Studies on vitellogenesis in the American cockroach

J. Insect Physiol., 1966, Vol. 12, pp. 767 to 779. Pergamon Press Ltd.

Printed in Great Britain

STUDIES ON VITELLOGENESIS IN THE AMERICAN COCKROACH RICHARD R. MILLS, FORREST C. GREENSLADE, and ERNEST F. COUCH Department of Biology, Tulane University, New Orleans, La. 70118, U.S.A. (Received

26 Jammy

1966)

Abstract-An

oiitheca is produced approximately every 6 days by the female American cockroach. Feeding takes place on the first 2 or 3 days of this 6 day cycle and the blood protein level peaks on the fourth day. Most of the yolk protein is deposited on the last 2 days of the cycle. Total RNA concentrations have been determined for both the midgut and fat body during the cycle, and the results of these experiments suggest that both organs take part in blood protein synthesis. Ultrastructural examination of these two tissues support this contention. Cauterization of the medial neurosecretory cells results in a decrease of blood protein levels. Allatectomy causes a rise in blood protein and prevents vitellogenesis. Implantation of three active corpora allata restores both the normal blood protein level and vitellogenesis. A humoral control of blood protein synthesis is postulated and the hormonal and/or nervous control of vitellogenesis and subsequent titheca production is discussed.

INTRODUCTION

THE endocrine

control of reproduction in insects has been extensively investigated during the preceding decades (WIGGLESWORTH,1964; DE WILDE, 1964). In the American cockroach it has been shown that the corpora allata is necessary for vitellogenesis (GIRARDIE,1962; CHEN et aZ., 1962), and it may influence the deposition of lipid in the fat body (BODENSTEIN,1953). VROMANet al. (1965) using Cl4 acetate as a precursor has found that allatectomized cockroaches incorporate twice the amount of label into the triglyceride fraction. In addition, the metabolism of fat appears to be affected since the turnover of both triglyceride and phospholipid is slowed by corpora allata removal (VROMA.N et al., 1965). The corpora allata may also influence protein synthesis in the American cockroach. Studies by WANG and DIXON (1960) h ave shown that muscle transaminase activity is reduced in allatectomized animals. MENON (1963), after subjecting haemolymph to paper electrophoresis, found four distinct negatively charged bands in normal cockroaches. Allatectomy produced distortion of the bands which would indicate some control by the allata. 767

768

FL R. MILLS, F. C. GREENSLADE,AND E. F. COUCH

The object of the present work was to elucidate the mechanisms by which the brain and corpora allata control vitellogenesis in the American cockroach Periplaneta americana (L.). Feeding cycles, blood protein levels, and some aspects of protein synthesis have been studied in relation to humoral control. METHODS

Adult female cockroaches from a standard laboratory culture were used in all experiments. Individuals were held in quart jars containing a piece of folded screen wire. Food and water were available at all times (unless otherwise noted) and a male was always included in long-term experiments. The amount of food ingested each day was determined by drying and then weighing the dog food pellet (Purina) on an analytical balance. Blood protein was obtained by the capillary method of MILLS (1966). Protein was determined by the procedure of LOWRYet al. (1951), and bovine serum albumen (Calbiochem) was used as a standard. The tip of a hot file (1 mm wide) was used to cauterize the region of the brain containing the medial neurosecretory cells. Sham operations involved the burning of the cuticle on the back of the head. To obtain intact brains an incision was made from the neck to the vertex of the head and after pulling the cuticle back, the brain tissue was lifted out with forceps. Extracts were prepared as previously described for the terminal abdominal ganglion (MILLS et al., 1965). Corpora allata were cauterized or removed through an incision in the anterior region of the neck. Usually the trachea had to be extirpated in order to ensure the success of the operation. The wound was closed by touching a hot probe to the edges. Sham operations were identical to the experimentals except the organs were left intact. The nucleic acids were determined by following the general procedure of OGUR and ROSEN(1950) as modified by CHEN (1960) and BRISTOWand DEUCHER (1964) and is as follows: (1) Samples were extracted for 3 hr in cold ethanol, centrifuged at 12,000 xg, and the supernatant discarded. The pellet was re-extracted in ethanol-ethyl ether (3 : 1) for 12 hr and centrifuged again. (2) The resulting pellet was extracted: (a) for 1 hr in 0.3 N perchloric acid (PCA) at 2°C (acidsoluble protein), (b) for 3.5 hr in 0.8 N PCA at 25°C (RNA), and for 20 min in 1.6 N PCA at 60°C (DNA). Quantitation of protein, RNA, and DNA was accomplished by the use of a Beckman DB spectrophotometer and followed the method of SCOTT et al. (1956). Microanalysis was done by the utilization of two sets of Beckman microcuvettes. One pair with a path length of 10 mm and a capacity of 60 ~1 was employed in order to determine concentrations in the range of 1 to 5 pg/lOO ~1, whereas another pair with a path length of 3 mm and a capacity of 300 ~1 was used for concentrations of 10 to 90 pg/lOO /.&I. Blanks of the appropriate normality of PCA were used for each of the three fractions and the difference in optical density of the maxima and minima gave the concentration according to concurrently determined standard curves. RNA was

VITELLOGENlLsIS IN THEAMBRICAN COCKROACH

769

obtained from Schwartz Bioresearch Inc. and DNA from Nutritional Biochemicals Corporation. Midgut and fat body tissue was fixed in situ at 4°C in phosphate-buffered 1% osmium tetraoxide (MILLONIG, 1961) for electron microscope studies. Fixation was continued for 90 min after the dissection of the organs. The tissues were dehydrated through a graded series of ethyl alcohol followed by propylene oxide and embedded in Araldite 502, using DDSA as a plastisizer and DMP-30 as a catalyst (LUFT, 1961). The material was sectioned with a Porter-Blum-I ultramicrotome using a glass knife. Silver and gold sections were mounted on uncoated copper grids and strained with either uranyl acetate or lead citrate (REYNOLDS, 1963). The grids were viewed with either a RCA-EMU-2 or RCA-EMU-SF For orientation purposes sections 0.5 to 1 p thick were electron microscope. stained with azure-B, methylene-blue, or toluidine-blue 0 and observed with the light microscope. RESULTS The normal sequence of events during vitellogenesis

After metamorphosis, female cockroaches complete egg maturation and an oijtheca is produced in approximately 13 days. Allatectomy in the last instar abolishes egg maturation which confirms the observations of GIRARDIE(1962) and CHEN et al. (1962) (Table 1). After the initial cycle, ootheca are produced on an average of every 6 days. During these 6 days certain changes take place and involve alteration of the feeding behaviour, changes in haemolymph protein titre, and changes in nucleic acid concentration. Correlation of feeding with vitellogenesik

After measuring the daily food ingestion of 70 female cockroaches, it was found that the animals usually eat only on the first 2 or 3 days of the 6 day cycle. There was a great deal of variation between the different females during this experiment, but almost all the animals ate during the first part of the cycle and then fasted until the oiitheca was produced. The average values of food consumed during the 6 day cycle are shown in Fig. 1. Allatectomy and/or brain cautery caused the animals to stop feeding so these experiments were inconclusive. Changes in blood protein levels during vitellogenesis

In the initial stages of the 6 day cycle the blood protein level is low. It increases until a peak is reached on the fourth day and declines rapidly until a minimum is reached midway through the fifth day (Fig. 2). Apparently: (I) food is ingested, (2) protein is synthesized and released into the haemolymph, and (3) the blood protein is incorporated into the yolk. Since the blood protein levels were cyclic in nature it was postulated that perhaps these changes are under hormonal control. Removal of the corpora allata

770

R. R. MILLS, F. C. GREENSLADE, AND E. F. COUCH

30

. -/’ . . : \ .

l1.

1

/

I

’

123456 TIME

IN DAYS

FIG. 1. Food consumption during the 6 day cycle. The amount of food eaten varied greatly for each female. Therefore each point is the average of at least 60 determinations.

.

60 / 70

,/.I’

\ . \ .I

. ./

1234567 TIME

IN DAYS

FIG. 2. Rise and fall of haemolymph protein concentration during the 6 day cycle.

Each point is the average of 10 observations.

771

VITELLOGSNESIS IN THE AMERICAN COCKROACH

increased the level of blood protein while brain cautery caused a decrease (Table 2). Starvation also caused a decrease in blood protein level when it was prolonged for as much as 7 days. TABLE

I-EFFE~TOFALLATECTOMYON

THEFIRSTPREOVIPOSITION

CYCLE

Operation

Number of animals

Number surviving

Normal animal Allatectomy in last instar

80 37

74 16

71 -

13.1 -

Sham-operated in last instar Allatectomy of adult

12

10

9

15.1

24

9

Sham-operated adult

24

21

17

29.0

Number Preoviposition ovipositing (days)

-

Maximum variation in the preoviposition period was f

-

5 days.

TABLE~-THEEFFECTOFALLATECTOMY,BRAINCAUTERY,ANDSTARVATIONONBLOODPROTEIN CONCENTRATION

Experiment Normal animal Sham-operated controls Allatectomy Brain cautery Allatectomy and brain cautery Starvation (7 days) (10 days)

Number of

Animals

animals

surviving

Blood protein level OLgIcLl)(average)

18 18 18 18 24

18 15 10 13 14

66 63 87 42 47

18 12

17 12

39 37

Ali animals that had undergone an operation were assayed for blood protein on the tenth day after the operation. The amount of food ingested was not determined. Normal animals were assayed after being starved for 5 days and then allowed to feed for 5 days. Maximum variation was + 8 pg protein per ~1.

The eflect of neck ligation on vitellogeneti

Since it appeared that the feeding response and blood protein levels were integrated in the yolk deposition cycle, neck ligation experiments were utilized to see if these changes were controlled by humoral or nervous stimuli originating in the head. A capillary tube was placed in the oesophagus (for water), and the neck was ligated with a piece of thread at various times during the cycle. Egg maturation was stopped if the ligatures were placed during the first 3 to 4 days of the cycle.

772

R. R. MILLS, F. C.

G-WE,

ANDE. F.

COUCH

After this time vitellogenesis continued to completion although in some cases a number of the oocytes were absorbed. These experiments are summarized in Fig. 3.

DAY OF

LIGATION

FIG. 3. The effect of neck ligation on the production of o6theca and oticyte absorption. O--O, o6cytes absorbed; l 0, o6theca produced. Each point is the average of at least 7 experiments.

The eflect of allatectomy on vitellogenesis

The ligation experiments suggested that some region in the head had the ability to influence vitellogenesis. Both GIRARLUE(1962) and CHEN et al. (1962) have demonstrated that the corpora allata were required for yolk deposition. Therefore a series of experiments were devised so as to show the effect of allatectomy and implantation on vitellogenesis. Removal of the corpora allata completely abolished vitellogenesis although occasionally females would produce an oijtheca before terminating the cycle. Implantation of one or two active glands failed to restore the process to normal. However, the addition of three or four pairs of glands allowed maturation to proceed normally. The insertion of an active pair of allata every 3 days for 12 days (GIRARDIE, 1962) also permitted maturation but the mortality was higher. These data are shown in Table 3. The size of the terminal oocyte varied greatly but ranged from O-3 mm in allatectomized cockroaches to 3.9 mm in some controls. Active corpora allata were also implanted into normal animals. Very little difference between the experimentals and controls were noted although the average length of the cycle appeared to be shortened by 12-15 hr.

VITRLLOGENESIS

IN

THB

AMERICAN

773

COCKROACH

The general condition of the fat body was determined during these experiments by observing tissue smears under the light microscope. The tissue was denoted as hypertrophied if there appeared a greater concentration of fat droplets than normal (Table 3). TABLE

~--THE

EFFECT

OF REMOVING

AND

REIMPLANTING

THE

CORPORA

ALLATA

ON

VITELLOGEI’ESIS

Condition Normal (day 1) CACA-+lCA CA-+2CA CA- + 3CA CA++lCA CA+ + 2CA CA+ + 3CA

Number of animals

Animals surviving

Yolk deposition

Blood protein &g/PO

18

17

+10

66

Normal

18 15 14 16 15 14 16

10 8 8 8 15 12 13

87 88 82 71 67 66 62

Hypertrophied Hypertrophied Hypertrophied Normal Normal Normal Normal

Normal animals were sham-operated. two segments of the abdomen.

Reabsorbed Reabsorbed +2 +8 +lO +10 +10

Condition of fat body

Active corpora allata were implanted in the anterior

Cauterization of the medial neurosecretory cells

When the region of the brain containing the medial neurosecretory cells was cauterized, some of the oiicytes were absorbed although vitellogenesis could not be completely abolished. If the animals lived through three consecutive cycles the third cycle was usually impaired. Implantation of ‘active’ brains within the animals failed to exert any effect. In one group of experiments 17 brain-cauterized animals lived through the first cycle and were injected daily with brain hormone extract during the second cycle. Three of these survived. Each of the three deposited yolk in the oiicytes during the third cycle and produced an oiitheca which was found later to contain viable eggs. This does not offer conclusive proof that the brain is implicated in the control of vitellogenesis but it does tend to indicate that the brain has some function in the process. Nucleic acid changes during the cycle The fat body becomes hypertrophied when the corpora allata is removed. This suggests that the tissue is retaining various products that would normally be used for yolk deposition. It can be seen from the electron micrograph that both lipid droplets and alpha glycogen particles are concentrated in the fat body and make up a large percentage of its bulk (Fig. 8). It has not been shown conclusively, but it is generally believed that the fat body is also responsible for the synthesis of the blood

774

R. R. MILLS, F. C. GREENSLADE, ANDE. F.

COUCH

protein which is incorporated unchanged into the yolk. This would indicate that the total RNA of the fat body would increase prior to an increase in blood protein. The RNA, however, does not conform to this pattern. From Fig. 4 it can be seen that the RNA rises on the second day but declines until the middle of the fourth day. A sharp increase is then noted, and a maximum value is attained during the fifth day. When the changes in RNA concentration are plotted with the changes of blood protein it can be seen that the fat body RNA is declining at the very time blood protein is rising (Fig. 5). On the other hand, the RNA in the midgut showed a gradual increase from day 2 to day 4 with a sharp jump on the fifth day (Fig. 4).

,

,

123456

TIME

IN DAYS

FIG. 4. The concentration of total RNA in the fat body and midgut during the 6 day cycle. Most of the fat body was extirpated, washed, dried, and weighed. The tissue was free of haemolymph and other contaminant except that it was impossible to remove all of the Malpighian tubules.

This suggests that the increase in blood protein from the second to fourth day is due to its synthesis in the gut. The sharp initial rise in blood protein appears to be due to synthesis by both organs. In addition, both organs appear to reach a maximum synthetic rate on the fifth day at the greatest period of yolk deposition. The DNA analysis was impaired by the finding of protein contamination as evidenced by a shift in the peak toward 280 mp. Thus, actual DNA values cannot be determined. However, the data do yield an interesting phenomenon. At the end of the vitellogenesis cycle the relative concentration of DNA (and its contaminating protein(s)) decrease to a value approaching zero in both the fat body and the midgut. This suggests a cyclic turnover of organ tissue and indicates that the two organs may be supplying the oijcyte at their own expense.

VITRLLOGRNESIS

IN THE

AMRRICAN

775

COCKROACH

- 80

TIME IN DAYS FIG. 5. A correlation between food ingested, blood protein levels, and the rise and fall of total RNA in the midgut and fat body. O0,midgut; l0, fat body; x ___...x , food eaten (mg); A- -A, blood protein @g/PI).

Correlation of ultrastructure and protein synthesis Since it appeared that both the fat body and midgut epithelium were involved in protein synthesis the ultrastructure of these organs was examined to see if the necessary cytological machinery was present. From Figs. 6 and 7 (the midgut) it can be seen that: (1) rough endoplasmic reticulum (RER) is in abundance and is lined up in distinct parallel rows, (2) Golgi apparatus are present and a number of vesicles can be seen, (3) secretory granules are in the cytoplasm, and (4) the cell membrane bordering the haemolymph contains a network of tubes and/or smooth endoplasmic reticulum (SER). Th us, the midgut epithelial cells display a morphology which is in keeping with the usual characteristics associated with cells capable of protein synthesis. It is, of course, still unknown as to which direction (i.e. cell to haemolymph or cell to lumen) the major portion of the protein is being secreted. Digestive enzymes are undoubtedly released into the lumen of the gut, and from ultrastructural examination it appears the cell is also capable of releasing protein into the haemolymph. The fat body was not examined in detail but Fig. 8 shows a cell containing RER (presumably for protein synthesis) glycogen particles and lipid droplets.

DISCUSSION

The results of this investigation show that a 6 day cycle of oiicyte maturation occurs in Pera@meta americana. The animals feed during the first part of the cycle and appear to fast until the oiitheca is constructed. This feeding pattern is similar to the initial ovarian cycle found in Lmcophaea (ENGELMANN and RAU, 1965).

776

R. R. MI=,

F. C. GREENSLAIX, AND E. F.

COUCH

Blood protein levels rise until the fourth day and then decline rapidly. This decline corresponds with the first morphological indication that large quantities of yolk are being deposited in the primary oocyte. The concentration of protein in the blood decreased after brain cauterization and increased after allatectomy. These findings are similar to results obtained by other workers (HILL, 1962; HIGHNAM et al., 1963). These data suggest that the brain controls blood protein synthesis and that the corpora allata are responsible for the utilization of the protein. It is known that the synthesis of proteases can be inhibited by brain impairment

(THOMSENand MPILLER,1959, 1963; DADD, 1961), and it is possible that the brain (by either endocrine or nervous control) can regulate the ingestion and/or catabolism of needed foodstuffs. Studies on a number of insects have revealed that ovariectomy results in blood protein accumulation (TELFER, 1954; HILL, 1962; MENON, 1963; ORR, 1964; COLES,1965). Thus, the corpora allata could be directly concerned with the absorption of blood protein by the oiicytes. Since it has been shown that protein passes through the otilemma in pinocytotic caveolae (ANDERSON,1964; ROTH and PORTER, 1964) it is possible that secretions from the corpora allata may influence this process. The corpora allata also appear to affect the utilization of fat body reserves. BODENSTEIN(1953) h as shown fat body hypertrophy after allatectomy which has been substantiated in this laboratory and ORR (1964) has found regulation of the fat body by the corpora allata in Phormia. Recently, VROMANet al. (1965) have demonstrated that both phospholipid and triglyceride turnover are affected by allatectomy. In addition, it has been shown that muscle transaminases of Peri-

planeta are affected by allatectomy (WANG and DIXON, 1960), and it is believed that the overall metabolism is influenced by the allata (WIGGLESWORTH,1964). The corpora allata may also enhance RNA synthesis. VANDERBERG (1963) has presented evidence of increased RNA synthesis in the ovary. Although, it has been found that specific blood proteins are incorporated into the yolk unchanged (TELFER, 1954; COLES, 1965) it is possible that the ovaries do make some small phanges in absorbed proteins. Further evidence that the corpora allata may affect crotein synthesis has been found by L’HELIAS (1953) in Dtippus. BLOOD PROTEIN SYNTHESIS The total RNA extracted from the midgut rises rapidly during the first day of the cycle, continues to increase at a reduced rate and peaks on the fifth day during This suggests that protein is being synthesized the height of vitellogenesis. throughout this period although it must be emphasized that this is total RNA which has not yet been subjected to ultracentrifugal analysis. However, ultrastructural examination of the epithelial cells showed extensive rough endoplasmic reticulum which indicates that much of the RNA is indeed polysomal. On the other hand, it is well known that a number of digestive enzymes are made by the midgut epithelial cells and it can be argued that the RNA is primarily concerned with the synthesis of these enzymes. However, there is very little food eaten during the latter part of the cycle, and it has been shown that enzyme

FIG.

6.

Epithelium

of the midgut showing rough endoplasmic Golgi (G), and secretory granules (S).

reticulum

(ER),

FIG. 7. Portion of the midgut epithelium bordering the haemolymph. folds of the basement membrane (BM) can be seen. Smooth endoplasmic (SER) and mitochondria (M) are also present.

Numerous reticulum

FIG. 8.

Fat

body of the cockroach showing glycogen particles (F), and rough endoplasmic reticulum.

(Gl),

fat droplets

VITELLOGENESIS

IN THE AMERICAN COCKROACH

777

production is not extensive during starvation (DAY and POWNING, 1949; RAFIGKHANand FORD, 1962). Since the highest values for gut RNA are obtained on the fifth day when food uptake is minimal it appears that digestive enzyme production cannot account for the increased RNA content at this time. ROTH and PORTER(1964) h ave shown that Ha-leucine fed to female mosquitoes accumulates in the oocyte before any appreciable label is found in the fat body. This suggests that the midgut epithelium is responsible for the synthesis of at least part of the yolk protein. In addition, their study shows that the necessary cytological machinery for protein synthesis and secretion is present in the midgut of Aedes. Similar cytological evidence has been presented in the present study for Peripkmeta. The RNA content in the fat body is somewhat cyclic in nature. The steep initial rise peaks on the second day and is followed by a decrease until vitellogenesis begins on the fourth day. A maximal value is reached on day 5 during the time that the oijcyte is taking up the greatest amount of protein. Thus, it appears that the fat body is contributing to the blood protein level both at the beginning and the end of the cycle. This agrees with the general concept that protein synthesis takes place in the fat body (SHIGEMATSU,1958; HILL, 1963). When the RNA content of the midgut and fat body (which implies protein synthesis) are examined collectively, it appears that both organs undergo an increased rate of synthesis at the start of the 6 day cycle. At this time structural and (in the case of the midgut) digestive protein production probably occurs. Blood protein

O&T E MATURATION

06T

1 HECA

FIG. 9. A hypothetical diagram depicting the probable relationships between the various organs.

778

R. R. MILLS, F. C. GRENSLADE, ANDE. F. COUCH

is then made by the midgut until the fourth day when out of necessity both organs must contribute to the synthesis of the yolk. The control over these cyclic changes appears to be hormonal. A general diagram depicting the relationship of the various organs is shown in Fig. 9. Some of these relationships have not been elucidated for Periplaneta but have been found in other cockroaches. For example, ROTH (1964) has shown that in Nauphoeta &era, the presence of an ootheca causes inhibition of the brain via the central nervous system. Also, in both N. tinera and L. maderae mating and feeding influence the activation of the corpora allata (JOHANNSON, 1955; ROTH, 1964). Acknowledgements--We would like to thank Dr. DONALDG. COCHRAN of the Virginia Polytechnic Institute for a critical reading of the manuscript and Dr. RICHARDD. LUMSDEN of Tulane University for many helpful discussions. Appreciation is also extended to Mr. GABE PERJESSYand Mr. C. RAYMONDLAKE for technical assistance. REFERENCES ANDERSONE. (1964) Oocyte differentiation and vitellogenesis in the cockroach Periplunetu americana. J. Cell Biol. 20, 131-155. BODENSTEIND. (1953) Studies on the humoral mechanisms in growth and metamorphosis of the cockroach Peripluneta ame*icunu--III. Humoral effects on metabolism. r. exp. Zool. 12q 105-115. BRISTOWD. A. and DEUCHERE. M. (1964) Changes in nucleic acid concentration during the development of Xerwpus laevis embryos. Expl Cell Res. 35, 580-589. CHEN P. S. (1960) Changes in DNA and RNA during embryonic urodele development. Expl Cell Res. 21, 523-534. CHEND. H., ROBBINSW. E., and MONROER. E. (1962) The gonadotrophic action of cecropia extracts in allatectomized American cockroaches. Experientia 18, 577. COLES G. C. (1965) Studies on the hormonal control of metabolism in Rhodnius prolixus StHl-I. The adult female. J. Insect Physiol. 11, 1325-1330. DADD R. H. (1961) Evidence for humoral regulation of digestive secretion in the beetle Tenebrio molitor. J. exp. Biol. 38, 259-266. DAY M. F. and PO~NING R. F. (1949) A study of the processes of digestion in certain insects. Amt. J. Gent. Res. (B) 2, 175-215. ENGELMANNF. and RAu I. (1965) A correlation between feeding and the sexual cycle in Leucophaea maderae (Blattaria). J. Insect Physiol. 11, 53-64. GIRARDIEA. (1962) Etude biometrique de la croissance ovarienne apres ablation et implantation de corpora allata chez Periplaneta americana. g. Insect Physiol. 8, 199-204. HIGHNAMK. C., LUSIS D., and HILL L. (1963) The role of the corpora allata during oiicyte growth in the desert locust Schistocerca gregaria Fork&l. J. Insect Physiol. 9, 587-596. HILL L. (1962) Neurosecretory control of haemolymph protein concentration during ovarian development in the desert locust. J. Insect Physiol. 8, 609-619. HILL L. (1963) Endocrine control of protein synthesis in female desert locusts, Schistocerca gregariu. J. Endocr. 26, 17-18. JOHANNSON A. S. (1955) The relationship between corpora allata and reproductive organs in starved female Leucophaea maderae (Blattaria). Biol. Bull., Woods Hole, 108, 40-44. L’H~LIAS C. (1953) Etude comparee de l’azote total et de l’azote non proteinique chez le phasme LXxippus moroSuS apres ablation des corpora allata. C.R. Acad. Sci., Paris 236, 2489-2491. LOWRY 0. H., ROSEBROUGH N. J., FARR A. H., and RANDALLR. J. (1951) Protein measurement with the Folin-Wu phenol reagent. J. biol. Chem. 193, 265-275.

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LUFT J. H. (1961) Improvements in epoxy resin embedding methods. J. biophys. biochem. Cytol. 9, 409414. MENON M. (1963) Endocrine influences on protein and fat in the haemolymph of the cockroach, Peripluneta americana. Proc. 16th int. Congr. Zool. 1, 297. MILLONIG G. (1961) The advantages of a phosphate buffer for 0~0~ solutions in fixation. J. appl. Phys. 32,1637. Hormone MILLS R. R. (1966) Hormonal control of tanning in the American cockroach-III. stability and post-ecdysal changes in hormone titre. g. Insect Physiol. 12, 275-280. MILLS R. R., MATHURR. B., and GUERRAA. A. (1965) Studies on the hormonal control of tanning in the American cockroach-I. Release of an activation factor from the terminal abdominal ganglion. J. Insect Physiol. 11, 1047-1053. OGUR M. and ROSEN G. (1950) The nucleic acids of plant tissue-I. The extraction and estimation of desoxypentose, nucleic acid, and pentose nucleic acid. Archs Biochem. 25,262. ORR C. W. M. (1964) The influence of nutritional and hormonal factors on the chemistry of the fat body, blood, and ovaries of the blowfly Phormiu regina (Meig.). J. Insect Physiol. 10,103-119. RAFIGKIUN M. and FORD J. B. (1962) Studies on digestive enzyme production and its relationship to the cytology of the midgut epithelium in Dysdercus fasicatus Sign. J. Insect Physiol. 8, 597-608. REYNOLDS E. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. ROTH L. M. (1964) Control of reproduction in female cockroaches with special reference to Nauphoeta cinerea-I. First preoviposition period. J. Insect Physiol. 10, 915-945. ROTH T. F. and PORTERK. R. (1964) Yolk protein uptake in the oiicyte of the mosquito Aedes aegypti L. J. Cell Biol. 20, 313-332. SCOTT S. F., FRACCASTORO A. P., and TAFT E. B. (1956) Studies in histochemistry-I. Determination of nucleic acids inmicrogram amounts of tissue. J. Histochem. Cytochem. 4, l-10. SHIGBMATSUH. (1958) Synthesis of blood protein by the fat body in the silkworm, Bombyx mori L. Nature, Lond. 182,880-882. STRANGEWAYS-DIXON J. (1961) The relationships between nutrition, hormones and reproduction in the blowfly Calliphora erythrocephala (Meig.)-II. The effect of removing the ovaries, the corpus allatum and the median neurosecretory cells upon selective feeding, and the demonstration of the corpus allatum cycle. J. exp. Biol. 38,637-646. TELFER W. H. (1954) Immunological studies of insect metamorphosis-II. The role of a sex-limited blood protein in egg formation by the cecropia silkworm. J. gen Physiol. 37, 539-558. THOMSENE. and MILLER I. (1959) Neurosecretion and intestinal proteinase activity in an insect Calliphora erythrocephalu (Meig.). Nature, Lond. 183, 1401-1402. THOM~BNE. and MBLLER I. (1963) Influence of neurosecretory cells and of corpus allatum on intestinal protease activity in the adult Calliphora erythrocephala (Meig.). r. exp. Biol. 40, 301-321. VANDBRBBRG J. P. (1963) The role of the gonodotrophic hormone in the synthesis of protein and RNA in Rhodnius prolixus (Hemiptera). Biol. Bull., Woods Hole 125, 576-581. VROMANH. E., KAPLANISJ. N., and ROBBINSW. E. (1965) Effect of allatectomy on lipid biosynthesis and turnover in the female American cockroach, Periplaneta americana (L.). J. fnrect PhysioZ. 11, 897-904. WANG S. and DIXON S. E. (1960) Studies in the transaminase activity of muscle tissue from allatectomized roaches, Pera@neta americana. Can.J. 2001. 38, 275-283. WIGGLESWORTHV. B. (1964) The hormonal regulation of growth and reproduction in insects. Adv. Insect Physiol. 2, 247-336. DE WILDE J. (1964) Reproduction-endocrine control. In Physiology of the Insecta 1, 59-90. (Ed. by ROCICS~EIN M.) Academic Press, New York. 48