Project description:Honey bee queens undergo dramatic behavioral (e.g., reduced sexual receptivity), physiological (e.g., ovary activation, ovulation, and modulation of pheromone production) and molecular changes after they complete mating. To elucidate how queen post-mating changes are influenced by seminal fluid, a non-spermatozoa-containing component of semen, we injected queens with semen or seminal fluid alone. We assessed queen sexual receptivity, ovary development, worker retinue response (which is influenced by queen pheromone production), and transcriptional changes in queen abdominal fat body and brain tissues. Injection with either seminal fluid or semen resulted in decreased sexual receptivity, increased attractiveness of queens to workers, and altered expression of several genes that are also regulated in naturally mated queens. The post-mating and transcriptional changes of queens receiving seminal fluid were not significantly different from queens treated with seminal fluid, suggesting that components in seminal fluid, such as seminal fluid proteins, are largely responsible for stimulating post-mating changes in queens.
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:Mating is a complex process that causes many behavioral and physiological changes, but the factors triggering these changes and the underlying molecular processes are not well characterized. Honey bee queens provide a convenient system for dissecting these factors (e.g., physical manipulation, insemination volume, insemination substance) via instrumental insemination. We examined the effects of carbon dioxide (CO2), a commonly used anesthetic in instrumental insemination that causes changes similar to those observed after mating, and physical manipulation, which presumably mimics the act of copulation, on the brain transcriptional changes in honey bee queens. We found significant gene overlap between our study and previous mating studies in honey bee queens and Drosophila. This suggests that molecular pathways regulating the mating process are conserved across different mating regimes of honey bees as well as across insect orders.
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.
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:Adult honey bee queens and workers drastically differ in ovary size. This adult ovary phenotype difference becomes established during the last two larval instars, when massive programmed cell death in the ovaries of worker larvae leads to the degeneration and removal of 95-99% of the ovariole anlagen. The higher juvenile hormone (JH) levels in queen larvae protect their ovaries against such degeneration. To gain insights into the molecular architecture underlying this divergence critical for adult caste fate we performed a microarray analysis contrasting RNA extracts from fourth and early fifth instar queen and worker ovaries. While for the fourth instar we found differential expression (log2FC > 1.0) for only nine genes, the number of differentially represented transcripts (DRTs) increased to 56 in early fifth instar ovaries. From these, 18 had their expression levels further analyzed by real-time PCR (RT-qPCR). For 13 of these the expression differed significantly between queen and worker ovaries at least one time point in development, and interestingly, genes with enzyme functions were overexpressed in workers, while genes related to transcription and signaling were so in queens. For the RT-qPCR confirmed genes we further analyzed their response to JH, revealing a significant up-regulation for two genes, one encoding a short chain dehydrogenase (SDR) and the other heat shock protein 90 (Hsp90). Five other genes, including Hsp60 and hexamerin 70b, were significantly down-regulated by JH. As SDR genes have previously come up as differentially expressed in different transcriptome assays in honey bee larvae, and heat shock proteins are involved in hormone responses, these are interesting candidates for further functional assays.
Project description:The microsporidia Nosema ceranae are intracellular parasites that proliferate in the midgut epithelial cells of honey bees (Apis mellifera). To analyze the pathological effects of those microsporidia, we orally infected honey bee workers 7 days after their emergence. Bees were flash frozen 15 days after the infection. Then, the effects on the gut ventriculi were analyzed and compared to non-infected (control) bees.
Project description:Here we present the first characterisation of small RNAs in honey bee reproductive tissues. We conclude that small RNAs are likely to play an integral role in honey bee gametogenesis and reproduction and provide a plausible mechanism for parent-of origin-effects on gene expression and reproductive physiology. present in honey bee reproductive tissues: ovaries, spermatheca, semen, fertilised and unfertilised eggs, and testes.
Project description:In honey bees, Vitellogenin (Vg) is hypothesized to be a major factor affecting hormone signaling, food-related behavior, immunity, stress resistance and lifespan. Likewise microRNAs play important roles in posttranscriptional gene regulation and affect many biological processes thereby showing many parallels to Vg functions. The molecular basis of Vg and microRNA interactions is largely unknown. Here, we exploited the well-established RNA interference (RNAi) protocol for Vg knockdown to investigate its effects on microRNA population in honey bee forager’s brain and fat body tissue. To identify microRNAs that are differentially expressed between tissues in control and knockdown foragers, we used µParaflo® microfluidic oligonucleotide microRNA microarrays. Our results show 76 and 74 miRNAs were expressed in the brain of control and knockdown foragers whereas 66 and 69 miRNAs were expressed in the fat body of control and knockdown foragers respectively. Target prediction identified potential seed matches for differentially expressed subset of microRNAs affected by Vg knockdown. These candidate genes are involved in a broad range of biological processes including insulin signaling, juvenile hormone (JH) and ecdysteroid signaling previously shown to affect foraging behavior. Thus, here we demonstrate a causal link between Vg expression-variation and variation in the abundance of microRNAs in different tissues with possible consequences for regulation of foraging behavior.