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Influence of juvenile hormone on fat body metabolism in ovariolectomized queens of the bumblebee, Bombus terrestris
Insect Biochem. Vol. 18, No. 6, pp. 557-563, 1988 Printed in Great Britain. All rights reserved
0020-1790/88 $3.00 + 0.00 Copyright © 1988 Pergamon Press plc
INFLUENCE OF JUVENILE HORMONE ON FAT BODY METABOLISM IN OVARIOLECTOMIZED QUEENS OF THE BUMBLEBEE, BOMBUS TERRESTRIS PETER-FRANK ROSELER and INGEBORG ROSELER Zoologisches Institut (II) der Universitiit, R6ntgenring 10, 8700 Wiirzburg, F.R.G. (Received 20 November 1987; revised and accepted 22 March 1988)
Abstract--Bumblebee queens of Bombus terrestris were ovariolectomized immediately after emergence. The influence of juvenile hormone I on metabolism in fat body [glycogen content, activity of UDP-glucose: glycogen 4-~t-D-glucosyltransferase (EC 2.4.1.11), lipid content, vitellogenesis] was studied in operated queens as well as in intact queens on day 5 after eclosion. In both groups no energy reserves were accumulated after juvenile hormone treatment, but synthesi§ of vitellogenin was induced. The results indicate that the change in glycogen and lipid metabolism is not directly caused by juvenile hormone, but more likely a consequence of stimulated vitellogenesis. Key Word Index: bumblebees, fat body metabolism, juvenile hormone
INTRODUCTION
MATERIALS AND METHODS
In bumblebees, queens and workers are separated by several characteristics. A fundamental physiological distinction between castes is that only queens are able to overwinter. After emergence queens and w o r k e r s differ in metabolism of fat body. Queens prepare for hibernation accumulating glycogen and fat (Alford, 1969; Marilleau et al., 1974; Pouvreau, 1976; R6seler, 1988). They do not participate in colony tasks, but leave the nest after some days. Previously we have analysed the caste specific differences in glycogen metabolism and found that glycogen accumulation in queens is brought about by a higher activity of UDP-glucose: glycogen 4-~-D-glucosyltransferase (EC 2.4.1.11; glycogen synthase), whereas the activity of glycogen phosphorylase (EC 2.4.1.1) does not differ between both castes (R6seler and R6seler, 1986). The preparation for diapause depends on a low juvenile hormone titre in haemolymph, which is about tenfold lower in queens than in workers on day 3 after eclosion (Rfseler, 1977a). After injection of juvenile hormone into newly emerged queens the activity of glucosyltransferase does not increase and no glycogen is accumulated, but it remains on a low level as in workers (R6seler and R6seler, 1986). By juvenile hormone treatment oogenesis is also induced, and queens start egg-laying approx. 1 week after emergence. It remained, therefore, unclear whether the alterations in fat body metabolism are directly affected by juvenile hormone or whether they are consequences of the hormonally induced eggformation. To gain more detailed information about the regulation of fat body physiology, we have studied the influence of juvenile h o r m o n e on fat body metabolism (glycogen, lipid, vitellogenesis) in unmutilated and in ovariolectomized queens on day 5 after emergence.
Bumblebees We have used Bombus terrestris sassaricus Tourn. (queens collected in Sardegna), because colonies of this subspecies produce many queens. Four colonies were started in captivity by single queens and kept at 28-30°C. Newly emerged queens were individually marked by colour spots. On day 5 the queens were killed by rapidly deep-freezing them on solid CO2 and maintained at -80°C until analysed. Ovariolectomy At first we ectomized the whole ovaries. However, this surgery resulted in a change of metabolism like that caused by injection of juvenile hormone. Possibly this effect was due to wounding. Therefore, many efforts were necessary to find a suitable technique. Finally, we did not eetomize the whole ovaries, but we removed only the ovarioles using the following method. Newly emerged queens were cooled at 2-3°C for 1 h. The ectomy was performed by two persons under a microscope using sterilized and siliconized forceps. A small incision was made on each side in the membrane between sternit 4 and 5. The oviduct was pulled out a little and the ovarioles were removed by small holes. The surrounding muscular coat of the ovaries, the connective tissue sheath, and the tracheoles remained in situ. All the protruded haemolymph which came in contact to air, was absorbed by soft paper. The wound was not sealed. This ovariolectomy process lasts about 3-5 min. After operation the queens were again kept cooled for 15 min, and transferred back into the colonies when they had recovered from cooling. In the nests they had continuous access to the honey and pollen storage so that they could eat ad libitum. Application of juvenile hormone To avoid any additional damage by injection, l0 #g of juvenile hormone I (Sigma) dissolved in 2 gl acetone was applied on the abdominal tergites of newly emerged queens or of ovariolectomized queens immediately after operation. In both groups some queens were again treated on day 2. Controls only received acetone.
557
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PETER-FRANK ROSELERand INGEBORG RI3SELER
Preparation Fat body was quickly removed in ice-cold Tris-HClbuffer (pH 7.4). The length of all terminal oocytes was measured under a microscope. Ovaries of operated queens were controlled for complete ovariolectomy. Determination of glycogen Pieces of fat body were homogenized in Ringer solution. After centrifugation (4000g, I°C, 10min), 10#1 of the supernatant was used for protein determination, and the centrifugate thoroughly vortexed to again obtain an emulsion. To 150/,1 of the emulsion 150/~1 NaOH (2 M) was added. Glycogen was then determined according to Roe and Dailey (1966), modified for small amounts (R6seler and R6seler, 1986). The glycogen content is expressed as #g glycogen/mg soluble protein. Glucosyltransferase assay The activity of UDP-glucose:glycogen 4
RESULTS
Effects o f ovariolectomy We ovariolectomized 81 newly emerged queens. O f these, 15 queens (18.5%) died after operation, 5 of them on the same day and 8 on the following day. The behaviour of the surviving operated queens did not differ from unmutilated queens, most of them left the nest for the first time on day 3. In untreated queens, the fat body became voluminous, it appeared whitish, and the cells contained numerous fatty globules on day 5 after eclosion. When we had removed the whole ovaries, fat body did not develop, but it remained a sparse layer of cells as in workers. The colour was yellow to brownish. N o fat and glycogen were accumulated. The same effect often occurred when we had made only two incisions in the intersegmental membrane and manipulated with forceps within the abdomen without removing the ovaries. We suggest, therefore, that the effect is generated by the damage of tissue (wound effect) or by a trauma of tissue exposed to air, instruments etc. (shock activation, Ziegler et al., 1979) during operation, but we have not investigated this problem more precisely. By removing only the ovarioles we obtained numerous queens not affected by operation. Only 11 of 29 ovariolectomized queens (38%) showed the altered physiology of fat body. Therefore, we suppose that the same percentage of queens with that operation effect also exists in groups treated with juvenile (17)
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Determination of protein Concentrations of soluble proteins of the supernatant were determined with bovine serum albumin as standard by the method of Lowry et al. (1951). Statistics Data were analysed using the t-test.
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lx
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lO,ug JH Fig. 1. Length of terminal oocytes in untreated (controls) and in juvenile hormone (JH) treated bumblebee queens on day 5 after eclosion (1 x 10/ag JH: application after emergence; 2 x 10/~g JH: repeated application on day 2). Vertical bars indicate SD. Number of queens in parentheses.
Influence of juvenile hormone on fat body metabolism
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Fig. 2. Fat body glycogen content in unoperated and in ovariolectomized bumblebee queens on day 5 after eclosion (controls: unoperated and ovariolectomized queens not treated with juvenile hormone (JH); 1 x 10/~g JH: application after emergence; 2 x 10 ;zg JH; repeated application on day 2; operation effect: queens affected by ectomy). Vertical bars indicate SD. Number of queens in parentheses. hormone after operation. All these treated queens, however, showed changes in fat body physiology, and, moreover, single and 2-fold hormone treatment yielded different effects. Thus, we can be sure that results on fat body in ovariolectomized queens treated with juvenile hormone were really caused by juvenile hormone and not by the operation.
Oogenesis In previous experiments we injected juvenile hormone into newly emerged queens (R6seler and Rfseler, 1986). In order to avoid any injury additional to operation we applied juvenile hormone onto the abdomen. In untreated queens the terminal oocytes grew slowly to about 0.45 mm on day 5 (Fig. 1). After a single application of 10 #g juvenile hormone to newly emerged queens, the oocytes increased to 0.7 mm (t = 5.46, P < 0.001). A second application on day 2 resulted in an increase to 1.5 mm on day 5 ( t = 2 5 . 1 , P < 0 . 0 0 1 ) . The difference between both treatments is also highly significant (t = 11.54, P < 0.001).
Glycogen The glycogen content in the fat body of unmutilated control queens increased to about 310 #g/mg protein on day 5 after eclosion (Fig. 2). A single application of juvenile hormone yielded a lowered glycogen content only in some queens on day 5. The mean value of 177 #g/mg protein is lower than in controls, but the difference is not significant. A second application on day 2 resulted in a strikingly lower glycogen amount (85#g/rag protein) in fat body than in controls (t = 4.61, P < 0.001) and in queens treated once (t = 2.08, P < 0.05). Ovariolectomized queens had a mean glycogen concentration of 220 #g/mg protein in fat body, the difference from unmutilated control queens is not
significant. Single and 2-fold application of juvenile hormone yielded the same glycogen content of about 90/~g/mg protein. This is significantly different (t = 3.39 resp. 3.27, P < 0.01) from glycogen content in untreated ovariolectomized queens. Hormone treatment resulted in a similar low glycogen content as in intact queens treated twice, the difference is not significant. The mean glycogen concentration in queens affected by operation was 190/~g/mg protein, the difference from unmutilated controls is significant on the P < 0.01 (t = 3.07) level.
Glucosyltransferase Figure 3 shows the specific activities of total glucosyltransferase a + b measured in the presence of I mM glucose-6-P on day 5 after eclosion. In unoperated controls and in ovariolectomized queens, the enzyme activity was found to be on the same level of about 47 nmol UDP-glucose/mg protein, min-1. Application of juvenile hormone to unoperated queens resulted in a significant lower enzyme level than in controls (t = 4.17 resp. 4.64, P < 0.001). The same effect we obtained in ovariolectomized queens (t =4.9 resp. 7.69, P <0.001). The difference between single and 2-fold hormone treatment is not significant in both groups. In queens which reacted to surgery, we found the lowest enzyme activity of < 20 nmol UDP-glucose/mg protein.min- ~ in the mean. This is statistically different from the enzyme level after a single application of juvenile hormone to unoperated queens (t = 2.21, P <0.05), but not different from the activity after a 2-fold hormone treatment. The activity of glucosyltransferase a was determined without glucose-6-P (Fig. 4). The level in intact controls as well as in ovariolectomized queens on day 5 is about 27 nmol UDP-glucose/mg protein, min-1. After application of juvenile hormone, the activity in
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Fig. 3. Specific activities of fat body glucosyltransferease a + b in unoperated and in ovariolectomized bumblebee queens on day 5 after eclosion (controls: unoperated and ovariolectomized queens not treated with juvenile hormone (JH) 1 x 10 #g JH: application after emergence; 2 x I0/zg JH: repeated application on day 2; operation effect: queens affected by ectomy). Vertical bars indicate SD. Number of queens in parentheses. unoperated queens was decreased, in queens treated once to about 20nmol (t =2.13, P < 0.05), and in queens treated twice, to about 17nmol (t =2.73, P < 0.02). In ovariolectomized queens we obtained similar results after hormone treatment, single application: 21 nmol (ns), 2-fold application: 19nmol (t = 2.82, P < 0.01). We found the lowest activity of glucosyltransferase a in queens affected by the operation (15.7nmol UDP-glucose/mg protein min-~; t = 2.42, P < 0.05). The percentage of the a-form was also influenced by juvenile hormone treatment; it increased from 57% up to more than 80%.
Lipid Queens accumulate large amounts of lipids in fat body prior to overwintering (R6seler, 1988). In newly emerged queens, the lipid concentration is about 0.5mg/mg protein and it increases to about 20 mg/mg protein during the prehibernation period. On day 5 the mean lipid concentration of fat body in unmutilated controls was 17 mg/mg protein (Fig. 5). Hormone treatment resulted in a significantly lower lipid concentration than in controls, single application: 3.7mg/mg protein (t = 7.78, P <0.001), 2-fold application: 1.1mg/mg protein ( t = 12.14,
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Fig. 4. Specific activities of fat body glucosyltransferase a in unoperated and in ovariolectomized bumblebee queens on day 5 after eelosion (controls: unoperated and ovariolectomized queens not treated with juvenile hormone (JH); 1 x 10/ag JH: application after emergence; 2 x 10/zg JH: repeated application on day 2; operation effect: queens affected by ectomy). % = Proportion of glucosyltransferase a. Vertical bars indictate SD. Number of queens in parentheses.
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Fig. 5. Fat body lipid content in unoperated and in ovariolectomized bumblebee queens on day 5 after eclosion (controls: unoperated and ovariolectomized queens not treated with juvenile hormone (JH); 1 x I0/lg JH: application after emergence; 2 x 10 pg JH: repeated application on day 2; operation effect: queens affected by ectomy). Vertical bars indicate SD. Number of queens in parentheses. P <0,001). In ovariolectomized queens, the lipid concentration was approx. 15 mg/mg protein, this is not statistically different from unmutilated queens. Application of juvenile hormone also yielded a clear effect in ovariolectomized queens: single application: 2.9 mg/mg protein (t = 10.25, P < 0.001), 2-fold application: 1.1 mg/mg protein (t = 14,0, P < 0.001). The difference between single and 2-fold treatment is significant in both groups (intact queens: t = 3.39, P < 0.01; ovariolectomized queens: t = 2.95, P < 0.01). In queens affected by ectomy the lipid concentration of fat body was as low as in queens treated twice with juvenile hormone.
Haernolymph proteins Bumblebee queens differ from workers in the banding pattern of haemolymph proteins (R6seler and R6seler, 1973, 1974). On day 2 after emergence a specific protein fraction (C/D) occurs which disappears after hibernation, when eggs are formed. After juvenile hormone treatment the concentration of this fraction remains low as in workers (R6seler, 1976). The electrophoretic separation of haemolymph proteins of 5-day-old queens is shown in Fig. 6. In intact queens (gel 1) as well as in ovariolectomized queens (gel 3) the C/D-fraction is dark stained, the concentration of vitellogenins H/J-fraction, in contrast, is low. After application of juvenile hormone the C/D-band vanishes and vitellogenins appear in high concentrations in both groups. The effect of a 2-fold hormone treatment on vitellogenin concentration in ovariolectomized queens (gel 5) is greater than that of a single application (gel 4). In unoperated queens we have only studied the effect of 2-fold applications (gel 2). In queens affected by operation (gel 6 ) t h e
C/D-band also disappeared and vitellogenins were present as after a single application of juvenile hormone. Moreover, ovariolectomy resulted in specific changes of banding pattern, e.g. in all operated queens fraction F occurred (gels 3~). D I S C U S S I O N
In bumblebees, the caste specific metabolism after emergence is controlled by a different endocrine program switched on during pre-imaginal differentiation of queens and workers. A characteristic of queens is the obligatory diapause which they enter approx. 2 weeks after emergence. Preparation for diap.ause depends on a low juvenile hormone titre in haemolymph. After treatment with juvenile hormone no reserves of fat and glycogen are accumulated, but eggs are formed (R6seler and R6seler, 1986; R6seler, 1988). Ectomy of whole ovaries and sometimes other operation methods as well affected fat body metabolism in the same way as juvenile hormone treatment. However, we do not know whether this alteration was directly induced by the operation methods (e.g. wound effect) or mediated by corpora allata activated by the operation. In bumblebees, activity of corpora allata is correlated to gland size (R6seler, 1977b). In operated queens, however, we have not observed an increase in gland size. This is in contrast to previous investigations on B. terrestris queens (R6seler and R6seler, 1984). Sham operation of Pars intercerebralis resulted in egg-formation and decline in lipid content, but these alterations were related to an increase in corpora allata volume. Topical application of juvenile hormone was not as effective as injection of hormone. Only a second application of 10 ~g juvenile hormone on day 2 after
562
PETER-FRANK ROSELER and INGEBORG ROSELER
C/D F H/J
1
2
3
4
5
6
Fig. 6. Disc-electrophoretic separation of haemolymph proteins of bumblebee queens on day 5 after eclosion. 1: unoperated control; 2: unoperated + 2 x 10 ttg juvenile hormone (JH); 3: ovariolectomized control; 4: ovariolectomized + 1 x 10#g JH; 5: ovariolectomized + 2 x 10/~g JH; 6: operation effect (queens affected by ectomy). 1 x 10/tg JH: appheation after emergence: 2 x 10/~g JH: repeated application on day 2. Capital letters indicate bands cited in text. eclosion had the same effect as a single injection of 5/~g juvenile hormone into newly emerged queens. Oocyte growth, lipogenesis and vitellogenesis reacted most sensitively to hormone treatment, they all showed significant effects even after a single application. We have also found vitellogenesis to be very sensitive to juvenile hormone in bumblebee workers. Low doses induced vitellogenin production, but no egg-formation (R6seler, 1977b) The responses resemble dose-dependent effects, but the different responses to single and 2-fold applications or to different doses are likely to be caused by a prolongation of hormone influence. Exogenous juvenile hormone is rapidly excreted in bumblebees. It is, therefore, unlikely that molecules were still present in queens on day 5 after a single treatment with 10 ~g at the time of eclosion (R6seler, 1977b; R6seler and Rfseler, 1978). It seems that growth of oocytes and production of vitellogenins were induced by single treatment, but were again stopped after the hormone has disappeared and lipids were formed. After a second application on day 2 as well as after injection of high doses, the hormonal influence is prolonged so that the effects on day 5 are stronger than after a single application. What we see on day 5 are snap-
shots, but we cannot deduce from the data whether oocytes are growing or resting and whether metabolites are accumulating or remaining on a certain level. In this study we have shown that ovaries are not involved in the control of fat body metabolism. Fat body of queens ovariolectomized immediately after eclosion and not affected by operation developed and accumulated reserves as in unoperated controls. It also responded to juvenile hormone treatment to the same extent: no glycogen and fat reserves were accumulated and the activity of glucosyltransferase remained low. Moreover, the protein fraction C/D in haemolymph did not appear. This protein characteristic of queens during the prehibernation period seems to be a "short day protein" (de Loof and de Wilde, 1970), of which the function is not clear. On the other hand, juvenile hormone induced synthesis and release of vitellogenins in fat body. The results indicate that the ovaries of bumblebees do not control vitellogenesis. It is generally accepted that vitellogenesis is mainly regulated by juvenile hormone (Engelmann, 1979). By contrast, in the honeybee queen, synthesis of vitellogenins seems to be controlled not by juvenile hormone, but probably by humoral factors (peptides?) from the head (Engels, 1987; Kaatz, 1987). In honeybee workers, however, Imboden et al. (1976) have found a juvenile hormonedependent vitellogenesis. As far as it is known, juvenile hormone does not directly influence carbohydrate metabolism in fat body, but it controls lipid metabolism and protein synthesis (Keeley, 1978; Beenakkers, 1983; Steele, 1983). Thus, a dual role of juvenile hormone in fat body metabolism was postulated: induction of protein (vitellogenin) synthesis and inhibition of lipogenesis. Our studies have not given clear evidence that juvenile hormone directly influences glycogen and lipid metabolism in the fat body because all the effects could be a consequence of induced vitellogenin synthesis. By the stimulated vitellogenesis the demand for energy increases so that no reserves can be stored. This might be additionally controlled by juvenile hormone suppressing lipogenesis. We assume, therefore, that in bumblebee queens juvenile hormone primarily induces synthesis of vitellogenins, whereas synthesis of "short day protein" is inhibited. Production of vitellogenins and formation of energy reserves seem to be mutually exclusive pathways. Acknowledgements--This work was supported by the Deutsche Forschungsgemeinschaft (Ro 242/8-3). We thank Mrs H. Foil for her excellent technical assistance.
REFERENCES
Alford D. V. (1969) Studies on the fat-body of adult bumblebees. J. apicult. Res. 8, 37~,8. Beenakkers A. M. T. (1983) Regulation of lipid metabolism. In Endocrinology oflnsects (Edited by Downer R. G. H. and Laufer H.), pp. 441450. Liss, New York. Engelmann F. (1979) Insect vitellogenin: Identification, biosynthesis, and role in vitellogenesis. Adv. Insect Physiol. 14, 49-108. Engels W. (1987) Reproduction and caste development in social bees. In Chemistry and Biology of Social Insects
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(Edited by Eder J. and Rembold H.), pp. 275-281. R6seler P.-F. (1977b) Juvenile hormone control of Peperny, Miinchen. oogenesis in bumblebee workers, Bombus terrestris. Imboden H., Willie H., Gerig L. and L/ischer M. (1976) Die J. Insect Physiol. 23, 985-992. Vitellogeninsynthese bei der Bienen-Arbeiterin (Apis mel- Rfseler P.-F. (1988) Role of morphogenetic hormones in lifera) und ihre Abh~ingigkeit yon Juvenilhormon (JH). caste polymorphism in bumblebees. In Morphogenetic Revue suisse Zool. 83, 928-933. Hormones of Arthropods (Edited by Gupta A. P.). Rutgers Kaatz H.-R. (1987) Regulation of vitellogenin synthesis in Univ. Press. In press. honey bee queens. In Chemistry and Biology of Social R6seler I. and R6seler P.-F. (1973) Anderungen im Muster Insects (Edited by Eder J. and Reinhold H.), pp. 317-318. der Haemolymphproteine von adulten K6niginnen der Peperny, Miinchen. Hummelart Bombus terrestris. J. Insect Physiol. 19, Keeley L. L. (1978) Endocrine regulation of fat body 1741-1752. development and function. A. Rev. Ent. 23, 329-352. Rbseler P.-F. and Rfseler I. (1974) Morphologiscbe und Loof A. de and Wilde J. de (1970) Hormonal control of physiologische Differenzierung der Kasten bei den Humsynthesis of vitellogenic female protein in the Colorado melarten Bombas hypnorum (L.) und Bombus terrestris beetle, Leptinotarsa decemlineata. J. Insect Physiol. 16, (L.). Zool. Jb. Physiol. 78, 175-198. 1455-1466. R6seler P.-F. and R6seler I. (1978) Studies on the regulation Lowry O. H., Rosebrough N. J., Farr A. L. and Randall of the juvenile hormone titre in bumblebee workers, R. J. (1951) Protein measurement with the Folin phenol Bombus terrestris. J. Insect Physiol. 24, 707-713. reagent. J. biol. Chem. 193, 265-275. R6seler P.-F. and R6.seler I. (1984) Der EinfluB von CO2 Marilleau R., Pouvreau A. and Bekaert A. (1974) Evolution und der Kauterisation der Pars intercerebralis auf quantitative des lipides chez les reines de bourdons die Aktivit/it der Corpora allata und die Eibildung bei (Hymenoptera, Apoidea, Bombinae, Bombus Latr,) au Hummeln (Bombus hypnorum und Bombus terrestris). cours de leur torpeur hivernale. C.r. Soc. Biol. 168, Zool. .lb. Physiol. 88, 237-246. 952-958. R6seler P.-F. and R6seler I. (1986) Caste specific differences Maurer H. R. (1968) Disk-Elektrophorese. De Gruyter, in fat body glycogen metabolism of the bumblebee, Berlin. Bombus terrestris. Insect Biochem. 16, 501-508. Ornstein L. and Davies B. J. (1962) Disc-Electrophoresis. Steele J. E. (1983) Endocrine control of carbohydrate Distillation Industries, Rochester, New York. metabolism in insects. In Endocrinolgy of Insects (Edited Pouvreau A. (1976) Contribution fi la biologie des by Downer R. G. H. and Laufer H.), pp. 427-439. Liss, bourdons. Etude de quelques param~tres ~cologiques et New York. physiologiques en relation avec l'hibernation des reines. Thomas J. A., Schlender K. K. and Larner J. (1968) A rapid Th~se, Paris. filter paper assay for UDP-glucose-glycogen glucosylRoe J. H. and Dailey R. R. (1966) Determination of transferase, including an improved biosynthesis for UDPglycogen with the anthrone reagent. Analyt. Biochem. 15, ~4C-glucose. Analyt. Biochem. 25, 486~99. 245-250. Ziegler R., Ashida M., Fallon A. M., Wimer L. T. Wyatt Rfseler P.-F. (1976) Juvenile hormone and queen rearing in S. S. and Wyatt G. R. (1979) Regulation of glycogen bumblebees. In Phase and Caste Determination in Insects. phosphorylase in fat body of Cecropia silkmoth pupae. Endocrine Aspects (Edited by Liischer M.), pp. 55-61. J. comp. Physiol. 131, 321-332. Pergamon Press, Oxford. Zfllner H. and Kirsch K. (1962) 12ber die quantitative Rfseler P.-F. (1977a) Endocrine control of polymorphism Bestimmung von Lipoiden (Mikromethode) mittels der in bumblebees. Proc. VIII Int. Cong. IUSSI, Wageningen, vielen natiirlichen Lipoiden (allen bekannten Plasmalipp. 22-23. poiden) gemeinsamen Sulphophosphovanillin-Reaktion. Z. ges. exp. Med. 135, 545-561.
0020-1790/88 $3.00 + 0.00 Copyright © 1988 Pergamon Press plc
INFLUENCE OF JUVENILE HORMONE ON FAT BODY METABOLISM IN OVARIOLECTOMIZED QUEENS OF THE BUMBLEBEE, BOMBUS TERRESTRIS PETER-FRANK ROSELER and INGEBORG ROSELER Zoologisches Institut (II) der Universitiit, R6ntgenring 10, 8700 Wiirzburg, F.R.G. (Received 20 November 1987; revised and accepted 22 March 1988)
Abstract--Bumblebee queens of Bombus terrestris were ovariolectomized immediately after emergence. The influence of juvenile hormone I on metabolism in fat body [glycogen content, activity of UDP-glucose: glycogen 4-~t-D-glucosyltransferase (EC 2.4.1.11), lipid content, vitellogenesis] was studied in operated queens as well as in intact queens on day 5 after eclosion. In both groups no energy reserves were accumulated after juvenile hormone treatment, but synthesi§ of vitellogenin was induced. The results indicate that the change in glycogen and lipid metabolism is not directly caused by juvenile hormone, but more likely a consequence of stimulated vitellogenesis. Key Word Index: bumblebees, fat body metabolism, juvenile hormone
INTRODUCTION
MATERIALS AND METHODS
In bumblebees, queens and workers are separated by several characteristics. A fundamental physiological distinction between castes is that only queens are able to overwinter. After emergence queens and w o r k e r s differ in metabolism of fat body. Queens prepare for hibernation accumulating glycogen and fat (Alford, 1969; Marilleau et al., 1974; Pouvreau, 1976; R6seler, 1988). They do not participate in colony tasks, but leave the nest after some days. Previously we have analysed the caste specific differences in glycogen metabolism and found that glycogen accumulation in queens is brought about by a higher activity of UDP-glucose: glycogen 4-~-D-glucosyltransferase (EC 2.4.1.11; glycogen synthase), whereas the activity of glycogen phosphorylase (EC 2.4.1.1) does not differ between both castes (R6seler and R6seler, 1986). The preparation for diapause depends on a low juvenile hormone titre in haemolymph, which is about tenfold lower in queens than in workers on day 3 after eclosion (Rfseler, 1977a). After injection of juvenile hormone into newly emerged queens the activity of glucosyltransferase does not increase and no glycogen is accumulated, but it remains on a low level as in workers (R6seler and R6seler, 1986). By juvenile hormone treatment oogenesis is also induced, and queens start egg-laying approx. 1 week after emergence. It remained, therefore, unclear whether the alterations in fat body metabolism are directly affected by juvenile hormone or whether they are consequences of the hormonally induced eggformation. To gain more detailed information about the regulation of fat body physiology, we have studied the influence of juvenile h o r m o n e on fat body metabolism (glycogen, lipid, vitellogenesis) in unmutilated and in ovariolectomized queens on day 5 after emergence.
Bumblebees We have used Bombus terrestris sassaricus Tourn. (queens collected in Sardegna), because colonies of this subspecies produce many queens. Four colonies were started in captivity by single queens and kept at 28-30°C. Newly emerged queens were individually marked by colour spots. On day 5 the queens were killed by rapidly deep-freezing them on solid CO2 and maintained at -80°C until analysed. Ovariolectomy At first we ectomized the whole ovaries. However, this surgery resulted in a change of metabolism like that caused by injection of juvenile hormone. Possibly this effect was due to wounding. Therefore, many efforts were necessary to find a suitable technique. Finally, we did not eetomize the whole ovaries, but we removed only the ovarioles using the following method. Newly emerged queens were cooled at 2-3°C for 1 h. The ectomy was performed by two persons under a microscope using sterilized and siliconized forceps. A small incision was made on each side in the membrane between sternit 4 and 5. The oviduct was pulled out a little and the ovarioles were removed by small holes. The surrounding muscular coat of the ovaries, the connective tissue sheath, and the tracheoles remained in situ. All the protruded haemolymph which came in contact to air, was absorbed by soft paper. The wound was not sealed. This ovariolectomy process lasts about 3-5 min. After operation the queens were again kept cooled for 15 min, and transferred back into the colonies when they had recovered from cooling. In the nests they had continuous access to the honey and pollen storage so that they could eat ad libitum. Application of juvenile hormone To avoid any additional damage by injection, l0 #g of juvenile hormone I (Sigma) dissolved in 2 gl acetone was applied on the abdominal tergites of newly emerged queens or of ovariolectomized queens immediately after operation. In both groups some queens were again treated on day 2. Controls only received acetone.
557
558
PETER-FRANK ROSELERand INGEBORG RI3SELER
Preparation Fat body was quickly removed in ice-cold Tris-HClbuffer (pH 7.4). The length of all terminal oocytes was measured under a microscope. Ovaries of operated queens were controlled for complete ovariolectomy. Determination of glycogen Pieces of fat body were homogenized in Ringer solution. After centrifugation (4000g, I°C, 10min), 10#1 of the supernatant was used for protein determination, and the centrifugate thoroughly vortexed to again obtain an emulsion. To 150/,1 of the emulsion 150/~1 NaOH (2 M) was added. Glycogen was then determined according to Roe and Dailey (1966), modified for small amounts (R6seler and R6seler, 1986). The glycogen content is expressed as #g glycogen/mg soluble protein. Glucosyltransferase assay The activity of UDP-glucose:glycogen 4
RESULTS
Effects o f ovariolectomy We ovariolectomized 81 newly emerged queens. O f these, 15 queens (18.5%) died after operation, 5 of them on the same day and 8 on the following day. The behaviour of the surviving operated queens did not differ from unmutilated queens, most of them left the nest for the first time on day 3. In untreated queens, the fat body became voluminous, it appeared whitish, and the cells contained numerous fatty globules on day 5 after eclosion. When we had removed the whole ovaries, fat body did not develop, but it remained a sparse layer of cells as in workers. The colour was yellow to brownish. N o fat and glycogen were accumulated. The same effect often occurred when we had made only two incisions in the intersegmental membrane and manipulated with forceps within the abdomen without removing the ovaries. We suggest, therefore, that the effect is generated by the damage of tissue (wound effect) or by a trauma of tissue exposed to air, instruments etc. (shock activation, Ziegler et al., 1979) during operation, but we have not investigated this problem more precisely. By removing only the ovarioles we obtained numerous queens not affected by operation. Only 11 of 29 ovariolectomized queens (38%) showed the altered physiology of fat body. Therefore, we suppose that the same percentage of queens with that operation effect also exists in groups treated with juvenile (17)
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Influence of juvenile hormone on fat body metabolism
559
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Fig. 2. Fat body glycogen content in unoperated and in ovariolectomized bumblebee queens on day 5 after eclosion (controls: unoperated and ovariolectomized queens not treated with juvenile hormone (JH); 1 x 10/~g JH: application after emergence; 2 x 10 ;zg JH; repeated application on day 2; operation effect: queens affected by ectomy). Vertical bars indicate SD. Number of queens in parentheses. hormone after operation. All these treated queens, however, showed changes in fat body physiology, and, moreover, single and 2-fold hormone treatment yielded different effects. Thus, we can be sure that results on fat body in ovariolectomized queens treated with juvenile hormone were really caused by juvenile hormone and not by the operation.
Oogenesis In previous experiments we injected juvenile hormone into newly emerged queens (R6seler and Rfseler, 1986). In order to avoid any injury additional to operation we applied juvenile hormone onto the abdomen. In untreated queens the terminal oocytes grew slowly to about 0.45 mm on day 5 (Fig. 1). After a single application of 10 #g juvenile hormone to newly emerged queens, the oocytes increased to 0.7 mm (t = 5.46, P < 0.001). A second application on day 2 resulted in an increase to 1.5 mm on day 5 ( t = 2 5 . 1 , P < 0 . 0 0 1 ) . The difference between both treatments is also highly significant (t = 11.54, P < 0.001).
Glycogen The glycogen content in the fat body of unmutilated control queens increased to about 310 #g/mg protein on day 5 after eclosion (Fig. 2). A single application of juvenile hormone yielded a lowered glycogen content only in some queens on day 5. The mean value of 177 #g/mg protein is lower than in controls, but the difference is not significant. A second application on day 2 resulted in a strikingly lower glycogen amount (85#g/rag protein) in fat body than in controls (t = 4.61, P < 0.001) and in queens treated once (t = 2.08, P < 0.05). Ovariolectomized queens had a mean glycogen concentration of 220 #g/mg protein in fat body, the difference from unmutilated control queens is not
significant. Single and 2-fold application of juvenile hormone yielded the same glycogen content of about 90/~g/mg protein. This is significantly different (t = 3.39 resp. 3.27, P < 0.01) from glycogen content in untreated ovariolectomized queens. Hormone treatment resulted in a similar low glycogen content as in intact queens treated twice, the difference is not significant. The mean glycogen concentration in queens affected by operation was 190/~g/mg protein, the difference from unmutilated controls is significant on the P < 0.01 (t = 3.07) level.
Glucosyltransferase Figure 3 shows the specific activities of total glucosyltransferase a + b measured in the presence of I mM glucose-6-P on day 5 after eclosion. In unoperated controls and in ovariolectomized queens, the enzyme activity was found to be on the same level of about 47 nmol UDP-glucose/mg protein, min-1. Application of juvenile hormone to unoperated queens resulted in a significant lower enzyme level than in controls (t = 4.17 resp. 4.64, P < 0.001). The same effect we obtained in ovariolectomized queens (t =4.9 resp. 7.69, P <0.001). The difference between single and 2-fold hormone treatment is not significant in both groups. In queens which reacted to surgery, we found the lowest enzyme activity of < 20 nmol UDP-glucose/mg protein.min- ~ in the mean. This is statistically different from the enzyme level after a single application of juvenile hormone to unoperated queens (t = 2.21, P <0.05), but not different from the activity after a 2-fold hormone treatment. The activity of glucosyltransferase a was determined without glucose-6-P (Fig. 4). The level in intact controls as well as in ovariolectomized queens on day 5 is about 27 nmol UDP-glucose/mg protein, min-1. After application of juvenile hormone, the activity in
560
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Fig. 3. Specific activities of fat body glucosyltransferease a + b in unoperated and in ovariolectomized bumblebee queens on day 5 after eclosion (controls: unoperated and ovariolectomized queens not treated with juvenile hormone (JH) 1 x 10 #g JH: application after emergence; 2 x I0/zg JH: repeated application on day 2; operation effect: queens affected by ectomy). Vertical bars indicate SD. Number of queens in parentheses. unoperated queens was decreased, in queens treated once to about 20nmol (t =2.13, P < 0.05), and in queens treated twice, to about 17nmol (t =2.73, P < 0.02). In ovariolectomized queens we obtained similar results after hormone treatment, single application: 21 nmol (ns), 2-fold application: 19nmol (t = 2.82, P < 0.01). We found the lowest activity of glucosyltransferase a in queens affected by the operation (15.7nmol UDP-glucose/mg protein min-~; t = 2.42, P < 0.05). The percentage of the a-form was also influenced by juvenile hormone treatment; it increased from 57% up to more than 80%.
Lipid Queens accumulate large amounts of lipids in fat body prior to overwintering (R6seler, 1988). In newly emerged queens, the lipid concentration is about 0.5mg/mg protein and it increases to about 20 mg/mg protein during the prehibernation period. On day 5 the mean lipid concentration of fat body in unmutilated controls was 17 mg/mg protein (Fig. 5). Hormone treatment resulted in a significantly lower lipid concentration than in controls, single application: 3.7mg/mg protein (t = 7.78, P <0.001), 2-fold application: 1.1mg/mg protein ( t = 12.14,
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Fig. 4. Specific activities of fat body glucosyltransferase a in unoperated and in ovariolectomized bumblebee queens on day 5 after eelosion (controls: unoperated and ovariolectomized queens not treated with juvenile hormone (JH); 1 x 10/ag JH: application after emergence; 2 x 10/zg JH: repeated application on day 2; operation effect: queens affected by ectomy). % = Proportion of glucosyltransferase a. Vertical bars indictate SD. Number of queens in parentheses.
Influence of juvenile hormone on fat body metabolism (14)
561
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Fig. 5. Fat body lipid content in unoperated and in ovariolectomized bumblebee queens on day 5 after eclosion (controls: unoperated and ovariolectomized queens not treated with juvenile hormone (JH); 1 x I0/lg JH: application after emergence; 2 x 10 pg JH: repeated application on day 2; operation effect: queens affected by ectomy). Vertical bars indicate SD. Number of queens in parentheses. P <0,001). In ovariolectomized queens, the lipid concentration was approx. 15 mg/mg protein, this is not statistically different from unmutilated queens. Application of juvenile hormone also yielded a clear effect in ovariolectomized queens: single application: 2.9 mg/mg protein (t = 10.25, P < 0.001), 2-fold application: 1.1 mg/mg protein (t = 14,0, P < 0.001). The difference between single and 2-fold treatment is significant in both groups (intact queens: t = 3.39, P < 0.01; ovariolectomized queens: t = 2.95, P < 0.01). In queens affected by ectomy the lipid concentration of fat body was as low as in queens treated twice with juvenile hormone.
Haernolymph proteins Bumblebee queens differ from workers in the banding pattern of haemolymph proteins (R6seler and R6seler, 1973, 1974). On day 2 after emergence a specific protein fraction (C/D) occurs which disappears after hibernation, when eggs are formed. After juvenile hormone treatment the concentration of this fraction remains low as in workers (R6seler, 1976). The electrophoretic separation of haemolymph proteins of 5-day-old queens is shown in Fig. 6. In intact queens (gel 1) as well as in ovariolectomized queens (gel 3) the C/D-fraction is dark stained, the concentration of vitellogenins H/J-fraction, in contrast, is low. After application of juvenile hormone the C/D-band vanishes and vitellogenins appear in high concentrations in both groups. The effect of a 2-fold hormone treatment on vitellogenin concentration in ovariolectomized queens (gel 5) is greater than that of a single application (gel 4). In unoperated queens we have only studied the effect of 2-fold applications (gel 2). In queens affected by operation (gel 6 ) t h e
C/D-band also disappeared and vitellogenins were present as after a single application of juvenile hormone. Moreover, ovariolectomy resulted in specific changes of banding pattern, e.g. in all operated queens fraction F occurred (gels 3~). D I S C U S S I O N
In bumblebees, the caste specific metabolism after emergence is controlled by a different endocrine program switched on during pre-imaginal differentiation of queens and workers. A characteristic of queens is the obligatory diapause which they enter approx. 2 weeks after emergence. Preparation for diap.ause depends on a low juvenile hormone titre in haemolymph. After treatment with juvenile hormone no reserves of fat and glycogen are accumulated, but eggs are formed (R6seler and R6seler, 1986; R6seler, 1988). Ectomy of whole ovaries and sometimes other operation methods as well affected fat body metabolism in the same way as juvenile hormone treatment. However, we do not know whether this alteration was directly induced by the operation methods (e.g. wound effect) or mediated by corpora allata activated by the operation. In bumblebees, activity of corpora allata is correlated to gland size (R6seler, 1977b). In operated queens, however, we have not observed an increase in gland size. This is in contrast to previous investigations on B. terrestris queens (R6seler and R6seler, 1984). Sham operation of Pars intercerebralis resulted in egg-formation and decline in lipid content, but these alterations were related to an increase in corpora allata volume. Topical application of juvenile hormone was not as effective as injection of hormone. Only a second application of 10 ~g juvenile hormone on day 2 after
562
PETER-FRANK ROSELER and INGEBORG ROSELER
C/D F H/J
1
2
3
4
5
6
Fig. 6. Disc-electrophoretic separation of haemolymph proteins of bumblebee queens on day 5 after eclosion. 1: unoperated control; 2: unoperated + 2 x 10 ttg juvenile hormone (JH); 3: ovariolectomized control; 4: ovariolectomized + 1 x 10#g JH; 5: ovariolectomized + 2 x 10/~g JH; 6: operation effect (queens affected by ectomy). 1 x 10/tg JH: appheation after emergence: 2 x 10/~g JH: repeated application on day 2. Capital letters indicate bands cited in text. eclosion had the same effect as a single injection of 5/~g juvenile hormone into newly emerged queens. Oocyte growth, lipogenesis and vitellogenesis reacted most sensitively to hormone treatment, they all showed significant effects even after a single application. We have also found vitellogenesis to be very sensitive to juvenile hormone in bumblebee workers. Low doses induced vitellogenin production, but no egg-formation (R6seler, 1977b) The responses resemble dose-dependent effects, but the different responses to single and 2-fold applications or to different doses are likely to be caused by a prolongation of hormone influence. Exogenous juvenile hormone is rapidly excreted in bumblebees. It is, therefore, unlikely that molecules were still present in queens on day 5 after a single treatment with 10 ~g at the time of eclosion (R6seler, 1977b; R6seler and Rfseler, 1978). It seems that growth of oocytes and production of vitellogenins were induced by single treatment, but were again stopped after the hormone has disappeared and lipids were formed. After a second application on day 2 as well as after injection of high doses, the hormonal influence is prolonged so that the effects on day 5 are stronger than after a single application. What we see on day 5 are snap-
shots, but we cannot deduce from the data whether oocytes are growing or resting and whether metabolites are accumulating or remaining on a certain level. In this study we have shown that ovaries are not involved in the control of fat body metabolism. Fat body of queens ovariolectomized immediately after eclosion and not affected by operation developed and accumulated reserves as in unoperated controls. It also responded to juvenile hormone treatment to the same extent: no glycogen and fat reserves were accumulated and the activity of glucosyltransferase remained low. Moreover, the protein fraction C/D in haemolymph did not appear. This protein characteristic of queens during the prehibernation period seems to be a "short day protein" (de Loof and de Wilde, 1970), of which the function is not clear. On the other hand, juvenile hormone induced synthesis and release of vitellogenins in fat body. The results indicate that the ovaries of bumblebees do not control vitellogenesis. It is generally accepted that vitellogenesis is mainly regulated by juvenile hormone (Engelmann, 1979). By contrast, in the honeybee queen, synthesis of vitellogenins seems to be controlled not by juvenile hormone, but probably by humoral factors (peptides?) from the head (Engels, 1987; Kaatz, 1987). In honeybee workers, however, Imboden et al. (1976) have found a juvenile hormonedependent vitellogenesis. As far as it is known, juvenile hormone does not directly influence carbohydrate metabolism in fat body, but it controls lipid metabolism and protein synthesis (Keeley, 1978; Beenakkers, 1983; Steele, 1983). Thus, a dual role of juvenile hormone in fat body metabolism was postulated: induction of protein (vitellogenin) synthesis and inhibition of lipogenesis. Our studies have not given clear evidence that juvenile hormone directly influences glycogen and lipid metabolism in the fat body because all the effects could be a consequence of induced vitellogenin synthesis. By the stimulated vitellogenesis the demand for energy increases so that no reserves can be stored. This might be additionally controlled by juvenile hormone suppressing lipogenesis. We assume, therefore, that in bumblebee queens juvenile hormone primarily induces synthesis of vitellogenins, whereas synthesis of "short day protein" is inhibited. Production of vitellogenins and formation of energy reserves seem to be mutually exclusive pathways. Acknowledgements--This work was supported by the Deutsche Forschungsgemeinschaft (Ro 242/8-3). We thank Mrs H. Foil for her excellent technical assistance.
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Alford D. V. (1969) Studies on the fat-body of adult bumblebees. J. apicult. Res. 8, 37~,8. Beenakkers A. M. T. (1983) Regulation of lipid metabolism. In Endocrinology oflnsects (Edited by Downer R. G. H. and Laufer H.), pp. 441450. Liss, New York. Engelmann F. (1979) Insect vitellogenin: Identification, biosynthesis, and role in vitellogenesis. Adv. Insect Physiol. 14, 49-108. Engels W. (1987) Reproduction and caste development in social bees. In Chemistry and Biology of Social Insects
Influence of juvenile hormone on fat body metabolism
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(Edited by Eder J. and Rembold H.), pp. 275-281. R6seler P.-F. (1977b) Juvenile hormone control of Peperny, Miinchen. oogenesis in bumblebee workers, Bombus terrestris. Imboden H., Willie H., Gerig L. and L/ischer M. (1976) Die J. Insect Physiol. 23, 985-992. Vitellogeninsynthese bei der Bienen-Arbeiterin (Apis mel- Rfseler P.-F. (1988) Role of morphogenetic hormones in lifera) und ihre Abh~ingigkeit yon Juvenilhormon (JH). caste polymorphism in bumblebees. In Morphogenetic Revue suisse Zool. 83, 928-933. Hormones of Arthropods (Edited by Gupta A. P.). Rutgers Kaatz H.-R. (1987) Regulation of vitellogenin synthesis in Univ. Press. In press. honey bee queens. In Chemistry and Biology of Social R6seler I. and R6seler P.-F. (1973) Anderungen im Muster Insects (Edited by Eder J. and Reinhold H.), pp. 317-318. der Haemolymphproteine von adulten K6niginnen der Peperny, Miinchen. Hummelart Bombus terrestris. J. Insect Physiol. 19, Keeley L. L. (1978) Endocrine regulation of fat body 1741-1752. development and function. A. Rev. Ent. 23, 329-352. Rbseler P.-F. and Rfseler I. (1974) Morphologiscbe und Loof A. de and Wilde J. de (1970) Hormonal control of physiologische Differenzierung der Kasten bei den Humsynthesis of vitellogenic female protein in the Colorado melarten Bombas hypnorum (L.) und Bombus terrestris beetle, Leptinotarsa decemlineata. J. Insect Physiol. 16, (L.). Zool. Jb. Physiol. 78, 175-198. 1455-1466. R6seler P.-F. and R6seler I. (1978) Studies on the regulation Lowry O. H., Rosebrough N. J., Farr A. L. and Randall of the juvenile hormone titre in bumblebee workers, R. J. (1951) Protein measurement with the Folin phenol Bombus terrestris. J. Insect Physiol. 24, 707-713. reagent. J. biol. Chem. 193, 265-275. R6seler P.-F. and R6.seler I. (1984) Der EinfluB von CO2 Marilleau R., Pouvreau A. and Bekaert A. (1974) Evolution und der Kauterisation der Pars intercerebralis auf quantitative des lipides chez les reines de bourdons die Aktivit/it der Corpora allata und die Eibildung bei (Hymenoptera, Apoidea, Bombinae, Bombus Latr,) au Hummeln (Bombus hypnorum und Bombus terrestris). cours de leur torpeur hivernale. C.r. Soc. Biol. 168, Zool. .lb. Physiol. 88, 237-246. 952-958. R6seler P.-F. and R6seler I. (1986) Caste specific differences Maurer H. R. (1968) Disk-Elektrophorese. De Gruyter, in fat body glycogen metabolism of the bumblebee, Berlin. Bombus terrestris. Insect Biochem. 16, 501-508. Ornstein L. and Davies B. J. (1962) Disc-Electrophoresis. Steele J. E. (1983) Endocrine control of carbohydrate Distillation Industries, Rochester, New York. metabolism in insects. In Endocrinolgy of Insects (Edited Pouvreau A. (1976) Contribution fi la biologie des by Downer R. G. H. and Laufer H.), pp. 427-439. Liss, bourdons. Etude de quelques param~tres ~cologiques et New York. physiologiques en relation avec l'hibernation des reines. Thomas J. A., Schlender K. K. and Larner J. (1968) A rapid Th~se, Paris. filter paper assay for UDP-glucose-glycogen glucosylRoe J. H. and Dailey R. R. (1966) Determination of transferase, including an improved biosynthesis for UDPglycogen with the anthrone reagent. Analyt. Biochem. 15, ~4C-glucose. Analyt. Biochem. 25, 486~99. 245-250. Ziegler R., Ashida M., Fallon A. M., Wimer L. T. Wyatt Rfseler P.-F. (1976) Juvenile hormone and queen rearing in S. S. and Wyatt G. R. (1979) Regulation of glycogen bumblebees. In Phase and Caste Determination in Insects. phosphorylase in fat body of Cecropia silkmoth pupae. Endocrine Aspects (Edited by Liischer M.), pp. 55-61. J. comp. Physiol. 131, 321-332. Pergamon Press, Oxford. Zfllner H. and Kirsch K. (1962) 12ber die quantitative Rfseler P.-F. (1977a) Endocrine control of polymorphism Bestimmung von Lipoiden (Mikromethode) mittels der in bumblebees. Proc. VIII Int. Cong. IUSSI, Wageningen, vielen natiirlichen Lipoiden (allen bekannten Plasmalipp. 22-23. poiden) gemeinsamen Sulphophosphovanillin-Reaktion. Z. ges. exp. Med. 135, 545-561.