Constant Topological Organization of the Coleopteran Metamorphosing Nervous System: Analysis of Persistent Elements in the Nervous System of Tenebrio molitor Olaf Breidbach lnstitut fur Angewandte Zoologie, D-5300 Bonn 1, F.R.G.
SUMMARY Evidence is provided that the topological organization of the larval neuropil is preserved during metamorphosis of Tenebrio molitor. Constancies in neuronal organization were due in part to persistence of individual neurons whose morphologies were individually followed. It ap-
pears that the phenomenologically static situation of the metamorphosing neuropil is achieved by stabilization and regulation due to cellular interactions. Comparisons are made with features of hemimetabolan postembryogenesis.
INTRODUCTION
they become “respecified” (Levine and Truman, 1985). Thus these neurons accomplish an adaptation to a functionally different organized body structure. Recent studies on the metamorphosing nervous tissue of I14undzm and of the mealwormbeetle Tenehrio outline the extent of such persistent neurons in holometabolous insects (Levine, 1984, 1986; Breidbach, 1987a, 1988a, 1990c; Kent and Levinc, 1988j. lt is likely that nearly all larval motoneurons persist throughout metamorphosis in these species to become incorporated in a functionally and structurally different imaginal motor system (Kent and Levine, 1988). Likewise all interneurons identified in larval l’cncbrzo so far have proved to persist throughout metamorphosis (Breidbach 1987b,c,d, 198Yb, 1 9 9 0 ~ ) . Thus a number ofquestions arise. First, how are we to describe the persistence of a neuron in detail, concerning both the quality of its structural preservation and the events that take place throughout pupal reorganization? Second, what is the extent of persisting elements, and how do they interact with newly forming neurons?
The concept of the identified neuron as originally stated in a neuroethological study of invertebrates is that a certain neuron outlines its special structural and physiological features repetitively in all individuals of one species (Strausfeld, 1976; Wilson, 1978: Goodman, Pearson, and Heitlcr, 1979). Its uniform structural organization, thereby is interpreted as being the result of functional specification (Hoyle, 1976; Davis, 1978; Kupferman and Wciss, 1978; Huber, 1989). What happens with such a functionally adapted neuron when the structural and functional organization of an individual is transformed during ontogenesis? Studies on the metamorphosis of the nervous system of the moth 1l4unu‘ut.u sextu show. that even complex reorganization of the muscular system does not necessarily result in a complete destruction of the respective motoneurons (Levine and Truman, 1985). As initially demonstrated by Truman and Reiss ( 1976 j certain larval motoneurons of A4andz.ic.a persist within metamorphosis of the moth. Therein, Rccewed June 20, 1990: accepted June 22. 1990 Journal ofNeurobiology. Vol. 21, No. 7. pp. 990-1001 (1990) 0 1990 John Wilcy & Sons, Inc. CCC 0022-3034/90/070990- 12$04.00
990
MATERIAL
The mealworm beetle, Tenehrio molitor, and the larger american tenebrionid, Zophobus morio,
C‘oletipterun Metamorphosing .h’ervous SJatem
possess freely moving larvae and “free” pupae, easily accessible for manipulation. In Tenebrio metamorphosis lasts for approximately 10 days at room temperature. PERSISTENCE OF MOTONEURONS
Studies of the metamorphosis of the abdominal nervous system of Munducu have shown that motoneurons persist throughout metamorphosis in spite of structural reorganization of the appropriate parts of the muscular system (Leyine, 1986). In the thorax, metamorphic reorganization of the muscular system is more extensive (Lee, 1964), especially in regard to thc newly developing appendages, that is, legs and wings. These originate from imaginal discs, which maintain their embryionic features until the onset of metamorphosis (Poodry and Schneiderman. 1970: Huet and Lcnoir-Rousseaux, 1976). To demonstrate whether. in spite of these alterations in the structural organization of the thoracic periphery, the thoracic motoneurons persist metamorphosis, three strategies were followed. Initially the motoneurons of different instars were mapped by retrograde labelling techniques, using either horseradish peroxidase (HRP). cobalt-, or nickelchloride (for technical details see Breidbach, 1987a, 1989a,b; Kutsch and Schneider, 1987). Application was performed by dye injection into the appropriate muscles or, in the case of the pupal instars where the muscles were cytolysed, into the appropriate nerves. These studies allowed an interpretation of the time course of neuronal development. To show the persistence of single neurons directly, HRP was injected into a larval/prepupal muscle and the individual was allowed to continue development (Breidbach. 1987a). In the late pupa or in the imaginal stage. the nervous system of such an individual was dissected out. Neurons identified as being labelled with HRP were interpreted as persisting neurons. These results were substantiated using the hydroxyurea (HU) technique. HU inhibits proliferation in the nervous tissue (Truman and Booker, 1986) and injection of it in the late larva or prepupal stages inhibits the formation of new neurons. Neurons stained at the adult stage in individuals that were HU-treated in the prepupa. therefore, were regarded as neurons that persist metamorphosis ( Breidbach, 1989b). In combining these results. it was possible to reconstruct the metamorphic fate of the thoracic motoneurons of the tenebrionid beetles.
991
Motoneurons that innervate the ventral and dorsal thoracic longitudinal muscles and the coxodorsal leg muscles persist throughout metamorphosis (Breidbach 1987a, 1988a,b). Comparison of the larval and the adult situation in Tenebrio showed, that all these motoneurons maintained their principal larval arborization patterns ( Breidbach 19881-3).For these neurons, serial homology was demonstrated within the thoracic ganglia. All exhibit a stereotyped ontogenetic development. These types consist of motoneurons that innervate the coxo-dorsal muwles (CDM), the ventral longitudinal muscles (VLM). and the dorsal longitudinal muscles ( DLM ) (Fig. 1 ) . The large VLMs of one hemisegment are innervated via the interconnective nerve by three persisting neurons that form two groups ( Breidbach 1987a). There is a contralaterally situated neuron in the postcrior part of the ganglion proximal to the interconnective nerve, and there is also a posterior cluster of two neurons situated contralaterally in the anterior part of the ganglion caudal to the interconnective nerve. These neurons do not delete the principal arborization structures of their larval dendritic trees, which form an essential ipsilateral projection. Rather. they extend their arborizations with onset of pupation forming large contralateral dendritic projection patterns. Thus these neurons build up the framework of the later adult structural characteristics in the prepupa before the later adult muscles arc differentiated. The varying structural and functional characteristics of the muscular system do not seem to be necessary for the establishment of the later adult structural characteristics of this set of neurons. The DLMs of one hemisegment are innervated by 1 1 neurons via the interconnective nerve (Breidbach and Kutsch, 1990). These are likewise separable into an anterior and posterior cluster. The antenor cluster consists of seven neurons, situated in the ipsilateral ganglion rostra1 to the interconnective nerve. The posterior cluster comprises four neurons. The somata of one of these is dorso-medially situated. One other neuron is ventro-medially situated, whereas the somata of the other two neurons are contralaterally localized, having both an ipsilateral and a contralateral dendntic arborization. These neurons likewise do not dclete their dendritic trees during metamorphosis, but maintain their larval structural characteristics throughout pupation. The metamorphosis of the more dorsal contralateral neuron of the posterior group was studied in more detail in Zophobus (Breidbach and Kutsch, 1990). This neuron
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SUMMARY Evidence is provided that the topological organization of the larval neuropil is preserved during metamorphosis of Tenebrio molitor. Constancies in neuronal organization were due in part to persistence of individual neurons whose morphologies were individually followed. It ap-
pears that the phenomenologically static situation of the metamorphosing neuropil is achieved by stabilization and regulation due to cellular interactions. Comparisons are made with features of hemimetabolan postembryogenesis.
INTRODUCTION
they become “respecified” (Levine and Truman, 1985). Thus these neurons accomplish an adaptation to a functionally different organized body structure. Recent studies on the metamorphosing nervous tissue of I14undzm and of the mealwormbeetle Tenehrio outline the extent of such persistent neurons in holometabolous insects (Levine, 1984, 1986; Breidbach, 1987a, 1988a, 1990c; Kent and Levinc, 1988j. lt is likely that nearly all larval motoneurons persist throughout metamorphosis in these species to become incorporated in a functionally and structurally different imaginal motor system (Kent and Levine, 1988). Likewise all interneurons identified in larval l’cncbrzo so far have proved to persist throughout metamorphosis (Breidbach 1987b,c,d, 198Yb, 1 9 9 0 ~ ) . Thus a number ofquestions arise. First, how are we to describe the persistence of a neuron in detail, concerning both the quality of its structural preservation and the events that take place throughout pupal reorganization? Second, what is the extent of persisting elements, and how do they interact with newly forming neurons?
The concept of the identified neuron as originally stated in a neuroethological study of invertebrates is that a certain neuron outlines its special structural and physiological features repetitively in all individuals of one species (Strausfeld, 1976; Wilson, 1978: Goodman, Pearson, and Heitlcr, 1979). Its uniform structural organization, thereby is interpreted as being the result of functional specification (Hoyle, 1976; Davis, 1978; Kupferman and Wciss, 1978; Huber, 1989). What happens with such a functionally adapted neuron when the structural and functional organization of an individual is transformed during ontogenesis? Studies on the metamorphosis of the nervous system of the moth 1l4unu‘ut.u sextu show. that even complex reorganization of the muscular system does not necessarily result in a complete destruction of the respective motoneurons (Levine and Truman, 1985). As initially demonstrated by Truman and Reiss ( 1976 j certain larval motoneurons of A4andz.ic.a persist within metamorphosis of the moth. Therein, Rccewed June 20, 1990: accepted June 22. 1990 Journal ofNeurobiology. Vol. 21, No. 7. pp. 990-1001 (1990) 0 1990 John Wilcy & Sons, Inc. CCC 0022-3034/90/070990- 12$04.00
990
MATERIAL
The mealworm beetle, Tenehrio molitor, and the larger american tenebrionid, Zophobus morio,
C‘oletipterun Metamorphosing .h’ervous SJatem
possess freely moving larvae and “free” pupae, easily accessible for manipulation. In Tenebrio metamorphosis lasts for approximately 10 days at room temperature. PERSISTENCE OF MOTONEURONS
Studies of the metamorphosis of the abdominal nervous system of Munducu have shown that motoneurons persist throughout metamorphosis in spite of structural reorganization of the appropriate parts of the muscular system (Leyine, 1986). In the thorax, metamorphic reorganization of the muscular system is more extensive (Lee, 1964), especially in regard to thc newly developing appendages, that is, legs and wings. These originate from imaginal discs, which maintain their embryionic features until the onset of metamorphosis (Poodry and Schneiderman. 1970: Huet and Lcnoir-Rousseaux, 1976). To demonstrate whether. in spite of these alterations in the structural organization of the thoracic periphery, the thoracic motoneurons persist metamorphosis, three strategies were followed. Initially the motoneurons of different instars were mapped by retrograde labelling techniques, using either horseradish peroxidase (HRP). cobalt-, or nickelchloride (for technical details see Breidbach, 1987a, 1989a,b; Kutsch and Schneider, 1987). Application was performed by dye injection into the appropriate muscles or, in the case of the pupal instars where the muscles were cytolysed, into the appropriate nerves. These studies allowed an interpretation of the time course of neuronal development. To show the persistence of single neurons directly, HRP was injected into a larval/prepupal muscle and the individual was allowed to continue development (Breidbach. 1987a). In the late pupa or in the imaginal stage. the nervous system of such an individual was dissected out. Neurons identified as being labelled with HRP were interpreted as persisting neurons. These results were substantiated using the hydroxyurea (HU) technique. HU inhibits proliferation in the nervous tissue (Truman and Booker, 1986) and injection of it in the late larva or prepupal stages inhibits the formation of new neurons. Neurons stained at the adult stage in individuals that were HU-treated in the prepupa. therefore, were regarded as neurons that persist metamorphosis ( Breidbach, 1989b). In combining these results. it was possible to reconstruct the metamorphic fate of the thoracic motoneurons of the tenebrionid beetles.
991
Motoneurons that innervate the ventral and dorsal thoracic longitudinal muscles and the coxodorsal leg muscles persist throughout metamorphosis (Breidbach 1987a, 1988a,b). Comparison of the larval and the adult situation in Tenebrio showed, that all these motoneurons maintained their principal larval arborization patterns ( Breidbach 19881-3).For these neurons, serial homology was demonstrated within the thoracic ganglia. All exhibit a stereotyped ontogenetic development. These types consist of motoneurons that innervate the coxo-dorsal muwles (CDM), the ventral longitudinal muscles (VLM). and the dorsal longitudinal muscles ( DLM ) (Fig. 1 ) . The large VLMs of one hemisegment are innervated via the interconnective nerve by three persisting neurons that form two groups ( Breidbach 1987a). There is a contralaterally situated neuron in the postcrior part of the ganglion proximal to the interconnective nerve, and there is also a posterior cluster of two neurons situated contralaterally in the anterior part of the ganglion caudal to the interconnective nerve. These neurons do not delete the principal arborization structures of their larval dendritic trees, which form an essential ipsilateral projection. Rather. they extend their arborizations with onset of pupation forming large contralateral dendritic projection patterns. Thus these neurons build up the framework of the later adult structural characteristics in the prepupa before the later adult muscles arc differentiated. The varying structural and functional characteristics of the muscular system do not seem to be necessary for the establishment of the later adult structural characteristics of this set of neurons. The DLMs of one hemisegment are innervated by 1 1 neurons via the interconnective nerve (Breidbach and Kutsch, 1990). These are likewise separable into an anterior and posterior cluster. The antenor cluster consists of seven neurons, situated in the ipsilateral ganglion rostra1 to the interconnective nerve. The posterior cluster comprises four neurons. The somata of one of these is dorso-medially situated. One other neuron is ventro-medially situated, whereas the somata of the other two neurons are contralaterally localized, having both an ipsilateral and a contralateral dendntic arborization. These neurons likewise do not dclete their dendritic trees during metamorphosis, but maintain their larval structural characteristics throughout pupation. The metamorphosis of the more dorsal contralateral neuron of the posterior group was studied in more detail in Zophobus (Breidbach and Kutsch, 1990). This neuron
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