Location: Southern Horticultural Research
Project Number: 6062-21430-003-22-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2015
End Date: Aug 31, 2020
Several external influences have been involved in Colony Collapse Disorder (CCD) in the United States. Abiotic or biotic stressors do not act in isolation; for example pesticide exposure can impair both detoxification mechanisms and immune responses, rendering the bees more susceptible to parasites. Therefore, this makes the antioxidant system of pollinator such as honey bee an attractive model to study the regulation of transcriptional responses during elevated oxidative stress caused by environmental stressors (acaricides, herbicides, pesticides). The overall goal is to investigate the fundamental biological question how genes are switched on and off in response to toxic environmental stressors in honey bee. It will allow the identification and characterization of key players at the molecular level to harness the honey bee immune response against stressors. Aim 1: Determine the transcriptional gene expression in response to environmental stressors to identify oxidative stress biomarkers in honey bees. Given the rich diversity of honey bee antioxidants genes (38 genes), selecting candidate antioxidant enzymes on which to focus is essential. To gain an insight into the global gene expression in response to environmental stressors (pesticides) RNA-Seq and proteome approach will be used. This experiment will provide a catalog of differentially expressed transcripts upon pesticide exposure. Aim 2: Define the functional role of the honey bee antioxidant genes involved in mitigating chronic pesticide-induced oxidative stress. In this study, the honey bees will be exposed to sub-lethal doses of pesticides which in turn generates more damaging reactive oxygen species (ROS) and results in oxidative damage, cytotoxicity, and even cell death. Cellular responses related to the redox status will be assessed using cytometry assays for free thiols and ROS levels. These experiments will provide an insight into pesticide induced antioxidant response in honey bees, and potential role of antioxidants in harnessing the bee health. Aim 3: A system biology approach will be used to elucidate Varoa destructor/ honey bee interaction, and the impact of acaricides and other pesticides on bee immune system. RNA-Seq and proteome approach will be used to shed light on the expression of parasite, and bee genes. In order to develop de novo transcriptome of Varroa mites, libraries will be made from whole female mites. This experiment will provide an indication of differential regulation of mite gene expression. The expression of the selected target genes will be confirmed by qRT-PCR assay. Additionally, to confirm the identity of differentially expressed or up-regulating genes a targeted proteome approach will be utilized. Parasitized honey bee hemolymph will be collected to perform LC-MS/MS to identify the bee immune system gene targets. To determine the functional role of newly identified mite genes responsible for the dysfunction of bee immune genes, experiments will be performed to examine the functional role of key target genes. Additional funds will be used for salary for new Post-doc, supported for 2 years.
The two specific aims are directed at a) understanding whether the antioxidants are the first lines of defense against high levels of ROS and b) how they mitigate chronic oxidative stress. Methods proposed in this cooperative research agreement have either been published or routinely used in the lab to study antioxidant machinery in arthropod model system. Aim 1: The total RNA from honey bees will be extracted using illustra RNAspin Mini RNA isolation kit (GE Healthcare). The quality of the RNA samples will be confirmed by lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies). The total RNA quantity will be estimated by Nanodrop, and the total RNA samples will be used for further cDNA synthesis. Briefly, the Clontech SmartPCR cDNA kit will be used to generate cDNA from total RNA. Restriction digestion was used to remove adaptor sequences, and the resulting cDNA will be fragmented, profiled using Agilent Bioanalyzer, and subjected to Illumina library preparation. Agilent Bioanalyzer 2100 will be used to assess the quality, quantity, and size distribution of the Illumina libraries. The libraries will then be submitted for sequencing. Paired-end 100 or 125 nucleotide reads will be generated and checked for data quality. Assembly of all reads will be done using the assemble programs Abyss and Soapdenovo-Trans. Resulting contigs will be re-assembled by a pipeline of BLASTn and cap3 assembler. Coding sequences will be extracted based on BLASTx results deriving from several database matches. Reads from all libraries will be mapped back into CDS by BLASTn. Reads will be mapped up to a maximum of five different CDS if the BLAST score will be the same for all matches. For heat map display of the CDS temporal expression, the number of the reads for each treatment will be normalized by multiplying it to the grand total number of reads deriving from all libraries and dividing the product by the total number of reads of the particular library. Differential gene expression clustering will be performed with the programs Expander version 6.5, using input read fragments per thousand nucleotide per million (FPKM) data and the click1 algorithm. All coding sequences and their reads will be available for browsing (public access/sharing) with hyperlinks to several databases. FPKM will be calculated for each library. Deducted coding sequences and their translations will be deposited at DDBJ/EMBL/GenBank for public access. We expect significant differences between control and pesticide treated groups. We anticipate identifying additional differentially expressed antioxidant candidates. A total of 38 antioxidant genes in the honey bee genome have been identified. However, the physiological roles of several honey bee antioxidants have yet to be established. The purpose of this experiment is to gain an insight into the antioxidant response of honey bees and its natural microbiota towards pesticide-induced stress. We will measure the spatiotemporal (time-dependent and tissue-dependent) gene expression of all target antioxidant genes, and total bacterial load using qRT-PCR or ddPCR assay.