Expression analysis of D. melanogaster selected for contrasting life histories
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
SUBMITTER: Silvia Remolina
PROVIDER: E-GEOD-38106 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:By combining an experimental evolution approach with genomic techniques, we investigated the effects of seminal fluid on female gene expression. In our study, we experimentally manipulated the mating system in replicate populations of D. melanogaster, by removing post-copulatory sexual selection, with the aim of testing differences in short term post-mating reaction of females evolved under different mating strategies. We show that monogamous females suffer decreased fecundity, regardless of the type of male they were mated with, and that their post-mating gene expression profiles differ significantly from promiscuous females, involving 1141 transcripts (9% of the genes tested). These transcripts are active in several tissues, mainly ovaries, neural tissues, midgut and spermathecae, and are involved in metabolic processes, reproduction and signaling pathways. Our results provide a list of candidate genes responsible for the decrease in female fecundity in the absence of post-copulatory sexual selection, and demonstrate how the female post-mating response can evolve under different mating systems over relatively short time frames. From an LHM base population, we created 8 replicate populations and maintained them under experimental evolution: 4 populations were allowed to mate only once every generation (monogamy), and the other 4 were kept under the standard mating protocol (promiscuous). After 46 generations, we crossed males and females within the same population and with individuals of the opposite treatment. Mated female flies were frozen 6 h after mating and RNA extracted. Two biological replicates per cross per population (2x2x8=32 samples).
2014-01-01 | E-GEOD-48385 | biostudies-arrayexpress
Project description:Drosophila melanogaster adult flies fed on food containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. We also examined flies’ locomotor behavior secondary to PTZ withdrawal. Interestingly, an increased climbing speed was found to develop seven days after withdrawal. Importantly, antiepileptic drugs showed effectiveness in the above fly model. Earlier, we submitted in GEO time series of fly head microarray gene expression profiling during chronic PTZ and PTZ withdrawal phase. We also submitted previously expression profiles associated with antiepileptic drug treatment. Here, we have undertaken a different line of work. Having developed a well characterized acquired model of behavioral and gene expression plasticity, we found an opportunity here to investigate if drug exposure to adult males could cause transgenerational effect. To probe this, we carried out a systematic study at both behavioral and microarray gene expression levels. In the latter, we asked the question that do F0 testis, F1 males’ head, F1 females’ head, F1 testis, F2 males’ head and F2 females’ head show gene expression changes if F0 male parents had a history of PTZ exposure? A total of 28 microarray slides were used in this study. Unless mentioned otherwise, standard method of fly handling and manipulation was used. Freshly generated isogenic D. melanogaster Oregon-R wild type flies were grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin. The cultures were grown at 24 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in food containing 8 mg/ml PTZ for seven days. Flies were then shifted to vials containing NF. Seven days after shifting, F0 males were mated to same age group females maintained on NF throughout, without any drug exposure. Males and females were housed in groups in vials containing NF. Eggs collected on NF were used to generate F1 individuals as unmated males and virgin females. F1 males were mated to same age group females which were independently collected and maintained on NF. These females had no history of drug exposure in prior generations. Males and females were housed in groups in vials containing NF. Eggs collected on NF were used to generate F2 individuals as unmated males and virgin females. F0 testis, F1 males’ head, F1 females’ head, F1 testis, F2 males’ head and F2 females’ head were used for RNA isolation. Total RNA was isolated from pools of heads or testis, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from extracted RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of control (no PTZ exposure of F0 males) and treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with control as Cy3- and experimental as Cy5- labeled sample, and the rest two as the opposite, control as Cy5- and experimental as Cy3- labeled sample. Two sets of microarrays were generated for F1 males. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10 mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10 mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D14Kv1, CDMC, Toronto, Canada). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10 µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The preprocessing and quantification of the 16 bit TIFF images were carried out using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations. Analysis was also carried out using fold-change criterion in Acuity 4.0 itself.
2009-03-25 | E-GEOD-15136 | biostudies-arrayexpress
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2014-03-17 | E-GEOD-50918 | biostudies-arrayexpress
Project description:Data from whole-body, head, thorax, abdomen, ovaries, testes and accessory glands was used to assess possible causes of a non-random distribution of sex-biased genes (sexually dimorphic expression). All samples were derived from virgin adult flies. We measured gene expression of male and female Drosophila serrata from 43 lines (whole-body) and multiple tissues sampled from outbred laboratory stock. All flies were originally samples from Brisbane, Queensland, Australia. Data from two replicates for each sex/line are presented, plus 3-5 replicates per sex/tissue. 24 adult whole-body samples were not used in analyses due to poor quality, giving a total of 176 arrays.
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2011-12-31 | E-GEOD-24923 | biostudies-arrayexpress
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Project description:Transcript abundance was measured in whole-body virgin male Drosophila serrata from 41 inbred lines that had diverged through 27 generations of mutation accumulation. Pleiotropic mutations are the ultimate source of genetic variation in complex traits, including many human diseases. However, the nature and extent of mutational pleiotropy remain largely unknown. Here, we investigate the variation in 11,604 gene expression traits among 41 mutation accumulation lines of Drosophila serrata, which had diverged for 27 generations. We detected significant mutational variance in 4.6% of ESTs, but 70% of ESTs were invariant among lines, allowing us to reject a null hypothesis of phenome-wide universal pleiotropy. Mutational covariance among ESTs was detected at a frequency of only 1 in 193 random pairs of variable EST, bu t was detected among random combinations of five ESTs in 1 in 5 cases, revealing that mutational covariance among multiple ESTs was common. The observed frequency of significant multivariate covariance among random ESTs implied that a substantial number of ESTs (>70) must be pleiotropically affected by at least some mutations. We measured gene expression of male Drosophila serrata from 41 mutation accumulation lines (whole-body). Data from two replicates for each line are presented.
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2013-11-01 | E-GEOD-51942 | biostudies-arrayexpress
Project description:We recently developed a fly model of pentylenetetrazol (PTZ)-induced long-term behavioral plasticity. Pharmacological validation suggests this model to be kindling-like. Hence, our model is of relevance in understanding the molecular pathogenesis of epileptogenesis as well as in screening potential antiepileptogenic agents. Vigabatrin (VGB) is an antiepileptic drug, and in the process of developing our fly model we generated gene expression profile of flies’ head secondary to treatment of the insects with VGB. Our results provide novel insights in to the drug’s mode of action. Oregon-R wild type D. melanogaster was grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin. Flies were cultured at 24 +/- 1ºC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Ten to eleven days old unmated adult males were grown in either normal food (NF) or food containing 24 mg/ml of VGB for three days. Each culture vials contained 30 flies. Flies frozen in liquid nitrogen were agitated and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four VGB treated individuals, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche), and the cDNA purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of NF control and VGB treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and VGB treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The 16 bit TIFF images were preprocessed and quantified using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 2.21, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations.
2007-10-04 | E-GEOD-7203 | biostudies-arrayexpress