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Control of cuticular tanning in the cockroach: Bursicon release by nervous stimulation
J. Insect Physiol., 1967, Vol. 13, pp. 815 to 820. Pergamon Press Ltd. Printed in Geat Britain
CONTROL OF CUTICULAR TANNING IN THE COCKROACH: BURSICON RELEASE BY NERVOUS STIMULATION RICHARD R. MILLS Department of Biology, Tulane University, New Orleans, Louisiana (Received 9 August 1966)
Abstract-Severing the central nerve chord of Periplaneta americana stops the release of the tanning hormone (bursicon) from the terminal abdominal ganglion. Disrupting the stomatogastricnervous system fails to alter the tanning response. 1n tivo stimulation of the nerve chord induces the terminal ganglion to release the hormone. The strength and/or duration of the stimulus have no apparent effect on the extent of secretion. INTRODUCTION
RECENTinvestigations on the hormonal and nervous control of tanning in the blowfly have revealed that the brain is responsible for the tanning process and that a blood-borne hormone is involved (FRAENKELand HSIAO, 1962; COTTRELL,1962a, b). Similar studies on the American cockroach have shown that the hormone is secreted from the terminal abdominal ganglion although it could not be determined if the brain was responsible for its release (MILLS et al., 1965). Severing the central nerve chord and the recurrent nerve in Calliphoru eliminates the tanning response (FRAENKELand HSIAO, 1962). This would imply that the hormone release is under nervous control. However, experiments using the cockroach have shown that isolated ganglia are capable of secreting bursicon (MILLS, 1965). These data suggest that in the cockroach, the hormonal release is not under nervous control, which would be in direct contrast to the work of FRAENKELand HSIAO (1962). The following experiments were designed to elucidate the mechanism by which hormone secretion is controlled and to explain why isolated terminal abdominal ganglia could tan small pieces of cuticle. All experiments were performed with the American cockroach Periplaneta americana (L.). METHODS Sixth- to last-instar larvae of the American cockroach were held in cages as previously described (MILLS, 1966b). Newly ecdysed animals were removed as needed and used in all experiments. Tanning assays were conducted by the isolated cuticle procedure (MILLS, 1965) and occasional checks were made with the ligated thorax method (MILLS et al, 1965). Electrical stimulation was accomplished by the procedure described in Table 3. 815
RICHARDR. MILLS
816
RESULTS
Severing the central nerve chord between the posterior two ganglia inhibited the releaseof the tanning hormone from the terminalabdominal ganglion. This was determined by assaying blood samples at different times after cutting the nerve chord. From Table 1 it can be seen that blood samples taken 20 and 30 min after TABLE ~-THE
EFFECTOF SEVERING THE CENTRALNERVECHORDON THE CONCENTRATION OF BURSICONIN THE HAEMOLYMPH
Time after ecdysis that nerve chord was cut (mm)
Time after cut that blood was taken (min)
15
Dilution of blood
Tanning score
0 10 20 30
Undiluted
2 2 0 0
30
0 10 20 30
Undiluted
6 6 4 4
60
0 10 20 30
Diluted Q
7 6 3 2-3
90
0 10 20 30
Diluted i
10 10 8 8
120
0 10 20 30
Diluted GJ
5 5 2 0
Bursicon concentration was determined by assaying the ability of the blood to darken small circles of washed cuticle (MILLS, 1965). The cuticle taken from the first two abdominal segments retained the epidermal cell layer but was devoid of muscle or fat-body tissue. The assay was conducted in small test-tubes (max. vol. 3 ml) containing the piece of cuticle and 20 ~1 of blood. Blood was obtained by splitting the coxa and allowing it to flow up a capillary tube of predetermined volume (MILLS, 1966a). Dilutions were necessary when the haemolymph was taken at 60, 90, and 120 min after severing the nerve chord because undiluted blood always gave a value of 10. The dilution media were as previously described (MILLS et al., 1965). White untanned’cuticle was arbitrarily assigned a value of 0 and normally tanned cuticle a value of 10. Intermediate shades denoted as 2, 4, 6, and 8 were set up in sequence and experimentally darkened cuticle was compared with this series.
CONTROL
817
OFCUTICULAR1;ANNINGINTHECOCKROACH
the operation show a decrease in activity. The two operations performed 30 and 60 min after ecdysis are particularly significant since there is normally a sharp rise in hormone titre at this point (MILLS, 1966a). A number of other incisions were made and the nerve chord was severed at several points throughout the central nervous system. Additional experiments involving the disruption of the stomatogastric nervous system were inconclusive. These data are summarized in Table 2. TABLET-THEEFFECTOFSBVERINGTHECENTRALNERVECHORD
ONHORMONERELIWE
Location of cut
Animals
Animals surviving
Inhibition (%I
Between second and third abdominal ganglia
11
10
80flO
Between third thoracic and first abdominal ganglia
12
10
8Oj:lO
Between first and second thoracic
12
10
90+5
Circumesophageal connectives
17
10
80flO
Recurrent nerve anterior to corpora cardiaca
-34
10
25k35
Recurrent nerve posterior to corpora allata
16
10
30240
Operations were performed 60 min after ecdysis. Blood was taken at the time of the operation and 30 min later. Zero time blood was denoted as 100 per cent and 30 min blood was determined,as a percentage of this. Both the ligated thorax and the isolated cuticle methods were used to assay tanning activity.
The data presented above show that the hormone secretion is terminated 20 to 40 min after the nerve chord is cut. This indicates that the terminal abdominal ganglion is no longer receiving the necessary stimuli or that some type of inhibitory mechanism becomes functional due to the operation. It is possible that the sites of secretion have some form of built-in inhibitor which is suppressed by the brain. It could then become active after the chord is severed and subsequently cut off the release of the hormone. In order to determine which of these hypotheses was correct, the central nerve chord was cut between the second and third abdominal ganglia and stimulating electrodes placed beneath the cut end. Recording electrodes were placed between the fifth and terminal ganglia to measure the strength of the stimulus going into the terminal ganglion. This also provided a check on changes in endogenous activity. The apparatus used is shown in Fig. 1.
818
RICHARDR. MILLS
FIG. 1. A schematic diagram illustrating the apparatus used for determining that the hormone release was by nervous stimulation. (1) Saline, (2) haemolymph, (3) stimulating electrodes, (4) toothpick, (5) nerve chord, (6) a ganglion, (7) recording electrodes, (8) glass capsule, (9) buffer solution, (10) piece of untanned cuticle, (11) terminal abdominal ganglion. A small piece of untanned cuticle was placed in the test-tube with the intact terminal abdominal ganglion. The final volume was adjusted to 0.5 ml with buffer. Stimulation of the cut ends of the nerve chord caused a release of the hormone from the terminal ganglion which activated the tanning of the piece of cuticle. TABLE ~-EFFECT
OF
ELECTRICALSTIMULATIONON THE
HORMONE
Stimulation
RELEASE
No. of animals
Strength (mv)
Duration (msec)
Hormone release (variation was _I 1)
Nerve chord not severed
7 6 6 4 4
1 1 1 3 3
2 4 6 4 6
7 7 8 8 8
Nerve chord severed
5 5 5 5 5 6 6 6
1 1 1 2 2 3 3 3
2 4 6 4 6 2 4 6
7 7 7 8 8 8 8 8
Non-operated controls
12
-
-
5
Operated controls
12
-
-
4
Operation
Stimulation was done once every 3 min over a Q hr period and the amount of hormone release was determined by the isolated cuticle method. Controls were not stimulated. Electrical stimulation was accomplished by the use of a Model S4G Grass Stimulator and SIU4B isolation unit. Recording was done with a Tetronix 502A dual-beam oscilloscope. A type FM 122 preamplifier powered by a FM 125 powerpack was connected
CONTROL
OF CUTICULARTANNING
IN THECOCKROACH
819
between the preparation and the oscilloscope. All electrodes were made of fine silver wire. The release of the hormone by stimulation was measured &zz&o in the following manner: the terminal abdominal ganglion was freed of connecting nerves and placed in a small gelatin or glass capsule containing a small piece of untanned cuticle and some buffer. A solution of saline (0.4% NaCl, 0+3% KC1 in O-1 M &&acetate buffer, pH 7-2) was used to keep the nerve chord wet. The chord was elevated during stimulation. Controls were prepared in an identical manner, but were never stimulated. This techhique is illustrated by Fig. 1.
Stimulation of the nerve chord produced a greater hormonal release in the experimentals than in the controls. The strength and/or duration of the stimulus did not significantly change the amount of hormone secretion as measured by the tam-ring of the isolated cuticle method. However, a more accurate analysis of hormone concentration may disprove this conclusion. The results of these experiments are summarized in Table 3.
DISCUSSION
The results obtained in this investigation show that stimuli carried posteriorly through the central nervous system are responsible for the release of the tanning hormone. The evidence is as follows: (1) Severing the nerve chord stops the secretion of bursicon. (2) Severing the stomatogastric nervous system appears to have little effect. (3) Stimulation of the cut ends of the central nerve chord causes a limited release of the hormone, therefore, eliminating the possibility of an inhibitory mechanism. This conclusion agrees with previously published observations that the hormonal secretion in Calliphora is under nervous control (FRAJINKEL and HSIAO, 1962). The results of this study also offers an explanation of why the isolated terminal abdominal ganglia are able to secrete tanning hormone in the absence of nervous stimuli. For 30 to 40 min after the connecting nerves have been removed, the ganglion continues to release bursicon. It is possible that nervous stimuli cause the transfer of the hormone from neurosecretory cell sites in the ganglion to ducts or canals from which it is released. Material already’ in the ducts could be discharged without the necessity of a stimulus from the central nervous system. Disruption of the stomatogastric nervous system appeared to have little effect on the tanning process. This is in contrast to results obtained for the blowfly Calliphora. However, bursicon is released from either the head or thorax in Calliphora (FRAENKEL and HSIAO, 1962) which is in close proximity to the stomatogastric nervous system. On the other hand, bursicon is released from the terminal abdominal ganglion in the American cockroach (MILLS et al., 1965) which is located in the seventh segment. Usually the stomadeum does not extend to this point in the cockroach and so would be devoid of connecting nerves. AcknowZe&ement-This work was supported in part by a grant from the National Science Foundation GB-4773.
820
RICHARDR. MILLS REFERENCES
COTTRELL C. B. (1962a) The imaginal ecdysis of blowflies. The control of cuticular hardening and darkening. g. exp. Biol. 39, 395-411. COTTFOZLLC. B. (1962b) The imaginal ecdysis of blowflies. Detection of the blood-borne darkening factor and determination of some of its properties. J. exp. Biol. 39,413-430. FRAENKELG. and HSIAO C. (1962) Hormonal and nervous control of tanning in the fly. Science, N.Y. i38, 27-29. MILLS R. R. (1965) Hormonal control of tanning in the American cockroach-II. Assay for the hormone and the effect of wound healing. J. Insect Physiol. 11,1268-1275. MILLS R. R., MATHURR. B., and GUERRAA. A. (1965) Studies on the hormonal control of tanning in the American cockroach-I. Release of an activation factor from the terminal abdominal ganglion. J. Insect Physiol. 11,1047-1053. MILLS R. R. (1966a) Hormonal control of tanning in the American cockroach-Hi. Hormone stability and post-ecdysial changes in hormone titre. J. Insect Physiol. 12, 275-280. MILLS R. R. (1966b) A cockroach rearing cage designed for the simple collection of oiitheca. 3. econ. Ent. 59, 490.
CONTROL OF CUTICULAR TANNING IN THE COCKROACH: BURSICON RELEASE BY NERVOUS STIMULATION RICHARD R. MILLS Department of Biology, Tulane University, New Orleans, Louisiana (Received 9 August 1966)
Abstract-Severing the central nerve chord of Periplaneta americana stops the release of the tanning hormone (bursicon) from the terminal abdominal ganglion. Disrupting the stomatogastricnervous system fails to alter the tanning response. 1n tivo stimulation of the nerve chord induces the terminal ganglion to release the hormone. The strength and/or duration of the stimulus have no apparent effect on the extent of secretion. INTRODUCTION
RECENTinvestigations on the hormonal and nervous control of tanning in the blowfly have revealed that the brain is responsible for the tanning process and that a blood-borne hormone is involved (FRAENKELand HSIAO, 1962; COTTRELL,1962a, b). Similar studies on the American cockroach have shown that the hormone is secreted from the terminal abdominal ganglion although it could not be determined if the brain was responsible for its release (MILLS et al., 1965). Severing the central nerve chord and the recurrent nerve in Calliphoru eliminates the tanning response (FRAENKELand HSIAO, 1962). This would imply that the hormone release is under nervous control. However, experiments using the cockroach have shown that isolated ganglia are capable of secreting bursicon (MILLS, 1965). These data suggest that in the cockroach, the hormonal release is not under nervous control, which would be in direct contrast to the work of FRAENKELand HSIAO (1962). The following experiments were designed to elucidate the mechanism by which hormone secretion is controlled and to explain why isolated terminal abdominal ganglia could tan small pieces of cuticle. All experiments were performed with the American cockroach Periplaneta americana (L.). METHODS Sixth- to last-instar larvae of the American cockroach were held in cages as previously described (MILLS, 1966b). Newly ecdysed animals were removed as needed and used in all experiments. Tanning assays were conducted by the isolated cuticle procedure (MILLS, 1965) and occasional checks were made with the ligated thorax method (MILLS et al, 1965). Electrical stimulation was accomplished by the procedure described in Table 3. 815
RICHARDR. MILLS
816
RESULTS
Severing the central nerve chord between the posterior two ganglia inhibited the releaseof the tanning hormone from the terminalabdominal ganglion. This was determined by assaying blood samples at different times after cutting the nerve chord. From Table 1 it can be seen that blood samples taken 20 and 30 min after TABLE ~-THE
EFFECTOF SEVERING THE CENTRALNERVECHORDON THE CONCENTRATION OF BURSICONIN THE HAEMOLYMPH
Time after ecdysis that nerve chord was cut (mm)
Time after cut that blood was taken (min)
15
Dilution of blood
Tanning score
0 10 20 30
Undiluted
2 2 0 0
30
0 10 20 30
Undiluted
6 6 4 4
60
0 10 20 30
Diluted Q
7 6 3 2-3
90
0 10 20 30
Diluted i
10 10 8 8
120
0 10 20 30
Diluted GJ
5 5 2 0
Bursicon concentration was determined by assaying the ability of the blood to darken small circles of washed cuticle (MILLS, 1965). The cuticle taken from the first two abdominal segments retained the epidermal cell layer but was devoid of muscle or fat-body tissue. The assay was conducted in small test-tubes (max. vol. 3 ml) containing the piece of cuticle and 20 ~1 of blood. Blood was obtained by splitting the coxa and allowing it to flow up a capillary tube of predetermined volume (MILLS, 1966a). Dilutions were necessary when the haemolymph was taken at 60, 90, and 120 min after severing the nerve chord because undiluted blood always gave a value of 10. The dilution media were as previously described (MILLS et al., 1965). White untanned’cuticle was arbitrarily assigned a value of 0 and normally tanned cuticle a value of 10. Intermediate shades denoted as 2, 4, 6, and 8 were set up in sequence and experimentally darkened cuticle was compared with this series.
CONTROL
817
OFCUTICULAR1;ANNINGINTHECOCKROACH
the operation show a decrease in activity. The two operations performed 30 and 60 min after ecdysis are particularly significant since there is normally a sharp rise in hormone titre at this point (MILLS, 1966a). A number of other incisions were made and the nerve chord was severed at several points throughout the central nervous system. Additional experiments involving the disruption of the stomatogastric nervous system were inconclusive. These data are summarized in Table 2. TABLET-THEEFFECTOFSBVERINGTHECENTRALNERVECHORD
ONHORMONERELIWE
Location of cut
Animals
Animals surviving
Inhibition (%I
Between second and third abdominal ganglia
11
10
80flO
Between third thoracic and first abdominal ganglia
12
10
8Oj:lO
Between first and second thoracic
12
10
90+5
Circumesophageal connectives
17
10
80flO
Recurrent nerve anterior to corpora cardiaca
-34
10
25k35
Recurrent nerve posterior to corpora allata
16
10
30240
Operations were performed 60 min after ecdysis. Blood was taken at the time of the operation and 30 min later. Zero time blood was denoted as 100 per cent and 30 min blood was determined,as a percentage of this. Both the ligated thorax and the isolated cuticle methods were used to assay tanning activity.
The data presented above show that the hormone secretion is terminated 20 to 40 min after the nerve chord is cut. This indicates that the terminal abdominal ganglion is no longer receiving the necessary stimuli or that some type of inhibitory mechanism becomes functional due to the operation. It is possible that the sites of secretion have some form of built-in inhibitor which is suppressed by the brain. It could then become active after the chord is severed and subsequently cut off the release of the hormone. In order to determine which of these hypotheses was correct, the central nerve chord was cut between the second and third abdominal ganglia and stimulating electrodes placed beneath the cut end. Recording electrodes were placed between the fifth and terminal ganglia to measure the strength of the stimulus going into the terminal ganglion. This also provided a check on changes in endogenous activity. The apparatus used is shown in Fig. 1.
818
RICHARDR. MILLS
FIG. 1. A schematic diagram illustrating the apparatus used for determining that the hormone release was by nervous stimulation. (1) Saline, (2) haemolymph, (3) stimulating electrodes, (4) toothpick, (5) nerve chord, (6) a ganglion, (7) recording electrodes, (8) glass capsule, (9) buffer solution, (10) piece of untanned cuticle, (11) terminal abdominal ganglion. A small piece of untanned cuticle was placed in the test-tube with the intact terminal abdominal ganglion. The final volume was adjusted to 0.5 ml with buffer. Stimulation of the cut ends of the nerve chord caused a release of the hormone from the terminal ganglion which activated the tanning of the piece of cuticle. TABLE ~-EFFECT
OF
ELECTRICALSTIMULATIONON THE
HORMONE
Stimulation
RELEASE
No. of animals
Strength (mv)
Duration (msec)
Hormone release (variation was _I 1)
Nerve chord not severed
7 6 6 4 4
1 1 1 3 3
2 4 6 4 6
7 7 8 8 8
Nerve chord severed
5 5 5 5 5 6 6 6
1 1 1 2 2 3 3 3
2 4 6 4 6 2 4 6
7 7 7 8 8 8 8 8
Non-operated controls
12
-
-
5
Operated controls
12
-
-
4
Operation
Stimulation was done once every 3 min over a Q hr period and the amount of hormone release was determined by the isolated cuticle method. Controls were not stimulated. Electrical stimulation was accomplished by the use of a Model S4G Grass Stimulator and SIU4B isolation unit. Recording was done with a Tetronix 502A dual-beam oscilloscope. A type FM 122 preamplifier powered by a FM 125 powerpack was connected
CONTROL
OF CUTICULARTANNING
IN THECOCKROACH
819
between the preparation and the oscilloscope. All electrodes were made of fine silver wire. The release of the hormone by stimulation was measured &zz&o in the following manner: the terminal abdominal ganglion was freed of connecting nerves and placed in a small gelatin or glass capsule containing a small piece of untanned cuticle and some buffer. A solution of saline (0.4% NaCl, 0+3% KC1 in O-1 M &&acetate buffer, pH 7-2) was used to keep the nerve chord wet. The chord was elevated during stimulation. Controls were prepared in an identical manner, but were never stimulated. This techhique is illustrated by Fig. 1.
Stimulation of the nerve chord produced a greater hormonal release in the experimentals than in the controls. The strength and/or duration of the stimulus did not significantly change the amount of hormone secretion as measured by the tam-ring of the isolated cuticle method. However, a more accurate analysis of hormone concentration may disprove this conclusion. The results of these experiments are summarized in Table 3.
DISCUSSION
The results obtained in this investigation show that stimuli carried posteriorly through the central nervous system are responsible for the release of the tanning hormone. The evidence is as follows: (1) Severing the nerve chord stops the secretion of bursicon. (2) Severing the stomatogastric nervous system appears to have little effect. (3) Stimulation of the cut ends of the central nerve chord causes a limited release of the hormone, therefore, eliminating the possibility of an inhibitory mechanism. This conclusion agrees with previously published observations that the hormonal secretion in Calliphora is under nervous control (FRAJINKEL and HSIAO, 1962). The results of this study also offers an explanation of why the isolated terminal abdominal ganglia are able to secrete tanning hormone in the absence of nervous stimuli. For 30 to 40 min after the connecting nerves have been removed, the ganglion continues to release bursicon. It is possible that nervous stimuli cause the transfer of the hormone from neurosecretory cell sites in the ganglion to ducts or canals from which it is released. Material already’ in the ducts could be discharged without the necessity of a stimulus from the central nervous system. Disruption of the stomatogastric nervous system appeared to have little effect on the tanning process. This is in contrast to results obtained for the blowfly Calliphora. However, bursicon is released from either the head or thorax in Calliphora (FRAENKEL and HSIAO, 1962) which is in close proximity to the stomatogastric nervous system. On the other hand, bursicon is released from the terminal abdominal ganglion in the American cockroach (MILLS et al., 1965) which is located in the seventh segment. Usually the stomadeum does not extend to this point in the cockroach and so would be devoid of connecting nerves. AcknowZe&ement-This work was supported in part by a grant from the National Science Foundation GB-4773.
820
RICHARDR. MILLS REFERENCES
COTTRELL C. B. (1962a) The imaginal ecdysis of blowflies. The control of cuticular hardening and darkening. g. exp. Biol. 39, 395-411. COTTFOZLLC. B. (1962b) The imaginal ecdysis of blowflies. Detection of the blood-borne darkening factor and determination of some of its properties. J. exp. Biol. 39,413-430. FRAENKELG. and HSIAO C. (1962) Hormonal and nervous control of tanning in the fly. Science, N.Y. i38, 27-29. MILLS R. R. (1965) Hormonal control of tanning in the American cockroach-II. Assay for the hormone and the effect of wound healing. J. Insect Physiol. 11,1268-1275. MILLS R. R., MATHURR. B., and GUERRAA. A. (1965) Studies on the hormonal control of tanning in the American cockroach-I. Release of an activation factor from the terminal abdominal ganglion. J. Insect Physiol. 11,1047-1053. MILLS R. R. (1966a) Hormonal control of tanning in the American cockroach-Hi. Hormone stability and post-ecdysial changes in hormone titre. J. Insect Physiol. 12, 275-280. MILLS R. R. (1966b) A cockroach rearing cage designed for the simple collection of oiitheca. 3. econ. Ent. 59, 490.