Arch. BioI. sa. Belgrade, 56 (1-2), 3P-4P, 2004.

VENTROMEDIAL NEUROSECRETORY NEURONS (TYPE A) IN THE SUBOESOPHAGEAL GAN­ GLION OF FUNEREUS MULS. (1869) DURING POSTEMBRYONIC DEVELOPMENT. Vera Nenadovic, Marija Mrdakovic and Larisa Ilijin, Department of Physiology and Biochemistry, "Sinisa Stankovic " Institute for Biological Research, 11060 Belgrade, and

UDC 595.768 : 591.341: 591.18

The presence of one pair ofventromedial neurosecretory The M..funereus larvae used in this experiment were neurons (vmnsn, type A) has been described in the suboesopha­ reared in laboratory conditions from hatching to sacrificing geal ganglion (SG) ofmany insect species from different orders (constant temperature of 23°C, relative humidity of 70%, ab­ (Raabe, 1982). These neurons synthesize arginine-vasopresin sence of light, and artificial diet according to Roberts, 1986). neuropeptides, which have a diuretic effect on the Malphigian Under these conditions, larval development lasts 6.5 months and tubules and/or inhibit water resorption in the rectum (Philipps, pupal development 15 days (Nenadovi c et a!., 1989; St anic 1983; Spring, 1990). Investigations of temperature stress in 2 eta!., 1989; Nenadov ic , 1992). Individuals were sacrificed on and 3 year old larvae of Morimus funereus confirmed the pres­ the fourth day after molting into the first, third, fourth, sixt, and ence ofone pair ofvmnsn SG and brain neurosecretory neurons eighth larval instars (the eighth being the last larval instar, LU); belonging to type A. Changes of their activity were noticed al­ in the prepupal and pupal stages; in pharat adults; and in 24-h ready after six hours of exposure to unfavorable temperature, adults. Each group consisted of five individuals. Head capsules which points to a significant role of its neurohormones in the were fixed in Bouin's fixative. Standard histological procedure processes of acclimation and acclimatization to thermal stress was used for embedding in paraffin. Serial paraffin sections of (Ivan o vi c eta!., 1975a, 1975b). 5urn were stained using the paraldehyde Thionine Phloxine and Investigations of differentiation of the cerebral neuroen­ Alcian Blue Phloxine techniques (Panov, 1980). Tissue analy­ docrine system during embrionic development of M funereus sis was performed using a light microscope. The size of peri­ showed that 6 to 7 day old embrios had a general morphologi­ carya was expressed as average values of the product of their cal plan of cerebral complex structure. A large number of par­ larger and smaller diameter. aldehyde-fuchsin positive (PF+) neurosecretory neurons (type A) in various phases of differentiation were found in the brain, The results presented on Fig.1 show increase in size of suboesophageal ganglion, and frontal ganglion. The highest vmnsn from the first to eighth larval instar (LU). The size of level of differentiation was noticed in SG, which underlines the these neurons decreases in the prepupa and pupa, i.e., at the be­ functional and evolutionary significance of its neurohormones ginning of and during metamorphosis. The size of vmnsn SG (Stan i c eta!., 1978;Nenadovic et al., 1986). increases in adults and they become larger than larval neurons. The present work gives the results of investigations of According to P anov (1968), the synthetic activity of neurose­ changes in the size and number ofvmnsn SG during postembri­ cretory tissues can increase by increase in number and/or size onic development of M.funereus. of their neurons. Both mechanisms have been described for protocerebral neurosecretory neurons of type A (AI' A , L" L The suboesophageal ganglion of newly hatched larvae is 2 2 still more developed than the brain. Brain lobes are feebly dif­ etc.) in Mfunereus (Nenadov i c , 1992). Similar results have ferentiated and distant. As flattened foils, they lean against the been obtained in Tenebrio molitor (Jankov i c-Hladni , 1971), gut in such way that their lateral parts are in a ventral position. which is in agreement with results obtained in other insect spe­ Corpora allata are well-developed, while the corpus cardiacum cies (Satija and Kaur, 1965; Weeks and Levine, 1990). is insufficiently differentiated and has a small number of visible A high number ofPF+ vmnsn SG is evident at the begin­ axons and intrinsic neurons, which is characteristic of embrios ning of the first larval instar (which lasts 6-7 days), but there (N e na do v ic et a!., 1978, 1987). were only three pairs of neurons (one pair of dorsal and two The suboesophageal ganglion of newly hatched larvae is pairs oflateral neurons) on the fourth day ofthe first instar. Their characterized by well-developed neuropil and a large number of number and position did not change during further postembri­ PF+ neurosecretory neurons. Embrionic traits are less expressed onic development. There are two possible causes of decrease in in SG than in the brain, i.e., neurons of SG are more differentiat­ number of vmnsn SG in the first instar of M. funereus larvae: ed than brain neurons (N enadovi c et al., 1985; N c nado v i c , death or migrations of neurons. Neuron death is best investi­ 1992). gated in insect metamorphosis (Singh and Srivastava, 1974; In view of the long postembrionic developmnet of M.fu­ Schwarc and Truman, 1982), but it is also known to happen nereus, the question arises as to how long cytodifferentiation of in specific phases ofembrionic and postembrionic development PF+ neurosecretory neurons ofits SG lasts. under the influence ofmany exogenous and neurohormonal fac-

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tors (Fahrbach, 1997). Migrations of PF+ neurons to other Belgrade. - Nenadovic, V (1992) PhLJ Thesis, Ur.iversity of Novi parts of central nervous system also appear to be possible, in Sad. - Nenadovic, V, Ivanovic, J. and Stanic, V (1989) XIX Yugoslav Entomological Meeting. - Nenadovic, V, Ivanovic, J., Stanic, V and view of the presence of arginine-vasopresin like neurons in Jankovic-Hladni, M. (1986) Acta entomologica Jugoslavica, 22, 1-2. the protocerebrum and first thoracic ganglion of some - Nenadovic, V, Ivanovic, J., Stanic, V and Jankovic-Hladni, M. (1985) (Girardie et al., 1984). Since diuresis in insects is under neu­ IBRO International Symposium on Neuroendocrinology "Peptide roendocrine control (Spring, 1990), further investigations on and Monoamine Neurohormones In Neuroendocrine Regulation", vrnnsn SG would be of great importance for solving both theo­ Leningrad. - Panov, A. A (1968) In: "Voprosi funkcionaljnoj mar­ retical and practical problems. fologii I embriologii nosekomnih", 93-132. - Panov, A. A. (1980) In: "Neuroanatomical techniques. Insect nervous system". Springer-Verlag, Acknowledgements - This work was supported by the Ministry of New York, Heidelberg, Berlin. - Phillips, 1.E. (1983) Endocrinology Science, Technology, and Development of Serbia, project No 1615. The ofInsects. Alan R. Liss, Inc., 411-425. - Raabe, M. (1982) In: Insect authors are grateful to Dr. Jelisaveta Ivanovic and Dr. Jelica Lazarevic Neurohormones. Plenum Press, New York, 1-352. - Roberts, D. B. for useful suggestions. (1986) In: Drosophila: a practical approach. IRL Press, Oxford, 15­ 19. - Satija, R. C. and Kaur, S. (1966) Research Bulletin (N.S.) ofthe References: Fahrbach, S. E. (1997) BioScience, 47,2,77-85. - Girardie, Punjab University, 17, III-IV, 353-365. - Schwarc, C. M. and Truman, A., Girardie, J., Proux, J., Remy, C. and Vieillemaringe, J. (1984) In: J. W. (1982) Science, 215, 1420-1421. - Singh, Y. N. and Srivastava, Biosynthesis, Metabolism andMode ofAction ofInvertebrate Hormones U. S. (1973) Inter. J. Insect. Morph. Embr., 2,169-175. - Spring, J. H. (ed. by J. Hoffmann and M. Porchet) Springer-Verlag, Berlin, (1990) J. Insect Physiol., 36, 13-22. - Stanic, v., Ivanovic, 1., Jankovic­ Heidelberg, 97-105. - Ivanovic, J., Jankovic-Hladni, M. and Milanovic, Hladni, M. and Milanovic, M. (1978) Gen. Camp. Endocrinol., 34, 107. M. (I975a) Camp. Biochem. Physiol. 50A, 125-130. - Ivanovic, 1. P., - Stanic, V, Ivanovic, J., Jankovic-Hladni, M. and Nenadovic, V (1989) Jankovic-Hladni, M. 1. and Milanovic, M. P. (1975b) J. Therm. Biol., XIX Yugoslav Entomological Meeting. - Weeks, 1. C. and Levine, R. B. 1, 53-58. - Jankovic-Hladni, M. (1971) PhD Thesis, University of (1990) Annual Review ofNeuroscience, 13, 183-194.

40 N::l, "1 §. :c 30 >< .!!. a: 1 w I-w 2°1 ::E < is 10t .---

1 1"11 IV VI LLI PP P FA A STAGE OF DEVELOPMENT

Fig. 1. Size of vmnsn SG during postembryouic development of Morimus funereus M. I, III, VI, LLI -larval stages, PP - prepupa, P _ pupa, PhA - pharat adult, A- adult. Fig. 2. Vmnsn SG in larval instar IV (x 640).