Pierre Charpin

Neurogenesis in adult insect mushroom bodies.

The occurrence of neurogenesis in mushroom bodies of adult insects belonging to several orthopteroid and coleopteran families is described. Using injections of 5‐bromo, T2′‐deoxy we showed that neuroblasts, which are progenitors of Kenyon cells during preimaginal instars continue to divide in adult Acheta domesticus. Their progeny constitute a central column in mushroom body cortices of 3‐week‐old females. Other Gryllidae, Gryllus bimaculatus and Gryllomorpha dalmatina, show the same pattern of neuroblast activity and migration of their progeny. Immunocytochemical staining of glial cells failed to reveal any immunoreactivity, either in proliferating regions or in the resulting cells.

Dual effect of ecdysone on adult cricket mushroom bodies

Mushroom bodies, which are the main integrative centre for insect sensorial information, play a critical role in associative olfactory learning and memory. This paired brain structure contains interneurons grouped in a cortex, sending their axons into organized neuropiles. In the house cricket (Acheta domesticus) brain, persistent neuroblasts proliferate throughout adult life. Juvenile hormone (JH) has been shown to stimulate this proliferation [Cayre, M., Strambi, C. & Strambi, A. (1994) Nature, 368, 57–59]. In the present study, the effect of morphogenetic hormones on mushroom body cells maintained in primary culture was examined. Whereas JH did not significantly affect neurite growth, ecdysone significantly stimulated neurite elongation. Moreover, ecdysone also acted on neuroblast proliferation, as demonstrated by the reduced number of cells labelled with 5‐bromodeoxyuridine following ecdysone application. Heterospecific antibodies raised against ecdysone receptor protein and ultraspiracle protein, the two heterodimers of ecdysteroid receptors, showed positive immunoreactivity in nervous tissue extracts and in nuclei of mushroom body cells, indicating the occurrence of putative ecdysteroid receptors in cricket mushroom body cells. These data indicate a dual role for ecdysone in adult cricket mushroom bodies: this hormone inhibits neuroblast proliferation and stimulates interneuron differentiation. These results suggest that a constant remodelling of mushroom body structure could result from physiological changes in hormone titres during adult life.

Ovaries and regulation of juvenile hormone titer in Acheta domesticus L. (Orthoptera)

A study was performed on females Acheta domesticus to examine the effects of various experimental conditions on the ovarian physiology. Using a radioimmunoassay to determine juvenile hormone (JH) titers as well as in vitro JH biosynthesis, we observed that retention of mature follicles in egg-retaining females, i.e., virgins or mated females not provided an egg-laying substrate, inhibits JH production and consequently oocyte development. Mating in intact as well as ovariectomized females does not affect corpora allata activity. It is only when mating is associated with egg laying that JH biosynthesis and hemolymph titers increased and oocyte development and fecundity are stimulated. Despite lower JH biosynthesis, ovariectomized females present enlarged corpora allata and the levels of JH observed in their hemolymph were intermediate between those of intact egg-laying and virgin females. In intact females, the hemolymph JH titers as well as the JH esterase activities were related to ovarian development. JH esterase activity was very high in ovariectomized animals. Several factors involved in ovarian development of A. domesticus are discussed.

Inhibition of polyamine biosynthesis alters oviposition behavior in female crickets

The role of polyamines in the expression of cricket oviposition, a juvenile hormone-dependent behavior, was investigated using a specific inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine (alpha-DFMO). The fat body of treated female house crickets (Acheta domesticus) did not show any putrescine and presented reduced levels of spermidine, whereas spermine titres were significantly enhanced. In nervous tissue, alpha-DFMO did not affect spermine titres but induced a severe drop in spermidine levels. In polyamine depleted females, the expression of egg-laying behavior was delayed and was expressed less frequently compared with controls. As drug treatment did not seem to affect juvenile hormone titres, the data suggest that juvenile hormone might act on behavior by way of polyamine metabolism. These results support the view that, in insects, as in vertebrates, the ornithine decarboxylase-polyamine system is involved in the maturation of complex behaviors.

Fate of neuroblast progeny during postembryonic development of mushroom bodies in the house cricket, Acheta domesticus.

Mushroom bodies represent the main sensory integrative center of the insect brain and probably play a major role in the adaptation of behavioral responses to the environment. Taking into account the continuous neurogenesis of cricket mushroom bodies, we investigated ontogenesis of this brain structure. Using BrdU labeling, we examined the fate of neuroblast progeny during the postembryonic development. Preimaginal Kenyon cells survived throughout larval and imaginal moults and persisted during adulthood. Our results indicate that the location of labelled Kenyon cells in the cortex of the adult cricket mainly depends upon the period when they were produced during development. The present data demonstrate that cricket mushroom bodies grow from the inside out and that, at any developmental stage, the center of the cortex contains the youngest Kenyon cells. This study also allowed us to observe the occurrence of quiescent neuroblasts. Kenyon cell death during postembryonic and adult life seems to be reduced. Although preimaginal Kenyon cells largely contribute to adult mushroom body structure, a permanent remodeling of the mushroom body occurs throughout the whole insect life due to the persistence of neurogenesis in the house cricket. Further studies are needed to understand the functional significance of these findings.

Effects of juvenile hormone on polyamines of the fat body and neural tissue of the cricket Acheta domesticus

Abstract The levels of polyamines and the activity of two of the rate limiting enzymes of their biosynthesis were studied in neural tissue and in the fat body of adult females of Acheta domesticus , submitted to different physiological experiments. Allatectomy, performed during the last larval instar, suppressed juvenile hormone production, resulted in the absence of the peak of hormone occurring in 3-day-old adult females and affected the normal pattern of polyamine distribution. As early as day 4 post-emergence, putrescine titres markedly decreased in the fat body, and spermidine levels dropped in the neural tissue. In the fat body, allatectomy depressed ornithine decarboxylase activity, but increased S -adenosylmethionine decarboxylase activity. In the neural tissue, lack of juvenile hormone depressed both ornithine decarboxylase and S -adenosylmethionine decarboxylase activities, resulting in a significant reduction of polyamine titres. Juvenile hormone injections into allatectomized females enhanced enzyme activities and increased polyamine titres. These findings suggest that the neural tissue, like the fat body, may be a target organ for juvenile hormone action. The present data reveal tissue-specific patterns of polyamine metabolism and demonstrate the action of juvenile hormone on polyamine metabolism.

Ecdysone deprivation affects polyamine metabolism in the house cricket Acheta domesticus

Abstract The basic importance of polyamines, which are involved in the regulation of DNA, RNA and protein synthesis, are underlined in numerous works. Using HPLC analysis, we determined polyamine levels in several tissues of 10-day old adult crickets subjected to different hormonal conditions. Ovariectomy performed during the last larval instar suppressed haemolymph ecdysteroids and significantly increased the spermidine titres in nervous tissue whereas it decreased the overall polyamine levels in the fat body. Ecdysone injections into ovariectomized females reduced the spermidine content in nervous tissue and markedly increased the overall fat body polyamine levels, an effect which was mimicked by saline injections in this last tissue. As compared to control values, the activity of ornithine decarboxylase (ODC), the first key enzyme in polyamine biosynthesis was significantly reduced by ovariectomy either in nervous tissue or in fat body. In ovariectomized females, ecdysone injections significantly stimulated only the fat body ODC activity. The effects of polyamines on protein phosphorylation was examined. The phosphorylation of two proteins (207 and 128 kDa respectively) present in the fat body and the nervous tissue of control and ovariectomized females decreased in the presence of polyamines. The phosphorylation of a 53.5 kDa phosphoprotein only present in the nervous tissue differed according to the hormonal status of the female and was strongly enhanced by polyamines. Polyamines also stimulated the phosphorylation of a 37.5 kDa phosphoprotein present in both nervous tissue and fat body. In this last tissue, the phosphorylation of a 20 kDa phosphoprotein present in ovariectomized females was inhibited by the addition of polyamines. This work demonstrates an action of ecdysone on polyamine levels in nervous tissue and underlines the ability of polyamines to modulate the phosphorylation of specific phosphoproteins which are under hormonal control. These data emphasize the importance of polyamines in the regulation of cell functions.

c-Fos-related antigens in the central nervous system of an insect, Acheta domesticus

Fos-related antigens (Fra) were detected in the nuclei of neurones in young adult Acheta domesticus female crickets by immunohistochemical analysis, using an antibody that recognizes the amino-acid sequence 127-152 of c-Fos protein. Specificity of Fra immunoreactivity was confirmed by Western blot analysis of nuclear extracts from neural tissues. A major immunoreactive doublet with an apparent molecular mass of 52,000/54,000 Da was detected in nuclear extracts. Immunostaining of the 52,000/54,000 Da doublet showed variations in intensity during the first 5 days following the imaginal molt. Staining was more intense between day 2 and day 4 when ecdysteroid titers were high. Expression of Fra was low in allatectomized (i.e., deprived of juvenile hormone and ecdysteroids) and ovariectomized (i.e., deprived of ecdysteroids) females as compared to control females. These results show the involvement of hormone-regulated process in expression of Fra. The effect of nociceptive stimulation on Fra expression was tested. Twenty minutes after removal of the ovipositor, a supplementary band with an apparent molecular mass of 70,000 Da appeared in the nuclear extracts, then decreased and disappeared totally after 45 min. Several other Fos-related antigens with different temporal patterns of expression were also detected.

CASEIN KINASE II ACTIVITY IN THE BRAIN OF AN INSECT, ACHETA DOMESTICUS : CHARACTERIZATION AND HORMONAL REGULATION

This study documented casein kinase II (CK II) activity in Acheta domesticus brain using specific antibodies and its regulation by polyamines. In control animals a transient decrease in CK II activity at day 3 after imaginal moult was observed in the brain but not in the fat body. If deprived of ecdysone by ovariectomy a different pattern was observed, with CK II activity being significantly higher on days 3 and 4 after emergence. After ecdysone injection in ovariectomized females, CK II activity decreased to levels similar to those in controls. The implications of ecdysone regulation of brain CK II activity are discussed.