Actions of the juvenile hormone, 20-hydroxy-ecdysone, and the oöstatic hormone during oögenesis in the flies Phormia regina and Sarcophaga bullata

0022-1910

ACTIONS

OF THE JUVENILE

ECDYSONE,

AND

OijGENESIS

HORMONE,

THE OijSTATIC

IN THE FLIES

SAKCOPHAGA

79 031)1-0105

S(l’.OO 0

ZO-HYDROXY-

HORMONE

DURING

PHOKMZA REGINA BULLA T.4

AND

GOTTFRIED FRAENKEL and MARGARET HOLLOWELL Department of Entomology, University of Illinois. Urbana.

IL 61801. U.S.A.

Abstract-Application of juvenile hormone (JH) to sugar-fed Phormiu flies leads to full ovarian development. i.e. the flies become autogenous. Application of JH to sugar+ liver-fed Phormnu leads to the simultaneous development of primary, secondary and tertiary oiicytes, suggesting the role of the oiistatic hormone is to shut off the action of the JH. This is consistent with the notlon that the oiistatic hormone may act on the corpus allatum either directly or through the neurosecretory system. Application of IO-hydroxyecdysone to Phormiuor Sawophuga. when primary oijcytes have started to develop (stage 4A). causes the primary oiicytes to degenerate. with concomitant development of the secondary okytes.

INTRODUCTION THE all-important role of thejuvenile hormone (JH) in controlling oiigenesis in many insect systems, including flies, has been amply established (for reviews see ENGELMANN, 1970; DOANE. 1972). In a foregoing paper (PAPPAS and FRAENKEL, 1978) we demonstrated that a product of the median neurosecretory cells in the brain stimulates the oijgenic action of the JH as was postulated long ago (THOMSEN, 1952), contrary to later claims about the JH stimulating the production of brain hormone (THOMSENand LEA, 1968). We also showed that the role of the oijstatic hormone (which inhibits development of subsequent oijcytes while the primary is developing (ADAMS et al., 1968) is to terminate the action of the JH, and that 20-hydroxyecdysone applied at the proper time causes the primary oticytes to degenerate. with concomitant development of the secondary oiicytes. The present investigation was undertaken to study the effect of repeated applications of JH where one application had given a definite, but limited response. This effort led to an extension of the previous study, in the following areas: (1) effect of the JH on oiigeoesis in the absence of a protein meal; (2) effect of the JH on the simultaneous development of several successive oijcytes; (3) degeneration of the primary oiicyte. caused by ecdysteroids.

MATERIALS

AND METHODS

The same fly species as in the foregoing papers (PAPPAS and FRAENKEL, 1977, 1978), Phormia regina Meig. and Sarcophaga bullata Park., were used. They were raised in laboratory cultures. These two species showed striking differences as to the importance of a protein meal for o(igenesis. Phormiu on water and sugar alone in former experiments had never shown any trace of yolk deposition and was considered

entirely anautogenous. In the present series of‘ experiments, Phormia in the absence of liver on occasion showed a very minute droplet of yolk in the primary oiicyte. far smaller than what was formerly scored as 4A. Sarcophagu on the former occasions. under the same condition. regularly showed initial yolk deposition up to stage 4A, then considered as ‘incipient’ autogeny. In our present experiments. Sarcophaga showed on many occasions full yolk deposition. Autogeny must have developed in our laboratory colony during the intervening 2 yr. The problem of autogeny is discussed in our former paper (PAPPAS and FRAENKEL. 1977), and below in the discussion. The ovaries were dissected out after a specified treatment and time, and the state of the ovaries was scored by a system formerly described (PAPPAS and FRAENKEL, 1977). Briefly. stages II 2 and 3 are before yolk deposition, 4A, 4B and 4C stand roughly for 114, l/2 and 3/4 of an oljcyte filled with yolk, and M for the oiicyte full with yolk. In almost all cases (for exceptlons see Table 4) the oiicytes in one ovary. and for that matter both ovaries, are in exactly the same state of development. The ages at which flies were treated or examined are stated as in our former publications. Day 0 is the day of emergence. One-dayold is between 24- and 4%hr-old, etc. The JH we used was the analogue JH/ZR-515 (Altosid). obtained from Zoecon. In the Faiority of cases it was dissolved in light mineral oil and applied topically and ventrally on the abdomen in doses of 2.5 ,ug!S ~1 or I &fly. The present investigation was in part sponsored by the desire to study the effect of several applications of JH, to see whether this would enhance effects formerly obtained with one application. It became clear immediately that repeated applications on 3 successive days led to very high mortalities, which not only would have required the use of inordinately high numbers of individual flies. but would have cast doubt on the

GOTTFRIEDFRAENKELAND MARGARETHOLLOWELL

306

Table I. Phormiu regha. Autogeny induced by JH (in acetone)

No. scored Control 2.5/1gJH Ixt 2SpgJH2xf 2.5 pg JH 3 x $ 1 pl acetone I x t 1 ~1 acetone 2 x 1 I ~1 acetone 3 x 8

20 20 22 23 I5 20 23

3

Oijgenic Primary cycle 4A 48 4C

in absence

ofliver feeding

phase in M

Secondary 3

20 I7 13 I2 I5 19 20

I 5

4

I

I 3 2

I

cycle*

5 4

5 4

I 3

* None beyond stage 3. t JH or acetone alone applied on day 3, ovaries inspected on day 7. : JH or acetone applied on day 3 and 4. ovaries inspected on day 7. $JH or acetone a In stages 1-3

applied

on days 3. 4. and 5, ovaries

significance of results obtained with the few survivors. This was somewhat remedied by reducing the applied volumes from 5 to 1~1, and the number of applications from 3 to 2. Application by injection, rather than topically, did not improve the situation substantially, though it appeared that two injections led to better survival than two topical applications. Consequently in many of the experiments we used only one topical application. Eventually (Table 1) we found that with acetone as the solvent, both applied topically or injected, survival was much better. Ecdysteroid was used in the form of 20-hydroxyecdysone, obtained from Rhoto Pharmaceutical Co., Osaka, Japan, and injected dissolved in water (5 pg/S pi/fly). Before application or injections the flies were immobilized on ice. Injections were made into the thorax, by means of finely drawn-out glass pipettes with a tip diameter of about 40 pm. RESULTS

(A) Stimulation oj’diigenesis by JH in the absence of’s protein meal

Autogeny-cgg development in the absence of a protein meal-occurs regularly in many fly species, and also irregularly in one and the same species. This shows that the function of dietary protein is not necessarily that of providing building stones, but may have that of triggering certain initial processes of Table 2. Phormiu regina. JH causing development of oiistatic

No. scored Control 2.5 pg JH* 1 ~1.oil* 2.5 pg JHt I jd. oil?

23 15 I8 4 2

3 2 6 9 I

*Topically applied. t Injected. The flies were fed liver from day 6.

Primary cycle 4C

I

M

inspected

on day 7

oiigenesis. One such function could conceivably be the mobilization of JH. With this possibility in mind, we tested the effect of application of JH on sugar-fed Phormia in the absence of a liver meal. No egg development occurred with one topical application of 2.5 pg dissolved in 1 ~1 mineral oil, and repeating this one and two days later led to such severe mortality as to make the results meaningless. Success was attained when JH was topically applied, dissolved in acetone (Table 1). With one application development proceeded in all cases beyond stage 3, and with 2 and 3 applications led to full development to maturity in a great number of instances. The controls. treated with acetone alone, only in a few instances showed development to stage 4A. The same experiments with Sarcophaga did not yield results. On almost all occasions the flies showed autogeny in the absence of the liver meal, and therefore could not be tested for the effect of JH on ohgenesis. (B) Effect ofJH on simultaneousdevelopment ofseveral oijcytes Formerly (PAPPAS and FRAENKEL, 1978) we showed that the application of JH during development of a primary oiicyte in Phormia caused very substantial simultaneous development of the secondary oiicytes, leading to full maturity in two specimens. This was interpreted to mean that the ‘ohstatic hormone (ADAMS et al., 1968) normally prevents development of of secondary/tertiary hormone

okytes

Stages of oiicytes Secondary cycle 3 4A 4B 4C

II 9 8 4

5

6

I

7

I

I

I

2 I

by abolishing

effect

M

Tertiary cycle 3 4A

6

3

4

3

I

2

I, JH or oil were applied on day 2, and the ovaries inspected on day

-a

- -b a

6

.\trWl,/Jlrclc(r M/r/l‘/ I-IS. I Plromitr rc:qir~u O\arioles from a lly treated with JH. each showing the tirst mature oiicyte (a). simultaneously with ~hc \ccond in about stase 4c‘. t b). and the third 111\tagc 3 (~1. I-IS. 2 f’ho~nirr W,I$/W (A) A normal ovary Hith I‘ullydacloped matureeggs. (B) Ovary liom a fly treated with JH Hherc every ovariole contains two mature eggs which makes the ovary correspondingly larger. f-19. 3. Plrorrnitr rc@rcr Treated uith XJ-hydrosy-ecdybonc. The primary oiicyte. still ofconsiderable size. is degenerating (;I). the secondary oiicyte shows beginning yolk deposition. th) and the third oiicytc I\ in stage 3. (c).

FIN. 4. Pltor~io rc’g~~ Treated with ‘O-h~droxy-ecdqso,,~ The primary oiicyte (a) is totally degenerate. IravIng onI> cl traceoftissue. thesecondary oiicyte(b)ihin ~ta.ge4C.and the tertiary oticyte (c) in a stage hetticcn -3 and 3A. tug. 5. .SUYO~/W~~U Mlurcr Treated Nith ecdysone. The primary oiicytesca) havedegenerated and are still attached to the developing secondary oiicytes (b) now in stage 4B A tertiary ociqte (c) in rtage 3 15 \tvhle Fig. 6. Phornliu r~‘g~tcr. Treated with 20-hqdroxq-ccd!~~,nc. An ovary has been torn apart. showing slmultaneousl! the presence of ovarioles with mature oiicytrs (a) and ovarmle\ with 06cytes in ‘;tage 4A th). It IS not clear whcthrr the\c ,~~c‘!tc4 arc prlmar! or \c~onii~~r~

succeeding okytes by inhibiting the action of the JH. The experiment was repeated with the intention of enhancing development of secondary oiicytes by the multiple application of JH. These efforts were mostly unsuccessful because of a high mortality. However, a repeat, with only one application, and dissolved in 1 ~1 mineral oil (as compared to 5 ~1 in former experiments), led to full success. Not only did almost all flies treated with JH have fully or almost fully mature secondary oiicytes, but the same flies also

showed a very substantial development up to 4A in the tertiary oocytes (Table 2 and Fig. 1). In this experiment a comparison was also made between the effect of JH when topically applied, or injected, and substantially the same results were obtained with both methods (Table 2). In flies with simultaneously developed primary and secondary oiicytes the ovaries were substantially enlarged (Fig. 2). Such flies had distended abdomens. In Surcophu~a no secondary development occurred

308

GOTTFRIED

FRAE~KELAND MARGARET HOLLOWELL

Table 3. Phormiu regina. 20-Hydroxy-ecdysone causes primary oijcytes to degenerate Stages of oiicytes ‘Secondary’ oljcyte ‘Primary’ oacyte 4C Degenerated 3 4A 48 Mature

No.

scored Control* Water+JH* ?O-Hydroxy-ecdysone ?O-Hydroxy-ecdysone 20-Hydroxy-ecdysone 20-Hydroxy-ecdysone 20-Hydroxy-ecdysone 20-Hydroxy-ecdysone

14 IS 19 15 16 15 14 17

24 hrt 48 hr: 72 hr* + JH 24 hrt + JH 48 hr$ + JH 72 hr*

*Ovaries inspected on t Ovaries inspected on : Ovaries inspected on The flies were fed liver were Inspected on day

day 6. day 4. day 5. from day 2.20-hvdroxv-ecdvsone 4. 5. or-6. . . _

(C) Degenerating effect of 20-hydroxy-ecdysone on the primary oiicyte We formerly reported (PAPPAS and FRAENKEL 1978) that injecting an ecdysteroid into flies with developing oiicytes caused the primary oiicytes to degenerate, with a corresponding development in the secondary oiicyte. The effect only occurred if the developing oijcytes were neither too young (less than stage 3), nor too old (over stage 4B). In a repeat of this experiment, Phormia was liver-fed on day 2, and the hormone was applied on day 3: the ovaries were inspected on days 4, 5 and 6, after having been subjected to the effect of 20hydroxy-ecdysone for 1, 2 or 3 days (Table 3). Most Effect of20-hydroxy-ecdysone

No. scored Water + oil* 20.Hydroxy-ecdysonet

17 13 12 12 8

2

6

7

9

2

I

2

II I 2

I 3

ovaries at the time of hormone injection were in stage 4A, the stage formerly found as the most favorable for responding to ecdysteroids. However, it is not possible to know with certainty for each individual fly in exactly which stage the oacytes had been at the time of injection. Of the flies inspected after 24 hr four contained mature primary oiicytes, and in 15 the primary oiicytes had degenerated. The secondary oiicytes had hardly developed beyond the stage reached in the controls. At the 48-hr point many of the secondary oijcytes had substantially developed to stage 4B, and 1 day later practically all the ‘primary’ oijcytes were mature, and none of the secondary ones had remained in stage 4B. The obvious conclusion is that about all ovarioles with degenerating oacytes by that time had the secondary oijcytes fully developed. The latter had the appearance of primary oiicytes because by that time there is hardly a visible trace left of the degenerated original primary o6cyte. Figures 3 and 4 are drawings of teased-apart ovarioles from Phormia treated with 20-hydroxyecdysone where the primary oiicytes have degenerated and secondary oijcytes developed to different degrees. with tertiary odcytes also beginning to develop. It was then of interest to know whether the

on degeneration okyte

4A

Primary 4B 4C

of primary

M

oijcyte. and development

Stages of oikytes No. of primary degenerated 3 _~___.~

of secondary

Secondary 4A 48 4C

12 4 104

,

3

6: 20-Hydroxy-ecdysone+JHt 20-Hydroxy-ecdysone+oil* 20-Hydroxy-ecdysone+JH*

14 IO

9 7 5

5 5 6 4 7 I 6

(5 pg,fly) or JH (2.5 pg.fly) was applied on day 3. and the ovaries

normally fed with sugar and liver. However, in another experiment when sugar-fed Phormiu were treated with JH (Table l), flies which had shown development to maturity simultaneously had the secondary oijcytes developed to stage 3, which represents a substantial development in a secondary oijcyte.

hulia~u

9 4 13

IS

4 14

with one application of 5 pg JH in oil, nor in the survivors from twice or three times repeated applications. In these experiments with Phormin the flies had been

Table 4. Sarcoplqqu

-

14 I5 4 6 16

M

6: r 12 8

6: -n 10 4

2 3

M

,

-

I

*Ovaries inspected on day 6. t Ovaries inspected on day 4. $ Six flies have within each ovary primary oiicytes at 4C-M stage showing no degeneration, and about equal number of primary degenerating oiicytes with secondary okytes at range of 4B-4C stages. 5 Primary oiicytes within each ovary ranged from 4A to 4C stage. These h8d presumably degenerated by &I 6. ’ Two ovaria had primary okytes degenerated. and secondary oiicytes at range of 4B-4C. The flies were fed liver from day 3,20-hydroxy-ecdysone. JH, oil or water were applied the same day, and the ovaries were inspected on day 4 or 6.

Hormonal

control

degenerating effect of 20-hydroxy-ecdysone could be counteracted by JH. Such an effect was suggested in our previous paper in one out of three experiments. In the experiment of Table 3 a set of tests was performed with JH applied at the time of ecdysteroid injection. The results with or without JH hormone are substantially the same, and do not suggest that JH in any way counteracted 20-hydroxy-ecdysone. A similar experiment performed with Sarcophagu, though in less detail, led to very similar results (Table 4 and Fig. 5). All primary oocytes from flies treated with the ecdysteroid degenerated, and there were ciear signs of developing secondary oocytes. Oiigenesis is slower in Sarcophaga and therefore did not show secondary oocytes developing to full maturity after 3 days. Essentially the same results were obtained in another experiment with Sarcophuga. On this occasion we observed and recorded a phenomenon of oocytes in different stages of development occurring in one and the same ovary (Table 4). Normally, developing oocytes in an ovary are remarkably well synchronized as to their stage of development. It appears that 20hydroxy-ecdysone upsets this synchrony and this is even extended to the secondary oocytes. A similar observation of an apparent absence of synchronization of development showed up in one experiment with ecdysteroid-treated Phormia where fully mature oocytes were present simultaneously with oocytes in stages between 4A and 4B (Fig. 6). It was impossible to tell from this preparation whether these stages represent primary or secondary oocytes. DISCUSSION JH and autogen?

of ohgenesis

I” flies

309

function of the protein meal is to trigger an action of JH (PAPPASand FRAENKEL,1977). The same experiment with Sarcophaga did not yield results, owing to a regular occurrence of autogeny, whereby full development occurred in the absence of a protein meal. Oijstatic hormone and JH

It has been known for a long time that in many fly species, including representatives of Calliphoridae, Sarcophagidae and Muscidae (but not in Drosophilidae) during development of the primary obcyte the subsequent oijcytes remain undeveloped until the first egg is laid. This led to the discovery by ADAMS er a/. (1968) of the existence of an oostatic hormone which inhibits development of the succeeding oocytes. The existence of an oostatic hormone explains certain phenomena, like the failure to induce oogenesis in sugar-fed flies by a repeated injection of haemolymph from specimens in the process of oogenesis where the nutritional and hormonal conditions in the haemolymph could be expected to be optimal for egg development (unpublished data). Or the discovery that in flies joined in parabjosis egg development takes place in the protein-fed, but not the unfed member (BENNETTOVAREZABOVA.

1972).

ADAMSet al. (1968) originally suggested that the oostatic hormone works by shutting off the activity of the corpus allatum. Our findings about simultaneous development of several oocytes in the presence of added JH certainly support this view. Subsequently ADAMSet al. (1975) considered the inhibition of the release of neurosecretory material as the primary function of the oostatic hormone. This is still entirely compatible with our view of the crucial importance of the JH in this context. PAPPASand FRAENKEL(1978) had shown that the release of JH is dependent on the action of neurosecretory material. The oostatic hormone could therefore act either directIy by shutting off the release of JH. or indirectly through inhibiting the release of neurosecretory material. Application of JH entirely circumvents the necessity of neurohormonal action. Results obtained in the present and a foregoing paper (PAPPASand FRAENKEL,1978) on the interaction

Although the importance of the JH in oogenesis in flies has been known for over 40 yr, and the phenomenon of autogeny also for very long, the question of whether anautogeny is caused more by a lack of JH than that of a protein meal per se has apparently not been directly tackled until very recently. MJENI and MORRISON(1976) showed that application of JH to sugar-fed Phormia regina caused a very slight initiation in ovarian development, sometimes leading to initial yolk deposition (4A, or less in our terminology). And more recently, SAKURAI (1977) reported vitellogenesis with a wide range of HORMONAL REGULATION manifestations in sugar-fed houseflies treated with JH. OF OOGENESIS IN f%orm/a Manifestations of autogeny in flies have been variously reported or discounted even in the same -----+ stlmulatory effects Protein me0 I species (see PAPPAS and FRAENKEL, 1977). One ---+ lnhibltory effects (oostotic hormone) possible explanation for the irregularity in results may i lie in the discovery of a photoperiodic effect on neurosecretory CA ‘c-, autogeny. autogeny being low in flies kept in darkness, moteria I *o and high in flies irradiated with UV light, in JH\ ‘O, Surcophagufalculuta (SCHLEIN,1977). Obviously most BRAIN “---either __ authors. including ourselves, paid no attention to the light conditions in their cultures. JH compensates for inhibition of oogenssis by early We regularly obtained full development in the first NSCor CAoocyte following application of JH to sugar-fed P. JH overrides effect of oostatic hormone regina, a species which in our experience is entirely JH stimulates oogenesls in absence of protein meol This suggests that anautogenous. certainly anautogeny in this species is in the last instance not due Fig. 7. Scheme of the effects and interactions of a protein to a lack of protein in the food, but a lack of JH in meal, neurosecretory hormone. JH, and oijstatic hormone in the regulation of oiigenesis in Phormiu r@nn available form, and further supports the idea that one

GOTTFRIEI) FRAIXNKEL ANI MARGAHLTHOLLOWILL

310

between neurosecretory effects, the JH, and oostatic hormone, are summarized in the scheme of Fig. 7, depicting the hormonal interactions during oogenesis in Phormiu regina. The crucial process in the initiation of oocyte development is stimulation of the ovary by JH released from the CA. In the normal course of events, this reaction originates with a stimulatory effect of a protein meal on neurosecretion which in turn stimulates the CA. This chain of events is interrupted by either the absence of a protein meal, or the (early) extirpation of the neurosecretory cells or the CA. Direct application of JH restores the defects brought about by any of these three manipulations. JH also overrides the effects of the oostatic hormone (which during development of an obcyte inhibits oiicytes) by development of the successive circumventing inhibition of CA activity brought about either directly, or indirectly via the neurosecretory system. Ecdysteroids and oiigenesis

The subject of a role of ecdysteroids, positive or negative, in oogenesis in insects has a curious history. For long it had been believed that the prothoracic glands degenerate in the adult, and play no role in oogenesis. ENGELMANN (1959), however, had shown that implantation of prothoracic glands, or even injection of crystalline ecdysone, inhibited corpus allatum activity and egg maturation in the cockroach, Leucophaea maderae. Subsequently, a degenerating effect of implanted ring glands on the ovary of the by DEORAS and housefly was demonstrated BHASKARAN (1967). The possibility of a role of moulting hormone in Drosophila in this context was recently raised by HODGETTS et al. (1977) who reported very low ecdysteroid titres in adults, though apparently significantly higher in the female. The recent discoveries of an important function of ecdysteroids in obgenesis of mosquitoes (e.g. HAGEDORN ef al., 1975) have re-opened this subject. Our data suggest a very specific degenerating effect in Phormia and Sarcophaga, so specific that it acted only on the primary oocyte during a brief developmental stage, and then caused development of subsequent oocytes. These effects manifested themselves with relatively high doses of the hormone (5 /*g, and possibly 1 pg/fly). There was no evidence of JH antagonizing this action. Even if this does not suggest a normal role of 20-hydroxy-ecdysone in egg development of flies, it points at least to a fundamental cytological effect of this hormone in this context. Acknowledgement-This

study

was

supported

by

Science 13383 and PCMll-24247.

successive National

Foundation

Grants

PCM74-

REFERENCES ADAMST. S., GRUGEL S.. ITTYCHERIAH P. I., OLSTADG. and CALDWELLJ. A. (1975) Interactions of the ring gland. ovaries, and juvenile hormone with brain neurosecretory cells in Musca domestica J. Insect Physiol. 21, 1027-1043. AOAMST. S.. HINTZA. M. and POMONIS J. G. (1968) Oostatic hormone production in houseflies, Musca domestica. with developing ovaries. J Insect Ph_rsiol. 14, 983-993. BENNETTOVA-REZABOVAB. (1972) The regulation of vitellogenesis by the central nervous system in the blowfly. Phormia regina. Acta en! BohemoslaLr. 69, 78-88. DEORAS G. S. and BHASKARAN Cl. (1967) Studies of the neuroendocrine system in the housefly, Musca nehulo--IV. Hormonal control of ovary development. J. L/nib*.Bomha~, 25, 73-87. DOANE W. W. (1972) The role of hormones in insect development. In Development .S_vsrems: Insects (Ed. by COLINCE S. J. and WADDINGTON C. W.). pp. 291-497. Academic Press, London. ENGELMANN F. (1959) ijber die Wirkung implantierter Prothoraxdriisen in adult Weibchen von Leucophaea maderae Z. vergl Physiol 41, 456-470. ENGELMANNF. (1970) The Physiology of Insecr Reproduction Pergamon Press. New York. HAGEDORN H. H.. O’CONNORJ. D.. FUCHSM. S.. SAGE B., SCHLAEGER D. A. and BOHM M. D. (I 975) The ovary as a source of ecdysone in an adult mosquito. Proc. natn Acad. Sci.. U S.A 72, 3255-3259. HODGETTS R. B., SAGE B. and O’CONNOR J. D. (1977) Ecdysone titers during post-embryonic development of Drosophila melanogaster. Devl. Biol. 60, 3 IO-3 17. MJENI A. M. and MORRISON P. E. (1976) Juvenile hormone analogue and egg development in the blowfly, Phormiu regino. Gen. camp. Endocr. 28, 17-23. PAPPAS C. and FRAENKEL G. (1977) Nutritional aspects of oogenesis in the flies. Phormia regina and Surcophaga bu[lata. Physiof Zool. 5, 237-246. _ PAPPAS C. and FRAENKEL G. (1978) , Hormonal asuects of oogenesis in the flies Phormia regina and Sarcophaga hullata J. Insecf Physiol. 24, 75-80. SAKURAI H. (1977) Endocrine control of oogenesis in the housetly. MUSM domesstica vicina. J Insect Physiol 23, 129>1302. SCHLEIN Y. (1977) Ultraviolet light and puparial weight as factors in the autogeny of the fleshfly, Sarcophaga lalculara. J. Insect Ph~siol. 23, 961-964. THOMSEN E. (1952) Functional significance of the neurosecretory brain cell and the corpus cardiacum in the female blowfly, Calliphora eryrhrocephala Meig. J. up Biol. 29, 137-l 72. THOMSEN E. and LEA A. 0. (1968) Control of the medial neurosecretory cells by the corpus allatum in Calliphora erythrocephala Gen camp. Endorr 12, 3 1-57.