Transcriptome Sequencing Analysis Reveals the Regulation of the Hypopharyngeal Glands in the Honey Bee, Apis mellifera carnica Pollmann
ABSTRACT: Transcriptome sequencing has become the main methodology for analyzing the relationship between genes and characteristics of interests, particularly those associated with diseases and economic traits. Because of its functional superiority, commercial royal jelly (RJ) and its production are major areas of focus in the field of apiculture. Multiple lines of evidence have demonstrated that many factors affect RJ output by activating or inhibiting various target genes and signaling pathways to augment their efficient replication. The coding sequences made available by the Honey Bee Genome Sequencing Consortium have permitted a pathway-based approach for investigating the development of the hypopharyngeal glands (HGs). In the present study, 3573941, 3562730, 3551541, 3524453, and 3615558 clean reads were obtained from the HGs of five full-sister honey bee samples using Solexa RNA sequencing technology. These reads were then assembled into 18378, 17785, 17065, 17105, and 17995 unigenes, respectively, and aligned to the DFCI Honey Bee Gene Index database. The differentially expressed genes (DEGs) data were also correlated with detailed morphological data for HGs acini. The results identify areas that warrant further study, including those that can be used to improve honey bee breeding techniques and help ensure stable yields of RJ with high quality traits. The 5 samples at given time (d3, d6, d9, d12, d16 after adult worker bees emergence from the comb) are in the critical stage of the RJ secretion and HGs developments indicated (triggered) the further caste differentiation (worker bees and queen) and task switch (nurse bees and foragers). 30 pooled heads of each samples were
Project description:The hypopharyngeal gland (HG) is the main site of the synthesis and secretion of royal jelly protein (RJP), and shows high plasticity. To identify differentially expressed genes (DEGs) that affect the development of HG and the synthesis and secretion of RJP, we performed a digital gene expression analysis of 9 d Apis mellifera under different conditions of nutrition and exposure to brood pheromone.Six RNA-seq libraries were generated using RNA extracted from 9 d bee HGs. A total of 2801 DEGs were identified on the basis of at least one pairwise comparison, among which 205, 1617 and 2328 genes were differentially expressed in comparisons between the Pollen group and the Honey group, the Brood group and Pollen group and the Brood group and Honey group respectively. The Brood group exhibited the highest number of DEGs, suggesting that brood pheromone plays a key role in the HG ontogeny. A total of 1991 genes were mapped to 129 canonical signaling pathways found in the Kyoto Encyclopedia of Genes and Genomes (KEGG), and the pathways associated with ribosome function and protein processing were significantly enriched. Hypopharyngeal gland mRNA profiles of 9-day old honeybees under three conditions (one control and two treatments) were generated by deep sequencing, in duplicate, using Illumina Hiseq 2500 platform.
Project description:Royal jelly (RJ) is a proteinaceous secretion of the hypopharyngeal glands (HGs) in the head of honeybee workers. It is a critical food for queen bees and young larvae that decides the fate of fertilized eggs in developing into either queen bees or worker bees during the early larval stages. RJ is also widely used in humans for health promotion as agent, such as antibacterial, antioxidant, and antiaging properties. To increase RJ yields, a stock of high RJ producing honeybees (RJBs) has genetically selected from Italian honeybees (ITBs) in China since 1980s. To date, one colony of RJBs can produce more than 10 kg of RJ per year, a yield that is 10-times greater than that produced by a colony of ITBs. To elucidate the mechanism of the enforced gland performance in producing RJ in RJBs, the spatio-temporal HG proteomes of newly emerged bees, nurse bees, and forager bees, were compared between the ITBs and RJBs. Proteins in the critical pathways that are implicated in the secretory activity of RJ in HGs are validated biochemically and biologically by manipulating the NBs into extended nursing periods and the FBs to revert into NBs. This will provide a novel mechanistic insight into the HGs achieving an enhanced biological mission of producing the valuable bee-product, RJ.
Project description:We characterized and compared hemolymph proteome of Royal Jelly bees (RJbs), a stock selected for increasing RJ output from Italian bees (ITbs) and ITbs across the larval and adult ages. Unprecedented depth of proteome was attained by identifying 3394 hemolymph proteins in both bee lines. The proteome supports the general function of hemolymph to drive development and immunity across different phases in both bees. However, age-specific proteome settings have adapted to prime the distinct physiology for larvae and adult bees. In larvae, proteome are thought to drive the temporal immunity, rapid organogenesis, and reorganization of larval structures. In adults, proteome play key roles to prompt tissues development and immune defense in NEBs, glands maturity in NBs and carbohydrate energy production in FBs. Comparing the proteome between the same aged larval and adult samples, RJbs and ITbs have tailored distinct hemolymph proteome programs to drive their physiology. Particularly, in day 4 larvae and NBs, a large number of highly abundant proteins enriched in protein synthesis and energy metabolism in RJbs relative to ITbs imply that RJb larvae and NBs have reprogrammed their proteome to initiate different developmental trajectory and high RJ secretion in response to the enhanced RJ production by selection. Our hitherto depth of proteome coverage gains novel sight on molecular details in driving hemolymph function and high RJ production by RJbs.
Project description:Background: Honey bee is a major insect used for pollination of a number of commercial crops worldwide. However, the number of managed honey bee colonies has recently declined in several countries, and a number of possible causes are proposed. Although the use of honey bees for pollination can be considered as disruption of the habitat, its effects on honey bees' physiology have never been addressed. In Japan, more than 100 thousands colonies are annually used for pollination, and intriguingly 80% of them are used in greenhouses. Recently, honey bee colonies have often collapsed when they are introduced into greenhouses. Thus, to suppress colony collapses and maintain the number of worker bees in the colonies are essential for successful long-term pollination in greenhouses and recycling honey bee colonies. Honey bee hives were installed into strawberry and eggplant greenhouses, and then the gene expression profiles of the honey bees were examined at the different time periods. Total 16 samples with two replicates were analyzed.
Project description:In this study we addressed whether the transcriptome profile in the honey bee brain is similar for two major parasites of honey bee, Varroa destructor and Nosema ceranae. Honey bees parasitized by these two parasites show accelerated behavioral maturation and deficiences in orientation and learning/memory that we hoped to characterized at the transcriptomic level. honey bee adults infested by Varroa destructor or Nosema ceranae compared to control bees, in duplicate
Project description:Flenniken - Honey bee gene expression microarray experimental design<br>To minimize variability between samples all arrayed bees were obtained from a single brood comb from a naturally mated queen, therefore all the bees were age-matched half-sisters. The bees selected for microarray analysis of virus (Sindbis-eGFP) co-injected with either virus-specific-dsRNA (vs-dsRNA) or non-specific dsRNA (ns-dsRNA) exhibited the reduced virus phenotype that was seen in the majority of the bees assayed. The five representative bees from each condition (v, v+vs-dsRNA, v+ns-dsRNA, dsRNA, and mock/injected with buffer) selected for microarray analysis were free of pre-existing conditions (assessed by APM analysis) (Runckel, Flenniken et al., 2011). To facilitate gene expression comparisons between multiple treatment groups we utilized a reference-design strategy in which each Cy5-labeled experimental sample was hybridized with a standardized Cy3-labeled reference sample. A complex RNA mixture representing hundreds of bees of various ages exposed to difference treatment groups, served as the reference RNA sample.
Project description:Experimental infection of (2 days old) adult honey bee workers (30 bees per replicates, 3 replicates per treatments, from 3 different colonies (one colony per cage for each treatment)) with 10^9 genome equivalent of Black Queen Cell Virus (BQCV) in 10µl of sugar solution and/or 10^5 fresh Nosema ceranae spores (control bees were given a similar bee extract in PBS, without pathogen). Bees were kept in cages of 30 bees in incubator (30°C/50%RH). At day 13 p.i., bees were flash frozen, and stored at -80°C. Brain mRNA profiles of 15 old bees were generated by deep sequencing, in triplicates except for bees infected by both Nosema ceranae and Black Queen Cell Virus (duplicates)
Project description:We studied the molecular mechanisms underlying the impact of pollen nutrients on honey bee (Apis mellifera) health and how those nutrients improve resistance to parasites. Using digital gene expression, we determined the changes in gene expression induced by pollen intake in worker bees parasitized or not by the mites Varroa destructor, known for suppressing immunity and decreasing lifespan of bees. bees with or without verroa, and fed or not fed pollen
Project description:Here, we examined the transcriptional and epigenetic (DNA methylation) responses to viral infection in honey bee workers. One-day old worker honey bees were fed solutions containing Israeli Acute Paralysis Virus (IAPV), a virus which causes muscle paralysis and death and has previously been associated with colony loss. Uninfected control and infected, symptomatic bees were collected within 20-24 hours after infection. Worker fat bodies, the primary tissue involved in metabolism, detoxification and immune responses, were collected for analysis. We performed transcriptome- and bisulfite-sequencing of the worker fat bodies to identify genome-wide gene expression and DNA methylation patterns associated with viral infection. There were 753 differentially expressed genes (FDR<0.05) in infected versus control bees, including several genes involved in epigenetic and antiviral pathways. DNA methylation status of 156 genes (FDR<0.1) changed significantly as a result of the infection, including those involved in antiviral responses in humans. There was no significant overlap between the significantly differentially expressed and significantly differentially methylated genes, and indeed, the genomic characteristics of these sets of genes were quite distinct. Our results indicate that honey bees have two distinct molecular pathways, mediated by transcription and methylation, that modulate protein levels and/or function in response to viral infections. Examination of epigenomic and transcriptomic antiviral responses to Israeli Acute Paralysis Virus in honey bees