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Suggested regulatory mechanisms for caudal regeneration in Allolobophora Molleri (Annelida: Oligochaeta)
Camp. Biochem. Physiol. Vol. SlA, No. 2, pp. 225-228, 1985
0300..9629/85 $3.00 + 0.00 Q 1985 PergamonPress Ltd
Printedin Great Britain
SUGGESTED REGULATORY MECHANISMS FOR CAUDAL REGENERATION IN ALLOLOBOPIrJORA MOLLERI (ANNELIDA: OLIGOCHAETA) M.
ALONSO-BEDATE
and E.
SEQUEROS
Departamento de Fisiologia Animal, Facultad de Ciencias Biologicas, Universidad Complutense, Madrid, Spain (Received 12 July 1984)
Abstract-l. In young Allolobophora molleri the brain is required for caudal regeneration. When the brain was present there was a high percentage (100%) of regenerated worms, but this percentage declined (2%) when the brain was removed. 2. A stimulatory factor or Growth Factor from the brain is probably required for release of a Regenerating Factor from suboesophageal ganglia or nerve cord neurosecretory cells. 3. In adult worms at clitellar stage the brain plays an inhibitory role on caudal regeneration. When the brain was present there was a low percentage (34%) of regenerated worms, but this percentage was higher when the brain was removed (80%). 4. Probably a Clitellum Factor from neurosecretory cells at supraoesophageal ganglia is responsible for inhibition of the neurosecretory cells activity that release a Regenerating Factor from suboesophageal ganglia or ventral nerve cord. 5. Finally, an intermediate situation could be observed in adult unclitellated worms.
INTRODUCTION
Since the first description Lumbricus
of neurosecretory cells in terrestris by Scharrer (1937) many in-
vestigations have been published dealing with the structure and function of these elements. In addition to the neurosecretory peptidergic cells, monoamine producing neurons were also demonstrated in the cerebral ganglion and ventral nerve cord in Lumbricus terrestris (Ehinger et aI., 1971; de Vries-Schoumacker, 1977). The introduction of immunocytochemical methods revealed the chemical composition of materials elaborated in the neurons of the central nervous system of the earthworms. Pancreatic polypeptide-like substance or vasoactive intestinal peptide, opiate-like materials, and endorphin and leucoenkephalin, substance P and ACTH-like activity could be detected (Sundler et al., 1977; Alumets et al., 1979; Remy et al., 1979; Aros et al., 1980). During the last twenty years, many works have been published about the possible function of neurosecretion on regeneration, reproduction and metabolism in Oligochaeta. It appears that in all earthworms which have been examined, the nervous system plays a role, either direct or indirect, in the control of regeneration (Hubl, 1956; Gallissian, 1973; Maissait, 1965). Herlant Meewis (1965, 1966) established the involvement of the ventral nerve cord in both anterior and posterior regeneration in Eisenia foetida. Removal of the brain of Eophila dollfusi followed by amputation of caudal segments induces diapause during which segment regeneration occurs (Gallissian, 1973). This might suggest that the brain produced a neurohormone which inhibits caudal regeneration. On the other hand some studies on Lumbricus argue for a stimulatory effect of the brain 225
neurosecretion on caudal regeneration (Chapron and Chapron, 1973). Marcel (1973) established the presence of specific inhibitory and trophic substances in the head and tail that are important in order to modify the activity of neurosecretory cells during regeneration. According to Moment (1979) the neurosecretory system plays an important role particularly in initiating regeneration in the earthworm Eisenia foetida and supposes that the neurosecretory cells are needful in the initial stages but not later, and are therefore not directly involved in the determination of the number of segments proliferated. Our present report is concerned with the role of the central nervous system on the phenomenon of caudal regeneration in Allolobophora molleri (Annelida: Oligochaeta). The possibility of a different mechanism for control of caudal regeneration depending on the animal age and reproductive stage in A. molleri has been studied. MATERIALS AND METHODS
Specimens of Allolobophora molleri were collected around Madrid from September to August. The animals were in clitellar and non-clitellar stages. The worms were starved for 24 hr prior to testing in order to clear the gut. They were not fed during the experiment because decerebrated and decapitated worms are unable to feed. All the animals were maintained at the laboratory temperature (22 k 3”C), in individual Petri plates with moist paper filter at the bottom. Dechlorinated fresh water was provided daily. The animals were maintained in dark conditions. Extirpation
of the supra and sub-oesophageal ganglia
The worms were anesthesized with 8% ethanol. Each worm was placed in a Petri dish with the body resting on a block of modeling clay. The supraoesophageal ganglia
M. ALONSO-BEDATE and E.
226
were removed under a dissecting microscope. Worms were placed dorsal side up. A small window was cut in the dorsal anterior portion of the head, between the first and second segments. The underlying muscles were gently pulled aside with an entomological pin. This allowed easy access to the supraoesophageal ganglia. The brain was lifted with forceps and removed. For extirpation of the infraoesophageal ganglia a small window was cut in the ventral portion of the head between the fifth and seventh segments. Then, the tissues were separated and the ganglia were removed. Finally, decapitation was accomplished by pulling the head (5-7 segments) away with fine dissecting scissors. Always, the amputation of about 40-50 caudal segments was made in the worms immediately after the extirpation. Caudal regenerations was examined was accomplished.
SEQUEROS
Table 1. Caudal regeneration (%) of debrained young worms of molleri. Effects of one brain implanted into the debrained worms
AIlolobophora
Operation Intact Brain removed One brain implanted
N
n
3 3 2
15 15 14
N, number of experiments; n, number experiment.
Regeneration
of worms used on the
were able to regenerate the caudal region (41%) without brain and suboesophageal ganglia. In the case of clitellated adult worms the effects of supraoesophageal, suboesophageal ganglia removal and decapitation on caudal regeneration and clitellum persistence are shown in Table 3. The results indicate that the brain has an inhibitory function on caudal regeneration mechanism, as it can be demonstrated’in intact animals (34%) and after brain removal (80%). On the other hand, the lowest percentage (11%) of regeneration was found when the suboesophageal ganglia were removed. In these worms the clitellum remains. Our results suggest that there is a major difference between young and clitellated worms.
25 or 30 days after operation
RESULTS In young A. molleri worms the effect of brain removal is shown in Table 1. It can be seen that the
percentage of caudal regeneration decreased to 2% when the brain was removed as compared to intact animals (1OO’A).Thus, in young specimens the brain is probably needful for caudal regeneration. Unfortunately, we do not know by the present time, whether our observation represents the presence of a metabolic factor or a regeneration factor or both. Our results indicate only that there is a really total regeneration when a young brain is implanted into debrained young worms (Table 1). The effects of supraoesophageal, suboesophageal ganglia removal and decapitation on caudal regeneration of unclitellated adult earthworms A. molleri are shown in Table 2. It can be seen that the percentage of regeneration has a tendency to increase (74%) in the specimens without brain. Thirty days after operation time a clitellum was not developed in these specimens. On the other hand, when suboesophageal ganglia are extirpated the number of regenerated specimens decrease (25%). Thirty days after operation time a well developed clitellum appeared in these specimens. Table 2 shows that in decapitated specimens a clitellum was not developed. Bur our experiments also indicate that the animals
DISCUSSION The endocrine role of the nervous system on caudal regeneration in some earthworms has been demonstrated by extirpation and grafting methods (Saussey, 1963; Michon et al., 1964; Gallissian, 1968, 1973; Chapron et al., 1972; Alonso-Bedate et al., 1983).
Our work demonstrates that when nervous system remains intact the percentage of caudally regenerated worms declines as the worm reaches the sexual maturity. When the worm is very small and young the brain neurosecretory cells are indispensable for cauda1 regeneration. We suggest that in a young worm a stimulatory factor or Growth Factor (GF) probably from neurosecretory cells of supraoesophageal ganglia is needful for regeneration. In fact, when the brain is extirpated the percentage of regeneration is only 2%. On the other hand the injection of one brain
Table 2. Effects of the brain removal, suboesophageal ganglion removal and decapitation on caudal regeneration (%) and secondary sexual features (Clitelhun) in unclitellated adult earthworm ANolobophora molleri
Operation Intact Brain removed Suboesophageal Decapitated
ganglia removed
N
n
Regeneration (%)
Clitellum appearance (%)
3 3 2 3
31 41 20 24
70 14 25 41
100 0 100 0
N, number of experiments; n, number of worms used on the experiment. Clitellum was observed 25-30 days after the operation.
Table 3. Effect of the brain removal, suboesophageal ganglion removal and decapitation on caudal regeneration (%) and secondary sexual features (Clitellum) in clitellated adult earthworm Aliolobouhora molkri Operation Intact Brain removed Suboesophaegeal ganglia removed Decaoitated
(%)
100 2 98
N
n
Regeneration (%)
Clitellum
2 1 2 2
31 15 24 19
34 80 11 41
Remain Disappear Remain Disaooear
N, number of experiments; n, number of worms used in the experiment. Clitellum was observed 25-30 days after the operation.
Caudal regeneration in Allolobophora molleri into brainless animals has a marked restorative effect on regeneration. This observation suggests the origin of a trophic substance in the brain of the young A. molleri.
On the contrary in adult worms in clitellar phase the presence of the supraoesophageal ganglia seems to exert an inhibitory influence in caudal regeneration. It could explain our results related with an increment of percentage of caudally regenerated worms following the ablation of the supraoesophageal ganglia (34% in intact worms and 80% in decerebrated worms). On the other hand we have observed in worms in clitellar phase a very important reduction of the percentage of caudal regeneration following ablation of the suboesophageal ganglia (11.3%) when compared with intact decerebrated and decapited (47%) worms. These results suggest that at least in A. molleri in clitellar phase, not only neurosecretory cells within the suboesophageal ganglia but also neurosecretory cells at nerve cord level, are activated by caudal amputation. This stimulatory effect is counteracted by the inhibitory effect of the supraoesophageal ganglia when worms are intact (34%). Finally, an antagonism has been often noted between sexuality, regeneration and diapause in earthworms. When a worm enters diapause, ovulation stops and reproductive organs begin to regress, but regeneration is possible. According to HerlantMeewis (1965) it seems probable that sexual reproduction and regeneration depend on the activity of different types of neurosecretory cells in the ventral ganglia that cannot function at the same time, one suppressing activity in the other. In A. molleri probably, a Clitellum Factor (CF) is synthesized by neurosecretory cells placed at supraoesophageal ganglia level. This CF should be only activated in adult
rG?
227
worms at clitellar phase. The role of CF could be the maintenance of the clitellum and also the inhibition of caudal regeneration during reproduction time. It could explain our observations related with clitellum disappearance following ablation of the supraoesophageal ganglia (10%) and with an increment of percentage of caudal regeneration in worms in clitellar phase (Fig. 1).
REFERENCES
Alonso-Bedate M. and Sequeros E. (1983) Neurosecretory phenomena in the cerebral ganglia of clitellated Allolobophora caliginosa. Acta Embryol. Morphol. exp. 4, 93-103. Alumets J., Hakanson R., Sundler F. and Thor& J. (1979) Neuronal localization of immunoreactive enkephalin and -endorphin in the earthworm. Nature, Lond. 279, 805-806. Aros B., Wenger T., Vigh B. and Vingh-Teichman I. (1980) Immunohistochemical localization of substance P and ACTH-like activity in the central nervous system of the earthworm Lumbricw terrestris. Acta Histochem. 66, 262-268. Chapron C. and Chapron J. (1972) Influence des amines biogenes et de leurs inhibiteurs sur la regeneration. Etude chez 1’Annelide Eisenia foetida. CR. Acad. Sci. Paris 274, 412-414. Chapron C. and Chapron J. (1973) Induction de la regeneration posterieure chez les Annelides du genre Lumbricus. C.R. Acad. Sci. Paris 276, 2691-2694. Ehinger B. and Myhrberg H. E. (1971) Neuronal localization of dopamine, noradrenaline and 5 Hydroxytryptamine in the central nervous system of Lumbricus terrestris. Histochemic. 28, 265-275. Gallissian A. (1968). Greffe des ganglions cerebroides chez le Lumbricide Eophila doll&i Tetry. Influence sur la diapause et la regeneration. C.R. Acad. Sci. Paris 267, 657-658.
a
b
Brain
Ul
VG
c---
E L.L
cc”
I
Fig. 1. Suggested regulatory mechanisms for caudal regeneration in the earthworm Allolobophora molleri. (a) In a young worm the activity of neurosecretory cells at suboesophageal ganglia (SG) and nerve cord ganglia (VG) are stimulated by a Stimulatory Factor (SF) or Growth Factor from the brain. A Regeneration Factor (Rg F) from Suboesophageal and nerve cord ganglia promotes segments proliferation. (b) In clitellated worms the active neurosecretory cells in the brain release an Inhibitory Factor (IF) or Clitellum Factor that inhibits the activity of suboesophageal and nerve cord ganglia. An intermediate situation could be observed in unclitellated worms.
228
M. ALONSO-BEDATE and E. SEQUEROS
Gallissian A. (1973) Role des cellules neurosecretrices dans Ie determinisme de la diapause et de la regeneration posterieure chez le Lombricide Eophila do&i Tetry C.R. Acad. Sci. Paris 276, 2721-2723. Herlan-Meewis H. (1965) Regeneration in Annelids. In Advances in Morphologenesis (Edited by Abercrombie M. and Brachet J.): Vol.- 4, pp. 155-215: Academic Press, New York. Herlant-Meewis H. (1966) Les cellules neurosecretrices de la chaine nerveuse d’Eisenia foetida. Z. ZelJforsch. 69, 319-325. Hub1 H. (1956) Uber die Beziehungen der Neurosekretion zum Regenerations geschehen bei Lumbriciden nebst Beschereibung eines neuartigen neurosekeretorischen Zelhypes in Unterschlundganglion. Arch. Entwicklungsmech Organismen. 149, 73-87. Maissait M. J. (1965) Influences de l’ablation des ganglions cerebroides sur la regeneration posterieure chez le Lombricien Allolobophora chlorotica Sav. Relations avec la diapause. C.R. Acad. Sci. Paris 261, 1749-1751. Marcel R. (1973) Action des substances inhibitrice et trophique sur les cycles scretoires des neurones de la chaine nerveuse ventrale au tours de la regeneration chez Eisenia foetida Sav. f. typica (Annelide, Oligochete). Gen. camp. Endocr. 21, 45-59. Michon J., Maissait J. and Angevain A. (1964) Contribution
a l’ttude de la neurosecretion chez le Lumbricien &eniella tetraedra. f. typica. C.R. Acad. Sci. Paris 259, 1248-1249. Moment G. B. (1979) Growth, posterior regeneration and segment number in Eisenia foetida. Megacariologica 3, 1677176. Remy Ch. and Dubois M. P. (1979) Localization par immunofluorescence de peptides analogues a l’endorphine dans les ganglion infra-oesophagiens du lombricide Dendrobaena subrubicunda. Eisen. Experientia 35, 137-138. Saussey M. (1963) Effects de la decerebration et de sur la I’amputation caudale spermatogenese d’dllolobophora icterica Savigny. (Oligochete, Lumbricidae). C.R. Acad. Sci. Paris 257, 511-513. Scharrer B. (1937) Uber sekretorisch Tltige neuerzellen bei wirvellosen Tieren. Naturwissenschaftten 25, 131-138. Sundler F., Hakanson R., Alumets J. and Walles B. (1977) Neuronal localization of pancreatic polypeptide (PP) and vasoactive intestinal peptide (VIP) immunoreactivity in the earthworm (Lumbricus terrestris). Brain Res. Bull. 2, 61-65. Vries-Schoumacker H. De. (1977) Fluorescence and ultrastructural localization of aminergic neurons in the nerve cord of Eisenia foetida (Annelida-Oligochaeta). Cell Tissue Res. 185, 351-360.
0300..9629/85 $3.00 + 0.00 Q 1985 PergamonPress Ltd
Printedin Great Britain
SUGGESTED REGULATORY MECHANISMS FOR CAUDAL REGENERATION IN ALLOLOBOPIrJORA MOLLERI (ANNELIDA: OLIGOCHAETA) M.
ALONSO-BEDATE
and E.
SEQUEROS
Departamento de Fisiologia Animal, Facultad de Ciencias Biologicas, Universidad Complutense, Madrid, Spain (Received 12 July 1984)
Abstract-l. In young Allolobophora molleri the brain is required for caudal regeneration. When the brain was present there was a high percentage (100%) of regenerated worms, but this percentage declined (2%) when the brain was removed. 2. A stimulatory factor or Growth Factor from the brain is probably required for release of a Regenerating Factor from suboesophageal ganglia or nerve cord neurosecretory cells. 3. In adult worms at clitellar stage the brain plays an inhibitory role on caudal regeneration. When the brain was present there was a low percentage (34%) of regenerated worms, but this percentage was higher when the brain was removed (80%). 4. Probably a Clitellum Factor from neurosecretory cells at supraoesophageal ganglia is responsible for inhibition of the neurosecretory cells activity that release a Regenerating Factor from suboesophageal ganglia or ventral nerve cord. 5. Finally, an intermediate situation could be observed in adult unclitellated worms.
INTRODUCTION
Since the first description Lumbricus
of neurosecretory cells in terrestris by Scharrer (1937) many in-
vestigations have been published dealing with the structure and function of these elements. In addition to the neurosecretory peptidergic cells, monoamine producing neurons were also demonstrated in the cerebral ganglion and ventral nerve cord in Lumbricus terrestris (Ehinger et aI., 1971; de Vries-Schoumacker, 1977). The introduction of immunocytochemical methods revealed the chemical composition of materials elaborated in the neurons of the central nervous system of the earthworms. Pancreatic polypeptide-like substance or vasoactive intestinal peptide, opiate-like materials, and endorphin and leucoenkephalin, substance P and ACTH-like activity could be detected (Sundler et al., 1977; Alumets et al., 1979; Remy et al., 1979; Aros et al., 1980). During the last twenty years, many works have been published about the possible function of neurosecretion on regeneration, reproduction and metabolism in Oligochaeta. It appears that in all earthworms which have been examined, the nervous system plays a role, either direct or indirect, in the control of regeneration (Hubl, 1956; Gallissian, 1973; Maissait, 1965). Herlant Meewis (1965, 1966) established the involvement of the ventral nerve cord in both anterior and posterior regeneration in Eisenia foetida. Removal of the brain of Eophila dollfusi followed by amputation of caudal segments induces diapause during which segment regeneration occurs (Gallissian, 1973). This might suggest that the brain produced a neurohormone which inhibits caudal regeneration. On the other hand some studies on Lumbricus argue for a stimulatory effect of the brain 225
neurosecretion on caudal regeneration (Chapron and Chapron, 1973). Marcel (1973) established the presence of specific inhibitory and trophic substances in the head and tail that are important in order to modify the activity of neurosecretory cells during regeneration. According to Moment (1979) the neurosecretory system plays an important role particularly in initiating regeneration in the earthworm Eisenia foetida and supposes that the neurosecretory cells are needful in the initial stages but not later, and are therefore not directly involved in the determination of the number of segments proliferated. Our present report is concerned with the role of the central nervous system on the phenomenon of caudal regeneration in Allolobophora molleri (Annelida: Oligochaeta). The possibility of a different mechanism for control of caudal regeneration depending on the animal age and reproductive stage in A. molleri has been studied. MATERIALS AND METHODS
Specimens of Allolobophora molleri were collected around Madrid from September to August. The animals were in clitellar and non-clitellar stages. The worms were starved for 24 hr prior to testing in order to clear the gut. They were not fed during the experiment because decerebrated and decapitated worms are unable to feed. All the animals were maintained at the laboratory temperature (22 k 3”C), in individual Petri plates with moist paper filter at the bottom. Dechlorinated fresh water was provided daily. The animals were maintained in dark conditions. Extirpation
of the supra and sub-oesophageal ganglia
The worms were anesthesized with 8% ethanol. Each worm was placed in a Petri dish with the body resting on a block of modeling clay. The supraoesophageal ganglia
M. ALONSO-BEDATE and E.
226
were removed under a dissecting microscope. Worms were placed dorsal side up. A small window was cut in the dorsal anterior portion of the head, between the first and second segments. The underlying muscles were gently pulled aside with an entomological pin. This allowed easy access to the supraoesophageal ganglia. The brain was lifted with forceps and removed. For extirpation of the infraoesophageal ganglia a small window was cut in the ventral portion of the head between the fifth and seventh segments. Then, the tissues were separated and the ganglia were removed. Finally, decapitation was accomplished by pulling the head (5-7 segments) away with fine dissecting scissors. Always, the amputation of about 40-50 caudal segments was made in the worms immediately after the extirpation. Caudal regenerations was examined was accomplished.
SEQUEROS
Table 1. Caudal regeneration (%) of debrained young worms of molleri. Effects of one brain implanted into the debrained worms
AIlolobophora
Operation Intact Brain removed One brain implanted
N
n
3 3 2
15 15 14
N, number of experiments; n, number experiment.
Regeneration
of worms used on the
were able to regenerate the caudal region (41%) without brain and suboesophageal ganglia. In the case of clitellated adult worms the effects of supraoesophageal, suboesophageal ganglia removal and decapitation on caudal regeneration and clitellum persistence are shown in Table 3. The results indicate that the brain has an inhibitory function on caudal regeneration mechanism, as it can be demonstrated’in intact animals (34%) and after brain removal (80%). On the other hand, the lowest percentage (11%) of regeneration was found when the suboesophageal ganglia were removed. In these worms the clitellum remains. Our results suggest that there is a major difference between young and clitellated worms.
25 or 30 days after operation
RESULTS In young A. molleri worms the effect of brain removal is shown in Table 1. It can be seen that the
percentage of caudal regeneration decreased to 2% when the brain was removed as compared to intact animals (1OO’A).Thus, in young specimens the brain is probably needful for caudal regeneration. Unfortunately, we do not know by the present time, whether our observation represents the presence of a metabolic factor or a regeneration factor or both. Our results indicate only that there is a really total regeneration when a young brain is implanted into debrained young worms (Table 1). The effects of supraoesophageal, suboesophageal ganglia removal and decapitation on caudal regeneration of unclitellated adult earthworms A. molleri are shown in Table 2. It can be seen that the percentage of regeneration has a tendency to increase (74%) in the specimens without brain. Thirty days after operation time a clitellum was not developed in these specimens. On the other hand, when suboesophageal ganglia are extirpated the number of regenerated specimens decrease (25%). Thirty days after operation time a well developed clitellum appeared in these specimens. Table 2 shows that in decapitated specimens a clitellum was not developed. Bur our experiments also indicate that the animals
DISCUSSION The endocrine role of the nervous system on caudal regeneration in some earthworms has been demonstrated by extirpation and grafting methods (Saussey, 1963; Michon et al., 1964; Gallissian, 1968, 1973; Chapron et al., 1972; Alonso-Bedate et al., 1983).
Our work demonstrates that when nervous system remains intact the percentage of caudally regenerated worms declines as the worm reaches the sexual maturity. When the worm is very small and young the brain neurosecretory cells are indispensable for cauda1 regeneration. We suggest that in a young worm a stimulatory factor or Growth Factor (GF) probably from neurosecretory cells of supraoesophageal ganglia is needful for regeneration. In fact, when the brain is extirpated the percentage of regeneration is only 2%. On the other hand the injection of one brain
Table 2. Effects of the brain removal, suboesophageal ganglion removal and decapitation on caudal regeneration (%) and secondary sexual features (Clitelhun) in unclitellated adult earthworm ANolobophora molleri
Operation Intact Brain removed Suboesophageal Decapitated
ganglia removed
N
n
Regeneration (%)
Clitellum appearance (%)
3 3 2 3
31 41 20 24
70 14 25 41
100 0 100 0
N, number of experiments; n, number of worms used on the experiment. Clitellum was observed 25-30 days after the operation.
Table 3. Effect of the brain removal, suboesophageal ganglion removal and decapitation on caudal regeneration (%) and secondary sexual features (Clitellum) in clitellated adult earthworm Aliolobouhora molkri Operation Intact Brain removed Suboesophaegeal ganglia removed Decaoitated
(%)
100 2 98
N
n
Regeneration (%)
Clitellum
2 1 2 2
31 15 24 19
34 80 11 41
Remain Disappear Remain Disaooear
N, number of experiments; n, number of worms used in the experiment. Clitellum was observed 25-30 days after the operation.
Caudal regeneration in Allolobophora molleri into brainless animals has a marked restorative effect on regeneration. This observation suggests the origin of a trophic substance in the brain of the young A. molleri.
On the contrary in adult worms in clitellar phase the presence of the supraoesophageal ganglia seems to exert an inhibitory influence in caudal regeneration. It could explain our results related with an increment of percentage of caudally regenerated worms following the ablation of the supraoesophageal ganglia (34% in intact worms and 80% in decerebrated worms). On the other hand we have observed in worms in clitellar phase a very important reduction of the percentage of caudal regeneration following ablation of the suboesophageal ganglia (11.3%) when compared with intact decerebrated and decapited (47%) worms. These results suggest that at least in A. molleri in clitellar phase, not only neurosecretory cells within the suboesophageal ganglia but also neurosecretory cells at nerve cord level, are activated by caudal amputation. This stimulatory effect is counteracted by the inhibitory effect of the supraoesophageal ganglia when worms are intact (34%). Finally, an antagonism has been often noted between sexuality, regeneration and diapause in earthworms. When a worm enters diapause, ovulation stops and reproductive organs begin to regress, but regeneration is possible. According to HerlantMeewis (1965) it seems probable that sexual reproduction and regeneration depend on the activity of different types of neurosecretory cells in the ventral ganglia that cannot function at the same time, one suppressing activity in the other. In A. molleri probably, a Clitellum Factor (CF) is synthesized by neurosecretory cells placed at supraoesophageal ganglia level. This CF should be only activated in adult
rG?
227
worms at clitellar phase. The role of CF could be the maintenance of the clitellum and also the inhibition of caudal regeneration during reproduction time. It could explain our observations related with clitellum disappearance following ablation of the supraoesophageal ganglia (10%) and with an increment of percentage of caudal regeneration in worms in clitellar phase (Fig. 1).
REFERENCES
Alonso-Bedate M. and Sequeros E. (1983) Neurosecretory phenomena in the cerebral ganglia of clitellated Allolobophora caliginosa. Acta Embryol. Morphol. exp. 4, 93-103. Alumets J., Hakanson R., Sundler F. and Thor& J. (1979) Neuronal localization of immunoreactive enkephalin and -endorphin in the earthworm. Nature, Lond. 279, 805-806. Aros B., Wenger T., Vigh B. and Vingh-Teichman I. (1980) Immunohistochemical localization of substance P and ACTH-like activity in the central nervous system of the earthworm Lumbricw terrestris. Acta Histochem. 66, 262-268. Chapron C. and Chapron J. (1972) Influence des amines biogenes et de leurs inhibiteurs sur la regeneration. Etude chez 1’Annelide Eisenia foetida. CR. Acad. Sci. Paris 274, 412-414. Chapron C. and Chapron J. (1973) Induction de la regeneration posterieure chez les Annelides du genre Lumbricus. C.R. Acad. Sci. Paris 276, 2691-2694. Ehinger B. and Myhrberg H. E. (1971) Neuronal localization of dopamine, noradrenaline and 5 Hydroxytryptamine in the central nervous system of Lumbricus terrestris. Histochemic. 28, 265-275. Gallissian A. (1968). Greffe des ganglions cerebroides chez le Lumbricide Eophila doll&i Tetry. Influence sur la diapause et la regeneration. C.R. Acad. Sci. Paris 267, 657-658.
a
b
Brain
Ul
VG
c---
E L.L
cc”
I
Fig. 1. Suggested regulatory mechanisms for caudal regeneration in the earthworm Allolobophora molleri. (a) In a young worm the activity of neurosecretory cells at suboesophageal ganglia (SG) and nerve cord ganglia (VG) are stimulated by a Stimulatory Factor (SF) or Growth Factor from the brain. A Regeneration Factor (Rg F) from Suboesophageal and nerve cord ganglia promotes segments proliferation. (b) In clitellated worms the active neurosecretory cells in the brain release an Inhibitory Factor (IF) or Clitellum Factor that inhibits the activity of suboesophageal and nerve cord ganglia. An intermediate situation could be observed in unclitellated worms.
228
M. ALONSO-BEDATE and E. SEQUEROS
Gallissian A. (1973) Role des cellules neurosecretrices dans Ie determinisme de la diapause et de la regeneration posterieure chez le Lombricide Eophila do&i Tetry C.R. Acad. Sci. Paris 276, 2721-2723. Herlan-Meewis H. (1965) Regeneration in Annelids. In Advances in Morphologenesis (Edited by Abercrombie M. and Brachet J.): Vol.- 4, pp. 155-215: Academic Press, New York. Herlant-Meewis H. (1966) Les cellules neurosecretrices de la chaine nerveuse d’Eisenia foetida. Z. ZelJforsch. 69, 319-325. Hub1 H. (1956) Uber die Beziehungen der Neurosekretion zum Regenerations geschehen bei Lumbriciden nebst Beschereibung eines neuartigen neurosekeretorischen Zelhypes in Unterschlundganglion. Arch. Entwicklungsmech Organismen. 149, 73-87. Maissait M. J. (1965) Influences de l’ablation des ganglions cerebroides sur la regeneration posterieure chez le Lombricien Allolobophora chlorotica Sav. Relations avec la diapause. C.R. Acad. Sci. Paris 261, 1749-1751. Marcel R. (1973) Action des substances inhibitrice et trophique sur les cycles scretoires des neurones de la chaine nerveuse ventrale au tours de la regeneration chez Eisenia foetida Sav. f. typica (Annelide, Oligochete). Gen. camp. Endocr. 21, 45-59. Michon J., Maissait J. and Angevain A. (1964) Contribution
a l’ttude de la neurosecretion chez le Lumbricien &eniella tetraedra. f. typica. C.R. Acad. Sci. Paris 259, 1248-1249. Moment G. B. (1979) Growth, posterior regeneration and segment number in Eisenia foetida. Megacariologica 3, 1677176. Remy Ch. and Dubois M. P. (1979) Localization par immunofluorescence de peptides analogues a l’endorphine dans les ganglion infra-oesophagiens du lombricide Dendrobaena subrubicunda. Eisen. Experientia 35, 137-138. Saussey M. (1963) Effects de la decerebration et de sur la I’amputation caudale spermatogenese d’dllolobophora icterica Savigny. (Oligochete, Lumbricidae). C.R. Acad. Sci. Paris 257, 511-513. Scharrer B. (1937) Uber sekretorisch Tltige neuerzellen bei wirvellosen Tieren. Naturwissenschaftten 25, 131-138. Sundler F., Hakanson R., Alumets J. and Walles B. (1977) Neuronal localization of pancreatic polypeptide (PP) and vasoactive intestinal peptide (VIP) immunoreactivity in the earthworm (Lumbricus terrestris). Brain Res. Bull. 2, 61-65. Vries-Schoumacker H. De. (1977) Fluorescence and ultrastructural localization of aminergic neurons in the nerve cord of Eisenia foetida (Annelida-Oligochaeta). Cell Tissue Res. 185, 351-360.