Regulation of juvenile hormone synthesis during pregnancy in the cockroach, Diploptera punctata

J. fnsecr Physiol. Vol. 31, No. 2. pp. 145-157. 1985 Printed in Great Britain. All rights reserved

Copyright

0022-1910185 $3.00 + 0.00 ctI 1985 Pergamon Press Ltd

REGULATION OF JUVENILE HORMONE SYNTHESIS DURING PREGNANCY IN THE COCKROACH, DIPLOPTERA SUSAN M.

Department

of Biology,

PUNCTATA

RANKIN*

and

University

BARBARA

of Iowa,

STAY

Iowa City, IA 52242, U.S.A.

(Received 3 1 May 1984; revised 24 July 1984) Abstract-Regulation of juvenile hormone synthesis during pregnancy was investigated after determining the normal rates of synthesis in pregnancy and the second gonadotrophic cycle in Diploptera punctata by direct in vitro radiochemical assay. The low rate of juvenile hormone synthesis during early pregnancy is maintained by three factors: (1) the small ovary which is incapable of eliciting increased rates of juvenile hormone synthesis (2) an inhibitory centre in the brain acting via intact nerves to the corpora allata (similar to that in virgin females) and (3) an inhibitory centre in the brain acting via the haemolymph (elicited by embryos in the brood sac). The existence of two inhibitory centres in the brain is supported by the additive effect of denervating the corpora allata and removing embryos. Whereas these operations alone activated the corpora allata in 54 and 31% of the females, respectively, together they activated 87%. similar to the 91% activated by denervation alone in late pregnancy. The inhibition which remains after denervation of the corpora allata can be removed by decapitation and restored by implantation of the protocerebrum from a pregnant female but not from one developing oocytes. The inhibition elicited by embryos in the brood sac can be overcome by introduction of a stimulatoq ovary and/or substitution of active corpora allata.

Key Word Index: Juvenile

hormone

synthesis.

pregnancy,

INTRODUCTION

in virgin females (Stay and Tobe, 1977; Riiegg et al., 1983). Thus, experimental analysis of possible neural and neuroendocrine components required for normal regulation of the sustained low activity during pregnancy, especially early pregnancy, may contribute to our understanding of the multiple factors which interact to regulate the corpora allata. In addition to sensory input from embryos via the central nervous system regulating the corpora allata, the ovary seems likely to be important and has been demonstrated to be so (Stay and Rankin, 1984; Buschor et al.. 1984). The first cycle of juvenile hormone synthesis requires the ovary (Stay and Tobe, 1978; Lanzrein et al., 1981b; Weaver, 1981). The developmental stages of basal oiicytes which can elicit increases in rates of juvenile hormone synthesis have been defined in D. punctata (Rankin and Stay, 1984). The ovaries of pregnant females are not stimulatory, nor are they able to prevent an increase in rates of juvenile hormone synthesis as can the ovary just prior to ovulation (Rankin and Stay, 1984). Hence it would seem important to investigate the role of the ovary as well as innervation of corpora allata and humoral factors from the brain in regulation of corpora allata during pregnancy. In this investigation we have used a direct in t’itro radiochemical assay to characterize the rates of juvenile hormone synthesis and oiicyte lengths as a cumulative measure of juvenile hormone titres. These parameters were measured during normal pregnancy following the first gonadotrophic cycle and the sec-

The mechanisms which restrain corpora allata during larval and adult periods are critical to the development of insects and adult reproductive success. The viviparous cockroach, Diploptera punctatu, provides a system for exploring factors which maintain low activity of corpora allata in that rates of juvenile hormone synthesis are low during the 60 days of pregnancy (Tobe and Stay, 1977) when the batch of embryos develops in the brood sac, the organ which supplies “milk” to embryos (Roth and Willis, 1955; Roth, 1967; Stay and Coop, 1973). The period of pregnancy in D. punctutu and in the ovoviviparous cockroaches Leucophaea maderae and Nauphoeta tin erea is especially intriguing because the conditions of early pregnancy are different from those of later pregnancy: embryo removal or severance of the ventral nerve cord in early pregnancy, which deprives the brain and thence the corpora allata of sensory stimuli emanating from the brood sac did not result in prompt increase in rates of juvenile hormone synthesis whereas those manoeuvres performed later in pregnancy did so (Engelmann. 1959, 1960; Roth and Stay, 1961; Buschor et al., 1984). Also neither denervation of the corpora allata (Stay and Tobe, 1977) nor cautery of the neurosecretory areas of the protocerebrum “activated” the corpora allata promptly (Riiegg et al.. 1983) whereas these manoeuvres did so *To whom

correspondence

should

Diploptera punctata

be addressed. I45

146

SUSAN M. RANKIN and

ond gonadotrophic cycle of remated and not-remated females. This provided a basis for evaluating the results of the following experiments on the regulation of the corpora allata in pregnant females. We tested whether the inhibition of corpora allata resulting from embryos in the brood sac occurs exclusively via intact nerves to the corpora allata by denervating the corpora allata and removing embryos, separately and in combination, We tested for factors from the brain acting on the corpora allata via the haemolymph by decapitating and implanting parts of brains. Finally, we tested the ability of the ovary to stimulate the corpora allata of pregnant females and the ability of active corpora allata to stimulate growth in ovaries of pregnant females. .MATERIALS AND METHODS

Animals were maintained at 27°C as described previously (Rankin and Stay, 1983). Females in the second gonadotrophic cycle were obtained by isolating 69- to 7l-day old pregnant animals, then checking daily for births. Age at parturition ranged from 7&75 days, and typically occurred on day 72. The age at second oviposition was 3 days after parturition and typically occurred on day 75. Ages expressed as days before parturition were estimates based on the mean age at parturition. Surgeq* Ovariectomy. decapitation and implantation procedures were as described by Rankin and Stay (1983); denervation was performed as described by Stay et al. (1983). Embryo removal was accomplished by applying gentle pressure from anterior to posterior of the abdomen of chilled animals. After embryo removal. brood sacs were restored to proper orientation with a small glass rod lubricated with saline. Brains for implantation were dissected from the head in Ringer’s solution (Yeager, 1939) [hereafter referred to as saline] and cut with irridectomy scissors at the optic lobes and between the deuto- and tritocerebra. The protocerebral segment, therefore, contained lateral as well as medial neurosecretory cells. The optic lobes, deuto- and tritocerebra were injected as controls.

BARBARA STAY

oocytes was measured using an ocular micrometer. Student’s t-test and X2 test were used in statistical analyses. RESULTS

The second cycle: rates of juvenile and oiicyte development

hormone synthesis

Whereas mating is a prerequisite for the first gonadotrophic cycle (Engelmann, 1959; Roth and Stay. 196 1; Stay and Tobe. 1977) it is not required for subsequent cycles although females normally mate after parturition. We determined the effect of mating on the day of parturition on rates of oiicyte development and juvenile hormone synthesis during the second cycle. The results are shown in Fig. 1. Rapid oiicyte development began before parturition. Two days before parturition mean oiicyte length exceeded that of a female at adult emergence (0.60 mm on day 0); one day before parturition, basal oocytes, 0.90 mm long, had already become vitellogenic (viteliogenesis starts at about 0.80 mm). At parturition. oijcytes were about 1.25 mm long. Chorionation began in 2 of mated females and a of non-mated females 2 days after parturition; 3 days after parturition all had begun or completed chorionation. Oocyte development for females mated on the day of parturition was the same as that for animals not mated and similar to a normal first-cycle (Fig. I. upper left; data derived from Johnson et al., 1984). Figure I (lower) shows rates of juvenile hormone

,?-*i i’

t

Histolog? At assay, implanted brains were recovered, fixed and stained for neurosecretory material with performic acid-resorcin fuchsin (Ittycheriah and Marks, 1971). Brain tissue was embedded in paraffin and cut into 5 pm serial sections for observation. ., 1

Rates of juvenile hormone synthesis of individual pairs of corpora allata were determined using the in vitro radiochemical assay as described by Tobe and Pratt (1974) [Fig. 91 or as modified by Feyereisen and Tobe (198 1) [Figs I-81. In the latter technique corpora allata were removed from the medium before juvenile hormone extraction and scintillation counting of radioactive hormone. Rates of juvenile hormone synthesis shown in Fig. 9 have been modified as described by Feyereisen and Tobe (1981) to correspond to those shown in Figs 1-8. Length of basal

3

5

7

18

I

39

58

fr Age(days) 0

70

72

t P

74

u 0

Fig. I. Mean rates of juvenile hormone synthesis and lengths of basal oijcytes during gestation and the second gonadotrophic cycle in females mated (-•-_) or unmated (--O--j on the day of parturition. For comparison are shown rates of juvenile hormone synthesis and lengths of basal oiicytes during the first gonadotrophic cycle (data taken from Johnson et al., 1984). 0 indicates oviposition; P, parturition. Each point is the mean of individual measurements shown beside points in the lower graph. Vertical bars represent standard errors of the means (SEM).

Corpora allata regulation in pregnancy Age

at

treatment(days158

1

CA

38

::

Sham

IS

‘:

On Dayo

147

denervated operated

[li,i,l

:, 4

6 Days

S 7 s 9 10 atter danervation

11

12

l

Days after

Operation

Fig. 2. Mean rates of juvenile hormone synthesis and lengths of basal oijcytes in females with corpora allata denervated (. n . . .), (--A--), (-a---) or sham denervated (..‘O’..), (--A--), (-_O-) on days 18. 38 and 58. respectively. Each point is the mean of individual measurements shown beside points in the lower graph. Vertical bars represent SEM. Inset, number of females aborting embryos on days after treatment on day 18 (m), 38 (m) or 58 (W). respectively.

synthesis for females whose oijcyte lengths are shown above. On the day before parturition the mean rate of juvenile hormone synthesis was lower than that of first cycle females with basal oijcytes of similar length. The maximal rate of juvenile hormone synthesis occurred on the day after parturition. Females mated on the day of parturition had a maximal rate of juvenile hormone synthesis significantly higher than that of unmated animals (P < 0.01). Nevertheless, the second cycles of juvenile hormone synthesis for both mated and unmated animals were lower than the first, although the rates of oocyte maturation were similar. The changing milieu of the pregnant female During pregnancy, juvenile hormone synthesis was low and the oocytes grew gradually (Fig. 1). The effect of denervation of corpora allata on rates of juvenile hormone synthesis and oiicyte growth was assessed in females at 3 stages of pregnancy: at 18 days (“early pregnancy”) when the embryos are within 2 days of dorsal closure and the onset of milk uptake; at 38 days (“mid-pregnancy”) when eye colour appears; at 58 days (“late pregnancy”) about

14 days before parturition. Because denervation has an increasingly rapid effect as pregnancy progresses females denervated on day 18 or 38 were assayed on days 9-13 after treatment; those denervated on day 58 were assayed on days 4-9 after treatment. Table 1 presents the percentage of females in which basal oiicytes were growing after treatment; oiicytes were scored as growing if lengths exceeded 0.50, 0.53 or 0.65 mm for females denervated on days 18, 38 or 58, respectively. These lengths were 0.02mm above the maxima1 length of oocytes in sham-operated animals (with the exception of one sham-operated animal in which oljcytes grew). Table 1 (upper) shows that (1) at all stages denervation activated an ovarian growth cycle in some animals, (2) the percentage of cycles activated increased with the age of females, from 54 to 91:/b in the groups denervated on days 18 and 58 respectively, and (3) there was a decrease in the interval from denervation to oviposition as pregnancy progressed. Table 1 (lower) shows results from sham-operated animals. In only one of the controls did oiicytes begin to develop, and these reached (13 days after treatment) only the size of those of a typical O-day female.

148

M.

SUSAN

Table

1, Elkct

of denervation of

RANKIN

and

of corpora

allata

on days

and

oviposition*

oacyte

growth

*.,, Females

CA

18

CA

Minimum

with

denervation

growth

58 on initiation

days

from

to oviposition

?h

53’

I3

38

21

x2**

12

denervated,

Day

,v

Treatmellt Sham Day Day Day *This

growth

oBcyte

length

(mm)

30

3*

0 63

23

o**

0.50

denervated. 38

Sham

o&yte

Maximal

wtth

denervated. IR

Sham

9

Yl***

33

58

‘I,, Females

denervated,

table lengths = corpora

(J***

27

58

on days CA

IS, 38 and

denervated.

Day

The

STAY

denervated,

Day

Ca

okyte

x

Treatment

BARBARA

is a summary of oBcytes 9-13:

of data

scored

**>0.53mm

from

as growing on

days

0.63 Fig. and

2. the days

9-13;

of assay were:

***>0.65mm

* > 0.50 mm

on days

&9.

allata.

Figure 2 depicts the daily determinations of juvenile hormone synthesis (lower) and oiicyte length (upper) after denervation on days IS,38 and 58 from which Table 1 was derived. Odcyte length of shamoperated animals increased only slightly as pregnancy progressed. Within the experimental animals, mean oiicyte length of those denervated on day 58 increased sharply and rather steadily after treatment (a had begun chorionation 7 days after denervation, and by 9 days after treatment, 2 were chorionated), and the accompanying cycle in rates of juvenile hormone synthesis was roughly similar to a normal second cycle. Females denervated on day 38 showed a wider variation in oiicyte sizes (z were chorionated 9 days after denervation, and i on 12 days after denervation), and an accompanying cycle of juvenile hormone synthesis which appeared lower than normal when expressed as a function of days after treatment. The animals denervated on day 18 also displayed a wide variation in oiicyte development and no cycle in rates of juvenile hormone synthesis was discernible although rates were higher than shamoperated controls (and one animal had chorionated eggs on day 13). However. when rates of juvenile hormone synthesis were expressed as a function of oiicyte length. cycles in rates of synthesis were seen in all three groups (Fig. 3). Those of females denervated on days 58 and 38 were almost identical and had females from the group denervated on day 18 been assayed when their basal oacytes were about 1.3 mm, the maximal rate of juvenile hormone synthesis would likely have been similar to the others, about 70 pmol h ’ per pair, somewhat higher than normal mated females in second cycle (Fig. 1). In the course of these experiments some females aborted their embryos. Abortions occurred generally when basal oiicytes reached 1.25-l .30 mm in length, the length typically found in normal second cycle temaies at parturition (Fig. 1). The number of females aborting on days 4-12 after denervation of the corpora allata is shown in Fig. 2 (inset). Of the females denervated on day 18, only one female had basal oiicytes which exceeded 1.3 mm at assay; she aborted 12 days after treatment.

Regulation of corpora allata in the early pregnant female: embryo removal and denervation of corpora allata The relatively low percentage of females responding to denervation of corpora allata with a cycle of juvenile hormone synthesis (Table 1; Fig. 2) suggested that factors other than those reaching the corpora allata by intact nervi corporis allati might contribute to the regulation of juvenile hormone synthesis in 18-day females. We therefore investigated whether removal of embryos on day 18 would be equivalent to denervating the corpora allata with respect to the rates of juvenile hormone synthesis ensuing and what the effect of these manoeuvres performed simultaneously would be.

70

-

60

-

50

-

40

-

30

-

20

-

10

-

CA

den.

l

On

Days

18

.

l

38

58

I// ’

I

1

I

1

1

!

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Basal

Oocyte

Length

(mm)

3. Mean rates of juvenile hormone synthesis as a function of length of basal oiicytes for pregnant females with corpora allata denervated on day 18 (...B...), 38 (--A--) or 58 (-•-). Each point is the mean of individual measurements shown beside points. Vertical bars repreFig.

sent SEM.

Corpora

allata

regulation

in pregnancy

149

Table 2. Effects of embryo removal and denervation of corpora allata on o&zyte growth in l&day pregnant females Treatment Embryos removed, Day 18 CA denervated. Day IS** Embryos removed, Day 18; CA denervated. Day 20 Sham removal of embryos, Day 18 Sham denervation of CA, Day 18**

N

y(, Females with occyte growth*

Maximal oiicyte length (mm)

25 26

31 54

1.55 1.50

23

87

1.55

31

0

0.48

30

3

0.63

All assays were 9-13 days after operation. *Lengths above 0.50 mm were scored as growing. **Taken from Table I. CA = corpora allata.

Rates of juvenile hormone synthesis and oiicyte growth were monitored on each day from 9 to 13 after the operations on day 18. Table 2 shows the percentage of females in which oiicytes grew within this period. After embryo removal alone ovarian growth occurred in 31% of the experimental animals but in none of the controls. The maximal length of basal oiicytes in experimental animals was 1.55mm, l.

sham

0

-Embryos

A,

sham

a

-Embryos

h CA den.

CA +z E c G

1.6

-

: A 0 ; :: 0

1.2

-

0.8

-

0.4

-

den.

.

TG : m

L

I

’

I

I

I

1

i

N c-

A-

_--

,

<.::. 0.. .

L_I ’

27

Capacity of the ovary from an early pregnant ,female to grow and to stimulate juvenile hormone synthesis in first cycle females

,*-_i ,

LA,’

-..*... ..+x.::$

I

I

I

I

28

29

30

31

Age

and in one female chorion formation had begun. No females had oviposited this second batch of oiicytes. When embryos were removed on day 18, and the corpora allata denervated or sham-operated on day 20 (the period between the 2 operations was allowed for recovery from abortion and to assess success of retention of the brood sac) basal oiicytes grew in 877; of the animals. Figure 4 shows daily rates of juvenile hormone synthesis and oiicyte lengths on 9-13 days after the operations i.e. days 27-31 of adult life. The animals experiencing both embryo removal and denervation of corpora allata had mean lengths of basal oiicytes which were significantly greater (P < 0.05) on days 30 and 31 than those after embryo removal or denervation alone or sham-operated animals. Rates of juvenile hormone synthesis by corpora allata from animals having both operations were not significantly higher than those with only one operation. likely because some ovaries had achieved an inhibitory size in the former group. We conclude from the additive effect of embryo removal and denervation of corpora allata in the promotion of increases in rates of juvenile hormone synthesis and ovarian growth that the inhibitory effect of the embryos does not act entirely via the intact nerves to the corpora allata.

(days)

Fig. 4. Mean rates of juvenile hormone synthesis and lengths of basal oiicytes on days 27-31 from females in which, on day 18, corpora allata were denervated (--a--) or sham denervated (...O,..); embryos removed (--A--) or sham removed (,.,A...); or embryos removed and on day 20, corpora allata denervated (-W-). The mean number of individual measurements for each point is 5.2, and the range is 4-7, except for a mean of 3 measurements on days 27 and 31 for embryos removed and corpora allata denervated and on day 31 for sham embryos removed (measurements for l&day denervated and sham-denervated animals are redrawn from Fig. 2). The vertical bars represent SEM.

The rate of growth of an ovary from an 18-day pregnant female and the rates of juvenile hormone synthesis associated with it were followed after implanting such an ovary into an ovariectomized mated 2-day old female. Corpora allata of this host are capable of responding to the presence of stimulatory ovaries by increasing their rates of juvenile hormone synthesis (Rankin and Stay, 1984). Mean lengths of basal oiicytes remained below 0.51 mm for 4 days, then grew slowly to 0.80 mm by 6 days and rapidly to 1.33 mm by 8 days after implantation (Fig. 5, upper). The accompanying mean rates of juvenile hormone synthesis were 2&25 pmol h ’ per pair for 4 days, then began a gradual increase in activity to 46.9 f 6.5 pmol h _ ’ per pair when mean oiicyte length was 1.33 f 0.10 mm (Fig. 5, lower). When rates of juvenile hormone synthesis are expressed as a function of basal oiicyte length the highest rate of synthesis is 68.7 pmol hi- ’

IS0

SUSAN M. RANKIN and BARBARA STAY

per pair, accompanying basal oocytes 1.24mm in length. Thus, the ovary of an l&day female requires a period of 4 days before it elicits an increase in rate of juvenile hormone synthesis. Without resorting to ovariectomy in an l&day pregnant female, 4 days is the maximal time available to test for stimulation of juvenile hormone synthesis by factors independent of the ovary. A corollary to this is that if we wished to assess ovarian effects in less than 4 days, we would need to implant a larger ovary.

Capacit), of an oour~~,from a 0-day,fkmale to stimulate juvenile hormone synthesis in early pregnant .females with denervated corporu allata One might hypothesize that the difference in response of denervated corpora allata between 1% and %-day females (Table I; Fig. 2) resides in the difference in ovarian development, including the delay of 4 days before ovaries from early pregnant females can increase in length (Fig. 5). Whether or not an ovary capable of immediate growth could mimic the conditions of late pregnancy was examined in the following way: corpora allata were denervated in 1S-day females, then on the following day an ovary with basal oijcytes 0.60mm long (basal oiicytes are 0.56 k 0.01 mm by day 62 in controls, Fig. 1) was implanted. Animals were assayed on days 20, 22, 24, 25 and 27 (i.e. 1, 3, 5,6 and 8 days after implantation) for rates of juvenile hormone synthesis and development of the implanted and host ovaries. Results are shown in Fig. 6 along with results from the experiment described in the next section. Both implanted and host ovaries displayed a wide variation in rate of oiicyte maturation (Fig. 6, upper), and there was an accompanying range in rates of juvenile hormone synthesis by host corpora allata (Fig. 6, lower). Females with denervated corpora allata and an implanted ovary were not different from those with denervated corpora allata and no ovary-implant with respect to growth of oiicytes or rates of juvenile 1.4

I_’ J -0”

1.0

0.e

I

9%

+O”

I

;

I

20 Adult

22

24

26

Age ( days )

Fig. 6. Mean rates of juvenile hormone synthesis by corpora allata denervated on day I8 and assayed on days 20. 22. 24. 25 and 27 after implanting, on day 19, an ovary with basal oiicytes 0.6 mm long (0) or such an ovary and a brain from a parturition-day female (0). Mean lengths of basal oiicytes of host (-) and implanted (---) ovaries are also shown (upper). Each point is the mean of individual measurements shown beside points in the lower graph. Inset, mean oiicyte lengths plotted against corresponding mean rates of juvenile hormone synthesis. Individual lengths of basal oiicytes were grouped in 0. I mm increments except for lengths greater than I .OOmm which were combined in one group. The number of individual measurements is shown beside each point. The vertical bars represent SEM.

hormone synthesis on the 9th day after the denervation (compare Fig. 6, day 27, open circles, and Fig. 2, day 9 after the operation, closed circles). Implanted ovaries grew in about half of the test animals (Table 3, treatment l), similar to the percentage of animals in which host ovaries grew after denervation of corpora allata (Table 1). When the rate of juvenile hormone synthesis is shown as a function of the length of basal oiicytes of the implanted ovaries (Fig. 6, inset) an increase in rates of juvenile hormone synthesis is associated with the increase in basal oljcyte length (see also Fig. 3). Role of the brain in regulating rates of juvenile mone synthesis in pregnant females

8

Adult Age (days

‘16

s

10

1

Fig. 5. Mean rates of juvenile hormone synthesis on days 3-10 by corpora allata from females ovariectomized as larvae and, as 2-day mated adults, implanted with ovaries from l&day, pregnant females. Mean basal oocyte lengths of implanted ovaries are shown above. Each point is the mean of individual measurements shown beside points in lower graph. The vertical bars represent SEM.

hor-

Implantation of supernumerary brains. Since an ovary with 0.6 mm basal oijcytes did not in all cases result in increased rates ofjuvenile hormone synthesis in pregnant females we tested whether some neurosecretion from an active brain might suffice to do so. The protocerebra from parturition-day females were chosen because the rates of juvenile hormone synthesis by corpora allata from those donors had just begun to escalate for the second gonadotrophic cycle. T’hus, it seemed plausible that this might be a “stimulatory” brain. Protocerebra from parturition-day females were implanted along with ovaries containing basal oijcytes 0.60 mm long into 19-day females in which the corpora allata had been denervated on day

Corpora

allata regulation in pregnancy

151

Table 3. Effects of decapitation and/or braint implantation on growth of previtellogenic ovaries* implanted into pregnant females with denervated corpora allata

Treatment

_

CA denervated 2. CA denervated + bram” 3. CA denervated; starved 4. Decapitated + (CA and brain)** I.

5. Decapitated + CA** 6. Decapitated + (CA + brain parts)** 7. Decapitated + CA** + brain***

Fig. 7 Symbol _~ 0 : A : 0

N -. 11 II II 15

Mean rates of JH synthesis (pmol h-’ pr-‘) + SEM :__ _ 18.3 * 4.9 18.3 f 2.4 22.4 f 4.3 20.2 * 3.4

Mean lengths of implanted okytes (mm) + SEM

7; OOcytes 20.80 mm (vitellogenic)

OxiOkytes > I.1 mm (vitellogenic and stimulatory)

0.08 0.07 0.06 0.07

45 55 64 60

27 21 27 20

IS I2 9

19.1 f 3.0 23.5 k 5.3 25.2 f 5.2

I .04 f 0.07 I .09 f 0.06

71 92 89

43 58 44

0.93 0.91 0.91 0.91

+ * f *

1.04 & 0.08

This table is a summary of measurements on Day 5 after implantation from Figs 6 and 7. tBram indicates protocerebrum; brain parts indicates opuc lobes, deutocerebrum and tritocerebrum. *from O-dav females: **from 18-dav meenant females: ***from Darturition-day females. X’ test P ;O.OOl for Treatments 114’~~Treatments 5-7. CA = corpora allata; JH = juvenile hormone.

18. Animals with denervated corpora allata and implanted -ovaries but without implanted brains (see above) served as controls for this experiment and results are also shown in Fig. 6. Growth of oiicytes (Fig. 6, upper) and rates of juvenile hormone synthesis (Fig. 6, lower) as a function of age and juvenile hormone synthesis as a function of oiicyte length (Fig. 6, lower, inset) were not different from those in hosts which did not receive brains along with ovaries. From these data we cannot show that the brain had a stimulatory, humoral effect on rates of juvenile hormone synthesis. Starvation

qf’pregnant

no effect on rates of juvenile hormone synthesis by denervated corpora allata in these pregnant females (compare treatments 1 and 3 in Table 3).

. .

.

. .

females

Since animals of the above experiment retained their own brains it was conceivable that a stimulatory effect of the implanted protocerebrum was masked by an inhibitory signal from the brain of the host. This possibility could be explored by decapitation, and implantation of a test brain; however. interpretation of the results of such experiments would require knowledge of the effects of starvation and inanition as well as the effects of decapitation on rates of juvenile hormone synthesis by denervated corpora allata. The protocol for experiments in which l&day females were starved or decapitated (see next experiment) included the implantation of a 0.6 mm ovary so that assays could be done after a relatively short interval; and implantation of corpora allata from 1&day females into the abdomen in both decapitated and starved animals. Table 3 and Fig. 7 show the results of these experiments. Removal of food and corpora allata on day 18 followed on day 19 with implantation of corpora allata and an ovary resulted on day 24 (5 days after implantation) in a mean rate of juvenile hormone synthesis of 22.4 + 4.3 pmol h I per pair, and a mean length of implanted oacytes of 0.91 f 0.06 mm (Table 3. treatment 3). Of the implanted ovaries, 647; contained basal o6cytes which had reached a vitellogenic length (> 0.80 mm) and 27% had reached a stimulatory vitellogenic size ( > 1.1mm) [Table 3, treatment 31. Maximal basal oiicyte length was 1.15 mm (Fig. 7, lower 0). These results are statistically the same as those obtained in fed animals in which corpora allata were denervated on day 18, an ovary was implanted on day 19 and assays carried out on day 24 (Table 3, treatment 1; Fig. 7, lower 0). Starvation alone had

.

!. .

l ?v

n

.

7

n

I

i’

!<9

I 06

OS

Length

1 1

13

of Implanted Oocytes

15

( mm 1

Fig. 7. Rates of juvenile hormone synthesis by corpora allata as a function of the length of basal oocytes assayed 5 days after ovaries with basal oiicytes 0.6mm long were implanted into 18-day pregnant females in which corpora allata were denervated. Lower graph represents all animals which retained or were implanted with protocerebra of lb-day pregnant females: starved animals (Cl): fed animals (0) [redrawn from Fig. 61; fed and implanted with protocerebra from parturition-day animals (0) [redrawn from Fig. 61; decapitated and implanted with protocerebra from 18-day animals (A). Upper graph represents animals from which protocerebra had been removed: animals decapitated (m); decapitated and implanted with protocerebra from parturition-day females (a); or decapitated and implanted with deutocerebra, tritocerebra and optic lobes from I&day females (V). The vertical line at 0.80 mm indicates rhe onset of vitellogenesis; the vertical line at I. 1mm indicates the size at which implanted oijcytes have been demonstrated to elicit increased rates of juvenile hormone synthesis (Rankin and Stay, 1984).

152

Decapitated pregnant f&ales tissue

SUSANM. RANKINand BARBAKASTAR implanted hxith brain

Decapitation of 18-day females followed on day 19 with implantation both of corpora allata from 18-day females and ovaries from O-day females (but no replacement of brains) resulted. 5 days after implantation, in a mean rate of synthesis by implanted corpora allata of 19. I k 3.0 pmol h ’ per pair and a mean basal oiicyte length in implanted ovaries of I .04 + 0.07 mm (Table 3, treatment 5). 7 I”,, of the implanted ovaries had vitellogenic ( > 0.80 mm) basal oacytes. and in 43”” the basal oijcyte length exceeded 1.1 mm (Table 3, treatment 5). A range of basal oiicyte lengths from 0.75 to a mature length of I .63 mm was observed in this group of animals (Fig. 7, upper n ). Clearly, the brain is not required for denervated corpora allata of low activity to respond to the presence of a O-day ovary and support complete development of implanted oiicytes. Comparison of these animals with starved ones (which retained their brains) suggests that brains from pregnant females have a humorally transmitted inhibitory effect on rates of juvenile hormone synthesis. The capacity of a brain from a pregnant female to inhibit rates of juvenile hormone synthesis was further investigated by decapitating animals on day I8 and implanting protocerebra and corpora allata from 18-day females and ovaries with basal olicytes 0.6mm long on day 19. Controls received pieces of neural tissue from the deutocerebrum, tritocerebrum and optic lobes. Implantation of protocerebra from 18-day pregnant females reduced the percentage of implanted ovaries in which basal oiicytes were vitellogenic from 92”,, in control animals that received brain parts other than the protocerebra (Table 3, treatment 6) to 60”,, (Table 3, treatment 4). The percentage of implanted ovaries in which basal oiicytes reached the vitellogenic and stimulatory size of I.1 mm was reduced by one-half. Thus, implanted brains from pregnant females appear to retain a capacity to prevent increases in rates of juvenile hormone synthesis by denervated corpora allata in the presence of a O-day ovary. The capacity of brains from parturition-day females to stimulate rates of juvenile hormone synthesis was tested by implanting such brains along with corpora allata from 18-day females and ovaries with basal oocytes 0.6 mm long into l9-day animals which had been decapitated on day 18. The results (Table 3. treatment 7; Fig. 7, upper 0) are the same as those for animals lacking protocerebra (i.e. decapitated animals or decapitated animals implanted with brain tissue other than protocerebra). Thus, brains from parturition-day females could not be shown to stimulate rates of juvenile hormone synthesis in these decapitated animals; however, since they likewise do not inhibit corpora allata activity, they differ qualitatively from those of pregnant females. In all cases in which protocerebra were implanted they were recovered and found to have retained their gross morphology. Samples which were processed for the demonstration of neurosecretory material and sectioned showed few if any pycnotic nuclei or invading blood cells. Neurosecretory material was prominent in brains from both pregnant and post-

partum animals and no obvious differences the two types of brains were observed.

between

Cupacity qf u stirnulatory ovary to overcome the inhihitorJ,
female Since ovaries with I. I mm basal o&ytes are more stimulatory than those with 0.60mm oiicytes when implanted into a Z-day. ovariectomized female (Rankin and Stay. 1984), we tested the capacity of an ovary with basal odcytes I.1 mm long (from a 4-day female) to overcome the inhibitory factor from the brain and increase juvenile hormone synthesis by denervated corpora allata in day-18 pregnant females. Controls had denervated corpora allata and saline injections. Assays were performed daily for 5 days after implantation (i.e. days 18-24 of host age). Denervation alone doubled the rate of juvenile hormone synthesis (from 5.5 k 1.0 on day 18 to 10.4-1_ 3.3 pmol h-’ per pair on day 19, Fig. 8. lower). The group implanted with ovaries showed a significantly higher rate than that of controls on day 22 (P cc 0.05). The mean growth of the implanted ovaries was slow until day 22. but i had begun chorion formation by then (Fig. 8, upper). Also on day 22, host oacytes were significantly larger than those of controls (P
of'juvenke hormone synthesis and @ct

Corpora allata from 2-day females are considerably more active (39.3 k 6.2 pmol h ’ per pair, Fig. I ) than those of I S-day pregnant females. The capac-

_a

L/1

18

’

’

20 Adult

1

22 Age ( days

24 )

Fig. 8. Mean rates of juvenile hormone synthesis by corpora allata denervated on day 18 and assayed on days 18-24. Females containing these corpora allata were implanted on day length (0) or lengths of basal ovaries are also

19 with an ovary of I.1 mm basal oiicyte injected with saline solution (0). Mean odcytes of host (---_) and implanted (---) shown (upper). Each point is the mean of individual measurements shown beside points in the lower graph. Vertical bars represent SEM. C indicates the fraction of ovaries in which basal okytes had begun or completed chorion formation.

Corpora allata regulation in pregnancy

153

rate of juvenile hormone synthesis declined from the presumed rate of synthesis at implant to 19.6 f 4.5 pmol h- ’ per pair then increased for 2 days before declining again; the implanted oiicytes had increased in length by day 24 and continued to grow to 1.54 f 0.09 mm by day 27, and $ had begun or completed chorion formation. The ovaries of the host also grew to 1.34 + 0.13 mm by day 27. and $ had begun chorion formation. In contrast, by day 24 in controls, rates of juvenile hormone synthesis had declined at the same rate as those in experimental animals, from the presumed rate of juvenile hormone synthesis at implant, to below 10 pmol h ’ per pair, but rates remained at about that level until day 27. when they increased slightly in activity. By day 27. basal obcyte lengths reached 0.81 + 0.09 mm, a juvenile hormone-dependent, vitellogenic size, but only about half the length of a mature okyte; none had begun

chorionation.

Thus

the

previtellogenic

ovary

supported greater juvenile hormone synthesis than was found in controls. The protocol for testing 2-day corpora allata in pregnant females with a vitellogenic ovary was as follows: females allatectomized on day 18 were im+CA

+ 0”

+CA

+

l 0

Saline

I,

10

ia

24 Adult

Age

25

20

+

27

(days)

Fig. 9. Mean rates of juvenile hormone synthesis by corpora allata from 2-day mated females which replaced glands of 19-day females and were assayed on days 24-27. Host females had been implanted on day 18 with a O-day ovary (0) or injected with saline solution (0). The mean rate of juvenile hormone synthesis for day 19 is the presumed rate for 2 day glands at implantation based on basal oiicyte length (derived from Johnson et al., 1984). Mean lengths of basal oiicytes from host (--) and implanted (---) ovaries are shown (upper). Each point is the mean of individual measurements shown beside points in the lower graph. Vertical bars represent SEM. C indicates fraction of ovaries in which basal oiicytes had begun or completed chorion formation.

ity of this “activated” pair of corpora allata to overcome the effects of the brain and to promptly produce a gonadotrophic cycle of activity in a pregnant female was tested when implanted alone and with a previtellogenic or vitellogenic ovary. The protocol for testing 2-day corpora allata with a previtellogenic ovary was as follows: after implanting an ovary of basal oiicytes 0.60mm long (from a O-day female) or injecting saline on day 18, the corpora allata of the host were removed and replaced with glands from 2-day females on day 19. Mean basal oiicyte lengths of the corpora allata donors for experimental animals and for controls were 0.84 f 0.02 mm and 0.79 f 0.01 mm respectively; rates of juvenile hormone synthesis typically associated with these oiicyte lengths are 45.7 + 5.6 and 33.0 k 5.7 pmol h-’ per pair respectively (calculated from Johnson et al., 1984). These rates and the rates of synthesis assayed on days 2&27 (i.e. 5-8 days after implantation and when implanted ovaries were 5-8-days old) are shown in Fig. 9. In the experimental animals on day 24, the

OL ’ 18

I

I

I

I

I

19

20

21

22

23

Adult

Age

( days

1

Fig. IO. Mean rates of juvenile hormone synthesis by corpora allata from 2-day mated females implanted with (a) or without (0) an ovary of 1. I mm basal oiicyte length into 19-day females which had been allatectomized on day 18. The presumed mean rate of juvenile hormone synthesis for day 19 was determined as described in Fig. 9. Mean basal oijcyte lengths of host (---) and implanted (---) ovaries are shown (upper). C indicates fraction of ovaries with chorionation of basal oijcytes in progress or completed. Each point is the mean of individual measurements shown beside points in the lower graph. The vertical bars represent SEM.

154

SUSAN M. RANKIN and BARBARASTAY

planted on day 19 either with a pair of 2-day corpora allata alone or 2-day corpora allata and an ovary in which basal oiicytes were 1.1 mm long (from 4-day females). Mean basal oiicyte length of the corpora allata donors was 0.80 + 0.02 mm for both experimental and control groups, and the presumed mean rate of juvenile hormone synthesis for the corpora allata from females with such oiicytes is 39.2 + 6.2 pmol h ’ per pair. These rates and those assayed on days 20-23 (i.e. 14 days after implantation of ovaries; thus implanted ovaries were 5-8days old) are shown in Fig. 10. In the experimental group the rates of juvenile hormone synthesis remained at the level at which they had been introduced for about 2 days, then increased to a peak on day 22 as the basal oiicytes of implanted ovaries reached 1.33 k 0.03 mm. By day 24, all implanted oiicytes had begun chorionation. Intact host oiicytes did not show an increase in basal oiicyte length until day 23. In contrast, in the control group, rates of juvenile hormone synthesis declined gradually from the time of implant for 2 days, then sharply on day 22 and remained low on day 23. As in the experimental group. intact host ovaries began to grow by day 23. but did not reach a juvenile hormonedependent length. DISCUSSION

The second gonadotrophin CJ& This study has shown by direct radiochemical measurements that the second cycle in rates of juvenile hormone synthesis is lower than the first (Fig. 1, lower), a prediction which Englemann (1959) made based on histological evidence of corpus allatum volume/million nuclei. We have also found that mating after parturition results in higher rates of juvenile hormone synthesis compared to females which are not mated at this time. This result is not surprising since mating is necessary for the first gonadotrophic cycle (Englemann. 1959; Roth and Stay, 1961; Stay and Tobe, 1977) yet not anticipated from the rate of odcyte maturation. Oiicyte maturation during the second cycle was the same for mated and not remated animals (Fig. 1; Englemann, 1959). We have also confirmed that the second cycle of oacyte maturation in D. punctata begins before parturition (Fig. 1. 1959; Roth and Stay, 1961; upper, Engelmann, Mundall et al., 1981). A second gonadotrophic cycle with a similar reduction in juvenile hormone synthesis accompanied by unaltered oacyte maturation has been shown in Schistocerca gregaria (Tobe and Pratt, 1975), whereas in Periplaneta americana. the maximal rates of synthesis in successive cycles are not reduced from that of the first (Weaver and Pratt, 1977). The role qf the ovary in regulating juvenile hormone synthesis No cycle in rates of juvenile hormone synthesis occurs in ovariectomized cockroaches of first-cycle age (Stay and Tobe, 1978: Lanzrein et al.. 1981a; Weaver, 1981; Stay et al., 1983); a cycle can be restored by implanting an ovary (Lanzrein er al.. 1981 b; Stay et al., 1983). When present and growing the ovary appears to have a changing capacity to

influence the corpora allata in that rates of juvenile hormone synthesis can be reliably predicted by stage of oijcyte development in many insects, e.g. the cockroaches P. americana (Weaver et al., 1975). N. cinerea (Lanzrein et al., 1981a.b) and D. punctata (Tobe and Stay, 1977. 1980; Stay ef al., 1983; Tobe, 1980; Feyereisen rt al., 1981). This has indeed been shown to be true in D. punctata by short term in viva incubation of corpora allata with ovaries of various sizes. Ovaries with basal oiicytes 0.38 mm did not elicit increased rates of juvenile hormone synthesis. nor did ovaries with basal oiicytes longer than I .5 mm. The rate of synthesis began to increase with exposure to 0.6-0.8 mm oiicytes and was significantly increased by exposure to oiicytes between 1.1 and 1.4 mm in length (Rankin and Stay, 1984). The basal oiicytes during normal pregnancy in D. punctuta grow gradually over a 50-day period from 0.38 to 0.60mm (Fig. 1). Yet this growth can occur after a delay of only a few (4-5) days if levels of juvenile hormone are increased, as we have shown in several ways: by implanting such an ovary into an ovariectomized first cycle female (Fig. 5): by denervating the corpora allata in pregnant females and implanting an additional stimulatory ovary (1.1 mm basal oacytes) [Fig. 81; or by implanting active corpora allata into pregnant females (Figs 9 and 10). Our results differ from those of Scheurer and Liischer (1966) who showed in Leucophaea maderae that ovaries from females early in pregnancy did not grow within 10 days after implantation into egg maturing females, nor did they grow within 9-12 days when “active” corpora allata and an immature ovary from a first cycle female were implanted into the early pregnant female (although the implanted ovary grew in all cases). Ovaries of N. cinerea females in early pregnancy also appear to require at least 12 days to grow after embryo removal coupled with injection of exogenous hormone, although vitellogenin titres rise within 24 h after application of the hormone (Buschor et al., 1984). The delay in response of the ovary to juvenile hormone in these cockroach species may provide a mechanism to ensure that a slight elevation of juvenile hormone synthesis for a short duration (which might occur. for example, in response to injury) would not jeopardize a pregnancy by stimulating an early oijcyte cycle and abortion of the embryos. As long as the basal oiicytes remain below a stimulatory size no cycle in rates of juvenile hormone synthesis ensues. Ovaries implanted into early pregnant females elicited increased juvenile hormone synthesis by denervated corpora allata and completed development (1) only in about SO’, of the cases when the ovary had basal oijcytes 0.6 mm long at implantation (Table 1). a size similar to that of a female in late pregnancy (Fig. 1), (2) always when the ovary had basal oiicytes 1.I mm long, but the cycle in rates of juvenile hormone synthesis was greatly reduced (Fig. 8). (3) always when an “active” pair of corpora allata replaced those of the host and accompanied an ovary with basal oiicytes either 0.6 mm (Fig. 9) or 1.1 mm long (Fig. IO); a normal second cycle in rates of juvenile hormone synthesis ensued. The cycle in rates of juvenile hormone synthesis observed after denervation of corpora allata and implantation of

Corpora allata regulation in pregnancy ovaries began only after a delay of at least 2 days. This delay appears to result from an inhibitory brain factor (see below). The role of the nervous system in regulating rates of juvenile hormone synthesis

The low rates of juvenile hormone synthesis during early pregnancy are maintained in part by inhibition from the brain via intact nerves to the corpora allata and in part by factors from the brain elicited by the presence of embryos which may travel via the haemolymph. Since experimental removal of these two inhibitions in early pregnancy promptly initiates a second gonadotrophic cycle in 87% of the test animals (Table 2) these two mechanisms may account for most, if not all, of the inhibition of the corpora allata in pregnant females. Basal oiicytes less than 1.5 mm in length are not inhibitory (Rankin and Stay, in press). It is well documented for a variety of insects that intact nerves to the corpora allata may restrain juvenile hormone synthesis (e.g. Engelmann, 1959, 1960; Baehr et al., 1973; Bhaskaran, 1981; Lanzrein et al., 1981b; Pipa, 1982; Khan et al., 1983) including virgin adult females (Roth and Stay, 1961; Stay and Tobe, 1977) and early last-instar larvae of D. punctata (Szibbo.and Tobe. 1983). We have shown that intact nerves to the corpora allata regulate juvenile hormone synthesis in pregnant females in that denervated corpora allata showed higher rates of juvenile hormone synthesis than did those with intact nerves (Figs 2 and 3; Table 2). The percentage of corpora allata which were activated to support a second cycle of oocyte growth after denervation increased with the age of the female at operation (Table 1). Neural inhibition of corpora allata in pregnant D. punctata, i.e. that inhibition which remained after embryo removal, decreased as pregnancy progressed since removal of embryos at progressively later stages in gestation was followed by shorter intervals to the next oviposition (SPgesser, 1960; Roth and Stay, 1962a,b; Buschor et al., 1984). This neural inhibition, like that of virgins, can be reduced still further at the end of gestation by mating (Fig. 1). Engelmann (1959) found that mating within 46 days after removal of embryos (at an unspecified time probably early in pregnancy) resulted in oviposition within 9 days of mating, whereas unmated control females did not oviposit for at least 41 days. Denervation of corpora allata (Stay and Tobe, 1977) or cautery of medial and lateral neurosecretory areas of the brain (Riiegg et a/.. 1983) mimics mating in virgin females but fails to activate glands in young pregnant females within a similar time period (5 days). The presence of maturing embryos in the brood sac is detected by exposure of increasing numbers of sensory hairs within the brood sac (Greenberg and Stay, 1974) and relayed to the brain via the ventral nerve cord (Engelmann, 1959, 1960; Roth and Stay, 1961). Inhibition elicited by the presence of embryos in the brood sac, i.e. that inhibition which persisted after denervation of corpora allata, decreased as pregnancy progressed (Fig. 2, Table 1). That embryo removal combined with denervation of corpora allata produced greater activation of corpora allata than

155

either operation alone (Table 2; Fig. 4) clearly shows that the route by which embryos maintain reduced rates of juvenile hormone synthesis is not exclusively via intact nerves from the brain to the corpora allata and likely involves a second inhibitory centre in the brain. This was substantiated by the finding that animals with protocerebra (either their own or implanted ones from animals of similar age) were less likely to support growth of an implanted ovary with increased corpus allatum activity than were animals without protocerebra (decapitated and not replaced with protocerebra from pregnant females). The capacity of a brain factor acting via the haemolymph to reduce as well as maintain low rates of juvenile hormone synthesis would explain the marked reduction in synthesis which occurred when active corpora allata were implanted into early pregnant females either without an accompanying ovary (Figs 9 and 10) or with an ovary in which basal oijcytes were 0.6 mm long (Fig. 9). Indeed, even the robust combination of implanting active denervated corpora allata and a large ovary required 2 days to overcome the inhibition afforded by the haemolymph of pregnant females (Fig. 10). The presence of a humorally transmitted factor from the brain which inhibits the corpora allata has also been suggested by egg growth bioassay in Pyrrhocoris apterus (Hodkova, 1979), and Rhodnius prolixus (Baehr et al., 1973), by radiochemical assay in Leptinotarsa decemlineata (Khan et al., 1983) and by moulting bioassay and radioimmune assay in Manduca sexta (Bhaskaran, 1981; Granger et al., 1981). A capacity of implanted brains to stimulate rates of juvenile hormone synthesis as assayed by supernumerary moulting has been demonstrated in larvae of Galleria mellonella (Krishnakumaran, 1972; Granger and Sehnal, 1974; Pipa, 1976) and Manduca sexta (Bhaskaran, 1981) and as assayed radiochemically in corpora allata from adult female D. punctata implanted into male hosts (Tobe et al., 1981). However, from our results following implantation of parturition-day brains into pregnant females either with or without heads (Figs 6 and 7; Table 3) we cannot conclude that the brain had a stimulatory effect on rates of juvenile hormone synthesis. Thus, we have not substantiated a previous hypothesis (Stay and Tobe, 1977) or our previous report (Stay and Rankin, 1984) that they do so; neither can we dismiss that possibility, since (1) brains from parturition-day females may have been inappropriate or (2) release of the putative stimulatory factor may have been insufficient and would require multiple brain implants (e.g. Granger and Sehnal, 1974). The finding that parturition-day brains did not reduce rates of juvenile hormone synthesis clearly demonstrates that such brains lack or do not release the inhibitory factor that effectively emanates from brains of early pregnant animals (Fig. 7; Table 3). To conclude, many factors interact to release the corpora allata from multiple inhibitions culminating in parturition and the second gonadotrophic cycle. The inhibitory factor elicited by maturing embryos in the brood sac disappears by the end of gestation. The disappearance could be explained by sensory adaptation, but this remains to be determined. The mech-

156

SUSAN; M. RANKIN and BARBARA STAY

anism governing the reduced effectiveness of a second inhibition, the one requiring intact nerves, is likewise unknown; possibly it relies on long-term oscillations with integrative centres of the brain or a mating mimic due to stretching of the bursa copulatrix by the expanding brood sac. Diminution of one or both sources of inhibition might cause a slight elevation in rates of juvenile hormone synthesis. The slight elevation in titre of hormone would maintain 01 intensify rates of synthesis by positive feedback as has been demonstrated by application of small amounts of exogenous juvenile hormone analogue in 1st cycle D. punctata (Tobe and Stay, 1979) and after embryo removal in N. cineren (Buschor et cd.. 1984). Basal oiicytes, which by this time are competent to grow immediately (Fig. I), would respond to the slightly increased titre of juvenile hormone with growth and further stimulation of juvenile hormone synthesis necessary for complete ovarian development.

Aclinon~led~ements-We thank Kuen Ghan and Paul Hebl for assistance in staining and sectioning brain tissue. Thts work was supported by United States Public Health Service Grant No. AI 15330.

REFERENCES Baehr J. C.. Cassler P. and Fain-Maurel M. A. (1973) Contribution expkrimentale et infrastructurale B I‘&ude de la dynamique du corpus allatum de Rhodnius prolixus Stal. Influence de la nutrition, de I’activitP ovarienne. de la pars intercerebralis et de ses connexions. Archs Zool. rrp. gen. 114, 61 I-626. Bhaskaran G. (198I ) Regulation of corpus allatum activity in last instar Manduca .se.~talarvae. In Current Topics in Insect Endocrinology and Nutrition. (Ed. by Bhaskaran G.. Friedman S. and Rodriguez J. G.). pp. 53-82. Plenum Press. New York. Buschor J.. Beyeler P. and Lanzrein B. (1984) Factors responsible for the initiation of a second oiicyte maturation cycle in the ovoviviparous cockroach Nauphoeta cinereu.

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30, 341-249.

Engelmann

F. (1959) The control of reproduction in Diploptera punctata (Blattaria). Biol. Bull. mar. hiol. Lah.. Woods Hole 116, 406419. Engelmann F. (1960) Mechamsms controlling reproduction in two viviparous cockroaches (Blattaria). A. N. y. Acad. Sci. 89, 516536. Feyereisen R.. Friedel T. and Tobe S. S. (1981) Farnesoic acid stimulation of C,, juvenile hormone biosynthesis by corpora allata of adult female Diploprrra punctaia. Inseci Biorhem.

11, 40 I-409.

Feyereisen R. and Tobe S. S. (1981) A rapid partition assay for routine analysis of juvenile hormone release by insect corpora allata. Analj,t. Biochem. 111, 372-374. @anger N. A. and Sehnal F. (1974) Regulation of larval corpora allata in Galleria mellonella. Nature 251.415~11. Granger N. A., Bollenbacher W. E. and Gilbert L. 1. (1981) An in vitro approach for investigating the regulation of the corpora allata during lava]-pupal metamorphosis. In Curren; Topics in Insect &docrinology and Nutrition. (Ed. by Bhaskaran G., Friedman S. and Rodriguez J. G.). .on. . 82-105. Plenum Press, New York. Greenberg S. and Stay B. (1974) Distribution and innervation of hairs in the brood sac of the cockroach. Diplopteru punrtata (Eschscholtz) (Dictyoptera; Blaberidae). lnt. J. Insect morph. Embr.voi. 3, 127-135. Hodkovli M. (1979) Hormonal and nervous inhibition ot reproduction by brain in diapausing females of Pyr-

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Roth L. M. and Stay B. (1962b) A comparative study of oocyte development in the false ovoviviparous cockroaches. Psyche 69, 165-208. Roth L. M. and Willis E. R. (1955) Intra-uterine nutrition of the ‘beetleroach’ Diploptera dvtiscoides (Serv.) during embryogenesis with noiesbn its Biology in the laborator; (Blattaria: Diplonteridae). Psvche. 62. 55-68. Riiegg R. P.. L&o;0 D. J. and.Tobe S: S. (1983) Control of corpus allatum activity in Diploptera punctata: roles of the pars intercerebralis and pars lateralis. Experientia 39, 1329-1334. Slgesser H. (1960) ijber die Wirkung der Corpora Allata auf den Sauerstoffverbrauch bei der Schabe Leucophaea maderae J. Insect Phvsiol. 5. 264-285.

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allata

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Schemer R. and Liischer M. (1966) Die phasenspezifische Eireifungkompetenz der Ovarien von Leucophaea maderae. Rer. Suisse Zool. 13, 511-516. Stay B. and Coop A. (1973) Developmental stages and chemical composition in embryos of the cockroach, Diplopfera punctata. with observations on the effect of diet. J. Insect. Physiol. 19, 147-171. Stay B. and Rankin S. M. (1984) Regulation of juvenile hormone synthesis by the corpora allata during pregnancy in the viviparous cockroach, Diplopfera punctafa. In tnsecf Neurochemistry and Neurophysiology. (Ed. by Borkovec A. B. and Kelly T. J.), pp. 483485. Plenum Press, New York. Stay B. and Tobe S. S. (1977) Control of juvenile hormone biosynthesis during the reproductive cycle of a viviparous cockroach. I. Activation and inhibition of corpora allata. Gen. Camp. Endocr. 33, 531-540. Stay B. and Tobe S. S. (1978) Control of juvenile hormone biosynthesis during the reproductive cycle of a viviparous cockroach. II. Effects of unilateral allatectomy, implantation of supernumerary corpora allata, and ovariectomy. Gen. Camp. Endocr. 34, 276286. Stay B., Tobe S. S., Mundall E. C. and Rankin S. (1983) Ovarian stimulation of juvenile hormone biosynthesis in the viviparous cockroach. Diplaptera puncfaia. Gen. Comp. Endow. 52, 341-349. Szibbo C. M. and Tobe S. S. (1983) Nervous and humoral inhibition of C,, juvenile hormone synthesis in last instar females of the viviparous cockroach, Diplopfera puncfata. Gen. Camp. Endocr. 49, 437445. Tobe S. S. (1980)Regulation of the corpora allata in adult female insects. In Insect Biology of the Future: VBW 80. (Ed. by Locke M. and Smith D. S.). pp. 345-367. Academic Press, New York. Tobe S. S. and Pratt G. E. (1974) The influence of substrate concentrations on the rate of juvenile hormone biosynthesis by corpora allata of the desert locust in vitro. Biochrm. J. 144, 107-113.

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