Experimental acquisition and loss of allatostatin sensitivity by corpora allata of Diploptera punctata

0022-1910(95)00047-X

J. Insect Physiol. Vol. 41, No. II, pp. 975-980, 1995 Copyright 6 1995 Elsevier Saence Ltd Printed in Great Britain. All rights reserved 0022-1910/95 $9.50 + 0.00

Experimental Acquisition and Loss of Allatostatin Sensitivity by Corpora Allata of Diploptera punctata G. C. UNNITHAN,*

R. FEYEREISEN*t

Received 29 December 1994; revised 27 March 1995

In the cockroach, Diplopterapunctata, acquisition of sensitivity to a low concentration of an allatostatin, the tridecapeptide APSGAQRLYGFGL-amide, occurs in the corpora allata (CA) of mated females between day 5 and day 6, just before choriogenesis, and corresponds to the shift between peak and declining JH synthetic rates [Pratt et al., Mol. Cell. Endocr. 70, 185-195 (1990)]. We show that the acquisition of allatostatin sensitivity is not affected by denervation of the CA, but is dependent on a humoral factor. Transplantation of day 5 CA to previtellogenic females prevents the acquisition of allatostatin sensitivity, whereas day 1 CA transplanted into post-vitellogenic females become sensitive. Ovariectomy in vitellogenic females disrupts both JH synthesis and the acquisition of allatostatin sensitivity. Removal of both ovaries and embryos in pregnant females does not impair the acquisition of sensitivity of day 1 CA in the post-vitellogenic endocrine milieu. Denervation of the CA in virgin females leads to a rapid decrease in allatostatin sensitivity and a slower increase in JH synthetic rates, both of which do not occur in vitro. Our results demonstrate that the sensitivity to allatostatins can be experimentally manipulated and suggests the existence of humoral factor(s) responsible for the important changes of CA sensitivity to this tridecapeptide. Cockroach

Juvenile hormone

Biosynthesis

Inhibition

INTRODUCTION

inhibition is reminiscent of the decreased sensitivity of active CA to brain extracts (Feyereisen and Farnsworth, 1987a; Rankin and Stay, 1987) and to pharmacological effecters of second messenger cascades (Meller et al., 1985, Feyereisen and Farnsworth, 1987a,b). Because the potency of allatostatins as inhibitors of JH synthesis is dependent not only on the concentration of peptide but also on the degree of sensitivity of the glands, it was felt that a study of the latter would be of interest. In particular, a better understanding of CA sensitivity to allatostatins would be prerequisite to an evaluation of these peptides as potential prototypes for new insect control agents. Such a study would also shed light on the mechanism of CA regulation during a gonotrophic cycle. We report here our initial efforts aimed at manipulating CA sensitivity to allatostatin experimentally. Our results emphasize the role of humoral factor(s) in the control of CA sensitivity.

Cockroach allatostatins (Stay et al., 1994) are peptides which rapidly and reversibly inhibit juvenile hormone (JH) synthesis by the corpora allata (CA) at specific times during insect development (Pratt et al., 1990; Stay et al., 1991). During the peak of JH synthesis on day 5 in mated females of Diploptera punctata, a low concentration (10 nM) of the tridecapeptide APSGAQRLYGFGLamide (allatostatin 1, Woodhead et al., 1989; ASAL, Pratt et al., 1989) does not inhibit JH synthesis at all. One day later, the same concentration inhibits JH synthesis maximally (SS-90%) in a short-term assay. CA sensitivity to this peptide then wanes during post-vitellogenesis and pregnancy (Pratt et al., 1990). Significant differences in the pattern of CA sensitivity are observed with the octadecapeptide ASB2 (Pratt et al., 1991b). The CA are more sensitive to ASB2 during vitellogenesis, and remain more sensitive in pregnant females. However, the most notable change in sensitivity to the octadecapeptide ASB2 is also seen during the day 5day 6 transition (Pratt et al., 1991 b). The refractoriness of day 5 CA to allatostatin

MATERIALS AND METHODS Insects

*Department of Entomology and Center for Insect Science, University of Arizona, Tucson, AZ 85721, U.S.A. tTo whom all correspondence should be addressed.

Diplopterapunctata was reared as described previously (Meller et al., 1985). Virgin females were collected from 975

976

G. C. UNNITHAN

cages free of adult males. Mated females were collected within 4 h of molting and mated status was confirmed by the presence of a spermatophore. The length of the terminal follicle was measured for all experimental animals.

and R. FEYEREISEN

previously otherwise, specifically.

(Pratt et al., 1989, 1990). “allatostatin” refers to this

Unless stated tridecapeptide

RESULTS Denervation

of the CA, transplantation

and ovariectomy Acquisition

For denervation of the CA, the insect was chilled on crushed ice and held to a dissecting dish with the help of a rubber-band. The head and pronotum were wiped with 70% alcohol. The head was stretched forward, to expose the neck, and held in position with pins placed on either side. Through a small slit cut on the base of the head capsule, the CA with attached part of the corpora cardiaca (CC) complex was detached from the brain with fine forceps. The wound was covered with the flap of head capsule along with attached neck membrane, the head was pushed back to the original position, and a few crystals of streptomycin were placed on the surface of the wound. No mortality was observed after denervation or sham operation. For transplantation, the CA-CC complex was dissected out in sterile Hanks’ salt solution and the cut-ends of the CC were tied to a piece of dental floss fiber. The CA-CC complex was introduced into the haemocoel of the recipient through a slit between the 2nd and 3rd abdominal sternum, after surface sterilizing the ventral side of the abdomen. The piece of dental floss fiber tied to the CC helped transfer during transplantation and recovery of the glands. For ovariectomy, chilled females were placed in the well of a dissecting dish, the posterior end of the abdomen facing up. The abdominal tip was wiped with 70% alcohol. With the help of flat forceps the body wall between the last tergite and sternite was stretched. The ovaries were pulled out through slits made laterally between the tergite and sternite. The tergite and sternite were then released to their original position. A few crystals of streptomycin were placed on the wound. Ovariectomy and sham operation led to slight bleeding and low mortality rates. Success of ovariectomy was verified at the time of sacrifice. Embryos were extracted through the opening of the genital pouch, from chilled pregnant females.

Inhibition of JH synthesis by allatostatin was determined by the radiochemical assay for incorporation of label from [methyl-3H]methionine into juvenile hormone III as modified by Pratt et al. (1990). Each group of treated corpora allata (see legends for n of individual pairs of CA per group) included concurrent control incubations (no peptide) and gland-free incubations (partition assay blank). Synthetic

peptide

All experiments LYGFGL-amide

used the tridecapeptide APSGAQR(ASAL, allatostatin 1) as described

by insensitive

CA

Table 1 shows the results of in vivo and in vitro experiments designed to test the role of the nervous connections between the brain and the CA in the process which leads to a dramatic gain of allatostatin sensitivity shortly after day 5 in mated females (Pratt et af., 1990). Allatostatin sensitivity refers to the level of inhibition achieved by a 10 nM concentration of allatostatin during a 2-h incubation of the CA. Whereas day 5 CA were insensitive to allatostatin, maximal sensitivity was achieved on day 6 (Pratt et al., 1990), and on day 7 sensitivity had slightly declined to 72% (Table 1). Denervation of the glands on day 5 and assay two days later showed that severance of the nervous connections of the corpora allata did not prevent the acquisition of allatostatin sensitivity (Table 1). However, when corpora allata were removed from the insect on day 5 and incubated in vitro for 18 or 48 h before assay, only a slow and modest gain in sensitivity to allatostatin was observed. EfSect of the host endocrine sensitivity by transplanted CA

milieu

on acquisition

of

In order to test how the sensitivity of denervated CA would be influenced by the endocrine milieu, we transplanted CA from mated day 1 and day 5 females into hosts of different ages. CA from day 1 females are characterized by a low level of allatostatin sensitivity and they normally retain low sensitivity until the day S/day 6 transition (Pratt et al., 1990). In contrast, day 5 CA are within 24 h of that transition to high sensitivity. The results (Fig. 1) show that day 1 CA transplanted into day 3 females and assayed 2 days later had a low sensitivity to allatostatin. However, when day 1 CA were transplanted into day 7 females and assayed 2 days later, they had acquired allatostatin sensitivity (Fig. 1). TABLE

In vitro bioassay

of sensitivity

1. Acquisition

of allatostatin sensitivity vitellogenesis

by CA at the end of

% Inhibition of JH synthesis by 10 nM allatostatin (n) Day Day Day Day Day Day Day

5 6 7 7 7 5 5

CA CA* CA CA after sham operation on day 5t CA after denervation on day 5t CA after 18 hours in vitro incubation? CA after 48 h in vitro incubationt

* Data from Pratt t Means followed P = 0.05.

et al. (1990). by a different

letter

- 5.2 f 5.6 (20) 92.7 + 4.3 (12) 71.8 f 4.7 61.3 f 5.1 66.2 f 6.8a (14) 17.5 + 5.7b (19) 33.8&4.9c (14)

are significantly

different

at

ALLATOSTATIN

SENSITIVITY

a

T

6 7

tk

ld>3d

ld>7d

5d>2d

Age of transplant and host

6

1

5d>7d

FIGURE 1.Sensitivity to allatostatin of CA transplanted into hosts of different ages. The sensitivity of transplanted CA (open bars) and host CA (stippled bars), 2 days after transplantation is expressed as the % inhibition of JH synthesis by 10 nM allatostatin. Key: 1 d>3 d indicates the age of the transplanted CA and the age of the host at transplantation. Each bar represents the mean SEM of the number of assays indicated above each bar. Differences between transplanted glands and host glands were not significantly different (P > 0.05, c-test).

Consequently, even though the transplanted glands had not undergone a cycle of JH synthesis with a peak of activity on day 5, they must have received a signal from the host endocrine milieu that caused their sensitivity to allatostatin to increase. CA from day 5 females transplanted into day 2 hosts and assayed two days later had only modest allatostatin sensitivity as did the CA of their hosts (Fig. 1). When transplanted into day 7 hosts and assayed 2 days later, the day 5 CA had gained high sensitivity. Thus a 2 day exposure to the endocrine milieu of the host insect had a significant effect on the sensitivity to allatostatin of transplanted CA. These experiments also showed that denervation per se did not lead to, or prevent, an acquisition of allatostatin sensitivity (see also Table 1, row 4). Efect of the ovary sensitivity

on the acquisition

IN DIPLOPTERA

911

P < 0.05). These results suggested that the presence of the ovary was necessary for the normal day S/day 6 acquisition of allatostatin sensitivity. However, because of the complex role of the ovary in regulating JH biosynthesis, it was not clear whether the effect seen did not simply reflect perturbations in the cycle of JH biosynthesis. Eflect of sensitivity

the post-vitellogenic

ovary

on

allatostatin

We then turned to post-vitellogenic females, when the ovary no longer exerts a stimulatory activity on JH synthesis (Rankin and Stay, 1984) but rather contributes to the inhibition of JH synthesis (Stay et al., 1980; Rankin and Stay, 1985) that is maintained during pregnancy. Removal of the ovaries and embryos on day 8 (just after deposition in the brood sac) did not affect allatostatin sensitivity of the CA 2, 7 or 14 days after the operation (Table 2). Moreover, CA from day 1 females transplanted into normal pregnant females and CA transplanted into ovariectomized females became equally sensitive (Table 2). During pregnancy the CA progressively lost their sensitivity to allatostatin, and the host milieu of pregnant females progressively decreased its ability to confer sensitivity to transplanted CA. This effect was independent of the ovaries or embryos. Loss of allatostatin sensitivity following CA in virgin females

denervation

of the

The CA of virgin females are under neural inhibition in D. punctata (Engelmann, 1959) and do not undergo a cycle of JH synthesis unless they are denervated (Stay and Tobe, 1977). Such denervated CA maintained in vivo rapidly increase their JH synthetic potential, but retain their low level of activity when incubated in vitro (Tobe et al., 1981). We tested the hypothesis that the difference

of allatostatin

The ovary plays an important role, first stimulatory then inhibitory, in the control of JH synthesis throughout the gonotrophic cycle of D. punctata (Feyereisen, 1985), and the acquisition of sensitivity to allatostatin during the day S/day 6 transition is correlated with preparation for choriogenesis (Pratt et al., 1990). Therefore, we tested the effect of ovariectomy at different times during vitellogenesis on allatostatin sensitivity of the CA on day 7, i.e. at a time when the CA of unoperated females have acquired high allatostatin sensitivity (Fig. 2, sham-operated controls). When ovariectomy was performed on day 3, during early vitellogenesis, the CA did not acquire allatostatin sensitivity by day 7 (Fig. 2), and JH synthesis remained low (21.2 + 2.8 pmol/pair/h on day 7). Ovariectomy on day 5 did not prevent acquisition of allatostatin sensitivity by day 7 (Fig. 2), and JH synthetic rates were normal. Ovariectomy on day 4 had an intermediate effect, i.e. the gain in allatostatin sensitivity was significant but not maximal (48 vs 79% in controls,

5

90 80 .Y, g

70

f

60

5 5 s

50

f e

3.

= S

20

40

I

10

6

10 f

10

f

10

a

-II

3dfi’d

4dffd

Age at ovariectomylassay

10

1 5d/7d

FIGURE 2. Effect of ovariectomy on the sensitivity of CA to allatostatin. Ovariectomy was performed on day 3,4 or 5 and sensitivity to allatostatin was assayed for each group on day 7. The stage of basal oocyte development of the ovaries on day 3, 4 and 5 was 0.90 $- 0.02, 1.16 f 0.08 and 1.43 + 0.24 mm respectively. Sensitivity is expressed as % inhibition of JH synthesis (mean + SEM). The number of assays is indicated above each bar (ovariectomy, open bars; sham operated insects, stippled bars). Asterisk indicates a significant difference from the sham-operated controls (P < 0.05, t-test).

978

G. C. UNNITHAN

TABLE

2. Allatostatin

sensitivity

of host CA and implanted

and R. FEYEREISEN

CA in normal

insects and following

Controls Day of assay (host age) Day Day Day Day Day

1

7.6 75.9 77.5 53.6 44.3

8 JO 15 22

+ & k + k

Ovaries Day 1 CA implanted 48 h before assay

Host CA 13.0 (10) 6.3 (8) 2.5 (14) 10.3 (12) 12.6 (7)

ovariectomy

of postvitellogenic

and embryos

removed

on day 8

Day 1 CA implanted 48 h before assay

Host CA

60.1 k 4.2 (17) 35.2 k 10.9 (10) 29.0 f 9.7 (9)

females

64.5 + 6.7 (10) 54.0 f 9.8 (7) 37.0 f 12.2 (7)

42.9 + 11.8 (6) 35.7 k 8.3 (7) 15.0 k 9.4 (8)

Values represent allatostatin sensitivity expressed as % inhibition of JH synthesis by 10 nM allatostatin (means k SEM, n). On each day there was no significant difference between implanted CA and host CA except in day 10 controls, and there was no significant difference between controls and operated insects

in the response of the CA in vivo and in vitro might be related to changes in the sensitivity of the glands to allatostatin. The CA were denervated in day 2 virgin females, and were assayed immediately, or left in situ for up to 2 days before assay (Fig. 3). Figure 4 shows that allatostatin sensitivity was low in day 2 virgin females, with approx. 30% inhibition of JH synthesis. However, between 1 and 2 h after denervation, sensitivity to 10 nM allatostatin decreased. The loss of sensitivity was statistically significant at 4 and 48 h after denervation. This loss was not related to the increase in JH synthetic rate, which was more gradual over the 48 h period and was only apparent after 16 h (Fig. 3). In contrast, CA of day 2 virgins did not lose their low level of allatostatin sensitivity when incubated in vitro for up to 24 h (Table 3). The rate of JH synthesis increased only slightly in 24 h in vitro, whereas it doubled over that time in vivo after denervation. These experiments showed that the endocrine milieu of the virgin host was responsible for the loss of allatostatin sensitivity and that this phenomenon was not an intrinsic property of denervated CA. However, the loss of allatostatin sensitivity following denervation was not irreversible. Indeed, CA from denervated virgin females, after generating a cycle of JH synthesis, did become sensitive as would normal CA (day 9 sensitivity

after denervation on day 2: 84.9 + 4.4%; n = 9). Thus virgins with denervated CA behaved as mated females. We also measured allatostatin sensitivity of the CA of virgins that had been ovariectomized on day 0 and denervated on day 2. At 24 h after denervation, the CA of these insects produced 31.5 f 3.2 pmol JH/pair/h and had a sensitivity of 12.0 f 9.5% (n = 13). These values are not different from the values of denervated (but not ovariectomized) controls (Figs 3 and 4). Thus, the loss of allatostatin sensitivity in denervated virgins was not caused by an effect of the ovary in preparation for vitellogenesis. Because the CA of virgin females show two levels of response to allatostatin (Pratt et al., 1990) a low plateau of 30% inhibition seen at the diagnostic concentration of 10 nM and a maximal inhibition of about 80% at very high concentrations, we also measured the sensitivity of the CA to a 10 PM concentration of allatostatin. Figure 4 shows that there was also a loss of sensitivity to 10 PM allatostatin following denervation, but this loss was more gradual than that seen at 10 nM. Inhibition of JH synthesis by this high dose decreased from 80 to 40% in 2 days (Fig. 4). Sham-operated insects had an allatostatin sensitivity of 69.2 f 6.6% (n = 8) at 2 days after the

60 e .f P g _a .; E E $ 3

50 40 30 20 10 -0 0

1

2

4

8

16

24

48

Hours after denervation

FIGURE 3. Rate of JH synthesis in v’tro of CA from day 2 virgins following denervation. CA from day 2 trirgin females were denervated and left in situ for W8 h. The glands were then retrieved and assayed in vitro for JH synthesis in the absence (stippled bars) or presence (open bars) of 10 nM allatostatin at various times following denervation. JH synthesis + SEM is shown at each time for a number of controls and experimental glands shown above each set of bars.

4

8

12

16

20

24

28

32

36

40

44

48

Hours after denervation

FIGURE 4. Sensitivity to low (10 nM, open squares) and high (10 mM, solid squares) allatostatin concentrations of CA from day 2 virgins following denervation. CA were assayed for allatostatin sensitivity at various times following denervation. Sensitivity is expressed as % inhibition of JH synthesis f SEM for 9-15 assays at each time (for 10 PM). The number of assays for 10 nM is shown in Fig. 3. Means marked with the same letter are not significantly different from each other (SAS analysis of variance and t-tests).

ALLATOSTATIN TABLE

3. Allatostatin

hours in vitro prior to assay

n

0 4 24

13 17 15

sensitivity of CA from maintained in vitro Rate of JH synthesis (pmol/pair/h)

females

Allatostatin sensitivity (% inhibition of JH synthesis)

15.7 * 1.4a 17.9 f 1.2ab 22.2 + 2.2b

Means in each column followed different at P = 0.05.

day 2 virgin

SENSITIVITY

29.9 k 6.5a 23.5 k 4.9a 29.7 k 9.0a

by a different

letter are significantly

operation; this was significantly higher than the denervated insects and not significantly different from control, day 2 virgins.

DISCUSSION

Humoral sensitivity

factor

for

acquisition

of

high

allatostatin

The sensitivity of D. punctata CA was shown to increase markedly between day 5 and day 6 in mated females. This shift from no sensitivity to 10 nM allatostatin to maximal sensitivity on day 6 has been calculated to take approx. 5 h in vivo (Pratt et al., 1990). CA which did not receive the signal of impending decline in JH synthesis (between day 5 and day 6) or were not exposed to the post-vitellogenic endocrine milieu did not acquire maximal sensitivity. This was shown by two types of experiments: (1) the transplantation of the CA into previtellogenic females where they failed to gain maximal sensitivity (Fig. 1) and (2) the transfer of the CA to in vitro incubation where they maintained high JH synthetic rates and gained less than maximal sensitivity (Table 1). Denervation of the CA did not prevent the acquisition of maximal sensitivity (Table 1, Fig. 1). Exposure to a high JH titer was not necessary for subsequent acquisition of allatostatin sensitivity because the CA did not need to undergo a cycle of JH synthesis in order to acquire maximal sensitivity. Indeed, CA from previtellogenic females implanted into postvitellogenic females acquired the high sensitivity to allatostatins characteristic of the host. These experiments imply that the increase in allatostatin sensitivity is not a developmentally ordained process, but results from the interactions of the CA with its endocrine milieu. We postulate the existence of a humoral factor responsible for this transition. Role of the ovary Pratt et al. (1990) showed that the acquisition of high allatostatin sensitivity slightly precedes the loss of follicular patency in the ovary. Ovariectomy early in the gonotrophic cycle prevented both the rise in JH synthesis and the acquisition of allatostatin sensitivity. Early ovariectomy prevents the cycle of JH synthesis (Stay and Tobe, 1978). Later ovariectomy did not prevent (-ov 47 day expt) acquisition of allatostatin sensitivity. Thus we hypothesize that the humoral factor, whatever its

IN DIPLOPTERA

979

origin, is released only in response to a “normal” cycle of JH synthesis, or to a signal from the ovary that this occurred. In view of the complex role of the ovary in regulating the cycle of JH synthesis, other interpretations would be imprudent. It is not clear from our ovariectomy experiments whether the ovary is the source of the humoral factor that causes the day S/day 6 transition in allatostatin sensitivity. If it is, this factor (1) must be long-lived and/or have a slow mode of action and (2) must be released by day 4, because ovariectomy on day 4 cannot prevent the acquisition of sensitivity and day 5 CA maintained in vitro gained a low but significant degree of sensitivity. The ovary and embryos do not influence the endocrine milieu after ovulation. It appeared that the presence or absence of the ovary did not contribute to the sensitivity of the (implanted or host) CA during pregnancy, but there was an ovary-independent decline. Loss of sensitivity

to allatostatin,

possible mechanisms

We also report the results of experiments showing the loss of allatostatin sensitivity following denervation of the CA in virgin females. This loss of sensitivity was not observed in vitro, suggesting that a humoral factor may have influenced the sensitivity of the CA in virgins. The response of the CA of virgin females to denervation was clearly different when tested against low (10 nM) vs high (10 PM) doses of allatostatin. In the first case, rapid (< 2 h) loss of sensitivity was seen following denervation, whereas 2 days were needed to lose 50% of the sensitivity to the high dose. It is probable that this loss of sensitivity to the high dose of allatostatin was not a result of denervation per se, but simply reflected the fact that denervated virgin CA, now behaving as CA of mated females, lost sensitivity to the high allatostatin dose as a normal physiological process (see Fig. 3 of Pratt et al., 1990). The clear difference in response to low and high doses of allatostatin supports the hypothesis that the high dose of allatostatin ASAL (allatostatin 1) interacts with a receptor that is insensitive to low doses of that peptide (Pratt et al., 1990, 1991a) but is sensitive to low doses of the octadecapeptide ASB2 (Pratt et al., 1991b). Changes in sensitivity to allatostatins are peptide specific (Pratt et al., 1990, 1991b) and are not restricted to the adult stage (Stay et al., 1991). Because allatostatins are present and possibly released from peripheral tissues such as the midgut (Reichwald et al., 1994), and may affect physiological processes other than CA regulation (Hertel and Penzlin, 1992; Lange et al., 1993; Duve and Thorpe, 1994) the modulation of CA sensitivity provides the insect with a mechanism to decrease the influence of allatostatins at inopportune times. Note added in proof Whereas denervation of sensitive CA from day 8 pregnant fremales results in a slight increase in JH synthesis (39.7 + 6.9 pmol/pair/h) but no loss in allatostatin sensitivity (91.3 + 5.2%) transplantation of these sensitive CA into day 3 mated (vitellogenic) females

980

G. C. UNNITHAN

leads to increased rate of JH synthesis (88.0 f. 9.7 pmol/pair/h) and loss in sensitivity (24.1 + 13.3%) (experimental protocol as in Fig. 1). REFERENCES Duve H. and Thorpe A. (1994) Distribution and functional significance of Leu-callatostatins in the blowfly Calliphora uomitoria. Cell Tissue Res. 216, 367-379. Engelmann F. (1959) The control of reproduction in Diplopterapunctata (Blattaria). Biol. Bull. 116, 406419. Feyereisen R. (1985) Regulation ofjuvenile hormone titer: synthesis. In Comprehensive Inseci Physiology, Biochemistry and Pharmacology (Eds Kerkut G. A. and Gilbert L. I.) pp. 391429. Pergamon Press, Oxford. Feyereisen R. and Farnsworth D. E. (1987a) Comparison of the inhibitory effects of brain extract, high K+ and forskolin on juvenile hormone synthesis by Diplopiera punctata corpora allata. Insect Biochem. II, 939-942. Feyereisen R. and Farnsworth D. E. (1987b) Inhibition of insect juvenile hormone synthesis by phorbol 12-myristate 13-acetate. FEBS Let?. 222, 345-348. Hertel W. and Penzlin H. (1992) Function and modulation of the antenna1 heart of Periplaneta americana (L). Acia Biol. Hung. 43, 113-125. Lange A. B., Chan K. K. and Stay B. (1993) Effect of allatostatin and proctolin on antenna1 pulsatile organ and hindgut muscle in the cockroach, Diploptera punciata. Archs Insect Biochem. Physiol. 24, 79-92. Meller V. H., Aucoin R. R., Tobe S. S. and Feyereisen R. (1985) Evidence for an inhibitory role of cyclic AMP in the control ofjuvenile hormone biosynthesis by cockroach corpora allata. Mol. Cell. Endocr. 43, 155-163. Pratt G. E., Farnsworth D. E. and Feyereisen R. (1990) Changes in the sensitivity of adult cockroach corpora allata to a brain allatostatin. Mol. Cell. Endocr. 70, 185-195. Pratt G. E., Farnsworth D. E., Siegel N. R., Fok K. F., and Feyereisen R. (1989) Identification of an allatostatin from adult Diploptera punctata. Biochem. Biophys. Res. Commun. 163, 1243-1247. Pratt G. E., Farnsworth D. E., Siegel N. R., Kam F. F. and Feyereisen R. (1991a) Two types of allatostatic peptides from brains of the cockroach Diploptera punctata. In Insect Neuropeptides (Eds Menn .I. J., Kelly T. J. and Masler E. P.). ACS Symp. Series 453, 177-192. Pratt G. E., Farnsworth D. E., Fok K. F., Siegel N. R., McCormack A. L., Shabanowitz J., Hunt D. F. and Feyereisen R. (199lb) Identity of a second type of allatostatin from cockroach brains: an

and R. FEYEREISEN octadecapeptide amide with a tyrosine-rich address sequence. Proc. Natl Acad. Sci. U.S.A. 88, 2412-2416. Rankin S. M. and Stay B. (1984) The changing effect of the ovary on rates of juvenile hormone synthesis in Diploptera punctata. Gen. Comp. Endocr. 54, 382-388. Rankin S. M. and Stay B. (1985) Ovarian inhibition ofjuvenile hormone synthesis in the viviparous cockroach, Diploptera punctata. Gen. Comp. Endocr. 59, 236237. Rankin S. M. and Stay B. (1987) Distribution of allatostatin in the adult cockroach, Diplopterapunctata and effects on corpora allata in vitro. J. Insect Physiol. 33, 551-558. Reichwald K., Unnithan G. C., Davis N. T., Agricola H. and Feyereisen R. (1994) Expression of the allatostatin gene in endocrine cells of the cockroach midgut. Proc. Natl Acad. Sci. U.S.A. 91, 111894111898. 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. Comp. Endocr. 33, 53 l-540. Stay B. and Tobe 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. Comp. Endocr. 34,276286. Stay B., Joshi S. and Woodhead A. P. (1991) Sensitivity to allatostatins of corpora allata from larval and adult female Diploptera punctata. J. Insect Physiol. 37, 63-70. Stay B., Tobe S. S. and Bendena W. B. (1994) Allatostatins: Identification, primary structures, functions and distribution. Adv. Insect Physiol. 25, 267-337. Stay B., Friedel T., Tobe S. S. and Mundall E. C. (1980) Feedback control ofjuvenile hormone synthesis in cockroaches: possible role for ecdysterone. Science 207, 898-900. Tobe S. S., Stay B., Friedel T., Feyereisen R. and Paulson C. (1981) The role of the brain in regulation of the corpora allata in female Diplopterapunctata. In Juvenile Hormone Biochemistry (Eds Pratt G. E. and Brooks G. T.), pp. 161-174. Elsevier/North-Holland, Amsterdam. Woodhead A. P., Stay B., Siedel S. L., Khan M. A. and Tobe S. S. (1989) Primary structure of four allatostatins: neuropeptide inhibitors of juvenile hormone synthesis. Proc. Natl Acad. Sci. U.S.A. 86, 5997-6001.

Acknowledgements-This work was supported by NIH grant DK 34549. We thank Drs Jan Veenstra and Tara Sutherland for critical reading of the manuscript.