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Control of germarial activity and yolk deposition in non-terminal oöcytes of Lucilia cuprina
J. InsectPhysiol.,1971, Vol. 17, pp. 601 to 606.
Pergamon Press. Printed in Great Britain
CONTROL OF GERMARIAL ACTIVITY AND YOLK DEPOSITION IN NON-TERMINAL OiiCYTES OF
LUCILIA
CUPRINA
A. D. CLIFT Department of Agricultural Botany, Faculty of Agriculture, University of Sydney, Sydney 2006, Australia (Received 15 September 1970) Abstract-Evidence is presented for a feedback mechanism in Lucilia cuprina which regulates the total number and stage of development of the ovarian follicles in each ovariole. A new neurosecretory material is thought to be involved in the control of the development of squamous follicle cells over the nurse cells. The implication of this new system is considered with respect to generally recognized features of the endocrine control of vitellogenesis in muscoid diptera. INTRODUCTION ENCELMANN (1968) described ovarian development in viviparous cockroaches in which development is prevented when mature eggs are present in the oviduct. ADAMS et al. (1968) reported that the penultimate oijcyte in Mum domestica did not develop to maturity until the terminal oijcyte had been laid. They also showed that this inhibition was controlled by a hormone which they called the ‘ oastatic hormone ‘. ADAMSet al. (1968) and ADAMS (1970) attributed the action of oiistatic hormone to inhibition of release of juvenile hormone from the corpus allatum. TELFER (1965) h as reviewed the literature on mechanisms of yolk formation in insect oijcytes. The bulk of protein yolk is obtained from the haemolymph by pinocytosis at the oiicyte surface in most insects, and a certain specific configuration of follicle cells is required for this to occur. This configuration involves the columnar follicle cells over the oiicyte moving apart, remaining connected by fine cytoplasmic filaments to allow access of the haemolymph to the oijcyte surface. In the same paper Telfer stated that it is this process which seems to be affected by allatectomy. The r81e of the neurosecretory cells of the brain in CuZZiphoru (THOMSEN, 1952) was also assumed to be concerned in protein synthesis. However, WILKF.NS (1969) reported findings from Surcophaga that indicated the neurosecretory cells had a gonadotrophic function, and the corpus allatum controlled protein synthesis. This paper describes and discusses evidence obtained from L. cup&a, much of it indirect, indicating the possible mode of action of the oiistatic hormone. The 601 01
A.D.
CLIFT
problem of the roles of the neurosecretory partially resolved. MATERIALS
cells and corpus allatum has also been
AND
METHODS
Eggs were collected on sheep liver and larvae maintained on mutton stewing chops. A pupation medium in the form of vermiculite flakes was provided, from which the puparia are readily sieved. Adults emerge in the morning, usually before 11.00 a.m. under insectary conditions of 12 hr light : 12 hr dark at 27°C and r.h. 40 to 50%. For this series of experiments females were separated out within 24 hr of emergence. The virgin females were then placed in cages and provided with sugar, water, and liver at 24 and 48 hr after emergence. Under such conditions most females had mature eggs by 96 hr. Virgin females will not oviposit, so can be fed excess liver without any effect on the terminal oocyte. In one experiment liver was fed at 24, 48, and 72 hr after emergence to determine the effect of excess liver before the terminal oocyte was mature. Part of each ovary was dissected out into a drop of water on a slide, the individual ovarioles teased out and covered with a cover-slip. Females were killed in ice cold ethanol and rapidly transferred to a saline solution (THOMSEN, 1952). To examine the corpus cardiacurn the fly was mounted vertically in a block of Plasticine and the head bent forward to expose the cervical membrane. The whole block was then immersed in saline and examined. The cervical membrane was then removed and fat body and muscles cleared away to reveal the corpus cardiacurn. The neurosecretory material within was visible as a bluish-white substance constrained within 9 to 12 vesicles. RESULTS
AND
DISCUSSION
Females fed liver at 24 and 48 hr could be maintained for at least 3 weeks after the initial 96 hr without any yolk being deposited in the penultimate oocyte. Further, the germarium becomes inactive after producing a total of three ovarian follicles. A single liver meal after 96 hr results in an additional ovarian follicle, rarely two, being produced, and a small amount of yolk being deposited in the penultimate oijcyte of some flies. If the additional liver meal is given before 96 hr, then fewer flies produce additional follicles. The data is presented more fully in Table 1. In a separate experiment, 46 flies in which some yolk had been deposited in the penultimate follicle, 8 had four or more follicles per ovariole, 38 had three follicles. All of 25 flies in which no yolk had been deposited in the penultimate oocyte had three follicles per ovariole. At least two extra liver meals, i.e. four in all are required to cause complete development of the penultimate oiicyte. In such cases there are usually four or five ovarian follicles, but never more. Thus, the germarium may become active only if the penultimate oijcyte deposits at least some yolk. Yolk deposition on the other hand may occur without germarial activity.
GERMARIALACTIVITYAND YOLKDEPOSITION IN 0iiCYTE.S OF
LUCILIA CUPRINA
603
Two independent systems are postulated, the first suppresses germarial activity and yolk deposition, the second suppresses development of the penultimate oiicyte after a small amount of yolk has been deposited. TABLE ~-EFFECT OF AMOUNT AND TIMING OF PROTEIN MEALS ON NUMBER OF FOLLICLES PRODUCED PER OVARIOLE TWO WEEKS AFTER MATURATION OF TERMINAL 06CYll3
No. of follicles/ovariole 3 4 5 6
Fed liver 1,2 days after emergence
Fed liver 1, 2, 3 days after emergence
Fed liver 1, 2, 7 days after emergence
Fed liver 1, 2, 7,9 days after emergence
11 0 0 0
13 2 1 0
10 4 1 0
24 23 5 0
ADAMS et al. (1968) and ADAMS (1970) attributed the action of o&static hormone to inhibition of release of juvenile hormone from the corpus allaturn. The data mentioned above indicate that a more complex interplay of regulatory # mechanisms exist in L. cuprina The juvenile hormone is not known to have any effect on the germarium (ENGELMANN, 1968). However, the juvenile hormone can be implicated in prevention of yolk deposition and in the prevention of growth of the penultimate oiicyte after some yolk has been deposited. Both of these phenomena have been associated with allatectomy in muscoid diptera (ENGELMANN, 1968; ADAMS et al., 1969) and also in L. cup&a. The situation in L. cuprina with regard to the inhibition of the penultimate oijcyte is much more clear cut than results with surgical allatectomy, in that total inhibition of yolk deposition has not been claimed by any worker using muscoid diptera, yet in L. cup&a unlike Musca domestica it is possible to observe such inhibition in the penultimate oijcyte. Observations on Lucilia have shown that the follicle cell configuration required for pinocytosis to occur (TELFER, 1965) is attained when the follicle is 350 to 400 pm long, and the yolk may occupy up to one-seventh of the follicle. The penultimate follicles in L. cuprina are either inhibited before any yolk deposition begins, or before the required configuration is attained, but when some yolk is present. It was decided, on the basis of the abovementioned evidence to consider yolk deposition occurring in two phases separated by the change in appearance of the follicle cells. On this basis, inhibition of the penultimate follicle occurs either before phase I or at the end of phase I, and allatectomy will give similar results. It is known that neurosecretory material is stored in the corpus cardiacurn (E~~~~~~~~,1968)andthatthecorpuscardiacumcanpartiallyreplacethemedian
CaZZiphora(THOMSEN, 1952). In L. cuprina the corpus cardiacurn comprises 9 to 12 readily discernable vesicles. The appearance of these vesicles was studied in relation to the observations in oijcyte development. group of neurosecretory cellsin
604
A. D. CLIFT
It was observed that the secretion may, at different times be diffuse, opalescent, dense, or granular. If the proportion of vesicles with a diffuse appearance was higher than those with a dense appearance, a release of neurosecretory material was assumed to have occurred. Three releases were observed: (i) 2 to 5 hr after a liver meal; (ii) at the initiation of yolk deposition in the oocyte; (iii) at the attainment of the follicle cell configuration required for pinocytosis. In most cases gravid females were observed to have a preponderance of vesicles with a dense or granular appearance.
PROTE I N MEAL
GERMARIUM /
CORPUS
// /
FIG. 1. Schematic representation of control of cyclical ovarian development. (1) Mature ovum produces oastatic hormone which prevents growth of penultimate follicle. (2) Two yolkless follicles produce a substance which inhibits germarial activity. (3) A protein meal breaks (1) so that the penultimate oocyte resumes growth, and yolk &position may occur. (4) The corpus cardiacum is stimulated by a protein meal to release neurosecretory material which (5) stimulates the follicle cells to adopt the configuration required for vitellogenesis. Once some yolk deposition has occurred, the one yolkless follicle may not be able to produce enough inhibitor to prevent germarial activity.
GERMARIAL ACTIVITYAND YOLK DEPOSITION IN ObCYTESOF LUCILIA
CUpRlNA
605
It would appear from this indirect evidence that two independent systems are involved. (i) The oijstatic hormone preventing growth of ovarian tissue. It is suggested that the germarium can produce follicles at a constant rate. By the time three follicles have been produced, yolk deposition begins in the terminal oocyte. This follicle could then produce a substance, oiistatic hormone, which prevents further development of the penultimate oocyte, either directly or via the corpus allatum. The two yolkless follicles could also produce a substance that suppressed germarial activity. A protein meal could then break the inhibition of the penultimate follicle by the terminal follicle. If the penultimate oocyte deposits some yolk, there will then be only one yolkless oiicyte which in some cases may not produce enough inhibitor to prevent germarial activity so another follicle may be produced. (ii) A neurosecretory material which is stored in the corpus cardiacurn and which is required for the follicle cells to adopt the configuration required for pinocytosis and hence for vitellogenesis. The material is probably released in response to a protein meal. In the absence of such a meal the o&static hormone may inhibit the release of material from the corpus cardiacum. This is consistent with the effect of injecting oijstatic hormone (ADAMS, 1970). In view of the interrelationships between the corpus allatum and neurosecretory cells (LEA and THOMSEN, 1969) it is not unreasonable to suggest that the corpus allatum controls the synthesis of the abovementioned neurosecretory material and perhaps also its release from the corpus cardiacurn. Thus the oostatic hormone would mimic the effect of allatectomy in some circumstances because both the oijstatic hormone and the juvenile hormone affect the same general process. Both systems are represented diagrammatically in Fig. 1. Acknowledgements-I would like to express my thanks to Dr. D. GILMOUR, Senior Principal Research Scientist, C.S.I.R.O., for his many helpful suggestions. Also to Dr. D. T. ANDERSON, Department of Zoology, University of Sydney, for his advice on cytological changes in tigenesis and his encouragement in this work. Special thanks are due to Dr. F. J. D. MCDONALD, Lecturer in Agricultural Entomology, University of Sydney, who provided all the facilities used in this work and who critically read the manuscript.
REFERENCES ADAMST. S. (1970) Ovarian regulation of the corpus allatum in the housefly Musca domestica. J. Insect Physiol. 16, 369-381. ADAMST. S., HINTZ A. M., and POMONISJ. G. (1968) Oostatic hormone production in houseflies Musca domestica with developing ovaries. J, Insect Physiol. 14,983-993. ADAMST. S. and MULLA M. S. (1967) The reproductive biology of HippeZates coZZusor--II. Gametogenesis. Ann. ent. Sot. Am. 61, 368-372. ENCELMANNF. (1968) Endocrine control of reproduction in insects. A. Rew. Ent. 13, l-26. LEA A. 0. and THCIMSKNE. (1969) Size independent secretion by the corpus allatum of Calliphora erythrocephala. J. Insect Physiol. 15,447482.
606
A. D. CLIFT
W. H. (1965) The mechanism and control of yolk yolk formation. A. Rew. I&t. 10, 161-184. THOMSENE. (1952) Functional significance of the neurosecretory brain cells and the corpus cardiacum in the blowfly Calliphora erythrocephala. J. exp. Biol. 29,137-172. WILKENSJ. L. (1969) The endocrine control of protein metabolism as related to reproduction in the fleshfly Surcophagu bulluta. J. Insect Physiol. 15,1015-1024.
TELFER
Pergamon Press. Printed in Great Britain
CONTROL OF GERMARIAL ACTIVITY AND YOLK DEPOSITION IN NON-TERMINAL OiiCYTES OF
LUCILIA
CUPRINA
A. D. CLIFT Department of Agricultural Botany, Faculty of Agriculture, University of Sydney, Sydney 2006, Australia (Received 15 September 1970) Abstract-Evidence is presented for a feedback mechanism in Lucilia cuprina which regulates the total number and stage of development of the ovarian follicles in each ovariole. A new neurosecretory material is thought to be involved in the control of the development of squamous follicle cells over the nurse cells. The implication of this new system is considered with respect to generally recognized features of the endocrine control of vitellogenesis in muscoid diptera. INTRODUCTION ENCELMANN (1968) described ovarian development in viviparous cockroaches in which development is prevented when mature eggs are present in the oviduct. ADAMS et al. (1968) reported that the penultimate oijcyte in Mum domestica did not develop to maturity until the terminal oijcyte had been laid. They also showed that this inhibition was controlled by a hormone which they called the ‘ oastatic hormone ‘. ADAMSet al. (1968) and ADAMS (1970) attributed the action of oiistatic hormone to inhibition of release of juvenile hormone from the corpus allatum. TELFER (1965) h as reviewed the literature on mechanisms of yolk formation in insect oijcytes. The bulk of protein yolk is obtained from the haemolymph by pinocytosis at the oiicyte surface in most insects, and a certain specific configuration of follicle cells is required for this to occur. This configuration involves the columnar follicle cells over the oiicyte moving apart, remaining connected by fine cytoplasmic filaments to allow access of the haemolymph to the oijcyte surface. In the same paper Telfer stated that it is this process which seems to be affected by allatectomy. The r81e of the neurosecretory cells of the brain in CuZZiphoru (THOMSEN, 1952) was also assumed to be concerned in protein synthesis. However, WILKF.NS (1969) reported findings from Surcophaga that indicated the neurosecretory cells had a gonadotrophic function, and the corpus allatum controlled protein synthesis. This paper describes and discusses evidence obtained from L. cup&a, much of it indirect, indicating the possible mode of action of the oiistatic hormone. The 601 01
A.D.
CLIFT
problem of the roles of the neurosecretory partially resolved. MATERIALS
cells and corpus allatum has also been
AND
METHODS
Eggs were collected on sheep liver and larvae maintained on mutton stewing chops. A pupation medium in the form of vermiculite flakes was provided, from which the puparia are readily sieved. Adults emerge in the morning, usually before 11.00 a.m. under insectary conditions of 12 hr light : 12 hr dark at 27°C and r.h. 40 to 50%. For this series of experiments females were separated out within 24 hr of emergence. The virgin females were then placed in cages and provided with sugar, water, and liver at 24 and 48 hr after emergence. Under such conditions most females had mature eggs by 96 hr. Virgin females will not oviposit, so can be fed excess liver without any effect on the terminal oocyte. In one experiment liver was fed at 24, 48, and 72 hr after emergence to determine the effect of excess liver before the terminal oocyte was mature. Part of each ovary was dissected out into a drop of water on a slide, the individual ovarioles teased out and covered with a cover-slip. Females were killed in ice cold ethanol and rapidly transferred to a saline solution (THOMSEN, 1952). To examine the corpus cardiacurn the fly was mounted vertically in a block of Plasticine and the head bent forward to expose the cervical membrane. The whole block was then immersed in saline and examined. The cervical membrane was then removed and fat body and muscles cleared away to reveal the corpus cardiacurn. The neurosecretory material within was visible as a bluish-white substance constrained within 9 to 12 vesicles. RESULTS
AND
DISCUSSION
Females fed liver at 24 and 48 hr could be maintained for at least 3 weeks after the initial 96 hr without any yolk being deposited in the penultimate oocyte. Further, the germarium becomes inactive after producing a total of three ovarian follicles. A single liver meal after 96 hr results in an additional ovarian follicle, rarely two, being produced, and a small amount of yolk being deposited in the penultimate oijcyte of some flies. If the additional liver meal is given before 96 hr, then fewer flies produce additional follicles. The data is presented more fully in Table 1. In a separate experiment, 46 flies in which some yolk had been deposited in the penultimate follicle, 8 had four or more follicles per ovariole, 38 had three follicles. All of 25 flies in which no yolk had been deposited in the penultimate oocyte had three follicles per ovariole. At least two extra liver meals, i.e. four in all are required to cause complete development of the penultimate oiicyte. In such cases there are usually four or five ovarian follicles, but never more. Thus, the germarium may become active only if the penultimate oijcyte deposits at least some yolk. Yolk deposition on the other hand may occur without germarial activity.
GERMARIALACTIVITYAND YOLKDEPOSITION IN 0iiCYTE.S OF
LUCILIA CUPRINA
603
Two independent systems are postulated, the first suppresses germarial activity and yolk deposition, the second suppresses development of the penultimate oiicyte after a small amount of yolk has been deposited. TABLE ~-EFFECT OF AMOUNT AND TIMING OF PROTEIN MEALS ON NUMBER OF FOLLICLES PRODUCED PER OVARIOLE TWO WEEKS AFTER MATURATION OF TERMINAL 06CYll3
No. of follicles/ovariole 3 4 5 6
Fed liver 1,2 days after emergence
Fed liver 1, 2, 3 days after emergence
Fed liver 1, 2, 7 days after emergence
Fed liver 1, 2, 7,9 days after emergence
11 0 0 0
13 2 1 0
10 4 1 0
24 23 5 0
ADAMS et al. (1968) and ADAMS (1970) attributed the action of o&static hormone to inhibition of release of juvenile hormone from the corpus allaturn. The data mentioned above indicate that a more complex interplay of regulatory # mechanisms exist in L. cuprina The juvenile hormone is not known to have any effect on the germarium (ENGELMANN, 1968). However, the juvenile hormone can be implicated in prevention of yolk deposition and in the prevention of growth of the penultimate oiicyte after some yolk has been deposited. Both of these phenomena have been associated with allatectomy in muscoid diptera (ENGELMANN, 1968; ADAMS et al., 1969) and also in L. cup&a. The situation in L. cuprina with regard to the inhibition of the penultimate oijcyte is much more clear cut than results with surgical allatectomy, in that total inhibition of yolk deposition has not been claimed by any worker using muscoid diptera, yet in L. cup&a unlike Musca domestica it is possible to observe such inhibition in the penultimate oijcyte. Observations on Lucilia have shown that the follicle cell configuration required for pinocytosis to occur (TELFER, 1965) is attained when the follicle is 350 to 400 pm long, and the yolk may occupy up to one-seventh of the follicle. The penultimate follicles in L. cuprina are either inhibited before any yolk deposition begins, or before the required configuration is attained, but when some yolk is present. It was decided, on the basis of the abovementioned evidence to consider yolk deposition occurring in two phases separated by the change in appearance of the follicle cells. On this basis, inhibition of the penultimate follicle occurs either before phase I or at the end of phase I, and allatectomy will give similar results. It is known that neurosecretory material is stored in the corpus cardiacurn (E~~~~~~~~,1968)andthatthecorpuscardiacumcanpartiallyreplacethemedian
CaZZiphora(THOMSEN, 1952). In L. cuprina the corpus cardiacurn comprises 9 to 12 readily discernable vesicles. The appearance of these vesicles was studied in relation to the observations in oijcyte development. group of neurosecretory cellsin
604
A. D. CLIFT
It was observed that the secretion may, at different times be diffuse, opalescent, dense, or granular. If the proportion of vesicles with a diffuse appearance was higher than those with a dense appearance, a release of neurosecretory material was assumed to have occurred. Three releases were observed: (i) 2 to 5 hr after a liver meal; (ii) at the initiation of yolk deposition in the oocyte; (iii) at the attainment of the follicle cell configuration required for pinocytosis. In most cases gravid females were observed to have a preponderance of vesicles with a dense or granular appearance.
PROTE I N MEAL
GERMARIUM /
CORPUS
// /
FIG. 1. Schematic representation of control of cyclical ovarian development. (1) Mature ovum produces oastatic hormone which prevents growth of penultimate follicle. (2) Two yolkless follicles produce a substance which inhibits germarial activity. (3) A protein meal breaks (1) so that the penultimate oocyte resumes growth, and yolk &position may occur. (4) The corpus cardiacum is stimulated by a protein meal to release neurosecretory material which (5) stimulates the follicle cells to adopt the configuration required for vitellogenesis. Once some yolk deposition has occurred, the one yolkless follicle may not be able to produce enough inhibitor to prevent germarial activity.
GERMARIAL ACTIVITYAND YOLK DEPOSITION IN ObCYTESOF LUCILIA
CUpRlNA
605
It would appear from this indirect evidence that two independent systems are involved. (i) The oijstatic hormone preventing growth of ovarian tissue. It is suggested that the germarium can produce follicles at a constant rate. By the time three follicles have been produced, yolk deposition begins in the terminal oocyte. This follicle could then produce a substance, oiistatic hormone, which prevents further development of the penultimate oocyte, either directly or via the corpus allatum. The two yolkless follicles could also produce a substance that suppressed germarial activity. A protein meal could then break the inhibition of the penultimate follicle by the terminal follicle. If the penultimate oocyte deposits some yolk, there will then be only one yolkless oiicyte which in some cases may not produce enough inhibitor to prevent germarial activity so another follicle may be produced. (ii) A neurosecretory material which is stored in the corpus cardiacurn and which is required for the follicle cells to adopt the configuration required for pinocytosis and hence for vitellogenesis. The material is probably released in response to a protein meal. In the absence of such a meal the o&static hormone may inhibit the release of material from the corpus cardiacum. This is consistent with the effect of injecting oijstatic hormone (ADAMS, 1970). In view of the interrelationships between the corpus allatum and neurosecretory cells (LEA and THOMSEN, 1969) it is not unreasonable to suggest that the corpus allatum controls the synthesis of the abovementioned neurosecretory material and perhaps also its release from the corpus cardiacurn. Thus the oostatic hormone would mimic the effect of allatectomy in some circumstances because both the oijstatic hormone and the juvenile hormone affect the same general process. Both systems are represented diagrammatically in Fig. 1. Acknowledgements-I would like to express my thanks to Dr. D. GILMOUR, Senior Principal Research Scientist, C.S.I.R.O., for his many helpful suggestions. Also to Dr. D. T. ANDERSON, Department of Zoology, University of Sydney, for his advice on cytological changes in tigenesis and his encouragement in this work. Special thanks are due to Dr. F. J. D. MCDONALD, Lecturer in Agricultural Entomology, University of Sydney, who provided all the facilities used in this work and who critically read the manuscript.
REFERENCES ADAMST. S. (1970) Ovarian regulation of the corpus allatum in the housefly Musca domestica. J. Insect Physiol. 16, 369-381. ADAMST. S., HINTZ A. M., and POMONISJ. G. (1968) Oostatic hormone production in houseflies Musca domestica with developing ovaries. J, Insect Physiol. 14,983-993. ADAMST. S. and MULLA M. S. (1967) The reproductive biology of HippeZates coZZusor--II. Gametogenesis. Ann. ent. Sot. Am. 61, 368-372. ENCELMANNF. (1968) Endocrine control of reproduction in insects. A. Rew. Ent. 13, l-26. LEA A. 0. and THCIMSKNE. (1969) Size independent secretion by the corpus allatum of Calliphora erythrocephala. J. Insect Physiol. 15,447482.
606
A. D. CLIFT
W. H. (1965) The mechanism and control of yolk yolk formation. A. Rew. I&t. 10, 161-184. THOMSENE. (1952) Functional significance of the neurosecretory brain cells and the corpus cardiacum in the blowfly Calliphora erythrocephala. J. exp. Biol. 29,137-172. WILKENSJ. L. (1969) The endocrine control of protein metabolism as related to reproduction in the fleshfly Surcophagu bulluta. J. Insect Physiol. 15,1015-1024.
TELFER