Drosophila Melanogaster Canton-S Research Articles

Dietary protein-to-carbohydrate ratio effects development and metabolism in Drosophila larvae and imago

Background. Nutrition during growth and development affects various traits not only in larvae but also imago including lifespan, reproduction, feeding, metabolism, and stress resistance. In this study, we have tested the hypothesis of whether the dietary protein-to-carbohydrate (P:C) ratio in the developmental diet could be related to subsequent changes in metabolic profile and physiological parameters in Drosophila larvae and imago. Materials and Methods. Drosophila melanogaster Canton-S strain were used in this study. Larvae were fed diets with different P:C ratios. Experimental media were composed of either 2 % or 5 % dry yeast and 0 %, 1 %, or 10 % of sucrose. We tested developmental rate, wet or dry body weight and the levels of certain metabolites inclu­ding glucose, glycogen, triacylglycerides and total lipids. The developmental rate was assessed by counting the number of generated pupae every 6/6/12 hours. For wet or dry weight measurement, 20 larvae or flies were weighed and transferred to plastic vial with a cut bottom. The flies were dried at 60 °C with the subsequent weighing after two days. Another two-day flies cohort were separated by sex and frozen in liquid nitrogen for further biochemical assays. Hemolymph glucose, total lipid concentration, triacyl­glycerides (TAG), body glucose and glycogen contents were determined spectrophotometrically. Results. We found that a low 0.08 P:C ratio in the diet slowed down pupation by ~20 % and decreased body weight in larvae. Hemolymph glucose levels in both larvae and imago were inversely associated with dietary P:C. Larvae developing on a diet with a low P:C ratio displayed a lower level of glycogen pool, but a higher level of lipids. Developmental dietary P:C ratio also influences metabolic traits such as hemolymph glucose, glycogen, TAG and total lipids in male and female imago. A higher total protein intake combined with restriction of sucrose consumption had glucose-lowering and lipids-lowering effects. Conclusions. Our study demonstrated that nutritional conditions during larval development trigger adaptive changes that provide a level of regulation necessary to surpass dietary stress in Drosophila imago.

Astragalus membranaceus Extract Prevents Calcium Oxalate Crystallization and Extends Lifespan in a Drosophila Urolithiasis Model.

Approximately 1 in 20 people develops kidney stones at some point in their life. Although the surgical removal of stones is common, the recurrence rate remains high and it is therefore important to prevent the occurrence of kidney stones. We chose Astragalus membranaceus (AM), which is a traditional Chinese medicine, to study the prevention of urolithiasis using a Drosophila model based on our previous screening of traditional Chinese herbs. Wild-type Drosophila melanogaster Canton-S adult fruit flies were used in this study. Ethylene glycol (EG, 0.5%) was added to food as a lithogenic agent. The positive control agent (2% potassium citrate (K-citrate)) was then compared with AM (2, 8, and 16 mg/mL). After 21 days, the fruit flies were sacrificed under carbon dioxide narcotization, and the Malpighian tubules were dissected, removed, and processed for polarized light microscopy examination to observe calcium oxalate (CaOx) crystallization. Then, the ex vivo dissolution of crystals in the Malpighian tubules was compared between K-citrate and AM. Survival analysis of the EG, K-citrate, and AM groups was also performed. Both 2% K-citrate and AM (16 mg/mL) significantly inhibited EG-induced CaOx crystal formation. Mean lifespan was significantly reduced by the administration of EG, and the results were significantly reversed in the AM (8 and 16 mg/mL) groups. However, AM extract did not directly dissolve CaOx crystals in Drosophila Malpighian tubules ex vivo. In conclusion, AM extract decreased the ratio of CaOx crystallization in the Malpighian tubules and significantly ameliorated EG-induced reduction of lifespan. AM prevented CaOx crystal formation in the Drosophila model.

Anti-aging effects of Lasia spinosa L. stem extract on Drosophila melanogaster

Anti-aging effect is a stimulating topic that attracting serious consideration from many researchers. Medicinal plants with anti-aging properties thereby have become common research subjects in various fields of science. This study aims to determine phytochemical constituents, specifically the total of phenolic and flavonoid contents and evaluate the anti-aging activity of ethanolic extracts from the stem of Lasia spinosa L. The DPPH and ABTS assays were applied to investigate the in vitro antioxidant and free radical scavenging properties. Drosophila melanogaster Canton-S was used as the model organism to evaluate in vivo antioxidant activity, lifespan extension, climbing, and reproduction abilities. The results indicated that Lasia spinosa L. extract presented the phenolic and flavonoid contents, which were 271.65 mg GAE/g and 579.57 mg QE/g, respectively. The EC50 values of antioxidant activity was 23.80 µg/mL in DPPH assay and was 3.56 µg/mL for the assay of ABTS. The result also showed that the lifespan of adult fruit flies fed on extracts from Lasia spinosa L. was increased in oxidative stress conditions induced by H2O2. In lifespan assays, Drosophila melanogaster fed on the ethanolic extract from Lasia spinosa L. stem at 0.5 mg/mL of concentration in combination with standard food showed their mean lifespan increased to 22.9%, 50% survival increased to 22.2%, and the maximum lifespan of last flies increased to 20.7% compared with the control. The research results demonstrated a potential property among its natural compounds of Lasia spinosa L, which is capable of anti-aging through antioxidant activity in vitro and in vivo conditions.

Long-Term Dietary Restriction Leads to Development of Alternative Fighting Strategies.

In competition for food, mates and territory, most animal species display aggressive behavior through visual threats and/or physical attacks. Such naturally-complex social behaviors have been shaped by evolution. Environmental pressure, such as the one imposed by dietary regimes, forces animals to adapt to specific conditions and ultimately to develop alternative behavioral strategies. The quality of the food resource during contests influence animals' aggression levels. However, little is known regarding the effects of a long-term dietary restriction-based environmental pressure on the development of alternative fighting strategies. To address this, we employed two lines of the wild-type Drosophila melanogaster Canton-S (CS) which originated from the same population but raised under two distinct diets for years. One diet contained both proteins and sugar, while the second one was sugar-free. We set up male-male aggression assays using both CS lines and found differences in aggression levels and the fighting strategies employed to establish dominance relationships. CS males raised on a sugar-containing diet started fights with a physical attack and employed a high number of lunges for establishing dominance but displayed few wing threats throughout the fight. In contrast, the sugar-free-raised males favored wing threats as an initial aggressive demonstration and used fewer lunges to establish dominance, but displayed a higher number of wing threats. This study demonstrates that fruit flies that have been raised under different dietary conditions have adapted their patterns of aggressive behavior and developed distinct fighting strategies: one favoring physical attacks, while the other one favoring visual threats.

Regression of Survivability Variance on the Cohort Age in Drosophila melanogaster. I. Survivability: Reaction Norm and Its Inheritance

We analyze the regression of survivability variance by cohort age in several generations of Drosophila melanogaster Canton-S stock. This estimation reflects survivability reaction norm. Three phases were detected. First and second was approximated by exponential equation and the third - by logistic dose-response curve. Parameters of the equations remains stable for decades and thus must have high heritability.

Dietary alpha-ketoglutarate increases cold tolerance in Drosophila melanogaster and enhances protein pool and antioxidant defense in sex-specific manner

Alpha-ketoglutarate (AKG) is an important intermediate in Krebs cycle which bridges the metabolism of amino acids and carbohydrates. Its effects as a dietary supplement on cold tolerance were studied in Drosophila melanogaster Canton S. Two-day-old adult flies fed at larval and adult stages with AKG at moderate concentrations (5–10mM) recovered faster from chill coma (0°C for 15min or 3h) than control ones. The beneficial effect of AKG on chill coma recovery was not found at its higher concentrations, which suggests hormetic like action of this keto acid. Time of 50% observed mortality after 2h recovery from continuous cold exposure (−1°C for 3–31h) (LTi50) was higher for flies reared on 10mM AKG compared with control ones, showing that the diet with AKG enhanced insect cold tolerance. In parallel with enhancement of cold tolerance, dietary AKG improved fly locomotor activity. Metabolic effects of AKG differed partly in males and females. In males fed on AKG, there were no differences in total protein and free amino acid levels, but the total antioxidant capacity, catalase activity and low molecular mass thiol content were higher than in control animals. In females, dietary AKG promoted higher total antioxidant capacity and higher levels of proteins, total amino acids, proline and low molecular mass thiols. The levels of lipid peroxides were lower in both fly sexes reared on AKG as compared with control ones. We conclude that both enhancement of antioxidant system capacity and synthesis of amino acids can be important for AKG-promoted cold tolerance in D. melanogaster. The involvement of AKG in metabolic pathways of Drosophila males and females is discussed.

Discreteness of survival curves. I. Deviations from the Gompertz law in Drosophila melanogaster Canton-S strain

Analysis of the age-specific mortality dynamics in the male imago of the Drosophila melanogaster Canton-S strain demonstrated pronounced deviations from the Gompertz law. Four successive observations carried out in large drosophila cohorts made it possible to determine that the survival curves of the objects within the cohort lifetime consist of five discrete regions (phases) divided by sharp inflections. All of the indicated regions are linear. A rigid genetic determination for the disclosed character of the investigated curves was hypothesized.

CONTENT OF CYTOSKELETON PROTEINS IN OOCYTES OF THIRD GENERATION DROSOPHILA MELANOGASTER FRUIT FLIES OBTAINED AFTER A 44.5-DAY SPACE FLIGHT.

Third-stage larvae of the third generation of Drosophila melanogaster Canton S fruit flies were obtained after a 44.5-day flight of spacecraft a<Foton-M4> (Russia, 2014). In laboratory, the larvae grew into a third generation of flies. On the second day of emergence ovaries were extracted from female flies (group pF). Similarly, a third generation of flies (group C) was raised in the synchronous experiment. Real-time RT-PCR and Western blotting were used to determine the content of cytoskeletal proteins and mRNA of respective encoding genes. The content of mRNA genes encoding beta (Act57B and Act87E), gamma-actin (Act5C), acting-binding proteins (Arpc3, Tmod, Svil, Fim and Actn), and tubulin (Betatub85D) and tubulin-binding proteins (Msps, T-cpl, Cct5, T-cpleta) was less in group pF but not in the control group. The relative content of actin cytoskeleton components reduced significantly both in membrane and cytoplasmic fractions in group pF as compared to group C; however, reduction of the cytoplasmic fraction was more dramatic. On the contrary, content of beta-tubulin, a component of the tubulin cytoskeleton, reduced more significantly in the membrane fraction in group pF but not in group C. However, the acetylated tubulin content of ovarian membrane or cytoplasmic protein fraction was found unchanged both in the pF and C groups. The data suggest the presence in animal cells, oocytes specifically, of a mechanism regulating segregation of these proteins depending on external mechanical factors.

Context- and dose-dependent modulatory effects of naringenin on survival and development of Drosophila melanogaster.

Naringenin, the predominant bioflavonoid found in grapefruit and tomato has diverse bioactive properties that encompass anti-carcinogenic, anti-inflammatory, anti-atherogenic, anti-estrogenic, anti-hyperlipidemic and anti-hyperglycemic characteristics. Naringenin has not been explored for its pro-longevity traits in fruit flies. Therefore, the current study explores its influence on longevity, fecundity, feeding rate, larval development, resistance to starvation stress and body weight in male and female wild-type Drosophila melanogaster Canton-S flies. Flies were fed with normal and high fat diets respectively. The results implied hormetic effects of naringenin on longevity and development in flies. In flies fed with standard and high fat diets, lower concentrations of naringenin (200 and 400µM) augmented mean lifespan while higher concentrations (600 and 800µM) were consistently lethal. However, enhanced longevity seen at 400µM of naringenin was at the expense of reduced fecundity and food intake in flies. Larvae reared on standard diet having 200µM of naringenin exhibited elevated pupation and emergence as flies. Eclosion time was hastened in larvae reared on standard diet having 200µM of naringenin. Female flies fed with a standard diet having 200 and 400µM of naringenin were more resistant to starvation stress. Reduction in body weight was observed in male and female flies fed with a high fat diet supplemented with 200 and 400µM of naringenin respectively. Collectively, the results elucidated a context- and dose-dependent hormetic efficacy of naringenin that varied with gender, diet and stage of lifecycle in flies.

Sub-strains of Drosophila Canton-S differ markedly in their locomotor behavior.

We collected five sub-strains of the standard laboratory wild-type Drosophilamelanogaster Canton Special (CS) and analyzed their walking behavior in Buridan's paradigm using the CeTrAn software. According to twelve different aspects of their behavior, the sub-strains fit into three groups. The group separation appeared not to be correlated with the origin of the stocks. We conclude that founder effects but not laboratory selection likely influenced the gene pool of the sub-strains. The flies' stripe fixation was the parameter that varied most. Our results suggest that differences in the genome of laboratory stocks can render comparisons between nominally identical wild-type stocks meaningless. A single source for control strains may settle this problem.

Sub-strains of Drosophila Canton-S differ markedly in their locomotor behavior

We collected five sub-strains of the standard laboratory wild-type Drosophila melanogaster Canton Special (CS) and analyzed their walking behavior in Buridan's paradigm using the CeTrAn software. According to twelve different aspects of their behavior, the sub-strains fit into three groups. The group separation appeared not to be correlated with the origin of the stocks. We conclude that founder effects but not laboratory selection likely influenced the gene pool of the sub-strains. The flies’ stripe fixation was the parameter that varied most. Our results suggest that differences in the genome of laboratory stocks can render comparisons between nominally identical wild-type stocks meaningless. A single source for control strains may settle this problem.

Sub-strains of Drosophila Canton-S differ markedly in their locomotor behavior.

We collected five sub-strains of the standard laboratory wild-type Drosophila melanogaster Canton Special (CS) and analyzed their walking behavior in Buridan's paradigm using the CeTrAn software. According to twelve different aspects of their behavior, the sub-strains fit into three groups. The group separation appeared not to be correlated with the origin of the stocks. We conclude that founder effects but not laboratory selection likely influenced the gene pool of the sub-strains. The flies' stripe fixation was the parameter that varied most. Our results suggest that differences in the genome of laboratory stocks can render comparisons between nominally identical wild-type stocks meaningless. A single source for control strains may settle this problem.

Cloning and characterization of a gene encoding glutathione-regulated potassium-efflux system protein KefKL from the endosymbiont Wolbachia.

The maternally inherited intracellular symbiont Wolbachia is well known for inducing a variety of reproductive and developmental abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility (CI), parthenogenesis, feminization of genetic males and male killing in different hosts. However, the molecular mechanisms by which this fastidious bacterium causes these abnormalities have not yet been determined. In our study, representational difference analysis (RDA) was used to analyze the genomic difference between different Wolbachia strains. A gene encoding glutathione-regulated potassium-efflux system protein KefKL from Wolbachia in Drosophila simulans Riverside (w Ri) was isolated. The homologous genes from Wolbachia in Drosophila melanogaster yw67c23 (wMel) and Wolbachia in Drosophila melanogaster CantonS (wMelCS) were also cloned and sequenced. Sequence analysis showed that these deduced amino acid sequences contained two important motifs: Na+/H+ antiportor and NAD binding domain, which shared conserved sequences among different strains. Considering the crucial function of KefKL for ionic homeostasis, this gene might play an important role in Wolbachia physiology. Further study indicated that there was no homologue detected from Wolbachia in Drosophila simulans DSW/Mau (wMa) and Wolbachia in Drosophila simulans Noumea (wNo). Whether Wolbachia contained KefKL (or the homologous gene) was consistent with the phylogenetic studies using wsp sequences, which showed that wMa and wNo were grouped into one branch, while w Ri, wMel and wMelCS were more closely related.

Cloning and characterization of a gene encoding phage- related tail protein (PrTP) of endosymbiont Wolbachia

Wolbachia is an obligatory, maternally inherited intracellular bacterium, known to infect a wide range of arthropods. It has been implicated in causing cytoplasmic incompatibility (CI), parthenogenesis, the feminization of genetic males and male-killing in different hosts. However, the molecular mechanisms by which this fastidious bacterium causes these reproductive abnormalities have not yet been determined. In this study, we report on the cloning and characterization of the gene encoding phage-related tail protein (PrTP) from Wolbachia in Drosophila melanogaster CantonS (wMelCS) and from Wolbachia in Drosophila melanogaster yw67c23 (wMel) by representational difference analysis (RDA) and ligation-mediated PCR (LM-PCR). The functionality of a bipartite nuclear localization signal sequence (NLS) of the gene was also successfully tested in Drosophila S2 cells. PrTP expression in various strains of Wolbachia was investigated. Our results suggest that PrTP may not induce CI directly. However, the existence of prtp provided direct evidence of phage-mediated horizontal gene transfer (HGT) that might play an important role in a variety of reproductive abnormalities of Wolbachia.

Obstructive sleep apnoea syndrome — Results and conclusions of a principal component analysis

Alpha-ketoglutarate (AKG) is an important intermediate in Krebs cycle which bridges the metabolism of amino acids and carbohydrates. Its effects as a dietary supplement on cold tolerance were studied in Drosophila melanogaster Canton S. Two-day-old adult flies fed at larval and adult stages with AKG at moderate concentrations (5–10 mM) recovered faster from chill coma (0 °C for 15 min or 3 h) than control ones. The beneficial effect of AKG on chill coma recovery was not found at its higher concentrations, which suggests hormetic like action of this keto acid. Time of 50% observed mortality after 2 h recovery from continuous cold exposure (−1 °C for 3–31 h) (LTi50) was higher for flies reared on 10 mM AKG compared with control ones, showing that the diet with AKG enhanced insect cold tolerance. In parallel with enhancement of cold tolerance, dietary AKG improved fly locomotor activity. Metabolic effects of AKG differed partly in males and females. In males fed on AKG, there were no differences in total protein and free amino acid levels, but the total antioxidant capacity, catalase activity and low molecular mass thiol content were higher than in control animals. In females, dietary AKG promoted higher total antioxidant capacity and higher levels of proteins, total amino acids, proline and low molecular mass thiols. The levels of lipid peroxides were lower in both fly sexes reared on AKG as compared with control ones. We conclude that both enhancement of antioxidant system capacity and synthesis of amino acids can be important for AKG-promoted cold tolerance in D. melanogaster. The involvement of AKG in metabolic pathways of Drosophila males and females is discussed.

Expression of Drosophila melanogaster F elements in vivo.

Drosophila melanogaster F elements are mobile, oligo(A)-terminated DNA sequences that probably propagate by the retrotranscription of RNA intermediates. Polyadenylated transcripts corresponding in size to full-length (4.7 kb) family members were detected in the Drosophila melanogaster Canton-S strain from 2nd larval instar to the adult stage. RNA accumulation reached a maximum in pupae. In the adult, F elements are transcribed in both sexes. F expression is directed in vivo by the intragenic promoter (Fin) located at the 5' end of F. Whole-mount hybridizations were carried out to define the site of synthesis of full-length transcripts found in the ovary. Selective RNA accumulation was not detected in the cytoplasm of any specific cell type. Stained nuclear dots were observed in nurse cells from stage 2-3 to the end of oogenesis. RNase treatment of egg chambers prior to the addition of the probe led to disappearance of the nuclear dots and appearance of a cytoplasmic hybridization signal suggesting leakage of nuclear transcripts. Transgenic lines harbouring the chloramphenicol acetyltransferase (CAT) gene under the control of the Fin promoter were obtained. In independent lines, CAT enzyme levels mirror the ontogenetic profile of F expression drawn from Northern RNA blotting data. An antisense promoter (Fout) that is located downstream from the Fin promoter and transcribe too bords the 5' end of F seems to be constitutively expressed in the fly.

Molecular cloning of alpha-amylase genes from Drosophila melanogaster. II. Clone organization and verification.

Restriction maps of an alpha-amylase structural gene clone, lambda Dm65, and of four putative alpha-amylase pseudogene clones are presented. Two alpha-amylase structural genes, inverted with respect to each other, are contained in lambda Dm65. Subregions of internal DNA sequence homology within lambda Dm65 and of cross-homology between the presumptive pseudogene clones and lambda Dm65 were determined. Subregions of cross-homology between the Drosophila clones and the mouse alpha-amylase cDNA clone, pMSa104, were also determined. The presence of functional alpha-amylase structural genes in lambda Dm65 was verified by injection of appropriate subclones into the germinal vesicle of Xenopus oocytes, followed by incubation of the oocytes under conditions that allowed coupled transcription and translation of injected genes to occur. Subclones of the 3.8- and 5.6-kb EcoRI fragments of lambda Dm65 were shown to code for alpha-amylase isozymes 1 and 3, respectively, of Drosophila melanogaster Canton-S. Both subclones are homologous to RNA of a size sufficient to accommodate the alpha-amylase-coding information. No RNA species homologous to other subcloned EcoRI fragments of lambda Dm65 was detected.