing pupae of Sarcophaga peregrina. Dissertation, Masters Degree, Kanazawa University (in Japanese). Koolman J. (1980) Ecdysteroids in the blowfly, Calliphora vicina. In Progress in Ecdysone Research (Edited by Hoffmann J. A.), pp. 187-210. Elsevier, Amsterdam. Kurahashi H. and Kano R. (1984) Phylogeny and geographical distribution of the genus, Boettcherisca rohdendorf (Diptera: Sarcophagidae). Jpn. J. Med. Sci. Biol. 37, 27-34. Kurahashi H. and Ohtaki T. (1977) Crossing between nondiapausing and diapausing races of Sarcophaga peregrina. Experientia 33, 186-187. Kurahashi H. and Ohtaki T. (1979) Induction of pupal diapause and photoperiodic sensitivity during early development of Sarcophaga peregrina larvae. Jpn. J. Med. Sci. Biof. 32, 77-82. Kurahashi H. and Ohtaki T. (1988) Geographical variation in pupal diapause incidence among Asian and Oceanan species of the fleshfly, Boeffcherisca (Diptera: Sarcophagidae) (in preparation). Moribayashi A. and Ohtaki T. (1978) Inactivation of ecdysone and possible feedback control of the titre during
164
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pupation of Sarcophaga peregrina. J. Insect Physiol. 24, 279-285. Moribayashi A. and Ohtaki T. (1980) Inactivation and reactivation of 20-hydroxyecdysone during pupal-adult development of the fleshfly, Sarcophaga peregrina. Jpn. J. Med. Sci. Biol. 33, 189--20 I. Moribayashi A.. Kurahashi H. and Ohtaki T. (1985) Comparative studies on ecdysone metabolism between mature larvae and pharate pupae in the fleshfly, Sarcophaga oereprina. Arch. Insect Biochem. Phvsiol. 2. 237-250. OitakrT. and Takahashi M. (1972) induction and termination of pupal diapause in relation to the change of ecdysone titer in the fleshfly, Sarcophaga peregrina. Jpn. J. Med. Sci. Biol. 25, 369-376. Ohtaki T., Yamanaka F. and Sakurai S. (1986) Differential timing of pupal commitment in various tissues of the silkworm, Bombyx mori. J. Insect Physiol. 32, 635-642. Richard D. S., Warren J. T., Saunders D. S. and Gilbert L. I. (1987) Haemolymph ecdysteroid titre in diapauseand nondiapause-destined larvae and pupae of Sarcophaga argyrostoma. J. Insect Physiol. 33, 115-122.
Richard E. Lee Jr, Cheng-ping Chen, Mark H. Meacham and David L. Denlinger (1987) Ontogenetic patterns of cold-hardiness and glycerol production in Sarcophaga crassipalpis. J. Insect Physiol. 33, 587-592. Richards G. (1981) Insect hormone in development. Biol. Ret:. 56, 501-549. Roberts B. and Warren M. A. (1975) Diapause in the Australian flesh fly Tricholioprocfia impatiens. (Diptera: Sarcophagidae) A&t. J. Zooi. 23, 563-567. _ Saunders D. S. (19711 The temuerature comoensated ohotoperiodic clock “p;ogramming” development and-pupal diapause in the flesh-fly, Sarcophaga argyrostoma. J. Insect Physiol. 17, 801-812. Vinogradova E. B. (1976) Embryonic photoperiodic sensitivity in two species of fleshflies, Parasarcophaga similis and Boetrcherisco seprentrionalis. J. Insect Physiol. 22, 819-822. Walker G. P. and Denlinger D. L. (1980) Juvenile hormone and moulting hormone titres in diapause and nondiapause destined flesh flies. J. Insect Physiol. 26, 661-664.
0300-9629/88 163.00+ 0.00 0 1988 Pergamon Press plc
DIFFERENT PROFILES OF ECDYSONE SECRETION AND ITS METABOLISM BETWEEN DIAPAUSE- AND NONDIAPAUSE-DESTINED CULTURES OF THE FLESHFLY, BOETTCHERISCA PEREGRINA ATSUKO MORIBAYASHI*, HIROMU KURAHASHI and TETSUYA OHTAKI Department of Technology and Medical Entomology, National Institute of Health, 10-35, Kamiosaki 2-chome, Shinagawa-ku, Tokyo 141 and Department of Biology, Faculty of Science, Kanazawa University, I-1, Marunouchi, Kanazawa 920, Japan. Telephone: (444) 2 181 (Received 5 January 1988) Abstract--l. Change of ecdysteroid content in whole body and ecdysteroid metabolism in larval and pupal stages were studied in diapause- and nondiapause-destined individuals of Eoettcherisca peregrina. 2. In nondiapause-destined animals, a small and large peak of ecdysteroid level were observed 16 and 116 hr after pupariation, respectively. In diapause-destined animals, however, only one peak was detected during larval-pupal transformation, and thereafter ecdysteroid content remained at a very low level over 360 hr of the observation period. 3. Injected radioactive ecdysone was metabolized into ecdysteroids more polar than 20-hydroxyecdysone and very polar degraded products in developing pupae, while in diapausing pupae ecdysone was mainly converted to epimers of ecdysteroid without hydroxylation at C26.
INTRODUCTION In Boettcherisca
peregrina (Ohtaki and Takahashi, 1972; Kurahashi and Ohtaki, 1979), as in other species of the flesh fly, exposure of embryos and larvae to a short-day light regime results in diapause at the pupal stage (Fraenkel and Hsiao, 1968; Denlinger, 1971; Saunders, 1971; Roberts and Warren, 1975; Vinogradova, 1976). In many Lepidopteran and Dipteran species, environmental stimuli, such as short day-length and/or low temperature, induce pupal diapause. During pupal diapause brain remains in an inactive state and PTTH release is stopped. Eventually, prothoracic gland does not secrete moulting hormone which is responsible for adult development (Denlinger, 1984). In sarcophagid flies, injection of ecdysone into diapausing pupae effectively induced their adult development (Fraenkel and Hsiao, 1968) and ecdysone level in whole body determined by sarcophaga-assay rose to more than 100 rig/g body weight from 2 days after the puparium formation in developing pupae, but remained at a low level during the same period in diapausing pupae (Ohtaki and Takahashi, 1972). This is confirmed by bioassay and radioimmunoassay of ecdysone in S. crassipafpis (Walker and Denlinger, 1980) and S. argyrostoma (Richard et al., 1987). Change of ecdysteroid content in the whole body is based on the balance between the secretion of ecdysone and the degradation of secreted ecdysone. Difference of the content in nondiapause-destined individuals from that in diapause-destined ones must be derived from (1) a different pattern of ecdysone
*To whom all correspondence
should be addressed
secretion and (2) a different process of ecdysone metabolism. We have studied the inactivation process of ecdysone in the nondiapause-destined larvae and pharate pupae (Moribayashi and Ohtaki, 1978,198O; Moribayashi et al., 1985). In the present paper, we describe different profiles of ecdysone metabolism between nondiapause- and diapause-destined animals of B. peregrina and discuss the biological implications of the different endocrinological events between these two systems. MATERIALS AND
METHODS
Experimental animals
The colony of the temperate race of Boettcherisca perewas routinely maintained in our laborat&y.In-this-experiment, adults were reared at 20°C on a liehtdark cvcle of 1I : 13 or 9: 15 hr. Dianausedestined larvae and puiae were obtained by rearing-larvae continuously under the same environment, whereas nondiapause-destined ones were obtained by culturing them from embryonic through whole larval stage under a longday condition (lightdark cycle of 15 : 9 or 16 : 8 hr) at 20°C. To synchronize their developmental stage, both populations of mature larvae were kept in contact with water for another 2 days, when their crops became empty, in the same condition as they were in growing stage. A portion of these full-grown larvae of both cultures in wet conditions was used immediately as “mature larvae” in the present experiment. The remainder were then transferred to dry conditions and allowed to undergo metamorphosis. The animals 20 hr after pupariation were used as “pharate pupae”. The animals were staged again at the pupariation. In nondiapausing animals, ecdysteroid content was determined by radioimmunoassay every 4 or 8 hr up to 360 hr when adult eclosion occurs. Diapause-destined animals were also sampled for ecdysteroid radioimmunoassay as above until 72 hr after the pupariation, and thereafter sampled every 12 hr until 360 hr after the pupariation. ,qrina (Sarcophaga peregrina)
157
ATSUKOMORIBAYASHI et al
158 Chemicals
Radioactive [23,24--‘H(N)]ecdysone (specific activity 70.0 Ciimmol) was purchased from New England Nuclear, Boston. Unlabeled ecdysone was bought from Sigma, St. Louis. 20-Hydroxyecdysone and 25-deoxy, 20,26-dihydroxyecdysone (inokosterone) were obtained from Rhoto Pharmaceutical Chemistry, Osaka, Japan. Extraction and separation of the ecdysteroid metabolites
A certain amount of radioactive ecdysone dissolved in 1 ~1 of water was injected into each mature larva or pharate pupa of the diapause- and nondiapause-destined animals. Ecdysone metabolites were extracted with a mixture of acetone and ethanol (1: 1 v/v). and separated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) as previously described (Moribayashi et al., 1985). Two development solvents were used for TLC separation. One of them (CHCI,: CH,OH = 4: 1 v/v) (system A) was used for separation of very polar metabolites of ecdysone from free ecdysteroids with moderate polarity. The other solvent (CH,COOC2H5: C,H,OH :H,O = 2: 8: 1 v/v) (system B) was used for separation of very polar metabolites into two groups (OA and OB) (Moribayashi and Ohtaki, 1980). Free ecdysteroid metabolites were extracted from the TLC plates by methanol. The methanol solution was passed through Millipore filters (catalog No. SJHVOO4NS) and subjected to HPLC under the following conditions: column Zorbax Sil, 15 cm/4.6mm; mobile phase CHj(CH,), CH,:C,H,OH:CH,CN:CH,OH=85:15:3:3(v/v); flow rate 0.6 ml/min; column pressure 25 kg/cm*; UV detector set at 254nm. Identification of free ecdysteroid metabolites
In order to identify free ecdysteroid metabolites separated by HPLC, the compounds were acetylated by acetic anhydride and pyridine (1: 1, v/v). The acetylated derivatives of metabolites and the standard tri- and tetra-acetylated ecdysone and 20-hydroxyecdysone were run together on TLC with the solvent (CH,COOC,H,:CH,(CH,),CH, = 8: 2 v/v) (system C). Every 5 mm of silica gel of the plates was scraped into miniviais, and then 0.1 ml of methanol and 1.0 ml of Insta-Gel were added for counting radioactivity. Radioimmunoassay of ecdysteroid content in whole body
Three animals, each taken from diapause- and nondiapause-destined colonies at certain intervals, were weighed and homogenized with a hand-driven homogenizer in 0.5 ml methanol and the homogenates were centrifuged at 10,OOOrpm for 2 min at 0°C. The supernatant was transferred to another tube and 0.5 ml of petroleum ether was added to remove lipids. The delipidated samples were kept at - 20°C and graded aliquots of the samples were then transferred to small tubes. After solvent was evaporated, samples were subjected to radioimmunoassay (RIA) of ecdysteroid according to the method of Horn ef al. (1976)
(A)
0 1
30 40 I
I
60
90
I
I
f
0
30
i!
Pupariatian +
I
20
40
I 60
1 80
t
I I 100 120 h
Fig. 1. Time of pupariation of diapause-destined (0) and nondiapause-destined (0) cultures. Eash point represents average time of three trials using 100 individuals in each test. Bars show ranges of the standard error. and Carrow ef al. (1981). Further details were described in a previous paper (Ohtaki et al., 1986). RESULTS
Efect of different photoperiods on pupariation and pupation Almost all larvae reared in a short-day condition from embryonic stage entered pupal diapause. In these animals, 50% pupariation took place 38.5-41.5 hr after the transfer to dry conditions,
whereas that of nondiapause-destined larvae kept in a long-day condition during whole larval stage was 29.5-31.5 hr after the dry treatment (Fig. 1). There was approx. a 10 hr difference in 50% pupariation between these two cultures. Pupation time was determined by observing the head evagination of pharate pupa, on opening the anterior tip of the puparium every 2 hr. Pupation of diapause- and nondiapause-destined animals occurred 48-50 hr and 58-60 hr after the pupariation, respectively. The delay in pupariation of diapausedestined larvae was entirely recovered by a shorter duration between pupariation and pupation, so that pupation of both groups took place almost at the same time after the dry treatment (Fig. 2). Changes of ecdysteroid levels during pupal and adult development
The level of ecdysteroids from mature larvae through adult development determined by RIA was shown in Fig. 3. The ecdysteroid titre in mature larvae was approx. 20 pg ecdysone equivalent/mg fresh body weight.
390 I‘
h
I
t
Pupariatian
Pupation
(B) I
0
I
t
0
0
60
I
90
I f
Pupation
390 h
I, Adult emergence
Fig. 2. Comparison of the times of 50% pupariation and pupation between diapause-destined (A) and nondiapause-destined (B) cultures. For determination of pupation time 10 animals each for every 2 hr were observed in either of the cultures by removing anterior part of pupal shell.
Ecdysteroid in diapausing and nondiapausing flies
159
600-
400-
zoo-
I
aa t D
’
0
”
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46
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72
n
96
I
120
”
144
166
”
192
216
”
240
264
”
266
312
”
336
360
h
Fig. 3. Ecdysteroid content in whole body in diapause-inducing condition (A) and in nondiapause-inducing condition (B). See text for details.
In white puparial stage of nondiapause-destined animals, the titre was nearly 150pg/mg and this increased towards pupation. Sixteen hours after the pupariation the titre peaked at approx. 230pg/mg, and then decreased 40 hr after pupariation, showing a minimum at about lOOpg/mg. This small peak of ecdysone titre was also observed in diapause-destined pharate pupae. After that, only in nondiapausedestined pharate pupae, the titre again increased with nearly daily fluctuations and reached the second maximum of 1500 pg/mg at 116 hr after pupariation, and the titre then began to decrease toward adult eclosion in which only 80 pg/mg of ecdysteroids was found. In the later pharate adult stage ecdysone titre was determined in female and male separately, but no significant differences were observed. In diapause-destined animals, however, the peak of ecdysteroid titre was only one at the pharate pupal stage and that was approx. 200pg/mg fresh body weight. The titre then decreased to almost undetectable level several hours before pupation and remained at a low level (20-40 pg/mg) until the end of observation period (360 hr after pupariation).
Radioactive metabolites were extracted and separated into the following three groups by TLC: OA (mainly composed by conjugated ecdysteroids), OB (very polar final metabolites) and free ecdysteroid metabolites. When labeled ecdysone was given to mature larvae of both groups, almost equal amounts of OA, OB and free ecdysteroid metabolites were produced 72 hr after the injection. Amounts of OA produced in animals treated with ecdysone in larval stage was much more than that treated in pharate
cm 20000
r
Ecdysteroid metabolism
Radioactivity of labeled ecdysone injected into both diapause- and nondiapause-destined pharate pupae reduced rapidly, and only half of the radioactivity could be recovered 24 hr after the injection. Then. the labeled substance gradually reduced to about 45% of original activity in 72 hr, and there was no remarkable difference between both groups (Fig. 4).
4oooL--0
24
48
72
hr
Fig. 4. Time course of recovered radioactivity after injection of 3[H]ecdysone into diapause-destined (A) and nondiapause-destined (0) pharate pupae.
ATSUKO MORIBAYASHI et
160
pupal stage in both groups. As shown in Fig. 5, in diapause-destined pharate pupae, labeled metabolites were mainly a free ecdysteroid group, composed of approx. 80% of total radioactivity recovered, whereas OB and free ecdysteroids were produced almost equally in nondiapause-destined pharate pupae. At 96 hr, the amount of OA fractions were reduced, but no difference was observed between both cultures (Fig. 5). HPLC analysis of the free ecdysteroid metabolites Elution patterns of the free ecdysteroid metabolites recovered 48 hr and 72 hr after injection of ecdysone into both diapause- and nondiapause-destined pharate pupae are shown in Figs 6 and 7. In developing pupae, ecdysone was mainly metabolized to several compounds, which were temporarily
IO
20
30
al. OA
08
........... ................ ................................
48h
( N I pharate
pupae
72
h
I-
( D )pharate
48h 72 h
1
pupae
Free
; ............................... .... ................ : : : : : :.:.:.:.....................
u
Fig. 5. Percent radioactivity of three major groups of metabolites: OA (mainly conjugated ecdysteroids), OB (very polar final metabolites) and free ecdysteroids, in the extracts 72 and 96 hr after injection of 3[H]ecdysone into diapause-destined (D) and nondiapause-destined (N) larvae (upper), and in those 48 and 75 hr after injection of ‘[HJecdysone into diapause-destined (D) and nondiapausedestined (N) pharate pupae (lower).
40
50
60
min
50
60
min
Fig. 6. HPLC profiles of free ecdysteroid metabolites extracted 48 hr (A) and 72 hr (B) after injection of ‘[Hlecdysone into nondiapause-destined pharatae pupae. Fractions were collected every 30 set (tilled columns) from the 21st min to 40th min, or every 1 min for the rest of the time. For comparison the halves of open columns, that are equivalent to the radioactivity of 30 set collection, are filled. See text for further details.
Eedysteroid in diapausing and nondiapausing flies
161
IA)
10
CPm
1000 750
500
20
min
40
I 1
10
20
30
40
50
60
min
Fig. 7. HPLC profiles of free ecdysteroid metabolite extracted 48 hr (A) and 72 hr (B) after injection of ‘[Hlecdysone into diapause-destined pharate pupae, Explanations are the same as in Fig. 6.
identified as 20.hydroxyecdysone, 26-hydroxyecdysone, 20,26dihydroxyecdysone and the epimers of the last two by their elution time, being compared with those of the cochromatographed standards (Fig. 6). In diapausing pupae, however, mainly 3-epiecdysone, 3-epi-20-hydroxyecdysone were found, but 26hydroxy and 20,2&dihydroxy derivatives were scarcely in the free ecdysteroid group (Fig. 7). From 48 to 72 hr after injection, in developing pupae, almost all of unchanged ecdysone disappeared, but the amounts of 20-hydroxyecdysone, 26-hydroxylated and 20,26-dihydroxylated derivatives inreased considerably. Identification
of acetylated metaboiites
The free ecdysteroid metabolites were then subjected to acetylation for more detailed identification. Seven free ecdysteroid metabolites were isolated from the extracts of developing pupae in order of increasing elution time (compounds I to VII shown in Fig. 6). Three free ecdysteroid metabolites (compounds I to III shown in Fig. 7) were isolated from
diapausing pupae. All these isolated samples were acetylated and applied on TLC separation (System C, detailed in Materials and Methods). The gel on the TLC plates was scraped in 5 mm strips and the radioactivity was determined. As shown in Fig. 8, standard samples of tri-acetyl and tetra-acetyl ecdysone migrated to the positions shown as a(I) and n(2), and those of 20-hydroxyecdysone migrated to /$(I) and p(2). The R, value of tetra-acetyl inokosterone was almost the same as that of tetra-acetyl 20-hydroxyecdysone. Acetylated product of compound I, which was temporarily identified as 3-epi-ecdysone by HPLC, migrated to the same region of acetylated ecdysone, confirming the identification by HPLC analysis. Compound II obtained from developing pupae behaved the same as ecdysone. However, compound II extracted from diapausing pupae was identified as 3-epi-20-hydroxyecdysone, but not as ecdysone, by the acetylation test. Compound III obtained from the extract of both groups was identified as 20-hydroxyecdysone. Similarly, compounds IV and VI were identified as
ATSUKO MORIBAYASHI
162
(A)
@d
(I31
O&Q
1>
cm 3-Epi-ecdysone
400
@aa
@dO +31
cpm 1500
600
et al
(I)
1250
3-Epi-ecdysone
1000 200
750. 500 250
er n
Ill
t
(II) Ecdysone
?
( 3-Epi-20-hydroxyecdysone
500 250 -
em@da
em ed@
n
4001
*
(III,
4
(III)
20-Hydroxyecdysone
20-Hydroxyecdysone
750
500 250
0
cpml n
r’,g,”
Tbp
BOO 600
HI
cm, 3-Epi-26-hydroxyecdysone
600
(VI 3-Epi-20,26-dihydroxyecdysone
, “I, 26-Hydroxyecdysone
20,26-Dihydroxyecdyrone
To,
Fig. 8. Comparative radioactivity of acetylated derivatives of ecdysteroid metabolites separated by HPLC in nondiapause-destined pharate pupae (A) and diapause-destined pharate pupae (B). Shaded circles show the migrated positions of the following standard compounds co-chromatographed with tested samples (Moribayashi et al., 1985). a 0, triacetylated ecdysone; a 0, tetraacetylated ecdysone; p 0, triacetylated 20-hydroxyecdysone; b 0, tetraacetylated 20-hydroxyecdysone; I, tetraacetylated inokosterone. Numbers in the right shoulder of each chromatogram are those of fractions separated by HPLC shown in Figs 6 and 7. The substances in each fraction are temporarily identified as ecdysteroid listed under the number.
1
Ecdysteroid
in diapausing and nondiapausing flies
3-epi-26-hydroxyecdysone and 26-hydroxyecdysone, respectively. Amounts of compounds V and VII isolated from developing pupae were not enough to obtain definite data, but they could be temporarily identified as 3-epi-20,26-dihydroxyecdysone and 20,26-dihydroxyecdysone, with reference to data described in previous report (Moribayashi et al., 1985). DISCUSSION
The ecdysteroid content in the whole body determined by RIA peaked between pupariation and pupation at approx. 200 rig/g body weight in both diapause- and nondiapause-destined individuals of Boettcherisca peregrina and is well within the range of physiological level and of titre measured by sarcophaga-assay which was reported previously (Ohtaki and Takahashi, 1972). The second peak of ecdysteroid content was observed in developing pupae of several species of the fly by either bioassay or RIA (see review by Richards, 1981). But, in some cases, the content determined by RIA was represented as much higher than that measured by bioassay (Koolman, 1980). We also found a high level of ecdysteroid content during the adult development. This may be caused by both non-specificity of antisera of ecdysone used and determining ecdysteroid levels of the whole body, in which inactivated ecdysteroids were stored, but not of the haemolymph. The absence of the second peak in diapausing pupae was also reported, using bioassays and RIAs (Ohtaki and Takahashi, 1972; Walker and Denlinger, 1980; Richard, D. S. et al., 1987). In the present study we analysed and identified ecdysone metabolites by TLC, HPLC and chemical methods. Although definite identification of the compounds could not be attained by these methods, we found clear differences in ecdysteroid metabolism between diapause- and nondiapause-destined animals In nondiapause-destined animals, 20,26-dihydrovylation of ecdysone at larval stage, and in addition to that 26-hydroxylation and epimerization in pharate pupal stage, were shown to be main inactivation processes of ecdysone, confirming the results reported in the previous paper (Moribayashi et al., 1978, 1985). It is worthy to note, however, that 26-hydroxyl derivatives were rarely found in the free ecdysteroid metabolites in diapausing pupae. The main inactivated products belonging to free ecdysteroid groups in these animals were 3-epimers of ecdysone and 20-hydroxyecdysone. These results suggest that, in diapausing pupae, either 26-hydroxylation of ecdysone did not occur or 26-hydroxyecdysone produced was rapidly converted to more polar metabolites, such as ecdysonoic acid or conjugated ecdysteroids (Richard et al., 1987). However, the latter case may not be possible because both OB (very polar metabolites of ecdysone) and OA (mainly conjugates of ecdysone) were produced in diapausing pupae less than in developing pupae (Fig. 5). A short-day condition induces pupal diapause in which ecdysone release and probably foregoing
PTTH secretion are absent in adult development of the flesh flies. From an evolutionary point of view, this species of sarcophagid flies is thought to emerge
163
in some tropical region (Kurahashi and Kano, 1984; Denlinger, 1986). Among sarcophagid species, Boettcherisca peregrina has the widest distribution, covering tropical and temperate countries, including Japan, where its races with facultative diapause or continuously developing races are found (Kurahashi and Ohtaki, 1977, and in preparation). It could be suggested, therefore, that incorporation of a new set of genes, which is responsible for the change of the endocrinological mechanisms, including stoppage of the PTTH release and different metabolic pathway of ecdysone, should be involved in the expansion of their territory to the temperate zone. A sequential process between environmental stimuli and endocrinological events is still entirely unknown. Further studies should be put forward to elucidate this process together with other regulatory mechanisms of metabolisms, for instance the induction of a high content of glycerol in diapausing pupae (Kitoh, 1979; Richard, E. Lee Jr et al., 1987). Acknowledgement-This work was partly supported by a grant from the Ministry of Education, Science and Culture, Japan (No. 62540541) to T.O.
REFERENCES
Carrow G. M., Calabrese R. L. and Williams C. M. (1981) Spontaneous and evoked release of prothoracicotropin from multiple neurohumal organs of the tobacco hornworm. Proc. Natl. Acad. Sci., USA 78, 5866-5870. Denlinger D. L. (1971) Embryonic determination of pupal diapause in the flesh fly, Sarcophaga crasipalpis. J. Insect Physiol. 17, 1815-1822. Denlinger D. L. (1984) Hormonal control of diapause. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), pp. 354412. Pergamon Press, Oxford. Denlinger D. L. (1986) Dormancy in tropical insects. Ann. Rev. Entomol. 31, 239-264. Fraenkel G. and Hsiao C. (1968) Morphological and endocrinological aspects of pupal diapause in a fleshfly, Sarcophaga argyrostoma and S. bullata. J. Insect Physiol. 14, 689-705. Horn D. H. S., Wilkie J. S., Sage B. A. and O’Conner J. D. (1976) A high affinity antiserum specific for the ecdysone nucleus. J. Insect Physiol. 22, 901-905. Kitoh K. (1979) Glycerol content in diapausing and developing pupae of Sarcophaga peregrina. Dissertation, Masters Degree, Kanazawa University (in Japanese). Koolman J. (1980) Ecdysteroids in the blowfly, Calliphora vicina. In Progress in Ecdysone Research (Edited by Hoffmann J. A.), pp. 187-210. Elsevier, Amsterdam. Kurahashi H. and Kano R. (1984) Phylogeny and geographical distribution of the genus, Boettcherisca rohdendorf (Diptera: Sarcophagidae). Jpn. J. Med. Sci. Biol. 37, 27-34. Kurahashi H. and Ohtaki T. (1977) Crossing between nondiapausing and diapausing races of Sarcophaga peregrina. Experientia 33, 186-187. Kurahashi H. and Ohtaki T. (1979) Induction of pupal diapause and photoperiodic sensitivity during early development of Sarcophaga peregrina larvae. Jpn. J. Med. Sci. Biof. 32, 77-82. Kurahashi H. and Ohtaki T. (1988) Geographical variation in pupal diapause incidence among Asian and Oceanan species of the fleshfly, Boeffcherisca (Diptera: Sarcophagidae) (in preparation). Moribayashi A. and Ohtaki T. (1978) Inactivation of ecdysone and possible feedback control of the titre during
164
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pupation of Sarcophaga peregrina. J. Insect Physiol. 24, 279-285. Moribayashi A. and Ohtaki T. (1980) Inactivation and reactivation of 20-hydroxyecdysone during pupal-adult development of the fleshfly, Sarcophaga peregrina. Jpn. J. Med. Sci. Biol. 33, 189--20 I. Moribayashi A.. Kurahashi H. and Ohtaki T. (1985) Comparative studies on ecdysone metabolism between mature larvae and pharate pupae in the fleshfly, Sarcophaga oereprina. Arch. Insect Biochem. Phvsiol. 2. 237-250. OitakrT. and Takahashi M. (1972) induction and termination of pupal diapause in relation to the change of ecdysone titer in the fleshfly, Sarcophaga peregrina. Jpn. J. Med. Sci. Biol. 25, 369-376. Ohtaki T., Yamanaka F. and Sakurai S. (1986) Differential timing of pupal commitment in various tissues of the silkworm, Bombyx mori. J. Insect Physiol. 32, 635-642. Richard D. S., Warren J. T., Saunders D. S. and Gilbert L. I. (1987) Haemolymph ecdysteroid titre in diapauseand nondiapause-destined larvae and pupae of Sarcophaga argyrostoma. J. Insect Physiol. 33, 115-122.
Richard E. Lee Jr, Cheng-ping Chen, Mark H. Meacham and David L. Denlinger (1987) Ontogenetic patterns of cold-hardiness and glycerol production in Sarcophaga crassipalpis. J. Insect Physiol. 33, 587-592. Richards G. (1981) Insect hormone in development. Biol. Ret:. 56, 501-549. Roberts B. and Warren M. A. (1975) Diapause in the Australian flesh fly Tricholioprocfia impatiens. (Diptera: Sarcophagidae) A&t. J. Zooi. 23, 563-567. _ Saunders D. S. (19711 The temuerature comoensated ohotoperiodic clock “p;ogramming” development and-pupal diapause in the flesh-fly, Sarcophaga argyrostoma. J. Insect Physiol. 17, 801-812. Vinogradova E. B. (1976) Embryonic photoperiodic sensitivity in two species of fleshflies, Parasarcophaga similis and Boetrcherisco seprentrionalis. J. Insect Physiol. 22, 819-822. Walker G. P. and Denlinger D. L. (1980) Juvenile hormone and moulting hormone titres in diapause and nondiapause destined flesh flies. J. Insect Physiol. 26, 661-664.