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Overview

Project Highlights:

  • The bumblebee is one of the most ecologically and economically important pollinators.
  • Laboratory and field studies have found effects on the behaviour, mortality and colony performance of bees exposed to neonicotinoids.
  • In a small preliminary BS-seq we found over thirty genes with differential methylation due to neonicotinoid exposure.

This project will quantify, for the first time, the epigenetic effects of neonicotinoids on bumblebees, by examining treated bees' methylomes and transcriptomes. The social epigenetics lab at the University of Leicester has pioneered the study of bumblebee epigenetics. Neonicotinoid insecticides have been strongly implicated in the decline of bees affecting a number of important biological functions; an exciting possibility is that they do this by affecting the methylation system of bees.

The role of insect pollinators in feeding a growing population and their recent declines is explicitly mentioned in NERC’s “The business of the environment” (page 5). This project fits into two NERC research areas; 1) Ecotoxicology - Diagnostics, tolerance and adaption in relation to natural and anthropogenic toxic substances and 2) Environmental genomics - Understanding the response of organisms to their natural environment at the level of the genome.

Neonicotinoids are effective insecticides used on many important crops, often as seed dressing. They are systemic, meaning they are absorbed by the plant and transported to all tissues where they remain active for many weeks. However, their tissue distribution and stability, which are important for their efficacy, determines their lack of species-specificity and their negative effects on the environment. In 2013 the European Union imposed a partial restriction on their use.

Laboratory and field studies have found effects on the behaviour, mortality and colony performance of bees exposed to neonicotinoids. The effects are so broad as to be likely caused by mechanisms additional to the primary toxicity, namely the block of the nicotinic acetylcholine receptors (nAChRs) resulting in major dysfunction of neuronal transmission. In mice, nicotine reduces DNA methylation in AChRs cells suggesting epigenetic consequences following repeated activation of nAChRs.

 Methylation, the addition of a methyl group to a cytosine, has important effects on the biology of bees, including the control of reproductive status, task switching and memory. Methylation affects gene expression and alternative splicing in social insects. In a small preliminary BS-seq we found over thirty genes with differential methylation due to neonicotinoid exposure.

Probably Maurice Maeterlinck not Einstein

Methodology

Ten colonies of the buff tailed bumblebee Bombus terrestris audax will be used. Aged controlled callow workers will be collected and reared in Perspex boxes, 5 workers per box. Control groups will be fed with sugar water for five days whereas treatment groups will be fed with sugar water and the neonicotinoid, imidacloprid (10ppb).

When the bees are five days old, they will be sacrificed and their brains dissected. RNA and DNA will be extracted. The student will analyse whole methylome (BS-seq) libraries of the brains of three neonicotinoid exposed workers and three control workers from each of ten colonies in order to establish if neonicotinoids affect the methylation status of bumblebee workers.

They will then analyse transcriptome (RNA-seq) libraries of the above samples in order to discover the gene expression changes and alternative splicing associated with neonicotinoid exposure.

 

Training and Skills

CENTA students are required to complete 45 days training throughout their PhD including a 10 day placement. In the first year, students will be trained as a single cohort on environmental science, research methods and core skills. Throughout the PhD, training will progress from core skills sets to master classes specific to CENTA research themes.

The student will be provided with training, as required, in R, a powerful and increasing popular statistical programming language, Python, a general-purpose, high-level programming language widely used in bioinformatics, molecular biology and bee husbandry.

Training will also be provided in the preparation of both transcriptomic and methylomic NGS libraries. The student will also become conversant with general molecular biology techniques such as PCR, qPCR and cloning.

Timeline

Year 1: Bee husbandry. Collecting samples. Carrying out treatments. Production and sequencing of libraries. Begin analysis.

Year 2: Complete analysis of BS-seq and RNA-seq libraries. Publish resultant papers.

Year 3: Explore other avenues opened by previous two years. For example candidate gene approach looking at methylation and gene expression in bees in the field exposed to neonicotinoids.

Partners and collaboration (including CASE)

This is a collaborative project between the lead supervisor Mallon and co-supervisor Rosato. The supervisors have complimentary interests and expertise in ecotoxicology and social insects and of next generation sequencing techniques to investigating these areas. Mallon will provide specific expertise in the role of epigenetics and gene expression, while Rosato provides expertise in ecotoxicology and candidate gene molecular biology. This proposal will benefit greatly from the ongoing collaboration between M and R in co-supervising a current PhD student working on bumblebees.

Further Details

Please contact Eamonn Mallon, Department of genetics, University of Leicester, ebm3@le.ac.uk for further details.