The Role of 20-Hydroxyecdysone in the Control of Carbohydrate Levels in Drosophila melanogaster under Short-Term Heat Stress.

It is known that 20-hydroxyecdysone is one of the most important hormonal regulators of development, reproduction and adaptation to unfavorable conditions in insects. Here, we show for the first time that exogenous 20-hydroxyecdysone increases the content of two main insect carbohydrates, trehalose and glucose, in Drosophila melanogaster females both in normal conditions and under short-term heat stress. It is found that the levels of both trehalose and glucose increase after 39 min of heat exposure and return to their original levels after 1.5 h. A scheme of hormonal regulation of carbohydrate content under heat stress, involving 20-hydroxyecdysone, juvenile hormone, and dopamine, is suggested.

The role of insulin signalling in the endocrine stress response in Drosophila melanogaster: A mini-review.

The endocrine stress response in Drosophila includes catecholamines, juvenile hormone (JH), 20-hydroxyecdysone (20E) and the insulin/insulin-like growth factor signalling pathway (IIS). Several changes in the IIS and hormonal status that occur under unfavourable conditions are universal and do not depend on the nature of stress exposure. The reviewed studies on the impact of different element of the Drosophila IIS, such as insulin-like receptor, the homologue of its substrate, CHICO, the transcription factor dFOXO and insulin like peptide 6, on the hormonal status suggest that the IIS controls catecholamine metabolism indirectly via JH, and there is a feedback loop in the interaction of JH and IIS. Moreover, at least one of the ways in which the IIS is involved in the control of stress resistance is mediated through JH/dopamine signalling.

Insulin-like peptide DILP6 regulates juvenile hormone and dopamine metabolism in Drosophila females.

Insulin-like peptide DILP6 is a component of the insulin/insulin-like growth factor signalling pathway of Drosophila. Juvenile hormone (JH) and dopamine (DA) are involved in the stress response and in the control of reproduction. In this study, we investigate whether DILP6 regulates the JH and DA levels by studying the effect of a strong hypomorphic mutation dilp641 on JH and DA metabolism in D. melanogaster females. We show that DILP6 regulates JH and DA metabolism: the mutation dilp641 results in a reduction in JH-hydrolysing activity and an increase in the activities of DA synthesis enzymes (alkaline phosphatase (ALP) and tyrosine hydroxylase (TH)). In the mutant females, we also found increased fecundity in addition to the intensity of the response (stress reactivity) of ALP and TH to heat stress. As we showed previously, this suggests an increased level of JH synthesis. We confirm this suggestion by treating the mutant females with the JH inhibitor, precocene, which restors the activity and stress reactivity of ALP and TH as well as fecundity to levels similar to those in the control flies. The data suggest a feedback system in the interaction between JH and DILP6 in which DILP6 negatively regulates the JH titre via an increase in the hormone degradation and a decrease in its synthesis.

[Gene dilp6 regulates octopamine metabolism in Drosophila melanogaster].

The effect of strong hypomorphic mutation of the insulin-like protein gene (dilp6) on metabolism of octopamine (one of the main biogenic amines in insects) was studied in Drosophila melanogaster males and females. The activity of tyrosine decarboxylase (the key enzyme of octopamine synthesis) and the activity of octopamine-dependent N-acetyltransferase (the enzyme of its degradation) were measured. It was demonstrated that the activity of both studied enzymes is decreased under normal conditions in the dilp6 41 mutants (as we previously demonstrated, this is correlated with an increased level of octopamine). It was also found that hypomorphic mutation of the dilp6 gene decreases the intensity of tyrosine decarboxylase response to heat stress. Thus, it was demonstrated for the first time that insulin-like DILP6 protein in drosophila influences the level of octopamine (regulating the activity of the enzyme degrading octopamine).

The insulin-like receptor gene expression in the tissues synthesizing gonadotropic hormones at sexual maturation of Drosophila melanogaster females].

The insulin/insulin-like growth factor signaling pathway is involved in the regulation of the synthesis of insect gonadotropic hormones, juvenile (JH) and 20-hydroxyecdysone (20E). We carried out the immunohistochemical analysis of the insulin receptor (InR) expression in the corpus allatum (the JH-producing gland) and in the ovarian follicular cells (a site for the synthesis of 20E precursor, ecdysone) in the process of sexual maturation of D. melanogaster females and examined the influence of exogenous JH on the InR expression in these tissues. For the first time, it was demonstrated that InR was expressed in follicular cells and that its expression in corpus allatum and follicular cells of Drosophila females was stage-specific, i.e., the expression intensity in young females greatly exceeded that in mature individuals. We also found a negative feedback loop in the regulation of JH levels by the insulin signaling pathway in Drosophila adults: the experimental increase in the JH titers in young females dramatically reduced the InR expression intensity in corpus allatum and follicular cells.

Mutation in the Drosophila insulin-like receptor substrate, chico, affects the neuroendocrine stress-reaction development.

It is shown for the first time that the insulin receptor substrate gene chico controls the functioning of the systems of metabolism of dopamine and juvenile hormone in Drosophila melanogaster females under normal conditions and in thermal stress.

[dFOXO Transcription Factor Regulates Juvenile Hormone Metabolism in Drosophila melanogaster Females].

dFOXO transcription factor is a component of the insulin/insulin-like growth factor signaling pathway in Drosophila. Juvenile hormone negatively regulates dFOXO gene expression. In the present work, the effect of hypomorphic dFOXO mutation on juvenile hormone metabolism under normal and stressing conditions and on D. melanogaster female resistance to thermal stress was studied. It was demonstrated that dFOXO mutation in D. melanogaster females induces (1) an increase in the level of juvenile hormone degradation and in the intensity of the response of the juvenile hormone metabolism system to thermal stress and (2) a decrease in thermal stress resistance. These parameters are indicators of the level of juvenile hormone synthesis and indicate its decrease in females with decreased dFOXO expression. Thus, the presence of feedback in the regulation of dFOXO gene expression by juvenile hormone was established for the first time.

[InR gene expression and octopamine metabolism in Drosophila melanogaster females].

The influence of suppression of the expression of the Drosophila insulin-like receptor gene (InR) in corpus allatum (the gland-synthesizing juvenile hormone) on octopamine and juvenile hormone metabolism and on the development of the stress-reaction in Drosophila melanogaster females was studied. It was demonstrated that the suppression of InR gene expression in corpus allatum induces in D. melanogaster females an increase in the activity of the enzyme that limits the rate of octopamine synthesis (tyrosine decarboxylase), as well as in the level of juvenile hormone degradation and the intensity of the response of octopamine and juvenile hormone metabolism systems to heat stress. It was mentioned that a decrease in InR gene expression in corpus allatum does not influence the activity of OA-dependent N-acetyltransferase (the enzyme that degrades octopamine). It was established that the influence of suppression of the InR gene expression in corpus allatum on octopamine metabolism is mediated by juvenile hormone, since the processing of flies by exogenous juvenile hormone restores the activity oftyrosine decarboxylase in flies with decreased InR expression in corpus allatum up to the normal level.

Fertility differences between two wild-type Drosophila melanogaster lines correlate with differences in the expression of the Jheh1 gene, which codes for an enzyme degrading juvenile hormone.

Juvenile hormone plays a "status quo" role in Drosophila melanogaster larvae, preventing the untimely metamorphosis, and performs a gonadotropic function in imagoes, ensuring the ovaries' preparedness for vitellogenesis. The decreased level of juvenile hormone results in reproductive disorders in D. melanogaster females including a delay in the oviposition onset and a fertility decrease. Another factor that can affect the insect reproduction is an infection with the maternally inherited symbiotic α-proteobacterium Wolbachia. The present study is devoted to the analysis of the expression of two juvenile hormone metabolism genes encoding enzymes of its synthesis and degradation, juvenile hormone acid O-methyltransferase ( jhamt) and juvenile hormone epoxide hydrase (Jheh1), respectively, in four wild-type D. melanogaster lines, two of them being infected with Wolbachia. Lines w153 and Bi90 were both derived from an individual wild-caught females infected with Wolbachia, while lines w153T and Bi90T were derived from them by tetracycline treatment and are free of infection. Line Bi90 is known to be infected with the Wolbachia strain wMel, and line w153, with the Wolbachia strain wMelPlus belonging to the wMelCS genotype. It was found that infection with either Wolbachia strain does not affect the expression of the studied genes. At the same time, it was shown that the w153 and w153T lines differ from the Bi90 and Bi90T lines by an increased level of the Jheh1 gene expression and do not differ in the jhamt gene expression level. Analysis of the fertility of these four lines showed that it does not depend on Wolbachia infection either, but differs between lines with different nuclear genotypes: in w153 and w153T, it is significantly lower than in lines Bi90 and Bi90T. The data obtained allow us to reasonably propose that the inter-line D. melanogaster polymorphism in the metabolism of the juvenile hormone is determined by its degradation (not by its synthesis) and correlates with the fertility level.

[Ubiquitous downregulation of InR gene expression affects stress associated hormone metabolism in Drosophila females].

The effect of the ubiquitous downregulation of insulin receptor (InR) gene expression on the metabolism of juvenile hormone (JH) and dopamine (DA) in young females of D. melanogaster under normal conditions and heat stress is studied. The activity ofJH degradation and alkaline phosphatase (ALP, an enzyme regulating DA synthesis) were used as indicators of JH and DA levels, respectively. We demonstrated that, under normal conditions, the ubiquitous inhibition of the InR gene expression in D. melanogaster females induced an increase in the JH degradation and ALP activity. As we have already shown, this is indicative of the decrease in the concentration of the above hormones. It was also found that the total inactivation of InR does not affect the initiation ofJ H and DA metabolic system response to heat stress; however, it does affect its intensity. Thus, the involvement of the insulin signaling pathway in the regulation of the JH and DA metabolism in Drosophila females was demonstrated in vivo under normal and stress conditions.

[Role of DopR in the molecular mechanism of the dopamine control of juvenile hormone metabolism in female Drosophila].

The effect of a decreased availability of the D1-like dopamine receptor (DopR) in Drosophila (caused by DopR antagonist added into food) on the juvenile hormone (JH) synthesis rate in young female D. melanogaster has been studied. The JH degradation rate and the alkaline phosphatase (ALP) and tyrosine decarboxylase (TDC) activities were used as indicators of the JH synthesis rate. Treatment of the flies with butaclamol, a specific DopR antagonist, has been demonstrated to increase the JH degradation rate, and the stress reactivity of the system of JH metabolism and decrease the ALP activity and stress reactivity, and increase the TDC activity and stress reactivity. As shown earlier, all this indicates a decrease in the JH synthesis rate in young female drosophila with a decreased DopR availability. It is concluded that the activating effect of dopamine on JH synthesis in Drosophila is mediated by D1-like receptors.

[The DD2R gene expression level in the corpus allatum affects the fitness of female Drosophila melanogaster].

The effect of tissue-specific suppression of the dopamine D2-like receptor gene (DD2R) in the corpus allatum (CA), the gland that synthesizes juvenile hormone (JH) on the Drosophila melanogaster resistance to heat stress has been studied. A decreased expression of the DD2R gene in the CA has been found to substantially decrease the heat stress resistance of adult transgenic female, but not male, D. melanogaster compared to the control group, this phenomenon being weakly pronounced in juvenile flies. The effect of DD2R activation on the D. melanogaster reproductive function has been estimated. It has been shown that treatment of D. melanogaster with a synthetic specific agonist of DD2R decreases the fertility, the effect being considerably stronger in adult flies than in juvenile ones. It is concluded that the change in the number of DD2Rs in CA or their activation decreases the fitness of Drosophila.

[Inhibition of DD2R gene expression in the corpus allatum activates alkaline phosphatase in female Drosophila melanogaster].

Tissue-specific inhibition of the expression of the D2-like dopamine receptor gene (DD2R) in the corpus allatum (CA), which is a gland that synthesizes the juvenile hormone (JH), was tested for effect on alkaline phosphatase (AP) activity and the intensity of the AP response to heat stress (stress reactivity) in female Drosophila melanogaster. AP activity and AP stress reactivity in transgenic females with lower DD2R expression in the CA were higher than in control flies. A pharmacological elevation in JH increased AP activity in females of the control strains. DD2R was assumed to mediate the inhibitory effect of dopamine of JH synthesis in the CA of D. melanogaster.

The transcription factor dfoxo controls the expression of insulin pathway genes and lipids content under heat stress in Drosophila melanogaster.

The insulin/insulin-like growth factor signaling (IIS) pathway is one of the key elements in an organism's response to unfavourable conditions. The deep homology of this pathway and its evolutionary conservative role in controlling the carbohydrate and lipid metabolism make it possible to use Drosophila melanogaster for studying its functioning. To identify the properties of interaction of two key IIS pathway components under heat stress in D. melanogaster (the forkhead box O transcription factor (dfoxo) and insulin-like peptide 6 (dilp6), which intermediates the dfoxo signal sent from the fat body to the insulin-producing cells of the brain where DILPs1-5 are synthesized), we analysed the expression of the genes dilp6, dfoxo and insulin-like receptor gene (dInR) in females of strains carrying the hypomorphic mutation dilp6 41and hypofunctional mutation foxo BG01018. We found that neither mutation inf luenced dfoxo expression and its uprise under short-term heat stress, but both of them disrupted the stress response of the dilp6 and dInR genes. To reveal the role of identif ied disruptions in metabolism control and feeding behaviour, we analysed the effect of the dilp6 41 and foxo BG01018 mutations on total lipids content and capillary feeding intensity in imago under normal conditions and under short-term heat stress. Both mutations caused an increase in these parameters under normal conditions and prevented decrease in total lipids content following heat stress observed in the control strain. In mutants, feeding intensity was increased under normal conditions; and decreased following short-term heat stress in all studied strains for the f irst 24 h of observation, and in dilp6 41 strain, for 48 h. Thus, we may conclude that dfoxo takes part in regulating the IIS pathway response to heat stress as well as the changes in lipids content caused by heat stress, and this regulation is mediated by dilp6. At the same time, the feeding behaviour of imago might be controlled by dfoxo and dilp6 under normal conditions, but not under heat stress.

Effects of 20-hydroxyecdysone and juvenile hormone on octopamine metabolism in females of Drosophila.

The effect of exogenous 20-hydroxyecdysone (20E) and juvenile hormone (JH) on the activities of the tyrosine decarboxylase (TDC), the first enzyme in octopamine (OA) synthesis, has been studied in young females of wild type D. virilis and D. melanogaster under normal and heat stress (38 degrees C) conditions. Flies fed 20E expressed increased TDC activity in both species. JH application decreased TDC activity in both species. A rise in JH and 20E levels did not prevent a TDC response to heat stress, but changed the response intensity. A long-term increase in JH titre had no effect on the activity of main OA catabolyzing enzyme, arylalkylamine N-acetyltransferase, in females of both species. A possible mechanism of regulation of OA levels by 20E and JH in Drosophila females is discussed.

20-hydroxyecdysone and juvenile hormone influence tyrosine hydroxylase activity in Drosophila females under normal and heat stress conditions.

The effects of exogenous 20-hydroxyecdysone (20E) and the juvenile hormone (JH) on the activity of the tyrosine hydroxylase (TH), the first and rate-limiting DA biosynthetic enzyme, has been studied in young females of wild type D. virilis and D. melanogaster under normal conditions and under heat stress (38 degrees C). Both 20E feeding of the flies and JH application led to a substantial rise in TH activity. A rise in JH and 20E levels was found not to prevent the response of TH to heat stress, but to change the intensity of its response to the stress exposure. Putative mechanisms of regulation of DA level by 20E and JH in Drosophila females are discussed.

Interplay of JH, 20E and biogenic amines under normal and stress conditions and its effect on reproduction.

Juvenile hormone (JH) and 20-hydroxyecdysone (20E) are well known to play a gonadotropic role in adult insects. In Drosophila the mechanism of reciprocal regulation of JH and 20E is shown to be responsible for their proper balance. Dopamine is a mediator in this JH and 20E interplay. A proper balance between JH and 20E is crucial for the normal progress of oogenesis. An imbalance of gonadotropins leads to reproductive defects: a rise in JH titre leads to oviposition arrest, a rise in 20E level, to the degradation of vitellogenic oocytes. Upon a change in the level of one of the gonadotropins, the balance is restored owing to the relative change in the titre of the other.

[Effect of the apterous56f mutation on N-acetyltransferase and alkaline phosphatase activities in Drosophila melanogaster females].

Alkaline phosphatase (AP) and N-acetyltransferase (NAT) activities were studied in 1-day-old Drosophila melanogaster females of the apterous56f (ap56f) strain, having an elevated level of the juvenile hormone (JH) and a decreased level of dopamine as a result of the mutation, and in the Canton S ancestral wild-type strain in the normal conditions and upon an experimental increase in JH titer. The AP and NAT activities in ap56f females were significantly lower than in Canton S females in the norm. JH application increased the AP activity of mutant females to the level characteristic to JH-treated wild-type females.