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Intrinsic differences in juvenile hormone synthetic ability between corpora allata of males and females of the cockroach Diploptera punctata
GENERAL
AND
Intrinsic
COMPARATIVE
ENDOCRINOLOGY
533-540 (1982)
Differences in Juvenile Hormone Synthetic Ability between Corpora Allata of Males and Females of the Cockroach Diplop tera puncta ta CATHARINE
Department
46,
of Zoology,
University
M. SZIBBO’ of Toronto,
AND STEPHEN
25 Harhord
Street,
S. TOBE
Toronto
MSS
lA1,
Ontario,
Canudu
Accepted July 21, 1981 To study the basis for differences in juvenile hormone (JH) synthesis exhibited by corpora allata (CA) of males and females of the cockroach Diploptera punctata, the activity of male and female CA is compared after implantation into allatectomized adult females. Implanted male CA are able to support complete oocyte development, but at a reduced rate. Measurement ofjuvenile hormone biosynthesis by implanted CA with an in vitro radiochemical assay shows that implanted male CA synthesize JH at much lower rates than those of implanted female CA. The fact that male CA are smaller than female CA accounts in part for their lesser synthetic ability.
The pattern of synthesis of Cl6 juvenile hormone (C,,JH) by corpora allata (CA) differs in adult males and females of the viviparous cockroach Diplopteru punctata Eschscholtz. In adult females, the CA undergo a cycle of JH synthesis that is closely correlated to the gonotrophic cycle; relatively low rates (5 - 10 pmol JH hr-‘) are observed both immediately after ecdysis and after oviposition, whereas JH is synthesized at high rates (X0 pmol hr-‘) during the period of vitellogenic oocyte growth (Tobe and Stay, 1977; Tobe, 1980). In contrast, the rate of JH synthesis by CA of adult males is more constant, remaining at less than 8 pmol hr’ during the first 41 days of adult life (Tobe et al., 1979). Sexual dimorphism in CA activity has been suggested for other insects on the basis of differences in CA size (Johansson, 19.58; Girardie and Granier, 1973; Mendes, 1948), ultrastructure (Fain-Maurel and Cassier, 1975; Fukuda et al., 1966), and implantation bioassays (Pener, 1967; BrousseGaury and Cassier, 1975). It is not clear, however, to what extent intrinsic differences in CA size. cell number, and enzymatic
activity between males and females dictate the JH synthetic dimorphism in D. punctata, nor if male CA synthesize JH at low rates because of inhibitory signals arriving via nervous connectives or through the haemolymph. It is the object of this study to determine if male and female CA differ in their capacities to synthesize JH when sexual differences in the nervous and humoral environment are eliminated. Accordingly. we have implanted male CA into either allatectomized male or female hosts and compared their activity to that of female CA. Our results show that the CA of males are intrinsically less active than the CA of females. Although this is partly related to the fact that male CA have less hormoneproducing tissue, they also appear to lack the capability to respond to signals which cause single female CA to synthesize JH at high rates. MATERIALS
AND METHODS
Diploptera punctatu were maintained at 21” with a photoperiod of 12 hr L, 12 hr D. Under these conditions, mating occurs on Day 0, immediately after imaginal emergence, whereas oviposition occurs at Day 8 or 9.
’ To whom all correspondence should be addressed. 533
0016~6480/82/040533-08$01.00/O Copyright All rights
0 1982 by Academic Press. Inc. of reproduction in any form reserved.
534
SZIBBO
AND
Newly emerged adults were isolated from the colony O-7 hr after ecdysis. All operations were performed within 9 hr of ecdysis of both host and donor animals; animals were maintained in a 28” incubator without a light/dark cycle in total darkness thereafter. The effects of photoperiod on CA activity in D. punctara are unknown. Mated females were used in all experiments; all Day-O males were unmated. Age of animals referred to in text is the age from time of ecdysis. Allatectomy and implantation operations were performed as described by Stay and Tobe (1977, 1978). After anesthetizing cockroaches 5-10 min in vials on crushed ice, a triangle was removed from the pronotum, an incision made in the neck membrane, and two dorsal trachea removed. Under sterile citratefortified bathing medium, CA were removed one at a time by cutting the corpus cardiacum (CC) just anterior to the CA. Care was taken to disturb the oesophageal nerve (Willey, 1961) as little as possible. After allatectomy, the cut ends of the CC were examined to ensure that no CA tissue remained. To ensure easier retrieval for measurement of JH synthesis, glands to be implanted were removed from the donor as a pair then transferred directly to an allatectomized host. This practice prevented loss of one member of a pair of implants. After allatectomy, or allatectomy plus implantation, the flap of neck cuticle was replaced and a few crystals of streptomycin sulfate were applied to the wound, which was sealed by replacing the piece of pronotum. Mortality was less than 10%. The synthesis of JH by isolated CA was determined in vitro by a radiochemical assay based on the incorporation of the methyl moiety of [Me-‘4C]methionine (Amersham-Searle. final specific activity 1.3-1.4 GBq/mmol) (35-39 mCi/mmol) into CIJH (JH III) after a 3-hr incubation period (Pratt and Tobe, 1974; Tobe and Pratt, 1974). C, JH is the only juvenile hormone synthesized by CA of adult females of D. punctata in vitro (Tobe and Stay, 1977). The length of basal oocytes was measured at the time of measurement of JH synthesis. At emergence, basal oocytes are about 0.8 mm in length, whereas full-grown mature oocytes are 1.6- 1.7 mm. For histological studies on male CA, CA were fixed in Bouin’s fixative (Humason, 1979), embedded in paraffin (mp 56-58”), serially sectioned at 5 ym, and stained in Mallory-Heidenhain (Cason, 1950). CA volume was estimated from serial sections as described by Scharrer and von Harnack (1958) and Szibbo and Tobe (198la). The number of nuclei per CA was determined as outlined in Szibbo and Tobe (1981a) by tallying all possible nuclear profiles in alternate 5-pm CA sections. Nuclear size is 5.5-6.5 pm, thus the same nucleus will not be counted more than once (Szibbo and Tobe, 1981a).
TOBE
RESULTS 1. Volume and Cell Number of Male CA
The volume and cell number of CA from unoperated males were determined at 0, 3, 5, and 9 days after adult ecdysis and compared to volume and cell number for unoperated females from Szibbo and Tobe (1981a) (Fig. 1). At Days 0, 3, and 5 the volume of male CA was less than one-half that of female CA and remained relatively constant, in contrast to the almost threefold increase occurring in female CA at Day 5. Estimated cell number per CA was also much less for male CA and did not change significantly, although a few mitotic figures were observed in CA of Day-3 animals, indicating that some cell proliferation may occur at this time.
01 0
2
4
6
6
Age idor -5 x
lo-
d z P '-a 2
a-
B
-
6.
Z
% 4 ; 0
* . ... .. ... ... ... .&."'
E 2
0
_.....l - . ... ... .. ...._.._._.*
2 2
4
6
0
Age (dwl FIG. 1. Changes in (A) mean CA volume and (8) mean cell number as a function of adult age for male (u) and for female (0) CA. Data for female CA are from Szibbo and Tobe (1981a). The vertical bars represent SE (n 2 5), unless smaller than the dimensions of the point.
CORPORA
ALLATA
As determined by microscopic observations from representative CA sections, nuclear size was similar in male and female CA (approx 6 pm; Szibbo and Tobe, 19gla). No structural differences between male and female CA other than size were observed. 2. Time to Oviposition for Allatectomized Female Hosts Implanted with Male CA To compare the relative abilities of male and female CA to support oocyte development, Day-O male or female CA were implanted into mated, allatectomized Day-O adult female hosts. Ten allatectomized control females did not release spermatophores and did not show any oocyte growth by Day 20. In Fig. 2 it is shown that male CA were capable of supporting complete oocyte development in allatectomized females, albeit at a reduced rate. Hosts receiving male glands oviposited much later and showed more variability in the time to oviposition than those receiving female CA. One hundred percent of animals implanted with female CA oviposited by 10 days, whereas only 30% of animals implanted with male CA oviposited by this time.
OF
MALE
535
Dipioptvru
3. Oocyte Growth in Allatectomized Female Hosts Implanted with Male CA In Fig. 3 it is shown that allatectomized hosts receiving a pair of male CA displayed lower rates of oocyte growth than hosts with a pair of female CA. Hosts receiving a single male CA displayed very little growth. At any age, some animals receiving male CA showed little or no oocyte development but almost all hosts with female CA matured oocytes. 4. JH Synthesis of Male and Female CA Compared in Female Hosts; JH Synthesis by Male CA in Male Hosts The rates of JH synthesis by male and female CA implanted in hosts of Fig. 3 are shown in Fig. 4. Male CA synthesized JH at low, constant rates, whereas implanted female CA underwent a cycle of JH synthesis reaching peak rates at Day 5. There was no difference in the synthetic capability of male CA implanted in either allatectomized males or females (Fig. 4). Single female CA are known to increase their rates of JH synthesis after unilateral allatectomy so that oocyte development 1.8
1.6 F E 5
1.4
F f 1.2 2 >. P 0 1.0
0.8
0.6 4
5
6 Age
Age ldvl
Age Iday4
FIG. 2. Histograms showing percentage of total animals ovipositing as a function of age for allatectomized adult mated females implanted at Day 0 with either a pair of (A) Day-O female CA or (B) Day-O male CA.
(days
7
6
I
FIG. 3. Mean oocyte length as a function of age for Day-O allatectomized adult females implanted with a pair of Day-O adult female CA (O), a pair of Day-O adult male CA (0). or a single Day-O male CA (A). Sample size is given beside the vertical bars, which represent SE.
536
SZIBBO
AND
70
.-“7 ; f E 6 I, P v
30-
20.
10.
0..
,
4
7
5 Age
6
7
(days1
FIG. 4. Mean rate of JH synthesis by a pair of male CA implanted into Day-O male (A) or female (0) hosts as a function of adult age. As a control, a pair of female CA were implanted into allatectomized females (0). Sample size is given beside the vertical bars which represent SE.
takes place at a normal rate (Stay and Tobe, 1978; Tobe and Stay, 1980). To evaluate the response of male CA to a reduction in the number of implanted CA, JH biosynthesis by a single male CA in an allatectomized female was determined. A single male CA implanted into an allatectomized male synthesized JH at mean rates of 5.9 ? 0.7(SE) (n = 29) pmol hr-l from Day 4 to Day 8. A single male CA implanted into an allatectomized female synthesized JH at similar rates, 4.4 t 0.7(n = 30) pmol hr-l. These rates are significantly less than mean rates for pairs of male CA implanted into allatectomized females or males (P < 0.001, Student’s t test) and demonstrate that there is no compensatory increase in rate of JH synthesis by single male implants for a reduction in the number of CA. DISCUSSION I. Differences in CA Morphometrics Activity in Unoperated Insects
and
The sexual dimorphism in JH synthesis exhibited in unoperated insects (Tobe and Stay, 1977; Tobe et al., 1979) and after im-
TOBE
plantation (Fig. 4) and the lower rates of oocyte development supported by male CA (Fig. 2) may be attributed partly to differences in CA size between the sexes. The volume and cell number of male CA is much less than female CA (Fig. 1). Male D. punctata weigh less, have smaller head capsules (Szibbo and Tobe, unpublished results) and haemolymph volume (Mundall and Tobe, unpublished results), and pass through one less stadium than female D. punctata (Willis et al., 1958). Therefore volumetric differences in the CA are related to differences in total body size. In Leucophaea maderae, another cockroach, adult males are also smaller than adult females, and similarly, the CA of males are smaller in volume and cell number (Scharrer and von Harnack, 1958). In those insects where body size and haemolymph volume of the male is smaller, lower activity of their CA may not lead to a difference in the haemolymph titer of JH (Mendes, 1948). Measurement of JH titer by physicochemical methods would be required to confirm this for D. punctata. The relatively constant and low values for CA volume and cell number of CA in unoperated males correlate well with their low, constant rates of JH synthesis (Tobe et al., 1979). In unoperated females, increases and decreases in volume and cell number parallel, but are exceeded by larger changes in JH synthesis (Szibbo and Tobe, 1981a). Although our light microscope observations did not reveal any structural differences between male and female CA, it is possible that the differences in activity at times of peak synthesis are reflected in quantities of organelles believed involved in JH synthesis i.e., mitochondria, endoplasmic reticulum, and ribosomes (Tobe and Saleuddin, 1977). Such differences may be detectable at the ultrastructural level. 2. Oocyte Development Supported by Implanted CA
The CA of male D. punctuta have the ability to support complete oocyte matura-
CORPORA
ALLATA
tion and oviposition in allatectomized females, but at a reduced rate (Figs. 2 and 3) reflecting their lower rates of JH synthesis (Fig. 4). Although this is the first study relating directly the JH synthesis of male CA to their ability to support oocyte development in allatectomized females, it is well known that male CA can support ovarian maturation (e.g., Wigglesworth, 1936; Pener, 1967; Joly, 1945; de Wilde, 1964). In Blahera fusca, the rate of oocyte maturation supported by male CA is also less than that for female CA (Brousse-Gaury and Cassier, 1975). Our implantation studies of male CA suggest that although the high rate and well-defined cycle of JH synthesis observed in CA of mated females are probably required for rapid oocyte growth (Tobe and Stay, 1977; Tobe, 1980), they are not strictly necessary for oocyte maturation in D. punctata. The CA of virgin adult female D. punctata are known to synthesize JH at rates lower than those of mated females (Tobe and Stay, 1977; Stay and Tobe, 1977) and yet complete oocyte maturation can occur in some older animals (Engelmann, 1959; Roth and Stay, 1961). However, both these examples still support the concept that time to oviposition reflects the cumulative titer of JH and indirectly, the biosynthesis of JH (Tobe and Stay, 1977, 1980: Stay and Tobe, 1977, 1978). There may be a threshold rate of JH synthesis below which no oocyte development occurs. Many females implanted with male CA fail to show any oocyte development, whereas almost all with female CA implants mature oocytes. 3. Comparison of JH Synthesis by Implanted Male and Femule CA
There is no doubt that the smaller volume and cell number of CA of unoperated male D. punctatu account in part for their lower activity. At Day 5, however, the rate of JH synthesis by female CA is over eight times that of male CA (Tobe and Stay, 1977: Szibbo and Tobe, 1981a; Tobe et al., 1979), whereas volumetric and cellular differences
OF
MALE
Lliplopterci
537
are only three- to fourfold (Fig. 1). Therefore, there may be additional reasons for the low rates of JH synthesis by male CA in situ. For example, signals transmitted via the nerves may play a role in maintaining low levels of JH synthesis in unoperated insects. The rates of JH synthesis for male CA implants in allatectomized males (Fig. 4) are double those reported by Tobe et al. (1979) for unoperated male CA. After denervation of CA, there are detectable levels of vitellogenin in the haemolymph of some male D. punctatu (Mundall, Szibbo and Tobe, unpublished). Vitellogenin is not found in normal males (Mundall et al., 1979). In mated females, denervation does not cause significantly higher peak rates of JH synthesis (Stay and Tobe, 1978). Possibly, the CA of male D. punctata are normally inhibited by their nervous connectives. Measurement of JH synthesis by male CA after sham operation would be required to confirm this. Even if severing the nerves to the CA does stimulate JH synthesis, the possibility that inhibitory factors circulating in the hemolymph prevents rates of JH synthesis from rising even further still remains. The rates of JH synthesis by male CA, however, are identical when implanted in either allatectomized males or females (Fig. 4). Similarly, female CA can achieve high rates of JH synthesis when implanted into newly emerged males. Therefore, the humoral environment of the male does not contribute to the low output of male CA. When both male and female CA are freed of nervous connectives and presented with the same humoral environment, we find that rates of JH synthesis by male implants are only one-fourth of peak rates of JH synthesis by implanted female CA at Day 5 (Fig. 4). Female CA implants behave like CA in control females (Fig. 4; Tobe and Stay, 1977) undergoing a cycle in JH synthesis and reaching maximal levels of synthesis of over 50 pmol hr-l (Stay and Tobe, 1978). Johansson (1958) also found that
538
SZIBBO
AND
transplantation of CA of male Oncopeltus fusciatus into females failed to cause an increase in their activity as judged by CA volume. 4. JH Synthetic
Potential
of Male CA
We do not know if implanted CA undergo cellular and volumetric changes similar to those of unoperated insects, but if they do, then a three- to fourfold difference in CA volume and cell number between male and female CA at times of peak synthesis (Fig. 1) could be the main explanation for the difference in synthetic ability of implants. It is remarkable, however, that the activity of the CA of adult females is not limited by the available CA volume. Following unilateral allatectomy, a single CA of female D. punctata can synthesize JH at rates as high as those of a pair of CA without undergoing compensatory hypertrophy or cell proliferation (Stay and Tobe, 1978; Szibbo and Tobe, 1981a,b). This response, which presumably occurs in response to a lowered JH titer caused by removal of one CA (Stay and Tobe, 1978) does not require intact nervous connections to the single CA (Tobe and Stay, 1980). It follows that signals for this compensatory increase in JH synthesis should operate also on implanted CA of males and high rates of JH synthesis should be expected also from the small male CA, if the total volume of hormone-producing tissue at the time of implantation was the sole determinant of JH synthetic potential. That is, similar rates of JH synthesis should be expected from a pair of male CA and a single female CA which have at Day 0 total volume and cell numbers that differ only slightly (Fig. 1). Single female CA, however, whether innervated or denervated attain mean rates of JH synthesis that are over 50 pmol hr-l (Stay and Tobe, 1978; Tobe and Stay, 1980; Szibbo and Tobe, 1981b), but pairs of male CA attain values less than one-third of this (Fig. 4). Comparison of rates of JH synthesis by
TOBE
CA of different sizes is best done by comparison of average rates per cell. Figure 5 shows changes in the rate of JH synthesis of implanted male and female CA from Fig. 4 in relation to their cell numbers at the time of implantation (Day 0) (Fig. 1) using JH synthesis data for single female CA from Tobe and Stay (1980). Because a single male CA synthesizes JH at rates that are one-half that of a pair of male CA (Results, Section 4), but a single female CA compensates for the loss of the other CA, synthesizing JH at rates equal to that of a pair of female CA, the rates of JH synthesis per Day-O cell for male CA are not only low and constant but also identical for single and paired male CA. In contrast, single female CA undergo large increases in the rate of JH synthesis per Day-O cell. The differences in Fig. 5 between singled and paired female CA cannot be attributed to increase in cell
f *IA/+.. B
....._....... n
1J 4
5
6 Age
7
,
8
Idvl
5. Mean rate of JH synthesis per Day-O cell as a function of time after implantation into allatectomized females for single male CA (A), pairs of male CA (A), single female CA (O), and pairs of female CA (0). Values are calculated by dividing rates of JH synthesis from Fig. 4 by total cell number at Day 0 from Fig. 1. Rates of JH synthesis for single female CA are from Tobe and Stay (1980). FIG.
CORPORA ALLATA
number in the single female CA (Szibbo and Tobe, 1981b). Figure 5, therefore, indicates that male CA implants have a much lower potential than female CA for synthesizing JH after implantation. It is likely that the CA of male D. punctata have a lower JH synthesizing potential than CA of females because of their smaller size. They also appear to be refractory to signals causing volumetric and synthetic increases in single female CA, however, and unlike female CA, they are unable to modulate JH synthesis in response to the number of CA present. Perhaps the activity of male CA is below a threshold necessary to elicit feedback stimulation after implantation into females. Male CA may be intrinsically different from female CA, lacking the appropriate receptors for stimulatory signals, having lower rates of substrate uptake, and/or an unmodifiably lower concentration of rate-limiting enzymes in the JH synthetic pathway. Such sexual differences may be a reflection of the different developmental histories of each. An understanding of the sexual dimornhism in JH synthetic capabilities in D. punctata may elucidate the enzymatic and cellular mechanisms permitting drastic modifications in rates of JH synthesis in females. I
ACKNOWLEDGMENTS We would like to acknowledge Peter Turner’s assistance on initial histological investigations on male CA. We are also grateful to Dr. E. Mundall and Dr. R. Feyereisen for critically reading this manuscript and to Professor B. Stay for her helpful comments. This work was supported by an operating grant from the National Sciences and Engineering Research Council of Canada. C.M.S. acknowledges receipt of an NSERC postgraduate scholarship.
REFERENCES Brousse-Gamy, P., and Cassier, P. (1975). Contrihution a l’etude de la dynamique des corpora arlara chez Blabera fusca. InfIuence du jeune, du groupment et du regime photoperiodique. Bull. Biol. Fr. Belg.
109, 253-277.
Cason, J. E. (1950). A rapid one-step Mallory-Heidenhain stain for connective tissue. Stain Technol. 25, 225-226.
OF MALE
539
Diplopteru
de Wilde, J. (1964). Reproduction-Endocrine control. In “The Physiology of Insecta” (M. Rockstein, ed.), Vol. 1, pp. 59-90. Academic Press, New York. Engelmann, F. (1959). The control of reproduction in Diploptera punctata (Blattaria). Biol. Bull. Mar. Biol. Lab. Woods Hole 116, 406-419. Fain-Maurel, M. A., and Cassier, P. (1969). Pleomorphisme mitochondrial dans les corpora allata de Locusta migratoria migratoriodes (R. & F.) au tours de la vie imaginale. Z. Zellforsch. 102, 543-553. Fukuda, S., Eguchi, G., and Takeuchi, S. (1966). Histological and electron microscopical studies on sexual differences in structure of the corpora allata of the moth of the silkworm Bombyx mori. Embryologia 9, 123-158. Girardie, A., and Granier, S. (1973). Systeme endocrine et physiologie de la diapause imaginale chez le criquet egyptien, Anacridium aegyptium. J. Insect Physioi. 19, 2341-2358. Humason, G. L. (1979). “Animal Tissue Techniques,” 4th ed. Freeman. San Francisco. Johansson, A. S. (1958). Relation of nutrition to endocrine-reproductive functions in the milkweed bug Oncopeltus fasciatus (Dallas) (Heteroptera: Lygaeidae). Nytt Mag. 2001. 7, 1-132. Joly, P. (1945). La fonction ovarienne et son controle humoral chez les Dytiscides. Arch. Zoo/. Exp. Gen.
Notes
Rev.
84, 49-
164.
Mendes, M. (1948). Histology of the corpora allata of Melanoplus dijferentialis (Orthoptera: Saltatoria). jaltatoria). Biol. Bull. 194-207.
Mar.
Biol.
Lab.
Woods
Hole
94,
Mundall, E. C., Tobe, S. S., and Stay, B. (1979). Induction of vitellogenin and growth of implanted oocytes in male cockroaches. Nature (London) 282, 97-98.
Pener, M. P. (1967). Comparative studies on the reciprocal interchange of corpora allata between males and females of adult Schistocerca gregaria (ForSk&l) (Orthoptera: Acrididae). Proc. R. Entomol. Sot. Lond. (A) 42, 139-148. Pratt, G. E., and Tobe, S. S. (1974). Juvenile hormone radiobiosynthesized by corpora allata of adult female locusts in vitro. Life Sci. 14, 575-586. Roth, L. M., and Stay, B. (1961). Oocyte development in Diplopteru punctata (Eschscholtz) (Blattaria). J. Insect Physiol. 7, 186-202. Scharrer, B., and von Harnack, M. (1958). Histophysiological studies on the corpus allatum of Leucophaea maderue. I. Normal life cycle in male and female adults. Biol. Bull. Mur. Biof. Lab. Woods Hole 115, 508-520. Stay, B., and Tobe, S. S. (1977). Control of juvenile hormone biosynthesis during the reproductive cycle of a viviparous cockroach. I. Activation and inhibition. Cert. Comp. Endocrinol. 33, 531-540.
540
SZIBBO
Stay, B., and hormone cycle of unilateral numerary Comp.
AND
Tobe, S. S. (1978). Control of juvenile biosynthesis during the reproductive a viviparous cockroach. II. Effects of allatectomy, implantation of supercorpora allata and ovariectomy. Gen.
Endocrinol.
34, 276-286.
Stay, B., Friedel, T., Tobe, S. S., and Mundall, E. C. (1980). Feedback control of juvenile hormone synthesis in cockroaches: Possible role for ecdysterone. Science 207, 898-900. Szibbo, C. M., and Tobe, S. S. (1981a). Cellular and volumetric changes in relation to the activity cycle in corpora allata of Diploptera punctata. J. Insect Physiol. 27, 609-613. Szibbo, C. M., and Tobe, S. S. (1981b) The mechanism of compensation in juvenile hormone synthesis following unilateral allatectomy in Diploptera punctata.
J. Insect
Physiol.
27, 655-665.
Tobe, S. S. (1980). Regulation of the corpora allata in adult female insects. In “Insect Biology in the Future” (M. Locke and D. S. Smith, eds.), pp. 345-368. 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. Biochem. J. 144, 107- 113. Tobe, S. S., and Saleuddin, A. S. (1977) Ul-
TOBE
trastructural localization of juvenile hormone biosynthesis by insect corpora allata. Cell Tissue Res.
183, 25-32.
Tobe, S. S., and Stay, B. (1977). Corpus allatum activity in vitro during the reproductive cycle of the viviparous cockroach Diploptera punctata. Gen. Comp.
Endocrinol.
31, 138-147.
S. S., and Stay, B. (1980). Control of juvenile hormone biosynthesis during the reproductive cycle of a viviparous cockroach. III. Effects of denervation and age on compensation with unilateral allatectomy and supernumerary corpora allata. Gen. Comp. Endocrinol. 40, 89-98. Tobe, S. S., Musters, A., and Stay, B. (1979). Corpus allatum function during sexual maturation of male Tobe,
Diplaptera
punctata.
Physiol.
Entomol.
4,79-86.
Wigglesworth, V. B. (1936). The function of the corpus allatum in the growth and reproduction of Rhodnius pro&us (Hemiptera). Quart. J. Microstop. sci. 79, 91-121. Willey, R. B. (1961). The morphology of the stomatodeal nervous system in Periplanetu americana (L.) and other Blattaria, J. Morphol. 108, 219-261. Willis, E. R., Riser, C. R., and Roth, L. M. (1958). Observations on reproduction and development in cockroaches. Ann. Entomol. Sot. Amer. 51, 53 -69.
AND
Intrinsic
COMPARATIVE
ENDOCRINOLOGY
533-540 (1982)
Differences in Juvenile Hormone Synthetic Ability between Corpora Allata of Males and Females of the Cockroach Diplop tera puncta ta CATHARINE
Department
46,
of Zoology,
University
M. SZIBBO’ of Toronto,
AND STEPHEN
25 Harhord
Street,
S. TOBE
Toronto
MSS
lA1,
Ontario,
Canudu
Accepted July 21, 1981 To study the basis for differences in juvenile hormone (JH) synthesis exhibited by corpora allata (CA) of males and females of the cockroach Diploptera punctata, the activity of male and female CA is compared after implantation into allatectomized adult females. Implanted male CA are able to support complete oocyte development, but at a reduced rate. Measurement ofjuvenile hormone biosynthesis by implanted CA with an in vitro radiochemical assay shows that implanted male CA synthesize JH at much lower rates than those of implanted female CA. The fact that male CA are smaller than female CA accounts in part for their lesser synthetic ability.
The pattern of synthesis of Cl6 juvenile hormone (C,,JH) by corpora allata (CA) differs in adult males and females of the viviparous cockroach Diplopteru punctata Eschscholtz. In adult females, the CA undergo a cycle of JH synthesis that is closely correlated to the gonotrophic cycle; relatively low rates (5 - 10 pmol JH hr-‘) are observed both immediately after ecdysis and after oviposition, whereas JH is synthesized at high rates (X0 pmol hr-‘) during the period of vitellogenic oocyte growth (Tobe and Stay, 1977; Tobe, 1980). In contrast, the rate of JH synthesis by CA of adult males is more constant, remaining at less than 8 pmol hr’ during the first 41 days of adult life (Tobe et al., 1979). Sexual dimorphism in CA activity has been suggested for other insects on the basis of differences in CA size (Johansson, 19.58; Girardie and Granier, 1973; Mendes, 1948), ultrastructure (Fain-Maurel and Cassier, 1975; Fukuda et al., 1966), and implantation bioassays (Pener, 1967; BrousseGaury and Cassier, 1975). It is not clear, however, to what extent intrinsic differences in CA size. cell number, and enzymatic
activity between males and females dictate the JH synthetic dimorphism in D. punctata, nor if male CA synthesize JH at low rates because of inhibitory signals arriving via nervous connectives or through the haemolymph. It is the object of this study to determine if male and female CA differ in their capacities to synthesize JH when sexual differences in the nervous and humoral environment are eliminated. Accordingly. we have implanted male CA into either allatectomized male or female hosts and compared their activity to that of female CA. Our results show that the CA of males are intrinsically less active than the CA of females. Although this is partly related to the fact that male CA have less hormoneproducing tissue, they also appear to lack the capability to respond to signals which cause single female CA to synthesize JH at high rates. MATERIALS
AND METHODS
Diploptera punctatu were maintained at 21” with a photoperiod of 12 hr L, 12 hr D. Under these conditions, mating occurs on Day 0, immediately after imaginal emergence, whereas oviposition occurs at Day 8 or 9.
’ To whom all correspondence should be addressed. 533
0016~6480/82/040533-08$01.00/O Copyright All rights
0 1982 by Academic Press. Inc. of reproduction in any form reserved.
534
SZIBBO
AND
Newly emerged adults were isolated from the colony O-7 hr after ecdysis. All operations were performed within 9 hr of ecdysis of both host and donor animals; animals were maintained in a 28” incubator without a light/dark cycle in total darkness thereafter. The effects of photoperiod on CA activity in D. punctara are unknown. Mated females were used in all experiments; all Day-O males were unmated. Age of animals referred to in text is the age from time of ecdysis. Allatectomy and implantation operations were performed as described by Stay and Tobe (1977, 1978). After anesthetizing cockroaches 5-10 min in vials on crushed ice, a triangle was removed from the pronotum, an incision made in the neck membrane, and two dorsal trachea removed. Under sterile citratefortified bathing medium, CA were removed one at a time by cutting the corpus cardiacum (CC) just anterior to the CA. Care was taken to disturb the oesophageal nerve (Willey, 1961) as little as possible. After allatectomy, the cut ends of the CC were examined to ensure that no CA tissue remained. To ensure easier retrieval for measurement of JH synthesis, glands to be implanted were removed from the donor as a pair then transferred directly to an allatectomized host. This practice prevented loss of one member of a pair of implants. After allatectomy, or allatectomy plus implantation, the flap of neck cuticle was replaced and a few crystals of streptomycin sulfate were applied to the wound, which was sealed by replacing the piece of pronotum. Mortality was less than 10%. The synthesis of JH by isolated CA was determined in vitro by a radiochemical assay based on the incorporation of the methyl moiety of [Me-‘4C]methionine (Amersham-Searle. final specific activity 1.3-1.4 GBq/mmol) (35-39 mCi/mmol) into CIJH (JH III) after a 3-hr incubation period (Pratt and Tobe, 1974; Tobe and Pratt, 1974). C, JH is the only juvenile hormone synthesized by CA of adult females of D. punctata in vitro (Tobe and Stay, 1977). The length of basal oocytes was measured at the time of measurement of JH synthesis. At emergence, basal oocytes are about 0.8 mm in length, whereas full-grown mature oocytes are 1.6- 1.7 mm. For histological studies on male CA, CA were fixed in Bouin’s fixative (Humason, 1979), embedded in paraffin (mp 56-58”), serially sectioned at 5 ym, and stained in Mallory-Heidenhain (Cason, 1950). CA volume was estimated from serial sections as described by Scharrer and von Harnack (1958) and Szibbo and Tobe (198la). The number of nuclei per CA was determined as outlined in Szibbo and Tobe (1981a) by tallying all possible nuclear profiles in alternate 5-pm CA sections. Nuclear size is 5.5-6.5 pm, thus the same nucleus will not be counted more than once (Szibbo and Tobe, 1981a).
TOBE
RESULTS 1. Volume and Cell Number of Male CA
The volume and cell number of CA from unoperated males were determined at 0, 3, 5, and 9 days after adult ecdysis and compared to volume and cell number for unoperated females from Szibbo and Tobe (1981a) (Fig. 1). At Days 0, 3, and 5 the volume of male CA was less than one-half that of female CA and remained relatively constant, in contrast to the almost threefold increase occurring in female CA at Day 5. Estimated cell number per CA was also much less for male CA and did not change significantly, although a few mitotic figures were observed in CA of Day-3 animals, indicating that some cell proliferation may occur at this time.
01 0
2
4
6
6
Age idor -5 x
lo-
d z P '-a 2
a-
B
-
6.
Z
% 4 ; 0
* . ... .. ... ... ... .&."'
E 2
0
_.....l - . ... ... .. ...._.._._.*
2 2
4
6
0
Age (dwl FIG. 1. Changes in (A) mean CA volume and (8) mean cell number as a function of adult age for male (u) and for female (0) CA. Data for female CA are from Szibbo and Tobe (1981a). The vertical bars represent SE (n 2 5), unless smaller than the dimensions of the point.
CORPORA
ALLATA
As determined by microscopic observations from representative CA sections, nuclear size was similar in male and female CA (approx 6 pm; Szibbo and Tobe, 19gla). No structural differences between male and female CA other than size were observed. 2. Time to Oviposition for Allatectomized Female Hosts Implanted with Male CA To compare the relative abilities of male and female CA to support oocyte development, Day-O male or female CA were implanted into mated, allatectomized Day-O adult female hosts. Ten allatectomized control females did not release spermatophores and did not show any oocyte growth by Day 20. In Fig. 2 it is shown that male CA were capable of supporting complete oocyte development in allatectomized females, albeit at a reduced rate. Hosts receiving male glands oviposited much later and showed more variability in the time to oviposition than those receiving female CA. One hundred percent of animals implanted with female CA oviposited by 10 days, whereas only 30% of animals implanted with male CA oviposited by this time.
OF
MALE
535
Dipioptvru
3. Oocyte Growth in Allatectomized Female Hosts Implanted with Male CA In Fig. 3 it is shown that allatectomized hosts receiving a pair of male CA displayed lower rates of oocyte growth than hosts with a pair of female CA. Hosts receiving a single male CA displayed very little growth. At any age, some animals receiving male CA showed little or no oocyte development but almost all hosts with female CA matured oocytes. 4. JH Synthesis of Male and Female CA Compared in Female Hosts; JH Synthesis by Male CA in Male Hosts The rates of JH synthesis by male and female CA implanted in hosts of Fig. 3 are shown in Fig. 4. Male CA synthesized JH at low, constant rates, whereas implanted female CA underwent a cycle of JH synthesis reaching peak rates at Day 5. There was no difference in the synthetic capability of male CA implanted in either allatectomized males or females (Fig. 4). Single female CA are known to increase their rates of JH synthesis after unilateral allatectomy so that oocyte development 1.8
1.6 F E 5
1.4
F f 1.2 2 >. P 0 1.0
0.8
0.6 4
5
6 Age
Age ldvl
Age Iday4
FIG. 2. Histograms showing percentage of total animals ovipositing as a function of age for allatectomized adult mated females implanted at Day 0 with either a pair of (A) Day-O female CA or (B) Day-O male CA.
(days
7
6
I
FIG. 3. Mean oocyte length as a function of age for Day-O allatectomized adult females implanted with a pair of Day-O adult female CA (O), a pair of Day-O adult male CA (0). or a single Day-O male CA (A). Sample size is given beside the vertical bars, which represent SE.
536
SZIBBO
AND
70
.-“7 ; f E 6 I, P v
30-
20.
10.
0..
,
4
7
5 Age
6
7
(days1
FIG. 4. Mean rate of JH synthesis by a pair of male CA implanted into Day-O male (A) or female (0) hosts as a function of adult age. As a control, a pair of female CA were implanted into allatectomized females (0). Sample size is given beside the vertical bars which represent SE.
takes place at a normal rate (Stay and Tobe, 1978; Tobe and Stay, 1980). To evaluate the response of male CA to a reduction in the number of implanted CA, JH biosynthesis by a single male CA in an allatectomized female was determined. A single male CA implanted into an allatectomized male synthesized JH at mean rates of 5.9 ? 0.7(SE) (n = 29) pmol hr-l from Day 4 to Day 8. A single male CA implanted into an allatectomized female synthesized JH at similar rates, 4.4 t 0.7(n = 30) pmol hr-l. These rates are significantly less than mean rates for pairs of male CA implanted into allatectomized females or males (P < 0.001, Student’s t test) and demonstrate that there is no compensatory increase in rate of JH synthesis by single male implants for a reduction in the number of CA. DISCUSSION I. Differences in CA Morphometrics Activity in Unoperated Insects
and
The sexual dimorphism in JH synthesis exhibited in unoperated insects (Tobe and Stay, 1977; Tobe et al., 1979) and after im-
TOBE
plantation (Fig. 4) and the lower rates of oocyte development supported by male CA (Fig. 2) may be attributed partly to differences in CA size between the sexes. The volume and cell number of male CA is much less than female CA (Fig. 1). Male D. punctata weigh less, have smaller head capsules (Szibbo and Tobe, unpublished results) and haemolymph volume (Mundall and Tobe, unpublished results), and pass through one less stadium than female D. punctata (Willis et al., 1958). Therefore volumetric differences in the CA are related to differences in total body size. In Leucophaea maderae, another cockroach, adult males are also smaller than adult females, and similarly, the CA of males are smaller in volume and cell number (Scharrer and von Harnack, 1958). In those insects where body size and haemolymph volume of the male is smaller, lower activity of their CA may not lead to a difference in the haemolymph titer of JH (Mendes, 1948). Measurement of JH titer by physicochemical methods would be required to confirm this for D. punctata. The relatively constant and low values for CA volume and cell number of CA in unoperated males correlate well with their low, constant rates of JH synthesis (Tobe et al., 1979). In unoperated females, increases and decreases in volume and cell number parallel, but are exceeded by larger changes in JH synthesis (Szibbo and Tobe, 1981a). Although our light microscope observations did not reveal any structural differences between male and female CA, it is possible that the differences in activity at times of peak synthesis are reflected in quantities of organelles believed involved in JH synthesis i.e., mitochondria, endoplasmic reticulum, and ribosomes (Tobe and Saleuddin, 1977). Such differences may be detectable at the ultrastructural level. 2. Oocyte Development Supported by Implanted CA
The CA of male D. punctuta have the ability to support complete oocyte matura-
CORPORA
ALLATA
tion and oviposition in allatectomized females, but at a reduced rate (Figs. 2 and 3) reflecting their lower rates of JH synthesis (Fig. 4). Although this is the first study relating directly the JH synthesis of male CA to their ability to support oocyte development in allatectomized females, it is well known that male CA can support ovarian maturation (e.g., Wigglesworth, 1936; Pener, 1967; Joly, 1945; de Wilde, 1964). In Blahera fusca, the rate of oocyte maturation supported by male CA is also less than that for female CA (Brousse-Gaury and Cassier, 1975). Our implantation studies of male CA suggest that although the high rate and well-defined cycle of JH synthesis observed in CA of mated females are probably required for rapid oocyte growth (Tobe and Stay, 1977; Tobe, 1980), they are not strictly necessary for oocyte maturation in D. punctata. The CA of virgin adult female D. punctata are known to synthesize JH at rates lower than those of mated females (Tobe and Stay, 1977; Stay and Tobe, 1977) and yet complete oocyte maturation can occur in some older animals (Engelmann, 1959; Roth and Stay, 1961). However, both these examples still support the concept that time to oviposition reflects the cumulative titer of JH and indirectly, the biosynthesis of JH (Tobe and Stay, 1977, 1980: Stay and Tobe, 1977, 1978). There may be a threshold rate of JH synthesis below which no oocyte development occurs. Many females implanted with male CA fail to show any oocyte development, whereas almost all with female CA implants mature oocytes. 3. Comparison of JH Synthesis by Implanted Male and Femule CA
There is no doubt that the smaller volume and cell number of CA of unoperated male D. punctatu account in part for their lower activity. At Day 5, however, the rate of JH synthesis by female CA is over eight times that of male CA (Tobe and Stay, 1977: Szibbo and Tobe, 1981a; Tobe et al., 1979), whereas volumetric and cellular differences
OF
MALE
Lliplopterci
537
are only three- to fourfold (Fig. 1). Therefore, there may be additional reasons for the low rates of JH synthesis by male CA in situ. For example, signals transmitted via the nerves may play a role in maintaining low levels of JH synthesis in unoperated insects. The rates of JH synthesis for male CA implants in allatectomized males (Fig. 4) are double those reported by Tobe et al. (1979) for unoperated male CA. After denervation of CA, there are detectable levels of vitellogenin in the haemolymph of some male D. punctatu (Mundall, Szibbo and Tobe, unpublished). Vitellogenin is not found in normal males (Mundall et al., 1979). In mated females, denervation does not cause significantly higher peak rates of JH synthesis (Stay and Tobe, 1978). Possibly, the CA of male D. punctata are normally inhibited by their nervous connectives. Measurement of JH synthesis by male CA after sham operation would be required to confirm this. Even if severing the nerves to the CA does stimulate JH synthesis, the possibility that inhibitory factors circulating in the hemolymph prevents rates of JH synthesis from rising even further still remains. The rates of JH synthesis by male CA, however, are identical when implanted in either allatectomized males or females (Fig. 4). Similarly, female CA can achieve high rates of JH synthesis when implanted into newly emerged males. Therefore, the humoral environment of the male does not contribute to the low output of male CA. When both male and female CA are freed of nervous connectives and presented with the same humoral environment, we find that rates of JH synthesis by male implants are only one-fourth of peak rates of JH synthesis by implanted female CA at Day 5 (Fig. 4). Female CA implants behave like CA in control females (Fig. 4; Tobe and Stay, 1977) undergoing a cycle in JH synthesis and reaching maximal levels of synthesis of over 50 pmol hr-l (Stay and Tobe, 1978). Johansson (1958) also found that
538
SZIBBO
AND
transplantation of CA of male Oncopeltus fusciatus into females failed to cause an increase in their activity as judged by CA volume. 4. JH Synthetic
Potential
of Male CA
We do not know if implanted CA undergo cellular and volumetric changes similar to those of unoperated insects, but if they do, then a three- to fourfold difference in CA volume and cell number between male and female CA at times of peak synthesis (Fig. 1) could be the main explanation for the difference in synthetic ability of implants. It is remarkable, however, that the activity of the CA of adult females is not limited by the available CA volume. Following unilateral allatectomy, a single CA of female D. punctata can synthesize JH at rates as high as those of a pair of CA without undergoing compensatory hypertrophy or cell proliferation (Stay and Tobe, 1978; Szibbo and Tobe, 1981a,b). This response, which presumably occurs in response to a lowered JH titer caused by removal of one CA (Stay and Tobe, 1978) does not require intact nervous connections to the single CA (Tobe and Stay, 1980). It follows that signals for this compensatory increase in JH synthesis should operate also on implanted CA of males and high rates of JH synthesis should be expected also from the small male CA, if the total volume of hormone-producing tissue at the time of implantation was the sole determinant of JH synthetic potential. That is, similar rates of JH synthesis should be expected from a pair of male CA and a single female CA which have at Day 0 total volume and cell numbers that differ only slightly (Fig. 1). Single female CA, however, whether innervated or denervated attain mean rates of JH synthesis that are over 50 pmol hr-l (Stay and Tobe, 1978; Tobe and Stay, 1980; Szibbo and Tobe, 1981b), but pairs of male CA attain values less than one-third of this (Fig. 4). Comparison of rates of JH synthesis by
TOBE
CA of different sizes is best done by comparison of average rates per cell. Figure 5 shows changes in the rate of JH synthesis of implanted male and female CA from Fig. 4 in relation to their cell numbers at the time of implantation (Day 0) (Fig. 1) using JH synthesis data for single female CA from Tobe and Stay (1980). Because a single male CA synthesizes JH at rates that are one-half that of a pair of male CA (Results, Section 4), but a single female CA compensates for the loss of the other CA, synthesizing JH at rates equal to that of a pair of female CA, the rates of JH synthesis per Day-O cell for male CA are not only low and constant but also identical for single and paired male CA. In contrast, single female CA undergo large increases in the rate of JH synthesis per Day-O cell. The differences in Fig. 5 between singled and paired female CA cannot be attributed to increase in cell
f *IA/+.. B
....._....... n
1J 4
5
6 Age
7
,
8
Idvl
5. Mean rate of JH synthesis per Day-O cell as a function of time after implantation into allatectomized females for single male CA (A), pairs of male CA (A), single female CA (O), and pairs of female CA (0). Values are calculated by dividing rates of JH synthesis from Fig. 4 by total cell number at Day 0 from Fig. 1. Rates of JH synthesis for single female CA are from Tobe and Stay (1980). FIG.
CORPORA ALLATA
number in the single female CA (Szibbo and Tobe, 1981b). Figure 5, therefore, indicates that male CA implants have a much lower potential than female CA for synthesizing JH after implantation. It is likely that the CA of male D. punctata have a lower JH synthesizing potential than CA of females because of their smaller size. They also appear to be refractory to signals causing volumetric and synthetic increases in single female CA, however, and unlike female CA, they are unable to modulate JH synthesis in response to the number of CA present. Perhaps the activity of male CA is below a threshold necessary to elicit feedback stimulation after implantation into females. Male CA may be intrinsically different from female CA, lacking the appropriate receptors for stimulatory signals, having lower rates of substrate uptake, and/or an unmodifiably lower concentration of rate-limiting enzymes in the JH synthetic pathway. Such sexual differences may be a reflection of the different developmental histories of each. An understanding of the sexual dimornhism in JH synthetic capabilities in D. punctata may elucidate the enzymatic and cellular mechanisms permitting drastic modifications in rates of JH synthesis in females. I
ACKNOWLEDGMENTS We would like to acknowledge Peter Turner’s assistance on initial histological investigations on male CA. We are also grateful to Dr. E. Mundall and Dr. R. Feyereisen for critically reading this manuscript and to Professor B. Stay for her helpful comments. This work was supported by an operating grant from the National Sciences and Engineering Research Council of Canada. C.M.S. acknowledges receipt of an NSERC postgraduate scholarship.
REFERENCES Brousse-Gamy, P., and Cassier, P. (1975). Contrihution a l’etude de la dynamique des corpora arlara chez Blabera fusca. InfIuence du jeune, du groupment et du regime photoperiodique. Bull. Biol. Fr. Belg.
109, 253-277.
Cason, J. E. (1950). A rapid one-step Mallory-Heidenhain stain for connective tissue. Stain Technol. 25, 225-226.
OF MALE
539
Diplopteru
de Wilde, J. (1964). Reproduction-Endocrine control. In “The Physiology of Insecta” (M. Rockstein, ed.), Vol. 1, pp. 59-90. Academic Press, New York. Engelmann, F. (1959). The control of reproduction in Diploptera punctata (Blattaria). Biol. Bull. Mar. Biol. Lab. Woods Hole 116, 406-419. Fain-Maurel, M. A., and Cassier, P. (1969). Pleomorphisme mitochondrial dans les corpora allata de Locusta migratoria migratoriodes (R. & F.) au tours de la vie imaginale. Z. Zellforsch. 102, 543-553. Fukuda, S., Eguchi, G., and Takeuchi, S. (1966). Histological and electron microscopical studies on sexual differences in structure of the corpora allata of the moth of the silkworm Bombyx mori. Embryologia 9, 123-158. Girardie, A., and Granier, S. (1973). Systeme endocrine et physiologie de la diapause imaginale chez le criquet egyptien, Anacridium aegyptium. J. Insect Physioi. 19, 2341-2358. Humason, G. L. (1979). “Animal Tissue Techniques,” 4th ed. Freeman. San Francisco. Johansson, A. S. (1958). Relation of nutrition to endocrine-reproductive functions in the milkweed bug Oncopeltus fasciatus (Dallas) (Heteroptera: Lygaeidae). Nytt Mag. 2001. 7, 1-132. Joly, P. (1945). La fonction ovarienne et son controle humoral chez les Dytiscides. Arch. Zoo/. Exp. Gen.
Notes
Rev.
84, 49-
164.
Mendes, M. (1948). Histology of the corpora allata of Melanoplus dijferentialis (Orthoptera: Saltatoria). jaltatoria). Biol. Bull. 194-207.
Mar.
Biol.
Lab.
Woods
Hole
94,
Mundall, E. C., Tobe, S. S., and Stay, B. (1979). Induction of vitellogenin and growth of implanted oocytes in male cockroaches. Nature (London) 282, 97-98.
Pener, M. P. (1967). Comparative studies on the reciprocal interchange of corpora allata between males and females of adult Schistocerca gregaria (ForSk&l) (Orthoptera: Acrididae). Proc. R. Entomol. Sot. Lond. (A) 42, 139-148. Pratt, G. E., and Tobe, S. S. (1974). Juvenile hormone radiobiosynthesized by corpora allata of adult female locusts in vitro. Life Sci. 14, 575-586. Roth, L. M., and Stay, B. (1961). Oocyte development in Diplopteru punctata (Eschscholtz) (Blattaria). J. Insect Physiol. 7, 186-202. Scharrer, B., and von Harnack, M. (1958). Histophysiological studies on the corpus allatum of Leucophaea maderue. I. Normal life cycle in male and female adults. Biol. Bull. Mur. Biof. Lab. Woods Hole 115, 508-520. Stay, B., and Tobe, S. S. (1977). Control of juvenile hormone biosynthesis during the reproductive cycle of a viviparous cockroach. I. Activation and inhibition. Cert. Comp. Endocrinol. 33, 531-540.
540
SZIBBO
Stay, B., and hormone cycle of unilateral numerary Comp.
AND
Tobe, S. S. (1978). Control of juvenile biosynthesis during the reproductive a viviparous cockroach. II. Effects of allatectomy, implantation of supercorpora allata and ovariectomy. Gen.
Endocrinol.
34, 276-286.
Stay, B., Friedel, T., Tobe, S. S., and Mundall, E. C. (1980). Feedback control of juvenile hormone synthesis in cockroaches: Possible role for ecdysterone. Science 207, 898-900. Szibbo, C. M., and Tobe, S. S. (1981a). Cellular and volumetric changes in relation to the activity cycle in corpora allata of Diploptera punctata. J. Insect Physiol. 27, 609-613. Szibbo, C. M., and Tobe, S. S. (1981b) The mechanism of compensation in juvenile hormone synthesis following unilateral allatectomy in Diploptera punctata.
J. Insect
Physiol.
27, 655-665.
Tobe, S. S. (1980). Regulation of the corpora allata in adult female insects. In “Insect Biology in the Future” (M. Locke and D. S. Smith, eds.), pp. 345-368. 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. Biochem. J. 144, 107- 113. Tobe, S. S., and Saleuddin, A. S. (1977) Ul-
TOBE
trastructural localization of juvenile hormone biosynthesis by insect corpora allata. Cell Tissue Res.
183, 25-32.
Tobe, S. S., and Stay, B. (1977). Corpus allatum activity in vitro during the reproductive cycle of the viviparous cockroach Diploptera punctata. Gen. Comp.
Endocrinol.
31, 138-147.
S. S., and Stay, B. (1980). Control of juvenile hormone biosynthesis during the reproductive cycle of a viviparous cockroach. III. Effects of denervation and age on compensation with unilateral allatectomy and supernumerary corpora allata. Gen. Comp. Endocrinol. 40, 89-98. Tobe, S. S., Musters, A., and Stay, B. (1979). Corpus allatum function during sexual maturation of male Tobe,
Diplaptera
punctata.
Physiol.
Entomol.
4,79-86.
Wigglesworth, V. B. (1936). The function of the corpus allatum in the growth and reproduction of Rhodnius pro&us (Hemiptera). Quart. J. Microstop. sci. 79, 91-121. Willey, R. B. (1961). The morphology of the stomatodeal nervous system in Periplanetu americana (L.) and other Blattaria, J. Morphol. 108, 219-261. Willis, E. R., Riser, C. R., and Roth, L. M. (1958). Observations on reproduction and development in cockroaches. Ann. Entomol. Sot. Amer. 51, 53 -69.