Transcription profiling of honeey bee to assess the effects of mating and instrumental insemination on honey bee behavior physiology and brain gene expression
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ABSTRACT: Mating is fundamental to the success and reproduction of most organisms, although the physiological and transcriptional changes associated with this process have been largely characterized only in Drosophila. In this study, we use honey bees as a model system since their queens undergo massive and permanent physiological and behavioral changes following mating. Previous studies have identified changes associated with the transition from a virgin queen to a fully-mated, egg-laying queen. Here, we further uncouple the mating process to examine the effects of natural mating vs. instrumental insemination and saline vs. semen insemination. We observed significant overlap between our study and analogous studies in Drosophila, suggesting that some post-mating mechanisms are conserved across insect orders.
Project description:Will be added/updated once the manuscript is finalized. Cardiocondyla obscurior queens. Three treatments: virgin queens, queens mated by real males, queens sham-mated (by sterile males). Queens collected 1 week and 8 week after mating. seven loops for queens collected 1 week after mating; nine loops for queens collected 8 weeks after mating. Five direct comparisons (with dye-swaps... so 10 arrays) were done of between 1 week and 8 week samples of queens mated by real males. Each sample is RNA from two queens (from different colonies). Samples were hybridized against Solenopsis invicta microarrays (signal was detectable for most clones!)
Project description:Adult reproductive diapause is a powerful overwintering strategy for many continental insect species including bumblebees, which enables queens to survive several months through harsh winter conditions and then build new beehives in the following spring. There are few reports regarding the molecular regulatory mechanism of reproductive diapause in Bombus terrestris, which is an important pollinators of wild plants and crops, and our previous researches identified the conditions for reproductive diapause of year-round mass rearing. Here, we performed combined RNA sequencing transcriptomics and quantitative proteomic analyses in different development phases relate to reproductive diapause. According to the overall analysis, we found these differentially expressed proteins/genes act in the citrate cycle, insect hormone biosynthesis, insulin and mTOR signalling pathway. To get better sense of the reproductive diapause regulated mechanism, some genes regulated JH synthesis, insulin/ TOR signal pathway were detected, the BtRheb, BtTOR, BtVg and BtJHAMT had lower expression levels in diapause queens, and the JH III titers levels and some metabolic enzymes activities were significantly up-regulated in found post-diapause queens. After microinjected insulin-like peptides (ILPs) and JH analog (JHA), some indicators shows the significantly changes of hormones, cold tolerance substances, metabolic enzymes and reproduction. Along with other related researches, a reproductive diapause regulated model during B. terrestris year-round mass rearing process was establishment. This study contribute to a comprehensive view and the molecular regulate mechanism of productive diapause in eusocial insect.
Project description: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.
Project description:Here we show that regions of the honeybee brain involved in visual processing and learning and memory show a genomic response to distance information. Using a method that separates effects of perceived distance from effects of actual distance flown, we found that individuals forced to shift from a short to a perceived long distance to reach a feeding site showed differences in gene expression in the optic lobes and mushroom bodies relative to individuals that continued to perceive flying a short distance.
Project description:In many animals living in groups the reproductivestatus of individuals is determined by their social status. In specieswith social hierarchies, the death of dominant individuals typicallyupheaves the social hierarchy and provides an opportunity for subordinateindividuals to improve their social status. Such a phenomenon occursin the monogyne form of the fire ant\emph{, Solenopsis invicta, }wherecolonies typically contain a single wingless reproductive queen, thousandsof workers and hundreds of winged non-reproductive virgin queens.Upon the death of the mother queen, many virgin queens shed theirwings and initiate reproductive development instead of departing ona mating flight. Workers progressively execute almost all of themover the following weeks. The workers base their collective decisionon pheromonal cues associated with the onset of reproductive developmentof the virgin queens which occurs after orphaning. To examine the factors that determine which virgin queens are executed and which survive, we set up artificial competitions between queens from different colonies. Using microarrays, we found that queens from winning colonies showed higher mitochondrial as well as organ development activities 24 hours after orphaning than did queens from colonies that lost the competitions. Furthermore, queens from colonies where queens shed their wings faster after orphaning were more likely to survive competitions. Finally, higher wing shedding speed is linked to higher mitochondrial activity. Eight competitions were initially conducted between queens form pairs of colonies. Six competitions clearly identified one winning colony and one losing colony. We thus have microarray data for six competitions (ie biological replicates), with one winning and one losing colony within each competition. Thus 12 colonies total. For each colony, RNA extracted from five queens was pooled, and hybridized against an unrelated common reference RNA that had been made from a pool of many S. invicta individuals of all castes and developmental stages. Lab work was conducted in blocks (by competition) with randomized order within competition to avoid introducing bias. No technical replication was performed.
Project description:We used microarrays to monitor expression patterns of several thousand genes in the brains of same-aged (10 day old) virgin queens, sterile workers, and reproductive workers in honey bees (Apis mellifera).
Project description:Mating is a complex process, which is frequently associated with behavioural and physiological changes. However, understanding of the genetic underpinnings of these changes is limited. Honey bees are both a model system in behavioural genomics, and the dominant managed pollinator of human crops; consequently understanding the mating process has both pure and applied value. We used next-generation transcriptomics to probe changes in gene expression in the brains of honey bee queens, as they transition from virgin to mated reproductive status. In addition, we used CO2-narcosis, which induces oviposition without mating, as an experimental control for the mating process. Mating produced significant changes in the expression of vision, chemo-reception, metabolic, and immune-related genes. Differential expression of these genes maps clearly onto known behavioural and physiological changes that occur during the transition from being a virgin queen to a newly-mated queen. A subset of these changes in gene expression were also detected in CO2-treated queens, as predicted from previous physiological studies. In addition, we compared our results to previous studies that used microarray techniques across a range of experimental time-points. Changes in expression of immune- and vision-related genes were common to all studies, supporting an involvement of these groups of genes in the mating process. However, these comparisons also indicate the need to understand the temporal dynamics of gene expression across the entire mating and reproductive process. Brain RNA samples for 3 treatments: control (N=4), mated (N=4) and treated with carbon dioxide (N=4)
Project description:We performed a whole-genome transcriptomic analysis of pleometrotic queens infected by SINV-1, SINV-2 or co-infected with both viruses, to characterize patterns of gene expression associated with viral infection and identify genes responding differentially to the two viruses. We sampled fire ant queens in Gainesville, Florida, immediately after a mating flight. We arranged them in pairs based on having similar weights (range ±0.2 mg) to allow pleometrotic colony founding. We housed paired queens in nesting tubes in claustral conditions (no food or water) for one month. Thereafter, we collected queens in dry ice, we isolated total RNA from whole bodies and used it to quantify SINV-1 and SINV-2 viruses with real-time PCR. We selected 2 queens that were virus-free as controls, 3 SINV-1-infected, 4 SINV-2-infected and 11 co-infected by both viruses. These samples were processed for microarrays analysis.
Project description:In species with social hierarchies, the death of dominant individuals typically upheaves the social hierarchy and provides an opportunity for subordinate individuals to become reproductives. Such a phenomenon occurs in the monogyne form of the fire ant, Solenopsis invicta, where colonies typically contain a single wingless reproductive queen, thousands of workers and hundreds of winged non-reproductive virgin queens. Upon the death of the mother queen, many virgin queens shed their wings and initiate reproductive development instead of departing on a mating flight. Workers progressively execute almost all of them over the following weeks. To identify the molecular changes that occur in virgin queens as they perceive the loss of their mother queen and begin to compete for reproductive dominance, we collected virgin queens before the loss of their mother queen, six hours after orphaning and 24 hours after orphaning. Their RNA was extracted and hybridized against microarrays to examine the expression levels of approximately 10,000 genes. We identified 297 genes that were consistently differentially expressed after orphaning. These include genes that are putatively involved in the signaling and onset of reproductive development, as well as genes underlying major physiological changes in the young queens. 3 samples: 0h, 6h, 24; five queens pooled per samples; each replicate loop of 3 samples was taken from an independent colony; 8 biological replicates (colonies) used. Hybridization according to a Dye-balanced loop design (no technical replication)