Effects of Environmental Contaminants on Gene Expression, DNA Methylation and Gut Microbiota in Buff-Tailed Bumble Bee - Bombus Terrestris
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UNIVERSITYOF LEICESTER DOCTORAL THESIS Effects of environmental contaminants on gene expression, DNA methylation and gut microbiota in Buff-tailed Bumble bee - Bombus terrestris Author: Supervisor: Pshtiwan BEBANE Eamonn MALLON A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in the Department of Genetics and Genome Biology March 2019 Abstract Bee populations are increasingly at risk. In this thesis, I explore various mechanisms through which environmental contaminants, namely imidacloprid and black carbon, can affect bumble bee epigenetics, behaviour and gut microbiota. I found imidacloprid has numerous epigenetic effects on Bombus terrestris non reproductive workers. I analysed three whole methylome (BS-seq) libraries and seven RNA-seq libraries of the brains of imidacloprid exposed workers and three BS-seq libraries and nine RNA-seq li- braries from unexposed, control workers. I found 79, 86 and 16 genes differentially methylated at CpGs, CHHs and CHGs sites respectively between groups. I found CpG methylation much more focused in exon regions compared with methylation at CHH or CHG sites. I found 378 genes that were differentially expressed between imidacloprid treated and control bees. In ad- dition, I found 25 genes differentially alternatively spliced between control and imidacloprid samples. I used Drosophila melanogaster as a model for the behavioural effects of imidacloprid on in- sects. Imidacloprid did not affect flies’ periodicity. Low doses (2.5 ppb) of imidacloprid in- creased flies’ activity while high doses (20 ppb) decreased activity. Canton-S strain was more sensitive to imidacloprid during geotaxis assay than M1217. I proposed that a possible modulator of imidacloprid’s effects on insects is its effects on insects’ gut microbiota. I produced and analysed thirty libraries of 16S rRNA metagenomic data from bee guts dividing by control and imidacloprid. There were no differences in alpha and beta bacterial diversity between groups while the ratio of one species of latobacillus increased in bees exposed to imidacloprid. As an extension of this microbiota work, I researched the effect of black carbon on gut micro- biota in the bumble bee. Bacterial cultivation methods showed significant increases of species (CFU) in bees exposed to black carbon and this was also detected by a qPCR approach. How- ever, in a metagenomic analysis, I did not find significant differences in alpha and beta diversity richness between black carbon and control groups. Declaration of Authorship I, Pshtiwan BEBANE, declare that this thesis titled, ’Effects of environmental contami- nants on gene expression, DNA methylation and gut microbiota in buff-tailed bumble bee Bombus terrestris’ and the work presented in it are my own. I confirm that: ■ This work was done wholly or mainly while in candidature for a research degree at this University. ■ Where any part of this thesis has previously been submitted for a degree or any other qualification at this University or any other institution, this has been clearly stated. ■ Where I have consulted the published work of others, this is always clearly at- tributed. ■ Where I have quoted from the work of others, the source is always given. With the exception of such quotations, this thesis is entirely my own work. ■ I have acknowledged all main sources of help. ■ Where the thesis is based on work done by myself jointly with others, I have made clear exactly what was done by others and what I have contributed myself. Signed: Date: ii DEDICATION This thesis is dedicated to: • My beloved, dearest and brilliant wife “Jwan” who never got tired of encourag- ing me and who leads me through the valley of darkness with light of hope and support. • My lovely little guys, my sons “Yousif” and “Laween”, whom I can’t force myself to stop loving. • My great parents, who never stop giving of themselves in countless ways. • My beloved brothers (shaxawan, Baxwan, Sherwan, Nahrow) and sisters (Amena and Shermen). • My great Parents-in-law. • My beloved brothers-in-law (Aso, Karzan, Karwan) and sisters-in-law (Hero, Shena and Shema). • My friends who encourage and support me. • All of the people in my life who have touched my heart. Acknowledgements Alhamdulillah, thank you Allah for all the blessing, You had given me. Thank You again for giving me the opportunity, knowledge and courage to overcome challenges and fin- ish this PhD project. I would like to thank and express my sincere gratitude to my supervisor Prof Eamonn Mallon. Thank you for all your support and guidance throughout my PhD, for your pa- tience, motivation, and immense knowledge. I would like to thank my second supervi- sor Prof Ezio Rosato who kindly continuous encouragement and has a nice suggestion during my study. Besides my supervisors, I would like to thank my PRP members Dr Sinéad Drea and Dr Iain Barber for their intuitive comments, guidance, questions and also suggestion. I would like to express my deepest thanks to every member of our lab past and present, with particular nods to Dr Mirko Pegoraro, Dr.Benjamin Hunt, Alun Jones, Hollie Mar- shall, Boris Berkhout, Christian Thomas, Zoe Lonsdale, all of whom were generous with time, assistance and opinions. Special thanks to the lab technicians, particularly Lisa Bedder and all staff, students in lab 104 and 121. Also, I want to thank all people in Genetics and Genome Biology, lab mates and admin- istrative staff for their continuous support. Very special thanks also to the University of Leicester for giving me an opportunity to complete this study. I would like to thank my parents, father-in-law, brothers, relatives, Ministry of Higher Education in Kurdistan/HCDP and Koya University for sponsoring and financially sup- porting me throughout my PhD study. I would especially like to thank my family. My dearest wife, Jwan has been extremely supportive of me throughout this entire process and has made countless sacrifices to help me get to this point. My mum, dad, sisters and brothers deserve special thanks for their continued support and encouragement. Without such a team behind me, I doubt that I would be in this place today. i Contents Abstract i Declaration of Authorship ii Acknowledgementsi List of Figures vi List of Tables xiii Abbreviations xv 1 General introduction1 1.1 Interactions between plants-pollinators-pests-ecotoxicology........1 1.2 Bumble bees and their importance in nature..................3 1.3 Evidence for the decline in bee populations..................5 1.4 Factors affecting insects communities......................6 1.4.1 Black carbon.................................6 1.4.2 What are neonicotinoids?.........................7 1.4.2.1 Imidacloprid and environmental residues..........9 1.4.2.2 Lethal effect of neonicotinoids................. 10 1.4.2.3 Sub-lethal effects of neonicotinoids.............. 11 Non neural effects.......................... 12 Gut bacteria effect......................... 13 1.4.3 Epigenetic effects.............................. 14 1.5 Aim and justification................................ 15 2 The neonicotinoid, imidicloprid affects gene expression, alternative splicing and DNA methylation in Bombus terrestris 17 2.1 Introduction..................................... 18 2.2 Materials and methods............................... 21 2.2.1 Bee husbandry............................... 21 ii Contents iii 2.2.2 Neonicotinoid feeding and brain sampling............... 21 2.2.3 BS-seq.................................... 21 2.2.3.1 Genomic DNA extraction, sequencing and mapping.... 21 2.2.3.2 Methylation differences between treatments........ 22 2.2.3.3 Methylation differences in exon level............. 23 2.2.4 RNA-seq................................... 23 2.2.4.1 RNA extraction and Illumina sequencing.......... 23 2.2.4.2 Alignment and assemble transcripts............. 24 2.2.4.3 Differential gene expression analysis............. 24 2.2.4.4 Alternative splicing events................... 24 2.2.5 GO term enrichment and Kegg analysis................. 25 2.3 Results........................................ 26 2.3.1 Methylation................................. 26 2.3.1.1 Sequencing, alignment and Methylation analysis...... 26 2.3.1.2 Methylation differences between treatments........ 28 2.3.2 Methylation differences in exon level.................. 31 2.3.3 GO term and KEGG pathway analysis.................. 34 2.3.4 Differential expression analysis...................... 43 2.3.4.1 Differential gene expression.................. 45 2.3.4.2 Go terms and KEGG pathway................. 49 2.3.5 Differential splicing of isoforms..................... 51 2.3.5.1 GO analysis............................ 57 2.3.6 DNA methylation - Expression correlation............... 58 2.4 Discussion...................................... 61 2.5 Collaborative work statement........................... 63 3 The neonicotinoid, Imidacloprid affects gut bacterial community in Bombus terrestris workers. 64 3.1 Introduction..................................... 65 3.2 Methods....................................... 71 3.2.1 Neonicotinoid toxicity test........................ 71 3.2.2 Bee husbandry, imdicloprid exposure and gut sampling....... 71 3.2.3 DNA extraction and library preparation................. 72 3.2.4 Bioinformatic analysis........................... 73 3.2.5 Taxonomy analysis............................. 75 3.2.6 Differential abundance analysis..................... 75 3.3 Results........................................ 77 3.3.1 Neonicotinoid anti bacterial