Ovarian Ecdysteroid Research Articles

Effects of Pergularia tomentosa active fraction on the oogenesis of the desert locust, Schistocerca gregaria: ovarian biochemical composition, and effects on the ecdysteroid pathway

AbstractFractionation of the methanolic extract of Pergularia tomentosa, using column chromatography, resulted in the separation of 10 fractions and only the active one (that gave mortality >70%) was kept and denoted the active fraction (AFPt). Adult female Schistocerca gregaria were treated with two concentrations of AFPt, 0.24% and 0.96%, to investigate the effect on the female reproductive potential. Both concentrations of AFPt significantly increased the duration between adult emergence and first oviposition, as well as affected fecundity and fertility. Furthermore, the effects of AFPt on vitellogenesis and ecdysteroid titres were examined. Biochemical analyses revealed a significant decrease in protein, lipid and carbohydrate contents in ovaries. This study also showed that AFPt induced a significant decrease in the ovarian ecdysteroids levels, as well as the haemolymph ecdysteroid titres. The transcript levels of the ecdysteroid synthesis enzymes Spook and Shadow were significantly decreased when treated with the highest concentration of AFPt, while transcript levels of the ecdysteroid synthesis enzymes Phantom and Disembodied increased significantly in animals treated with AFPt. Treatment with AFPt also resulted in a significantly higher transcript level of both the ecdysone receptor (EcR) and its partner ultraspiracle/retinoid‐X‐receptor (USP/RXR).

Induction of vitellogenesis, methyl farnesoate synthesis and ecdysteroidogenesis in two edible crabs by arachidonic acid and prostaglandins.

The present study investigated the effect of arachidonic acid (AA) and selected prostaglandins on the regulation of vitellogenesis, ecdysteroidogenesis and methyl farnesoate (MF) synthesis in the freshwater crab Oziotelphusa senex senex and the giant mud crab, Scylla serrata Administration of AA and prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2) significantly increased ovarian index, oocyte diameter and ovarian vitellogenin levels and ecdysteroid and MF levels in the hemolymph of crabs. Secretions of MF and ecdysteroids from in vitro cultured mandibular organs (MO) and Y-organs (YO) isolated from intermolt crabs injected with AA, PGF2α and PGE2 were greater when compared with controls. In contrast, injection of prostaglandin D2 (PGD2) had no effect on vitellogenesis, ecdysteroid and MF levels in circulation. In vitro secretion of MF from MO explants isolated from avitellogenic crabs incubated with AA, PGF2α and PGE2 increased in a time-dependent manner. Conversely, incubation of YOs isolated from avitellogenic crabs with AA, PGF2α and PGE2 had no effect on secretion of ecdsyteroids. These results implicate prostaglandins in the regulation of reproduction by inducing the synthesis of MF and consequent ecdysteroid synthesis in brachyuran crabs, and provide an alternative molecular intervention mechanism to the traditional eyestalk ablation methodology to induce vitellogenesis and ovarian maturation in crustaceans.

Role of the venus kinase receptor in the female reproductive physiology of the desert locust, Schistocerca gregaria

Venus kinase receptors (VKR) are a subfamily of invertebrate receptor tyrosine kinases, which have only recently been discovered. They contain an intracellular tyrosine kinase domain and an extracellular Venus FlyTrap domain. VKRs have been functionally and pharmacologically characterized in only two invertebrate species, namely the human parasite Schistosoma mansoni and the mosquito Aedes aegypti, where they play a crucial role in oogenesis. Here, we report the characterization of a VKR in the desert locust, Schistocerca gregaria. We performed an in-depth profiling study of the SgVKR transcript levels in different tissues throughout the female adult stage. Using the RNA interference technique, the possible role of SgVKR was investigated. SgVKR knockdown had significant effects on ovarian ecdysteroid levels and on the size of oocytes during the vitellogenic stage. SgVKR is probably involved in the complex cross-talk between several important pathways regulating female reproductive physiology. Contrary to A. aegypti and S. mansoni, we cannot conclude that this receptor is essential for reproduction, since silencing SgVKR did not affect fecundity or fertility. Considering the evolutionary distance between A. aegypti and S. gregaria, as well as the differences in regulation of their female reproductive physiology, this article constitutes a valuable asset in better understanding VKRs.

Mating-Induced Increase in Germline Stem Cells via the Neuroendocrine System in Female Drosophila.

Mating and gametogenesis are two essential components of animal reproduction. Gametogenesis must be modulated by the need for gametes, yet little is known of how mating, a process that utilizes gametes, may modulate the process of gametogenesis. Here, we report that mating stimulates female germline stem cell (GSC) proliferation in Drosophila melanogaster. Mating-induced increase in GSC number is not simply owing to the indirect effect of emission of stored eggs, but rather is stimulated by a male-derived Sex Peptide (SP) and its receptor SPR, the components of a canonical neuronal pathway that induces a post-mating behavioral switch in females. We show that ecdysteroid, the major insect steroid hormone, regulates mating-induced GSC proliferation independently of insulin signaling. Ovarian ecdysteroid level increases after mating and transmits its signal directly through the ecdysone receptor expressed in the ovarian niche to increase the number of GSCs. Impairment of ovarian ecdysteroid biosynthesis disrupts mating-induced increase in GSCs as well as egg production. Importantly, feeding of ecdysteroid rescues the decrease in GSC number caused by impairment of neuronal SP signaling. Our study illustrates how female GSC activity is coordinately regulated by the neuroendocrine system to sustain reproductive success in response to mating.

The role of juvenile hormone in dominance behavior, reproduction and cuticular pheromone signaling in the caste-flexible epiponine wasp, Synoeca surinama.

BackgroundThe popular view on insect sociality is that of a harmonious division of labor among two morphologically distinct and functionally non-overlapping castes. But this is a highly derived state and not a prerequisite for a functional society. Rather, caste-flexibility is a central feature in many eusocial wasps, where adult females have the potential to become queens or workers, depending on the social environment. In non-swarming paper wasps (e.g., Polistes), prospective queens fight one another to assert their dominance, with losers becoming workers if they remain on the nest. This aggression is fueled by juvenile hormone (JH) and ecdysteroids, major factors involved in caste differentiation in most eusocial insects. We tested whether these hormones have conserved aggression-promoting functions in Synoeca surinama, a caste-flexible swarm-founding wasp (Epiponini) where reproductive competition is high and aggressive displays are common.ResultsWe observed the behavioral interactions of S. surinama females in field nests before and after we had removed the egg-laying queen(s). We measured the ovarian reproductive status, hemolymph JH and ecdysteroid titers, ovarian ecdysteroid content, and analyzed the cuticular hydrocarbon (CHC) composition of females engaged in competitive interactions in both queenright and queenless contexts. These data, in combination with hormone manipulation experiments, revealed that neither JH nor ecdysteroids are necessary for the expression of dominance behaviors in S. surinama. Instead, we show that JH likely functions as a gonadotropin and directly modifies the cuticular hydrocarbon blend of young workers to match that of a reproductive. Hemolymph ecdysteroids, in contrast, are not different between queens and workers despite great differences in ovarian ecdysteroid content.ConclusionsThe endocrine profile of S. surinama shows surprising differences from those of other caste-flexible wasps, although a rise in JH titers in replacement queens is a common theme. Extensive remodeling of hormone functions is also evident in the highly eusocial bees, which has been attributed to the evolution of morphologically defined castes. Our results show that hormones which regulate caste-plasticity can lose these roles even while caste-plasticity is preserved.Electronic supplementary materialThe online version of this article (doi:10.1186/s12983-014-0078-5) contains supplementary material, which is available to authorized users.

Reproductive status, endocrine physiology and chemical signaling in the Neotropical, swarm-founding eusocial wasp Polybia micans.

In the evolution of caste-based societies in Hymenoptera, the classical insect hormones juvenile hormone (JH) and ecdysteroids were co-opted into new functions. Social wasps, which show all levels of sociality and lifestyles, are an ideal group in which to study such functional changes. Virtually all studies on the physiological mechanisms underlying reproductive division of labor and caste functions in wasps have been done on independent-founding paper wasps, and the majority of these studies have focused on species specially adapted for overwintering. The relatively little-studied tropical swarm-founding wasps of the Epiponini (Vespidae) are a diverse group of permanently social wasps, with some species maintaining caste flexibility well into the adult phase. We investigated the behavior, reproductive status, JH and ecdysteroid titers in hemolymph, ecdysteroid content of the ovary and cuticular hydrocarbon (CHC) profiles in the caste-monomorphic, epiponine wasp Polybia micans Ducke. We found that the JH titer was not elevated in competing queens from established multiple-queen nests, but increased in lone queens that lack direct competition. In queenless colonies, JH titer rose transiently in young potential reproductives upon challenge by nestmates, suggesting that JH may prime the ovaries for further development. Ovarian ecdysteroids were very low in workers but higher and correlated with the number of vitellogenic oocytes in the queens. Hemolymph ecdysteroid levels were low and variable in both workers and queens. Profiles of P. micans CHCs reflected caste, age and reproductive status, but were not tightly linked to either hormone. These findings show a significant divergence in hormone function in swarm-founding wasps compared with independently founding ones.

Circadian regulation of hemolymph and ovarian ecdysteroids during egg development in the insect Rhodnius prolixus (Hemiptera)

Ecdysteroids, classically studied as the molting hormones of insects, occur at lower levels in the adult stage, but their source and significance in adult physiology is debated. In Rhodnius prolixus, a blood meal initiates a cycle of egg development and ecdysteroids appeared in the hemolymph within 1–2h of feeding. Systematic changes in hemolymph ecdysteroid titer then occurred during egg development. There was a clear circadian rhythm in the hemolymph ecdysteroid titer throughout egg development. This is the first report of an ecdysteroid rhythm in any adult female insect. Of various tissues examined in vitro, only ovaries released ecdysteroids. The amounts released were adequate to account for observed hemolymph ecdysteroid titers. Therefore, ovaries are the primary, perhaps sole, source of hemolymph ecdysteroids. Ovaries in vitro showed a circadian rhythm of changes in ecdysteroid content and release that was in synchrony with the hemolymph ecdysteroid rhythm, indicating that ovarian ecdysteroid release drives the rhythm seen in the hemolymph. Potential regulation of ovarian ecdysteroids is discussed in relation to the known rhythms in prothoracicotropic hormone and insulin-like peptides that occur during egg development. The prospect of circadian regulation of egg development itself by hormones and/or an ovarian clock is also discussed.

In vivo effect of Neuropeptide F on ecdysteroidogenesis in adult female desert locusts (Schistocerca gregaria)

Neuropeptides are important regulatory factors that mediate key life processes, both in vertebrates and invertebrates. Many insect neuropeptides display pleiotropic activities, which means that they can influence multiple aspects of insect physiology. In the fruit fly, Drosophila melanogaster, Neuropeptide F (NPF) mediates diverse physiological processes, such as learning, stress responses, feeding and male courtship behavior. In locusts, only a truncated form of the predicted “full-length” NPF, the nonapeptide “trNPF”, has been isolated. This nonapeptide previously proved to be biologically active, since it was shown to influence food intake and weight increase, as well as oocyte growth in adult female desert locusts (Schistocerca gregaria [Forskål]). In the present study, we have further analyzed the effect of trNPF on female reproductive physiology in S. gregaria. We confirmed that daily trNPF injections in adult females elicit an increase of oocyte size. In addition, an RNAi-mediated knockdown of the Schgr-NPF precursor transcript in adult female locusts resulted in the opposite effect, i.e. significantly smaller oocytes. Moreover, we discovered that daily injections of trNPF in adult female S. gregaria, caused higher ecdysteroid titers in the ovaries and accelerated the appearance of ecdysteroid peaks in the hemolymph of these animals. The RNAi-based knockdown of the Schgr-NPF precursor transcript clearly resulted in reduction of both hemolymph and ovarian ecdysteroid concentrations, confirming the stimulatory effects of trNPF injections on adult female ecdysteroid levels. The observed results are discussed in relation to previous reports on NPF activities in locusts and other insects.

Effects of a methanolic extract of the plant Haplophyllum tuberculatum and of teflubenzuron on female reproduction in the migratory locust, Locusta migratoria (Orthoptera: Oedipodinae)

The effects of a methanolic extract of the plant Haplophyllum tuberculatum (ME-Ht) and of teflubenzuron (TFB) were compared on several reproductive variables and ecdysteroid titers in the females of Locusta migratoria. The test products were administered orally to newly emerged females at doses of 1500 (ME-Ht) and 10μg/female (TFB). The methanolic extract and TFB had comparable effects on several of the variables examined. Both significantly delayed the first oviposition and reduced fecundity and fertility. ME-Ht and TFB also displayed similar effects on ovarian growth, vitellogenesis and ecdysteroid titers. Both treatments induced a drop in hemolymph protein levels as well as a reduction in vitellogenin uptake by oocytes. This delay in oogenesis was accompanied by a resorption of terminal oocytes. However, whereas TFB completely blocked egg hatch, ME-Ht only had a modest inhibitory effect on this variable. Hemolymph and ovarian ecdysteroid titers, as measured by radioimmunoassay, were similar and low in both control and treated females, except for a peak observed only in control females at the end of vitellogenesis. We discuss the functional significance of the observed effects in the context of the putative modes of action of the methanolic plant extract and TFB.

Diflubenzuron et évolution des taux des ecdystéroïdes dans les ovaires et dans les oeufs du Criquet pèlerin,Schistocerca gregaria(Forskål, 1775) (Orthoptera: Acrididae)

The effect of DIMILIN 25 WP, the common name of diflubenzuron, was first examined on the weight of ovaries and on the weight of eggs laid by females of Schistocerca gregaria. Treatment was made 24h earlier by ingestion at 30 mg/ml. DFB reduces the weight of ovaries at 8, 12 and 16 days, compared to control series of the same age. Moreover, the insecticidal treatment causes a significant reduction in the weight of eggs laid.DFB action on ecdysteroid levels in ovaries and in eggs in females was also evaluated. The compound reduced ovarian ecdysteroid titres of the first ovarian cycle.In laid eggs of S. gregaria females, we measured ecdysteroid content before and after enzymatic hydrolysis, to compare free and conjugated ecdysteroids in both control eggs and after DFB treatment during embryonic development. DFB significantly reduced the levels of both free and conjugated ecdysteroids compared to the control.

The Role of Ecdysteroid 22‐Kinase in the Accumulation of Ecdysteroids in Ovary of Silkworm Bombyx mori

In the silkworm Bombyx mori, ecdysteroid 22-kinase (EcKinase) has been shown to catalyze the conversion of free ecdysteroids to physiologically inactive ecdysteroid 22-phosphates. First, a phylogenetic analysis of EcKinase showed that many proteins homologous to B. mori EcKinase are found mainly in Ecdysozoa, including insects and nematodes. Next, by RT-PCR analysis combined with laser microdissection and by immunohistochemistry, we demonstrated that free ecdysteroids in the B. mori ovary are synthesized in follicle cells and transferred into the oocyte where they are phosphorylated by EcKinase whose mRNA originates from nurse cells and the oocyte itself.

Reproductive division of labor, dominance, and ecdysteroid levels in hemolymph and ovary of the bumble bee Bombus terrestris

To determine whether ecdysteroids are associated with reproductive division of labor in Bombus terrestris, we measured their levels in hemolymph and ovaries of queens and workers. Queens heading colonies had large active ovaries with high ecdysteroid content, whereas virgin gynes and mated queens before and after diapause had undeveloped ovaries with low ecdysteroid content. The hemolymph ecdysteroid titer was rather variable, but in a pooled analysis of mated queens before and after diapause versus colony-heading queens, ecdysteroid titers were higher in the latter group. In workers, agonistic behavior, ovarian activity, ovarian ecdysteroid content, and hemolymph ecdysteroid titers were positively correlated, and were lowest when a queen was present. In queenless workers, ecdysteroid levels were elevated in dominant workers, and were also influenced by the presence of brood and by group demography; hormone levels were higher in bees kept in larger groups. These findings are consistent with the premise that in B. terrestris the ovary is the primary site of ecdysteroid synthesis, and they show that ecdysteroids levels vary with the social environment.

Imidazole derivative KK-42 reduces ecdysteroid titers and interferes with reproductive processes in adult females of Tenebrio molitor

KK-42, an imidazole derivative considered as potent inhibitor of ecdysteroid biosynthesis, was tested on adult females of Tenebrio molitor. The compound was applied topically (0.1, 1, 5, and 10 μg/insect) on 0- or 2-day-old adult females or added to the culture medium (1 and 10 μM) of ovaries. To test the effect of KK-42 on the reproduction capacity, ovaries were explanted from 2- and 4-day-old females corresponding, respectively, to follicles at the beginning and the end of vitellogenesis. KK-42 treatment reduced the fecundity and the longevity of females, and in addition, it delayed oviposition and affected growth and development of oocytes as evidenced by measurements of the ovarian weight, the number of oocytes per ovaries, and the size of the basal follicle. Hence, the effects were stronger in newly emerged females compared to 2-day-old females. Interestingly, RH-0345, an ecdysteroid agonist, could partly restore the effects on reproductive events induced by KK-42. In a second series of experiments using an enzyme immunoassay to measure ecdysteroid titers, KK-42 reduced the amounts of ecdysteroids in ovaries after application on newly emerged adult females. In 2-day-old females, it caused a significant effect with the highest doses. In addition, the compound significantly reduced the amounts of ecdysteroid released into the culture medium by ovaries explanted from 2- or 4-day-old females. The results are discussed in relation to the mode of action of KK-42 on reproduction.

Hormonal coordination of molting and female reproduction by ecdysteroids in the mole crab Emerita asiatica (Milne Edwards)

The measurement of hemolymph ecdysteroids using radioimmunoassay (RIA) indicates a biphasic increase during intermolt and premolt stages of the mole crab Emerita asiatica. The gradual rise during intermolt stage corresponds to vitellogenic activities in the ovary; whereas a distinctive premolt peak is characteristic of molting crustaceans. Injection experiments with 20-hydroxyecdysone (20E) during different molt cycle stages revealed the onset of precocious premolt changes, as determined by the epidermal retraction and setal development. Injection of 20E augmented protein synthesis in the ovary, hepatopancreas and integumentary tissues. Quantification of ecdysteroids in different developmental stages of ovary also indicated a progressive increase of ovarian ecdysteroids. Interestingly, the ovarian ecdysteroids after reaching a peak at C 3 stage, start declining drastically to reach the lowest level at D 3–4 stage. This decline in the ovarian ecdysteroids is inversely related to rising hemolymph ecdysteroids during these active premolt stages. The hatching of the embryos, attached to the pleopods of the ovigerous females also occurs under a high titer of hemolymph ecdysteroids. In support, 20E injection at C 3 stage crabs indicated a significant reduction in time duration of pleopodal embryonic development leading to hatching of zoea larvae. Understandably, the augmented hemolymph ecdysteroid titer helps in the synchronization of embryo hatching and the premolt changes, as occurring under the normal premolt conditions.

Inhibition of ovarian steroidogenesis by cyclic GMP in a fly.

Previous investigations in the female blowfly Phormia regina have shown that 3-isobutyl-1-methylxanthine (IBMX), a broad spectrum inhibitor of phosphodiesterases (PDEs), fails to mimic the steroidogenic effects of cAMP on ovaries, although it efficiently increases the concentrations of this second messenger. In this study, experiments carried out to clear up this contradiction demonstrated that IBMX, besides its effect on cAMP, also increased cGMP concentrations in blowfly ovary and that these two cyclic nucleotides controlled ovarian steroidogenesis antagonistically. In particular, a selective inhibitor of cGMP-specific PDEs, unlike IBMX, had a very strong negative effect on ovarian steroidogenesis. Moreover, a cGMP analog was able to inhibit steroid biosynthesis in previtellogenic and vitellogenic ovaries, thus affecting basal and acute steroidogenesis respectively. Our observations also demonstrated that cGMP was always present in blowfly ovary, reaching its maximal levels at the end of vitellogenesis, in close correlation with the physiological decrease in ovarian steroidogenesis. Experiments using an inhibitor of protein kinase G clearly indicated that the effects of cGMP were mediated by this enzyme. On the contrary, these effects did not seem to involve cGMP-regulated PDEs or ion channels. Our results also indicated that ovarian cGMP concentrations were not controlled by brain factors, suggesting a probable involvement of paracrine/autocrine factors. Nitric oxide (NO) appeared to be a good candidate for such a control, because an NO donor was able to stimulate ovarian cGMP concentrations and to drastically decrease ovarian ecdysteroid biosynthesis in blowflies. These data thus demonstrate, for the first time in invertebrates, a potent role of cGMP in the negative control of ovarian steroidogenesis and suggest a possible co-regulation with NO.

Hemolymph ecdysteroids do not affect vitellogenesis in the lubber grasshopper.

The role of hemolymph ecdysteroids in the reproduction of non-dipteran insects is unclear. We examine the role(s) of hemolymph ecdysteroids during egg production in the lubber grasshopper, Romalea microptera. In all individuals, hemolymph ecdysteroids rose to a sharp peak with similar maxima and then fell to undetectable levels. The time from the adult molt to the maximum ecdysteroid titer (E(max) titer) varied in response to food availability, whereas the time from E(max) titer to oviposition was unrelated to food availability. Because both the timing of egg production and the timing of E(max) responded similarly to environmental changes, ecdysteroids may be involved in egg production. We hypothesized that this role is the stimulation of vitellogenesis. Ovariectomized females had vitellogenin but no ecdysteroids, so ecdysteroids are not necessary for vitellogenin production. In addition, treatment of females with ecdysteroids altered neither Vg titers nor ovarian growth. Ovarian ecdysteriods increased at the same age in development as hemolymph ecdysteroids. In contrast to hemolymph ecdysteroids, ovarian ecdysteroids persisted until oviposition. Despite this, [(3)H]ecdysone injected into the hemolymph was detected later only at very low levels in the ovary, suggesting that hemolymph ecdysteroids are not sequestered by the ovary. In summary, our studies indicate that hemolymph ecdysteroids in adult females of the lubber grasshopper are associated with the timing of egg production, but they neither regulate vitellogenesis nor act as a source of ecdysteroids for the ovary.

Expression of allatostatin in the oviducts of the cockroach Diploptera punctata

The cockroach allatostatins (Y/FXFGL/Ia ASTs) are a ubiquitous family of peptides in the invertebrates. They affect numerous physiological processes including the inhibition of juvenile hormone III (JH) biosynthesis, inhibition of muscle contraction, inhibition of ovarian ecdysteroid biosynthesis and inhibition of vitellogenin (Vg) release from the fat body. We have developed and optimized a sensitive and specific quantitative competitive reverse transcriptase polymerase chain reaction (QC-RT-PCR) method to quantify Diploptera punctata AST (Dippu-AST) expression. Using this technique we show that tissues of both lateral and common oviducts and the ovary express message for Dippu-AST. Moreover, the pattern of expression observed in the oviducts and ovary is strikingly similar with significant changes occurring during the reproductive cycle. Specifically, expression of AST is drastically reduced during the time of maximal vitellogenin (Vg) uptake, with higher levels measured prior to and following vitellogenesis. Furthermore, using immunocytochemistry, we have shown Dippu-AST-like-immunoreactivity in the terminal abdominal ganglion, as well as in ventral nerve 7, some branches of which innervate the common and lateral oviducts with other branches innervating the bursa copulatrix and brood sac of mated female D. punctata. The pattern of Dippu-AST expression and immunocytochemical staining suggests that ASTs function, in part, to regulate the cycle of vitellogenesis in mated female D. punctata.

Possible involvement of ecdysteroids in photoperiodically induced suppresion of ovarian development in a Japanese strain of the migratory locust, Locusta migratoria

In a Japanese population of Locusta migratoria, adult females become reproductively inactive under crowding and long days (LD) and reproductively active under crowding and short days (SD). The identity and titre of ecdysteroids in the haemolymph and ovaries from adult females reared under SD and LD were investigated by RIA/HPLC. The effects of exogenous juvenile hormone (JH) III treatments on the termination of such reproductive arrest and ecdysteroid contents in LD females were also examined. In general, ecdysteroid titres in both haemolymph and ovaries were significantly higher in reproductively active SD females than in reproductively inactive LD females. A clear difference was also observed in oocyte growth between SD and LD individuals. JH III applications (three consecutive topical applications, 150 μg per insect per day from day 3) stimulated ovarian development in LD females and significantly increased the haemolymph and ovarian ecdysteroids to a level comparable to that of reproductively active SD adult females.

Cyclic AMP-dependent and independent stimulations of ovarian steroidogenesis by brain factors in the blowfly, Phormia regina

The involvement of cyclic-AMP (cAMP) as a potential second messenger in the neurohormonal control of ovarian steroidogenesis was investigated in the adult female blowfly Phormia regina. Individual measurements of ovarian cAMP concentrations and of ovarian biosynthesis of ecdysteroids, stimulated after a protein meal, demonstrated that steroidogenesis is preceded by a peak of cAMP in the ovaries. In vitro, ovarian steroidogenesis was stimulated by cell-permeable analogues of cAMP and by forskolin. Crude brain extracts were also able to elicit a rise of cAMP in the ovaries in vitro and the secretion of ecdysteroids into the medium: such extracts were more active before than after the protein meal, suggesting a rapid release of neuroendocrine material after feeding. Extracts were then prepared from the dorso-medial part of the brain, containing the neurosecretory cells of the pars intercerebralis (PI): these extracts were again found to stimulate the ovarian ecdysteroid secretion, but surprisingly, they failed to trigger a rise of cAMP in the ovaries in vitro. However, extracts from the rest of the cephalic nervous mass, deprived of PI, were also steroidogenic and they increased ovarian cAMP. Experiments with Rp-cAMPS, a cAMP antagonist, were not able to prevent the ecdysteroid stimulation by PI extracts, but did so partly for the extracts deprived of PI. This study thus indicates that at least two different cephalic factors are able to stimulate ovarian steroidogenesis in the blowfly, one elaborated by PI and acting via a cAMP-independent mechanism, and the other elaborated outside PI and using cAMP as a second messenger.

Endocrine and neuroendocrine effects of Azadirachtin in adult females of the earwig Labidura riparia

In previous studies we have shown that injection of the insect growth regulator Azadirachtin (AZA) into young vitellogenic females induces inhibition of vitellogenesis in a dose-dependent manner. Juvenile hormone treatment rescues vitellogenin synthesis and ovarian growth. The cytopathological effects on ovaries and fat body are not linked to an inhibition of feeding. In this work we investigated the effects of AZA on the endocrine and neuroendocrine system. Enzyme immunoassay reveals that ovarian ecdysteroid levels are drastically reduced, in a dose-dependent fashion, by AZA. Ultrastructural study indicates that corpus allatum cells exhibit signs of inactivity and degenerative changes after AZA exposure. Using an antibody against allastostatin-3 of Blatella germanica (BLAST-3), we show the appearance of strong immunoreactivity of numerous cells and axons in the brain of AZA-injected females. We conclude that vitellogenesis inhibition by AZA consists of a direct cytotoxic effect as well as a generalized disruption of endocrine and neuroendocrine functions.