| A fundamental goal of sociobiology is to explain the regulation mechanism of complex social behavior, especially in social insects. As eusocial insects, the honey bees Apis are characterized by the extreme reproductive division of labor. The queen monopolizes reproduction, while workers refrain from the ability to reproduce in the presence of a queen. However, when the colony loses its queen, many workers can activate their ovaries and initiate laying eggs. Ovaries are the functional unit of the female honey bee reproductive organs, and ovary activation is one of the significant factors that influence the honey bee reproductive capacity. Identifying candidate genes associated with ovary development will provide new insights into the molecular mechanisms involved in ovary activation and reproductive division of labor. In this study, we used the next generation high-throughput RNA-Sequencing technology (Illumina HiSeqTM2000) to describe mRNAs and miRNAs expression differences between inactivated and activated ovaries of queens and workers A. mellifera ligustica. According to the regulation relationship between miRNAs and mRNAs, the targets of miRNAs that are associated with ovary activation were predicted.In a rare strain of European derived of A. mellifera "anarchistic" workers are reproductively active even in the presence of a laying queen, resulting in the majority of males are offspring of workers rather than the queen. Anarchy is a revealing counterpoint to the general situation in queenright colonies, especially in European A. mellifera, where worker reproduction is rare. These bees provide an experimental resource for investigating the proximate mechanisms underlying the regulation and control of worker reproduction in wild-type colony. In this study, we confirmed the reproduction traits of anarchistic colonies occurring in China. In order to understand the role of the worker mandibular gland secretions in the acquisition of reproductive status, we investigated the mandibular gland profiles of workers sampled in anarchistic (AC) colonies. The main results were summarized as follows:1. mRNAs associated with honey bee ovary activationOvarian activity not only influences fertility, but is also involved with the regulation of division of labor between reproductive and behavioral castes of female honey bees. In order to identify candidate genes associated with ovarian activity, we compared the gene expression patterns between inactivated and activated ovaries of queens and workers by means of high-throughput RNA-Sequencing technology. A total of1615differentially expressed genes (DEGs) were detected between ovaries of virgin and mated queens, and more than5300DEGs were detected between inactivated and activated worker ovaries. Intersection analysis of DEGs among five libraries revealed that a similar set of genes (824) participated in the ovary activation of both queens and workers. A large number of these DEGs were predominantly related to cellular, cell and cell part, binding, biological regulation and metabolic process. In addition, over1000DEGs were linked to more than230components of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including25signaling pathways. In particular, our findings reveal differential ovarian expression of genes encoding proteins involved in nutrition related-functions and hormonal signaling. These include genes encoding for major royal jelly proteins (MRJPs), yellow proteins, small chemosensory proteins (CSPs), enzymes involved in juvenile hormone (JH) and ecdysteroid biosynthesis as well as members of the insulin-IGF-1signaling pathway. The reliability of the RNA-Sequencing results was confirmed by means of quantitative RT-PCR in ten genes. Our results provide new insights into the molecular mechanisms involved in ovary activation and reproductive division of labor.2. miRNAs associated with honey bee ovary activationmiRNAs are endogenous small (19-24nt) non-coding RNAs, which are usually target to one or more target genes in3’non-coding region, regulating gene expression by targeting mRNAs for cleavage or translational repression. In order to identify candidate miRNAs as well as their target genes associated with ovarian activity, we compared miRNAs expression between inactivated and activated ovaries of queens and workers by means of high-throughput RNA-Sequencing technology. We discovered80,79,133,111and97known miRNAs for inactivated ovaries of virgin queens (VQI), activated ovaries of egg-laying queens (EQA), inactivated ovaries of queenright workers (WIQR), inactivated ovaries of queenless workers (WIQL) and activated ovaries of queenless workers (WAQL), respectively. Moreover,28,32,64,13and10novel miRNAs were identified in VQI, EQA, WIQR, WIQL and WAQL, respectively. We detected28known miRNAs were significantly different between ovaries of virgin and mated queens, and more than100known miRNAs were significantly different between inactivated and activated worker ovaries. Intersection analysis of differentially expressed known miRNAs among five libraries revealed that a similar set of known miRNAs (16) participated in the ovary activation of both queens and workers. To identify candidate known miRNAs that are associated with ovary activation, we predicted their target genes by combining with mRNAs expression profiles of ovaries.3. Reproduction traits of anarchistic honey beesAanarchistic colony is rare in the natural condition. In2012, we first saw anarchistic syndrome in A. mellifera L. in China. We confirmed three honeybee colonies obtained two typical traits of worker reproduction in the presence of the queen: significantly more workers (3%～21%) activated their ovaries and initiated egg-laying; workers evaded egg policing by laying more acceptable eggs and reducing selective removal of worker-laid eggs by policing workers. Here, we show that anarchistic workers could reduce inhibitory effects of the queen by increasing the spatial distance to the pheromone source. In order to further understand the role of the worker mandibular gland secretions in the acquisition of reproductive status, we investigated the mandibular gland profiles of workers with inactivated ovaries (IAWs) and workers with activated ovaries (AWs) sampled in these three anarchistic colonies. The total amounts of five identified compounds (HOB,9-ODA,9-HDA,10-HDA and10-HDAA) were significantly higher in AWs compared to IAWs. The mandibular gland profiles of IAWs is dominated by the "worker substances"10-HDA and its precursor10-HDAA, while AWs is dominated by9-HDA, the precursor of the major compound of the queen mandibular pheromone (9-ODA). Our results are similar to previous studies showing that the mandibular gland profiles of A. m. capensis workers is dominated by the9-HDA in the presence of their own queen. The ratios of9-ODA/(9-ODA+10-HDA+10-HDAA) were no significant differences between IAWs and AWs. The ratios of the precursor substance10-HDAA/9-HDA of IAWs were worker specific (>1.0) whereas AWs were queen specific (≤1.0). Our results first demonstrate the relationship between anarchistic worker mandibular gland pheromone and worker reproduction. |
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