A biochemical study of oögenesis in the housefly, Musca domestica

Insect Biochem., 1976, VoL 6, pp. 13 to 18. Pergamon Press. Printed in Great Britain.

A BIOCHEMICAL STUDY OF OOGENESIS IN THE HOUSEFLY, MUSCA DOMESTICA T E m ~ c E J. HALL, SUNNY M. SANDmm AND MICHAEL R. CUMMINGS* Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60680, U.S.A. (Received 24 May 1975) Abstract--Biochemical changes during ovarian growth and development have been studied in a wild type strain of the housefly, Musca domestics, where both intra- and inter-ovarian synchronous development can be achieved. At 29°C, the first gonotrophic cycle is complete 72 hr after eclosion, and the second cycle in an additional 40 hr. In the first cycle, ovarian D N A content increases rapidly between 48 to 60 hr after emergence. RNA content begins rising at 36 hr and reaches a maximum of 180 gg/ovary. In vitro labeling of ovaries with aH-uridine indicates that ribosomal RNA is the major species of RNA present during the previtellogenic and early vitellogenic stages. Changes in protein content are similar to those in D N A content. Analysis of active ribosomes indicates that ovarian protein synthesis peaks 48 hr after eclosion, and represents the combined activity of follicle cells, nurse cells and obcyte. INTRODUCTION

mixture of powdered milk, sugar, and powdered egg 6 : 6 : 1 (T. S. A~AMS, personal communication). Water was provided in bottles equipped with wicks of cotton or paper towel For raising flies to be used in studies of orgenesis, eggs were collected by using an ovipositional substrate consisting of a black cloth soaked in saturated ammonium carbonate placed over a beaker containing CSMA. Eggs collected over a 1 to 2 hr period were seeded into beakers containing a mixture of CSMA, molasses and yeast. Each beaker received approximately 100mg of eggs. The beakers were tightly covered with paper towels or cheesecloth and incubated at 29°C. An additional one-half inch of dry CSMA was added to provide a substrate for pupation. Pupae were collected and held until eclosion. For studies on ovarian development, eclosing flies were collected over a 2 hr period, sexed, and 200 females and 350 males were placed in each cage and provided with food and water. Females were collected from these cages at times ranging from 2 to 96 hr post-eclosion for staging and biochemical analysis.

THE PROCESS of o~genesis in the polytrophic, meroistic ovary of the housefly, Musca domestics offers several advantages for the study of ovarian development in an advanced insect. I n Musca, o6genesis is a synchronous event, with egg chambers in all ovarioles developing at the same rate. Ovarian synchrony (MORRISON, 1963) has been especially useful in the study of biosynthetic pathways (MILLER arid COLLINS, 1973), macromolecular synthesis (MILLER and COLLINS, 1970; HALL and CUMMINGS, 1975), macromolecular transport (BIm~, 1963a, 1965), and hormonal control of ovarian function (ADAMS et al., 1968; ADAMS, 1970; ADAMS, 1974). I n addition, adult nutritional requirements for gametogenesis are known (GooDMAN et al., 1968), a chemically defined diet is available ( M o m u s o N and DAVIES, 1964; PERRY and MILLER, 1965) and the morphology, histochemistry and ultrastructure of oSgenesis have been investigated (GooDMAN et al., 1968; CHIA and MOrmlSON, 1972; ADAMS, 1974). However, little if any biochemical information is available concerning the gonotrophic cycle. T h i s study describes biochemical changes during o6cyte development in order to provide a m o r e quantitative interpretation of the morphological events in oSgenesis and to serve the basis for future studies of macromolecular synthesis during oSgenesis.

Ovarian staging Ovaries were dissected from etherized flies in cold Insect Ringer's solution, transferred to microscope slides and stained with 2% aceto-orcein for one minute. The ovarioles were gently separated by pressure on the coverslip and examined using a Wild M-20 phase contrast microscope at 100×. Stages of oSgenesis were classified from 1-14 using the criteria adopted for Muses by GOODMANet al. (1968). Tissue analysis and assays For the analysis of DNA, RNA and protein content, the ovaries were processed as previously described (CuMMUCGS et al., 1971) using the techniques of SmBKO et aL (1967). D N A was determined by absorption at 260 mB and the method of BURTON (1956). RNA was determined by absorption at 260mtt and the orcinol reaction (MEIBAUM, 1939). Protein was determined using the method of Loway et aL (1951).

MATERIALS AND METHODS Culture conditions Musca domestics of the Orlando wild strain were maintained in cages at 29°C, 55% r.h. in a 12 : 12 lightdark photoperiod. Adults were fed ad libitum on a * To whom reprint requests should be sent. 13

14

TERRENCE J. HALL,SUNNY M. SANDERSAND MICHAEL R. CUMMINGS

The assay for active ribosomes utilized the method of which occurs 36 hr post-eclosion, the ovary underMAaTIN (1973). For this determination, 200 mg of goes rapid growth and differentiation, and mature ovaries were homogenized in 4.5ml of I / 2 T K M orcytes appear 65 to 72 hr after emergence. I n the (40 mM KCI, 7'5 mM MgCI~, 25 mM Tris, pH 7.6, I mM dithiothreitol) and 0"5 ml of 10% dexoycholate in housefly, yolk formation and maturation is restricted 10% Triton X-100. The homogenate was centrifuged to the most posterior egg chamber in each ovariole, at 10,000g for 10min at 4°C in a Sorvall RC-2B and all egg chambers in a single ovarian cycle centrifuge. 300 IXl of supernatant was added to 100 IXl mature synchronously, and females oviposit 80 to 125 eggs in a single batch. of 2.5 M KC1 and 10 ~tl of pancreatic ribonuclease (1 mg/ml, Sigma Chemical Co.) and incubated for 2 to 5 min. 100 to 200 IXl of the incubation mixture was Ovarian development layered on 10 to 30% sucrose gradients made up in Stage 4 egg chambers are present in the ovary of 0.8 M KC1, 15 mM MgCI~ and 50 mM Tris, pH 7"6. newly eclosed females, indicating that critical The gradients were centrifuged at 4°C using an SW56 events in orgenesis, including germ cell division rotor in a Beckman L2-65B ultracentrifuge at 218,000 g and orcyte determination occur during the prefor 1 hr. The gradients were analyzed using an ISCO density gradient fractionator at 254 mix at a flow speed adult stages. Egg maturation in Musca females raised under controlled conditions proceeds with of 1 ml/min. Optical density profiles were recorded using a Houston strip chart recorder, and active ribo- both intra- and inter-ovarian synchrony (Fig. 1). some fractions were calculated by planimetry. The incorporation of JH-uridine into RNA was 14 LLI determined using an in vitro incubation with eight O 12 pairs of ovaries dissected from females 24 hr post< eclosion. The ovaries were incubated in 200 ixl of Schneider's medium containing 100 units/ml penicillin, z 8 100 ixg/ml streptomycin and 200-250 ixCi/ml of 3He,- e < uridine (40 to 60 Ci/mM, Schwarz-Mann). Ovarian 0 4 ribosomal RNA was extracted using a modification of 2 Kirby's method II (KIaBY, 1965). Ribosomal RNA and I i " l I I I 1 its precursor components were separated by electro15 30 4,5 60 7,.5 90 105 phoresis on 2-2% acrylamide gels according to LOEa'qING HOURS POST-ECLOSION (1967). Gels were scanned at 260 nm in a Gilford 240 spectrophotometer equipped with a linear transport, Fig. 1. Rate of ovarian maturation during the first two and sliced into 1 mm slices on a Mickle gel slicer. The gonotrophic cycles. Data are mean values from three gel slices were incubated overnight in 3% NCS in separate experiments. 0"4% PPO/toluene and counted in a Beckman LS-250 scintillationcounter. Details of this procedure have been ViteUogenesis begins at stage 8, 36 hr after eclosion, and is complete at stage 12, some 2 4 h r later. presented elsewhere (HALL and CUMMINGS,1975). Postvitellogenic maturation (stages 12 to 14) occupies an additional 12 hr and mature oOeytes RESULTS of the first ovarian cycle are present 72 hr after Orgenesis in Musca and Drosophila is somewhat eclosion. During postvitellogenic maturation of the similar, and the staging criteria established for first cycle, the penultimate egg chamber in each Drosophila (KtNG et al., 1956; CUMMINCS and ovariole initiates maturation, and completes preKINo, 1969) have been adopted for Musca viteUogenie development about 72 to 74 hr after (GooDMaN et al., 1968). Although other schemes eclosion. If oOcytes produced in the first cycle are have been proposed for housefly oSgenesis (BmR, oviposited, egg chambers of the second cycle 1963b; ADAMS, 1974) they are somewhat inprecise initiate vitellogenesis, and the second round of in either permitting a large degree of overlap in o~Sgenesis is completed approximately 108 hr after stages, or omitting most pre-vitellogenic events. eclosion. Under the conditions employed in this Since it is hoped that future studies of oogenesis in study, oi3genesis proceeds somewhat more rapidly Musca will not be limited to vitellogenic stages, the in the second cycle, where maturation from stage 6 14 stages of ovarian development described in through 14 requires 36 hr, whereas in the first cycle, detail by GOODMANet al., (1968) will be used here. 48 hr are required for maturation through the I n Musca, as in many other higher Diptera, same stages. mature eggs are formed only after the adult ecloses, and only if a protein source is available. Ovaries of Nucleic acid content Figure 2 shows the D N A content per ovary for newly eclosed females contain egg chambers in females raised at 29°C. From eclosion, D N A early previtellogenic stages of development. The initiation of vitellogenesis is regulated by the content increases slowly for the first 36 hr. At corpus aUatum (ADAMSand HINTZ, 1969), and it has 36 hr, the D N A content begins to increase rapidly, been shown that juvenile hormone controls certain with the greatest increase occurring between 48 to morphogenetic events in the developing ovary 60 hr. D N A content peaks at 60 hr after eclosion (ADAMS, 1974). Following hormonal stimulation, and declines slightly thereafter. Figure 3 shows the

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O6genesis in the housefly

next 36 hr, reaching a peak of about 180 lag/ovary at 72 hr after eclosion. In vitro labeling of ovaries explanted from 24-hr-old females with SH-uridine indicates that label is incorporated principally into ribosomal RNA (Fig. 4). After a 90-rain incubation, peaks of radioactivity are observed in a peak of heterogeneous material at the top of the gel and in the 38S, 28S, and 18S RNA regions. Shoulders on the trailing edge of the 28S and 18S peaks represent ribosomal RNA intermediates which are unresolved under the conditions employed here. In vitro labeling of ovaries dissected from females 42 hr post-eclosion shows that incorporation of label into ribosomal RNA continues (Fig. 5) during the early stages of the rapid increase in ovarian RNA content.

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Fig. 2. DNA content per ovary during the first 3 days of adult life after emergence.

Protein content Changes in ovarian protein content are shown in Fig. 6, and are similar to those for D N A (Fig. 3).

Fig. 3. RNA content per ovary during the first 3 days of adult life after emergence. data for ovarian RNA content. The RNA content climbs steadily from eclosion, and reaches an initial peak of 25 gg/ovary at 24 hr. From 24 to 36 hr after emergence the ovarian RNA content declines to a level close to that of ovaries from newly eclosed flies and then increases rapidly for the

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Fig. 4. RNA labeling after incubation of eight pairs of ovaries from 24-hr-old females for 90 man at 22°C. Extracted RNA was electrophoresed for 2 hr on 2"2~0 polyacrylamide gels. Fig. 5. RNA labeling after incubation of ovaries from 42-hr-old females for 90 rain at 22°C using all- uridine (250la Ci/ml). RNA was extracted and analyzed by electrophoresis on 2'2~/o gels for 2 hr.

15 25 35 45 55 65 75 HOURS POST-ECLOSION

Fig. 6. Protein content per ovary from eclosion to 3 days later. Each ovary of newly eclosed flies contains about 4 lag of protein, a value which changes little over the first 36 hr of development. With the onset of vitellogenesis the protein content undergoes a rapid increase, reaching a peak of 140 lag per ovary at 52 hr after eclosion. During the 16 hr of vitellogenesis, the protein content increases 70 fold. Whether the increase in protein content results from a combination of intraovarian synthesis and the incorporation of extraovarian protein has not

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Fig. 7. Active ribosome profile of ovaries determined by the method of MARTIN (1973). Values presented are mean values of three determinations, each using 200 rag/ovaries.

16

TERRENCE J. HALL, SUNNY M. SANDERSAND MICHAEL R. CUMMINOS

been completely resolved. A method for determining intraovarian protein synthesis by measuring the proportion of cytoplasmic ribosomes present as polyribosomes is available (MARTIN, 1973). T h e active ribosome profile of ovaries at various times after eclosion is presented in Fig. 7. Some 24 hr after eclosion, the ovary exhibits negligible activity. T h e percentage of active ribosomes increases rapidly between 24 to 48 hr, reaching a peak at 48 hr, with some 550/0 of the ovarian ribosomes active. This level declines in later stages of development, reaching a base level for mature ovaries of 15 to 20% active ribosomes.

DISCUSSION DNA The development of ovarian egg chambers in Muses may be divided into three major phases. During the previtellogenic phase (stages 1 to 7) the oticyte and nurse cells grow at roughly identical rates (GooDMAN et al., 1968), and nurse cell nuclear volume increases slowly (ADAMS, 1974). Since the ovary of a newly eclosed female contains egg chambers in stages 1 to 4, the level of D N A in ovaries from 0 to 36 hr (Fig. 2) represents that of the previtellogenic ovary. In the second phase of development (stages 8 to 10) yolk deposition begins and the oocyte grows rapidly at the expense of the nurse cells (GooDMAN et al., 1968; ADAMS, 1974). The beginning of this phase is marked by an increase in nurse cell nuclear volume (AOAMS, 1974) and an increase in D N A content (Fig. 2). During the middle and latter part of the vitellogenic phase, the nurse cell nuclear volume (ADAMs, 1974) and ovarian D N A content (Fig. 2) increase rapidly. Since JACOB and SIRLIN (1959) have shown that nurse cell nuclear volume is an estimate of D N A content, the rise in ovarian D N A content seen in the vitellogenic stages is probably due to endomitotic replication of D N A in nurse cells (see also ADAMS, 1974). In the postviteUogenic phase of ovarian development (stages 11-14) the nurse cell nuclei disfintegrate (GooDMAN et al., 1968; ADAMS, 1974), and by stage 13 are no longer present in the egg chambers. T h e breakdown of the nurse cell nuclei is associated with a decrease in D N A content (Fig. 2) which is similar to a decline observed for a corresponding stage in Drosophila (CUMMINGSet al., 1971).

RNA Following eclosion, ovarian R N A content begins to increase, reaching a minor peak at 24 hr (Fig. 3), and declining thereafter to levels similar to those seen at eclosion. T h e rapid increase in R N A content which begins at 36 hr is associated with the appearance of vitellogenic stages.

Autoradiographic studies of R N A synthesis during o6genesis in Musca have shown that much of the R N A in mature o6cytes is synthesized in the nurse cells and transferred to the oScyte through the ring canals (BI~a, 1967; PETZELT and Bma, 1970), and that active transport is involved in the transfer to the o6cyte (BIER, 1965). The o6cyte nucleus may also be active in R N A synthesis to a slight degree (BIER et al., 1967), although transport of this R N A to the o6plasm has not been shown. In vitro labeling of ovaries 24 hr post-eclosion shows that the increase in R N A content which occurs during previtellogenesis is associated with ribosomal R N A synthesis (Fig. 4). Further, in vitro labeling of ovaries 42 hr post-eclosion shows that the increase in R N A content during vitellogenesis is also associated with ribosomal R N A synthesis. AL-AD1L and KILGORE (1973) have demonstrated that R N A synthesized during o5genesis consists mainly of ribosomal R N A with smaller amounts of transfer R N A present. GADALLAH and MAREI (1972) observed the synthesis of massive amounts of R N A during vitellogenesis and calculated that over 90% of this R N A is ribosomal RNA. It is apparent, therefore, that the increase in R N A content during previtellogenic and vitellogenic stages is due ~o the production of ribosomes, which in the mature, unfertilized egg are mostly present as monosomes (GADALLAH et al., 1970; KINNIBURGH, personal communication). Protein Ovarian protein content remains constant until the initiation of vitellogenesis 36 hr post-eclosion. ViteUogenic egg chambers have been shown to contain increasing amounts of protein through the use of several techniques including electrophoresis (BODNARYK and MORRISON, 1966), autoradiography (BIEa, 1963b, 1965; CHIA and MoaalSON, 1972), and uhrastructural observation of pinocytosis (CHIA and MOrRISON, 1972; CUMMINGS, unpublished). In the o/Scyte, the viteUogenic stages are characterized by the appearance of large numbers of protein-containing yolk spheres (GooDMAN et al., 1968), and it is obvious that most of the protein increase seen during ovarian maturation is due to the presence of yolk protein or vitellogenin. In Muses, the deposition of yolk protein in the oScyte has been shown to be diet dependent (GooDMAN et al., 1968), and presumably vitellogenin is synthesized from nutrients ingested during adult life. In many other insects, including the mosquito Aedes (HAGEDORN and JUDSON, 1972), the fat body has been shown to be the site of viteUogenin synthesis, and viteUogenin is present in the haemolymph during ovarian yolk deposition. In Musca, although a female-specific protein has been identified in the haemolymph (BoDNARYK and MorrlSON, 1968), the haemolymph of females

O6genesis in the housefly undergoing vitellogenesis contains no protein antigenically similar to yolk protein. The active ribosome profile indicates that ovarian protein synthesis occurs during the vitellogenic stages. The peak of activity reached at 48 hr posteclosion represents the combined activities of the follicle cells, nurse cells and orcyte. Organized rough endoplasmic reticulum appears in the follicle cells surrounding the orcyte in stage 8 (WAxT, unpublished observations) and the follicle cells subsequently synthesize vitelline membrane and chorion. Whether the orcyte engages in protein synthesis during the vitellogenic and post-vitellogenie stages has not been determined. The level of 15% active ribosomes in mature ovaries probably represents activity in mature orcytes since the follicle cells are no longer present in post-vitellogenic egg chambers at 72 hr, and because the mature o6cytes represent the bulk of ovarian weight. GADALLAHand MAaEt (1972) also demonstrated the presence of a small number of polysomes in mature unfertilized o6cytes using sucrose gradient centrifugation. Mature o~cytes may maintain low levels of protein synthesis before fertilization and oviposition. Acknowledgements--We would like to thank T. S. ADAMS for his advice, and ALAN J. KINNmURGH,RON LAW, and MART'~AS TRAKIS for their technical assistance. This work was supported by National Science Foundation grant GB 36861.

REFERENCES ADAMS T. S. (1970) Ovarian regulation of the corpus allatum in the housefly, Musca domestica. J. Insect Physiol. 18, 349-360. ADAMS T. S. (1974) The r61e of juvenile hormone in housefly ovarian follicle morphogenesis. J. Insect Physiol. 20, 263-274. ADAMST. S. and HXNTZA. M. (1969) Relationship of age, ovarian development and the corpus allatum to mating in the housefly, Musca domestica. J. Insect Physiol. 15, 201-215. ADAMs T. S., HINTZ A. M., and POMONISJ. G. (1968) Orstatic hormone production in houseflies, Musea domestiea, with developing ovaries. J. Insect Physiol. 14, 983-993. AL-ADIL K. and KILGOREW. W. (1973) Incorporation of orally administered radioactive precursors into nucleic acids and ribosomes of housefly ovaries. J. Insect Physiol. 19, 909-915. BIEa K. (1963a) Synthese interzelhiliirer Transport and Abhau von Ribonukleins~iure in Ovar der Stubenfliege, Musca domestica. J. Cell Biol. 16, 436--440. BIER K. (1963b) Autoradiographische Untersuchungen fiber die Leismngen des Follikelepithels tend der N~ihrzellen bie der Dotterbildung un Ediwelssynthese in Fleigenovar. Wilhelm Roux' Arch. Ent~oiekMech. Org. 154, 552-575. Blva~ K. (1965) ~ber den Transport zelleigener Makromolekiile durch die Kernmembran. I. RNS-syntheses und RNS-transport unter Sauerstoffmangel und bei herabgesetzter Temperatur. Chromosoma 16, 58-69.

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Bllm K. (1967) O6genese, das Waehstum yon Riesenzellen. Naturwiss 54, 189-194. BIEa K., KtrNz W., and Rrsnva~TD. (1967) Strukmr und Funktion der Oocytenchromosomen und Nukleolen sowie der Extra-DNS wahrend der Oogenese panoistiseher und meroistischer Insekten. Chromosoma 23, 214-254. BODNARYK R. P. and MORRISON P. E. (1966) The relationship between nutrition, haernolymph proteins, and ovarian development in Musca domestica. .7. Insect Physiol. 12, 963-976. B O D N A ~ R. P. and MoaaIsoN P. E. (1968) Immunochemical analysis of origin of a sex-specific accumulated blood protein in female houseflies. J. Insect Physiol. 14, 1141-1146. BUSTON K. (1956) A study of the conditions and mechanism of the diphanylamine reaction for the colorimetric estimation of desoxyribonueleic acid. Biochem. J. 82, 315-323 CmA W and MO~ISON P. E. (1972) Autoradiographic and ultrastructural studies on the origin of yolk protein in the housefly, Musca domestica. Can. J. Zool. 50, 1569-1576. CUMMINGSM. R. and KING R. C. (1969) The cytology of the vitellogenic stages of o6genesis in Drosophila melanogaster. I. General staging characteristics. ~t. Morph. 128~ 427-441. CUMMINGSM. R., ROBIN M., and GANETZKYB. (1971) Biochemical aspects of o6genesis in Drosophila mclanogaster. J. Insect Physiol. 17, 2105-2118. GADALLAHA., KILGOREW. W., MAREIN., and PAINTER R. (1970) Protein synthesis by ribosomes from fertilized and unfertilized eggs of houseflies, Musca domestica. Insect Biochem. 1, 385-390. GADALLAHA. and MAgi N. (1972) Characteristics of nucleic acids from ovaries of virgin and mated houseflies, Musca domestica. J. Insect Physiol. 18, 973-979. GOODMANT., MORRISONP. E., and DAVIm D. (1968) Cytological changes in the developing ovary of the housefly fed milk and other diets. Can. J. Zool. 46, 409-421. HAGEDORNH. H. and JUDSON C. L. (1972) Purification and site of synthesis of Aedes aegypti yolk proteins. J. exp. Zool. 182, 367-377. HALL T. and CUMMINGSM. (1975) In vitro synthesis and processing of ribosomal RNA in the ovary of the housefly. Dev. Biol. (in press). JACOB J. and S I ~ I ~ J. L. (1959) Cell function in the ovary of Drosophila. I. DNA classes in nurse cell nuclei as determined by autoradiography. Chromosoma 10, 210-228. KING R. C., RUBINSONA. C., and SMITH R. F. (1956) O6genesis in adult Drosophila melanogaster. Growth 20, 121-157. KIRBy K. S. (1965) Isolation and characterization of ribosomal ribonucleic acid. Biochem. ft. 96, 266-269. LO~'~ING U. (1967) Fractionation of high molecular weight ribonucleic and by polyaerylamide gel electrophoresis. Biochem. J. 102, 251-257. LowRY O. H., ROSSeaOUGH N. J., FARR A. L., and RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MARTIN T. E. (1973) A simple general method to determine the proportion of active ribosomes in eucaryotic cells. Exp. Cell Res. 80, 496-498.

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TERRACE J. HALL, SUNNY M. SANDERSAND MICHAEL R. CUMMINGS

MEJ~AUM W. (1939) ~ b e r die Bestimmung kleiner Pentosemengen insubesondere in Derivaten der Adenylsaiire. Hoppe-Seyler's Z. physiol. Chem. 268, 117-120. MILLF.R S. and COLLINSJ. (1970) D N A synthesis in the developing ovary of the housefly. Comp. Biochem. Physiol. 26, 559-567. MILLF.R S. and COLLINSJ. (1973) Metabolic purine pathways in the developing ovary of the housefly, Musca domestica. Comp. Biochem. Physiol. 44B, 1153-1163. MORRISON P. E. (1963) The first and subsequent ovarian cycles of the housefly, Musca domestica L., in relation to chemically defined nutritional requirements of the adult. Ph.D. Thesis, McMaster University, Hamilton, Ontario, Canada. MORRISON P. E. and DAVIES D. M. (1964) Feeding of dry, chemically defined diets, and egg production in the adult house-fly. Nature, Lond. 201, 104-105.

PERRY A. S. and MILLER S. (1965) The essential role of folic acid and the effect of anti-metabolites on growth and metamorphosis of housefly larvae Musca domestica. ~. Insect Physiol. 11, 1277-1287. PETZELTC. and BIER K. (1970) Hemmung und Induktion yon Proteinsynthesen durch Actinomycin in den waschsenden Oocyten yon Musca dornestica. Roux' Arch. EntwickMech. Org. 164, 341-358. SHIBKO S., KOIVlSTOINENP., TRATYNEK C., NEWHALL A., and FaIEDMAN L. (1967) A method for the sequential quantitative separation and determination of protein, RNA, DNA, lipid and glycogen from a single rat liver homogenate or from a subcellular fraction. Analyt. Biochem. 19, 415-528.

Key Word Index: Biochemistry, o6genesis, housefly, RNA, DNA, protein active ribosomes.