Effect of the apterous 56f 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 apterous ^56f (ap ^56f ) strain, having an decreased level of the juvenile hormone (JH) and a increased 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 ap ^56f 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. Original Russian Text ? E.V. Bogomolova, N.V. Adonyeva, N.E. Gruntenko, I.Yu. Rauschenbach, 2008, published in Genetika, 2008, Vol. 44, No. 5, pp. 710–712.     

The mechanism of the effect of apterous56f mutation on the reproductive function of Drosophila melanogaster

The effects of L-dihydroxyphenylalanine (L-DOPA) and 20-hydroxyecdysone (20E) were studied with respect to the content of dopamine (DA), intensity of the juvenile hormone (JH) degradation, and fecundity of the wildtype flies (Canton S) and JH-deficient apterous56f mutants (in young females, carrying this mutation, the levels of DA and 20E production were strongly increased). Fly feeding with L-DOPA proved to increase the level of DA in a dose-dependent manner and reduce JH degradation in 2-day-old females of both strains. Feeding with 20E produced the same effect. Treating the wild-type flies with 2.5 mg L-DOPA caused a 24-h delay in beginning of oviposition and reduction in fecundity throughout the experiment. An L-DOPA dose of 1 mg caused no such changes. An experimental increase in 20E titer led to reduced fecundity of the wild-type flies, though no delay in oviposition was observed. In mutant flies, an increase in DA and 20E levels accelerated beginning of oviposition and increased fecundity of young females, though the latter parameter was reduced in mature individuals. Thus, an increase in endogenous DA and 20E characteristic of young apterous56f females is assumed to be a compensatory response that leads to a higher JH titer and induction of vitellogenesis.     

The ecdysoneless1 gene regulates metabolism of the juvenile hormone and dopamine in Drosophila melanogaster

The dopamine (DA) content and the level of juvenile hormone (JH) degradation were studied in females of the wild-type Canton S strain and the ecdysoneless1 (ecd1) mutant, which does not produce ecdysone at a restrictive temperature (29 degrees C). Exposure at the restrictive temperature considerably increased the JH-hydrolyzing activity and the DA content in five-day ecd1 females compared with flies of both strains growing at 19 degrees C and Canton S females exposed at 29 degrees C. In one-day ecd1 females, the level of JH degradation also increased at the restrictive temperature, but the DA content was low. The effect of ecdysone deficiency on the stress response in Drosophila melanogaster females was studied using changes in DA content and JH degradation were used as indices. The ecd1 mutation did not prevent the initiation of the stress response in females exposed at the restrictive temperature, but changed its intensity (stress reactivity). The interaction of 20-hydroxyecdysone with JH and DA in regulating Drosophila reproduction under normal conditions and in stress is discussed.     

The mechanism of the effect of apterous 56f mutation on the reproductive function of Drosophila melanogaster

The effects of L-dihydroxyphenylalanine (L-DOPA) and 20-hydroxyecdysone (20E) were studied with respect to the content of dopamine (DA), intensity of the juvenile hormone (JH) degradation, and fecundity of the wild-type flies (Canton S) and JH-deficient apterous ^56f mutants (in young females, carrying this mutation, the levels of DA and 20E production were strongly increased). Fly feeding with L-DOPA proved to increase the level of DA in a dose-dependent manner and reduce JH degradation in 2-day-old females of both strains. Feeding with 20E produced the same effect. Treating the wild-type flies with 2.5 mg L-DOPA caused a 24-h delay in beginning of oviposition and reduction in fecundity throughout the experiment. An L-DOPA dose of 1 mg caused no such changes. An experimental increase in 20E titer led to reduced fecundity of the wild-type flies, though no delay in oviposition was observed. In mutant flies, an increase in DA and 20E levels accelerated beginning of oviposition and increased fecundity of young females, though the latter parameter was reduced in mature individuals. Thus, an increase in endogenous DA and 20E characteristic of young apterous ^56f females is assumed to be a compensatory response that leads to a higher JH titer in order to induce vitellogenesis.     

Ecdysteroids regulate yolk protein uptake by Drosophila melanogaster oocytes

Juvenile hormones (JHs) are thought to drive the regulation of yolk protein uptake by ovaries in Drosophila melanogaster. However, the level of JH production in a mutant stock (ap(56f)) is depressed yet the flies are normally vitellogenic. The production of ecdysteroids by these ap(56f) ovaries in vitro is elevated above that of wild-type ovaries. The incubation of wild-type ovaries in the presence of 0.1mM JHB(3) increased ecdysteroid biosynthesis only during the first 18h following eclosion. Female Drosophila melanogaster undergo a pre-vitellogenic reproductive diapause when exposed to low temperature (11 degrees C) and a short-day photoperiod (L12:D12). The rate of ecdysteroid synthesis by the ovaries, but not JH production, increased within 12h of a temperature upshift to 25 degrees C from a basal level of 20+/-1pg/10 pair of ovaries/5h to a sustained level of 150+/-20pg/10 pair/5h. Vitellogenic oocytes were noted in all females within 12h of this temperature upshift. Diapause was also terminated by the injection of 1&mgr;g of 20-hydroxyecdysone into the abdomens of diapausing females as determined by an increase in ovary size, and the appearance of vitellogenic oocytes as compared to controls. These results are consistent with a revised model for the regulation of yolk protein uptake by ovaries in which ecdysteroids, and not JHs, play the prominent role.     

Dopamine and octopamine regulate 20-hydroxyecdysone level in vivo in Drosophila

The effects of increased level of dopamine (DA) (feeding flies with DA precursor, L-dihydroxyphenylalanine, L-DOPA) on the level of 20-hydroxyecdysone (20E) and on juvenile hormone (JH) metabolism in young (2-day-old) wild type females (the strain wt) of Drosophila virilis have been studied. Feeding the flies with L-DOPA increased DA content by a factor of 2.5, and led to a considerable increase in 20E level and a decrease of JH degradation (an increase in JH level). We have also measured the levels of 20E in the young (1-day-old) octopamineless females of the strain Tbetah(nM18) and in wild type females, Canton S, of D. melanogaster. The absence of OA led to a considerable decrease in 20E level (earlier it was shown that in the Tbetah(nM18) females, JH degradation was sharply increased). We have studied the effects of JH application on 20E level in 2-day-old wt females of D. virilis and demonstrated that an increase in JH titre results in a steep increase of 20E level. The supposition that biogenic amines act as intermediary between JH and 20E in the control of Drosophila reproduction is discussed.     

The <Emphasis Type="Italic">ecdysoneless</Emphasis><Superscript>1</Superscript> Gene Regulates Metabolism of the Juvenile Hormone and Dopamine in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The dopamine (DA) content and the level of juvenile hormone (JH) degradation were studied in females of the wild-type Canton S strain and the ecdysoneless ¹ (ecd ¹) mutant, which does not produce ecdysone at a restrictive temperature (29°C). Exposure at the restrictive temperature considerably increased the JH-hydrolyzing activity and the DA content in five-day ecd ¹ females compared with flies of both strains growing at 19°C and Canton S females exposed at 29°C. In one-day ecd ¹ females, the level of JH degradation also increased at the restrictive temperature, but the DA content was low. The effect of ecdysone deficiency on the stress reaction in Drosophila melanogaster females was studied using changes in DA content and JH degradation as the reaction indicators. The ecd ¹ mutation did not prevent the initiation of the stress reaction in females exposed at the restrictive temperature, but changed its intensity (stress reactivity). The interaction of 20-hydroxyecdysone with JH and DA in regulating Drosophila reproduction under normal conditions and in stress is discussed.     

Effects of juvenile hormone and 20-hydroxyecdysone on alkaline phosphatase activity in Drosophila under normal and heat stress conditions

The effect of 20-hydroxyecdysone (20E) and the juvenile hormone (JH) on the activity of the alkaline phosphatase (ALP) has been studied in young females of wild-type Drosophila virilis and Drosophila 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 ALP activity. ALP activity was also measured in young females of a JH-deficient strain of D. melanogaster, apterous(56f). A decrease in the enzyme activity was observed in the mutant females as compared to wild type. A rise in JH and 20E levels was found not to prevent the response of ALP to heat stress, but to change its stress-reactivity. Mechanisms of regulation of dopamine (DA) level by gonadotropins in Drosophila are discussed.     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N(2) Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO(3)-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a "conventional" Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.     

Role of arylalkylamine <Emphasis Type="Italic">N</Emphasis>-acetyltransferase in regulation of biogenic amines levels by gonadotropins in <Emphasis Type="Italic">Drosophila</Emphasis>

The effect of 20-hydroxyecdysone (20E) and the juvenile hormone (JH) on the activity of the arylalkylamine N-acetyltransferase (AANAT) was studied in young females of wild-type D. virilis and D. melanogaster. 20E feeding of the flies led to a decrease in AANAT activity in both species when dopamine (DA) was used as substrate, but did not affect the enzyme activity when octopamine (OA) was used as substrate. JH application increased AANAT activity with DA as substrate in both species, but did not change it with OA as substrate. AANAT activity was also measured in young females of a JH-deficient strain of D. melanogaster, apterous ^56f . A decrease in the enzyme activity was observed in the mutant females as compared to wild-type. Mechanisms of regulation of DA level by gonadotropins in Drosophila are discussed.     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N2 Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO₃-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a “conventional” Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.     

A genetic role of isozyme types in plasma alkaline phosphatase activity in the young chicken

A genetic role of isozyme types in plasma alkaline phosphatase (AP) activity within dam families in the young chicken was investigated in a White Plymouth Rock strain kept in our laboratory since 1961. Plasma samples were obtained at 32 and 56 days of age and subjected to horizontal polyacrylamide gel electrophoresis and two methods of analysis.
A higher level of plasma AP activity of the fast (F) type relative to that of slow (S) type was re-confirmed. The F types of full-sib chicks had distinctly higher AP activity than the S types. Also within isozyme types, family differences were significant in the F type but not in the S type. The correlation of AP activities between 32 and 56 days of age was significant in the F type but not in the S type, which could be attributed to the effect of aging. The genetic control of plasma AP activity in young chickens were discussed under a hypothesis of two independent genetic systems, i.e. major genie and polygenic.     

A genetic role of isozyme types in plasma alkaline phosphatase activity in the young chicken.

A genetic role of isozyme types in plasma alkaline phosphatase (AP) activity within dam families in the young chicken was investigated in a White Plymouth Rock strain kept in our laboratory since 1961. Plasma samples were obtained at 32 and 56 days of age and subjected to horizontal polyacrylamide gel electrophoresis and two methods of analysis. A higher level of plasma AP activity of the fast (F) type relative to that of slow (S) type was re-confirmed. The F types of full-sib chicks had distinctly higher AP activity than the S types. Also within isozyme types, family differences were significant in the F type but not in the S type. The correlation of AP activities between 32 and 56 days of age was significant in the F type but not in the S type, which could be attributed to the effect of aging. The genetic control of plasma AP activity in young chickens were discussed under a hypothesis of two independent genetic systems, i.e. major genic and polygenic.     

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.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. V. Biochemical characterization of a strain (ebony) that is UV- and X-ray sensitive and deficient in photorepair

We investigated larval sensitivity to UV and repair of UV- and X-ray-induced lesions in the DNA of the ebony strain compared to a wild-type strain (Canton S). The ebony strain was previously characterized as being more sensitive to UV-induced killing of embryos than Canton S. Also the ebony strain is more sensitive to X-rays for induction of larval killing, dominant lethals and recessive lethals. In this paper it is demonstrated that (1) ebony larvae are more sensitive to killing by UV and less proficient in photoreactivation (PR) ability than Canton S larvae; (2) the ebony strain has a defect in PR repair of endonuclease-sensitive sites induced in the DNA of primary cell cultures by UV irradiation; (3) the ebony strain has a defect in the repair of single-strand breaks induced in the DNA by X-rays (again in primary cell cultures), at least early on in the repair incubation. A rough localization of the UV sensitivity and the PR ability is presented and the possible relevance of the biochemical to the genetic results is discussed.     

Decrease in juvenile hormone level as a result of genetic ablation of the corpus allatum cells affects the synthesis and metabolism of stress related hormones in Drosophila

Previous studies have shown that juvenile hormone (JH) regulates dopamine (DA) and octopamine (OA) content in Drosophila, and we have shown the influence of an increase in JH level on DA and OA metabolism in young females of Drosophila virilis and Drosophila melanogaster. Here we investigate the effects of genetic ablation of a subset of cells in the Corpusallatum (CA, endocrine gland synthesizing JH) on the DA levels and activities of alkaline phosphatase (ALP), tyrosine hydroxylase (TH), DA-dependent arylalkylamine N-acetyltransferase (DAT) and tyrosine decarboxylase (TDC) in young D. melanogaster females under normal conditions and upon heat stress (38°С). We show that ablation of СА cells causes: (1) a decrease in ALP, TH and DAT activities, (2) an increase in DA level and (3) an increase in TDC activity in young females. The CA ablation was also found to modulate ALP, TH and TDC responses to heat stress. Mechanisms of regulation of DA and OA levels by JH in Drosophila females are discussed.     

Interaction of hormones in controlling reproductive function of Drosophila females under stressful conditions is genetically determined

The effect of heat stress (38 degrees C) on the content of octopamine (OA) and 20-hydroxyecdysone (20HE) was studied under normal and stressful conditions in adult flies of Drosophila virilis lines contrasting in the level of the juvenile hormone (JH). The wild-type flies (line 101) exhibited a pronounced sex dimorphism for the content of both OA and 20HE, which was substantially lower in this line than in flies of the mutant line 147. The level of both hormones increased in flies of line 101 exposed to heat stress, whereas it remained unchanged in flies of line 147 under the same conditions. The effect of heat stress on the level of JH metabolism and fertility was also studied in D. melanogaster wild-type lines and lines carrying mutations in genes responsible for OA and DA syntheses. In octopamineless females of the T beta hnM18 line and in females of the Ste line characterized by a doubled content of DA, JH degradation differed from normal: it was increased in both young and mature T beta hnM18 females, while decreased in young and increased in mature Ste flies. Fertility was substantially lower in the Ste than in the wild-type line. Flies of all of the D. melanogaster lines produced a stress response; however, in mutant lines, both fertility and stress reactivity of the systems controlling JH metabolism differed significantly from that of the wild-type lines. The role of JH, 20HE, OA, and DA interaction in regulation of Drosophila reproduction under stressful conditions is discussed.     

Temporal profiles of juvenile hormone titers and egg production in virgin and mated females of Heliothis virescens (Noctuidae)

Juvenile hormones (JH) I, II, and III were monitored in hemolymph of virgin and mated females of various ages in Heliothis virescens. JH I was the predominant homologue followed by JH II, but JH II was present at a higher level in young virgin females. JH III was detectable only at a low level. In virgin females, hemolymph JH titers were low at emergence (2.2ng/ml-total amount of JH I and JH II), but increased thereafter and reached a maximum at 24h of age (53.5ng/ml). At 30h and 36h of age, JH titers dropped to a low level, but increased again in older virgin females. After mating, JH titers increased significantly. JH titers at 0h after uncoupling (137.4ng/ml) were nearly 3 times as high as those in 24-h-old virgin females. Within 6h after uncoupling, JH titers decreased slightly, but titers increased with age of mated females and reached a level of 320.2ng/ml hemolymph at 72h after uncoupling. The titer of JH I and JH II was correlated highly with total number of eggs produced (r(2)=0.70, P<0.001). Mating stimulated JH production, resulting in an increase in egg production.     

Mechanisms of age-specific regulation of dopamine metabolism by juvenile hormone and 20-hydroxyecdysone in <Emphasis Type="Italic">Drosophila</Emphasis> females

20-hydroxyecdysone (20E) and the juvenile hormone (JH) have an age-specific effect on total dopamine (DA) content in Drosophila (Gruntenko and Rauschenbach 2008). Earlier we studied the mechanism of influence of 20E and JH on DA metabolism in young females (Rauschenbach et al. in J Insect Physiol 53:587–591, 2007a: Arch Insect Biochem Physiol 65:95–102, 2008a; Gruntenko et al. in Arch Insect Biochem Physiol 72:263–269, 2009). Here we investigate the effects of 20E and JH on the activities of the alkaline phosphatase (ALP), tyrosine hydroxylase (TH) and DA-dependent arylalkylamine N-acetyltransferase (AANAT) in mature females of wild type D. virilis under normal conditions and under heat stress (38°C). 20E feeding of the flies led to a substantial decrease in ALP and TH activities and to an increase in AANAT activity in mature females. JH application resulted in an increasing of ALP and TH activities, but did not influence AANAT activity in mature females. A rise in JH and 20E levels was found to change ALP and TH stress reactivities. Mechanisms of age-specific regulation of DA level by 20E and JH in Drosophila females are discussed.