Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling by array of honey bee queens Apis mellifera L. to investigate the differential effects of insemination volume and substance on reproductive changes

ABSTRACT: Mating causes dramatic changes in female insects at the behavioral, physiological, and molecular levels. The factors driving these changes (e.g., seminal proteins, seminal volume) and the molecular pathways by which these factors are operating have been characterized only in a handful of insect species. Here we use instrumental insemination of honey bee queens to examine the role of the insemination substance (saline vs. semen) and volume (1 vs. 8 uL) in triggering post-mating changes. We also examine differences in gene expression patterns in the fat bodies of queens that have high ovary activation to determine if events during copulation can cause long-term changes in gene expression. We found that the instrumental insemination procedure alone caused cessation of mating flights and triggered ovary activation, with insemination volume contributing to increased ovary activation. Hierarchical clustering grouped queens primarily by insemination substance and then insemination volume, suggesting that while volume may trigger short-term physiological changes, substance plays a greater role in regulating long-term transcriptional changes. There was considerable but not a complete overlap in the gene pathways regulated by these two factors. Comparisons with gene lists from previous studies on queen mating revealed that several of the same biological processes and pathways were regulated, but only one gene (defensin) was found to be regulated in all studies. Our results suggest that both insemination substance and volume trigger molecular post-mating changes by altering overlapping gene pathways involved in honey bee reproduction.

ORGANISM(S): Apis mellifera

SUBMITTER: Elina Lastro

PROVIDER: E-MEXP-3681 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Differential effects of insemination volume and substance on reproductive changes in honey bee queens (Apis mellifera L.).

Niño E L EL Tarpy D R DR Grozinger C M CM

Insect molecular biology 20130217 3

Mating causes dramatic changes in female insects at the behavioural, physiological and molecular level. The factors driving these changes (e.g. seminal proteins, seminal volume) and the molecular pathways by which these factors are operating have been characterized only in a handful of insect species. In the present study, we use instrumental insemination of honey bee queens to examine the role of the insemination substance and volume in triggering post-mating changes. We also examine difference ...[more]

PMID: 23414204

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Project description:Mouse macrophages J774A.1 were pre-treated with the anti-inflammatory lipid eicosapenaenoic acid (EPA) or the pro-inflammatory lipid ceramide (Cer) for 3h. Macrophages were then infected with Mycobacterium smegmatis for 1h, and total RNA was collected. In a parallel experiment, infected macrophages were infected for 1h, 4h and 24h. At these time points, macrophages were lysed, bacteria was collected and quantified by the Colony Forming Units (CFU) Assay. This provided the kinetics of the killing of Mycobacteria inside mouse macrophages. CFU experiments revealed that cells pre-treated with EPA showed an increased number of bacteria inside macrophages, in contrast to cells pre-treated with Cer. To dissect the molecular mechanisms involved in the survival and killing of mycobacteria infected macrophages, mediated by lipids, gene expression studies were performed. Cultures of Mycobacterium smegmatis mc2155 harbouring a p19-(long lived) EGFP plasmid were grown to exponential growth phase. Bacteria were pelleted, washed in PBS and resuspended in medium DMEM with a multiplicity of infection (MOI) of 10 (10 bacteria per macrophage). Clumps of bacteria were removed by ultrasonic treatment of bacterial suspensions in an ultrasonic water bath for 15 minutes, followed by a low speed centrifugation for 2 minutes. Mouse macrophages J774A.1 were pre-.treated with the anti-inflammatory lipid eicosapenaenoic acid (EPA) or the pro-inflammatory lipid ceramide (Cer), 3h before infection. Mouse macrophages J774A.1 were infected with Mycobacterium smegmatis mc2155 for 1h, at 37M-BM-:C and 5% CO2. After 1h of infection, cells were washed with PBS. After washing, 1 ml Trizol was added per well, to collect total RNA. The experimental condition were: samples 1.1, 1.2, 1.3: Untreated macrophages; samples 2.1, 2.2, 2.3: Mock treated macrophages (ethanol 1ul/ml); samples 3.1, 3.2, 3.3: EPA (15uM) treated macrophages (ethanol 1ul/ml); samples 4.1, 4.2, 4.3: Cer (5ug/ml) treated macrophages (ethanol 1ul/ml); samples 5.1, 5.2, 5.3: Untreated macrophages, infected with Mycobacterium smegmatis mc2155 for 1h; samples 6.1, 6.2, 6.3: Mock treated macrophages (ethanol 1ul/ml) infected with Mycobacterium smegmatis mc2155 for 1h; samples 7.1, 7.2, 7.3: EPA (15uM) treated macrophages (ethanol 1ul/ml) infected with Mycobacterium smegmatis mc2155 for 1h; samples 8.1, 8.2, 8.3: Cer (5ug/ml) treated macrophages (ethanol 1ul/ml) infected with Mycobacterium smegmatis mc2155 for 1h; In a parallel experiment, infected macrophages remained with the bacteria for 1h, 4h and 24h after infection. After these time points, the macrophages were lysed, the bacteria was collected and quantified by the Colony Forming Units (CFU) Assay. This provided the kinetics of the killing of non-pathogenic intracellular bacteria inside mouse macrophages. For the CFU assay, after 1h of infection, cells were washed with PBS and Gentamicin (10ug/ml) in DMEM to kill the extracellular bacteria. At discrete time points, cells were washed with PBS and lysed with sterilized water. Quantitative cultures of bacteria were performed in a 10-fold serial dilutions, inoculated on 7H10 agar plates. 5ul were plated in triplicate and the number of colonies were counted after 48h.

Dataset Information

Transcription profiling by array of honey bee queens Apis mellifera L. to investigate the differential effects of insemination volume and substance on reproductive changes

Publications

Differential effects of insemination volume and substance on reproductive changes in honey bee queens (Apis mellifera L.).

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