Embryo development and sex determination in the Cassava whitefly (Bemisia tabaci) Rebecca Louise Corkill University of East Anglia A thesis submitted for the degree of Doctor of Philosophy September 2019 This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright law. In addition, any quotation or extract must include full attribution. Abstract The cassava whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), is a highly invasive and destructive agricultural pest, with a global distribution. B. tabaci insect has evolved resistance to multiple insecticides, and therefore, new control methods will have to be developed to control this insect. The self-limiting system, which produces a female-specific lethal heritable element and causes population suppression, works well for control of mosquitoes. The goal of this thesis is to assess the feasibility of creating a self-limiting system in B. tabaci. The self-limiting system requires generating transgenic insects, and this is done via injection of constructs into eggs at the pre-germ-line cell stage (around blastoderm formation), this enables stable germline transformation. However, there has been limited research into B. tabaci early embryogenesis stages, and therefore it is unclear when transformation constructs will have to be introduced. In this thesis, I conducted confocal microscopy studies to determine the timing of early embryogenesis stages in B. tabaci MED. Unexpectedly, I revealed that early embryogenesis in B. tabaci starts before the eggs are oviposited, unlike in mosquitoes and fruitflies. Therefore, injecting laid B. tabaci eggs is too late for obtaining stable transgenic whitefly lines, though somatic transgenics may be obtained. Past self-limiting transgenic insects used genes found in the sex determination pathway, for female-specific lethality, as these were expressed early and had sex-specific splicing. Genes of the sex determination pathways in B. tabaci and other hemipteran are largely unknown. Hence, I conducted a genome-wide search of 11 publicly available hemipteran genomes to identify their sex determination genes. Also, I conducted single-embryonic RNA-seq experiments in B. tabaci to assess if these genes undergo sex-specific splicing at the early embryogenesis stages in males and females. These results identified sex determination genes in B. tabaci and other hemipterans and revealed potential targets for future genetic control methods. ii “If we knew what it was we were doing, it would not be called research, would it?” - Albert Einstein iii Table of contents Abstract ................................................................................................................................... ii Table of contents .................................................................................................................... iv Acknowledgements ................................................................................................................. ix List of Figures ........................................................................................................................... x List of tables ......................................................................................................................... xiv List of abbreviations ............................................................................................................. xvii Chapter 1: Introduction ............................................................................................................ 1 1.1 Hemipteran ............................................................................................................................... 2 1.2. Bemisia tabaci .......................................................................................................................... 3 1.2.1 Life cycle and morphology of Bemisia tabaci .................................................................... 3 1.2.2 Cryptic species ................................................................................................................. 10 1.1.3 Why is controlling Bemisia tabaci so important? ............................................................ 11 1.2.3 Genomics of Bemisia tabaci ............................................................................................. 11 1.3 Sex-determination .................................................................................................................. 12 1.3.1 Overall structure of the sex determination cascade........................................................ 12 1.3.2 Sex determination in other insects .................................................................................. 13 1.3.3 Signalling element and ‘key’ genes .................................................................................. 13 1.3.4 Double switch genes ........................................................................................................ 17 1.3.5 Sex support genes ............................................................................................................ 19 1.3.6 Current knowledge of the sex determination pathway in Hemiptera ............................. 20 1.4 Control methods for B. tabaci................................................................................................. 22 1.4.1 Genetic control ................................................................................................................ 22 1.4.2 The self-limiting gene technique...................................................................................... 23 iv Aims of the project ................................................................................................................. 27 Overview of thesis contents ......................................................................................................... 27 Contributions to thesis .................................................................................................................. 29 Chapter 2: Materials and methods .......................................................................................... 30 2.1 Sex determination gene discovery .......................................................................................... 31 2.1.1 Pipeline for the bioinformatics ........................................................................................ 31 2.1.2 Genomes from the sex determination pipeline ............................................................... 32 2.1.3 Sex determination protein NCBI ...................................................................................... 34 2.1.4 Multiple sequence alignments ......................................................................................... 34 2.1.5 Full-length protein pairwise comparison ......................................................................... 34 2.1.6 Phylogenetic trees............................................................................................................ 35 2.2 Whole mount in-situ protocol for B.tabaci MED embryos ..................................................... 36 2.2.1 B. tabaci MED maintenance ............................................................................................. 36 2.2.2 B. tabaci MED virgin collection ........................................................................................ 36 2.2.3 B. tabaci MED non-virgin collection ................................................................................. 36 2.2.4 B. tabaci MED dissection.................................................................................................. 36 2.2.5 Original DAPI protocol with oviposited eggs ................................................................... 37 2.2.6 Propidium iodide staining ................................................................................................ 37 2.2.7 Final optimised DAPI-staining protocol............................................................................ 38 2.2.8 Confocal microscopy ........................................................................................................ 39 2.2.9 Embryo analysis in FIJI...................................................................................................... 39 2.3 Single embryo RNA-seq ........................................................................................................... 40 2.3.1 Sample preparation ......................................................................................................... 40 2.3.2 Library preparation method and quality control ............................................................. 40 2.3.3 Determining the sex of the samples ................................................................................ 40 2.3.4 Creating a transcriptome assembly ................................................................................. 40 2.3.5 Creating files for gene expression analysis ...................................................................... 41 v Chapter 3: Sex determination genes appear conserved across Hemiptera with diverse sexual lifecycles ................................................................................................................................ 43 3.1 Introduction ...........................................................................................................................