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DNA Methylation As a Mechanism for Caste System Determination in Solenopsis Invicta: Do the Genes Determine the Queen?

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DNA Methylation As a Mechanism for Caste System Determination in Solenopsis Invicta: Do the Genes Determine the Queen? DNA Methylation as a Mechanism for Caste System Determination in Solenopsis invicta: Do the Genes Determine the Queen? By Kristen E. Walker A thesis submitted in partial fulfillment of the requirements of the University Honors Program University of South Florida St. Petersburg May 3, 2017 Thesis Director: David John, Ph.D. Lecturer, College of Arts and Sciences University Honors Program University of South Florida St. Petersburg CERTIFICATE OF APPROVAL ___________________________ Honors Thesis ___________________________ This is to certify that the Honors Thesis of Kristen E. Walker has been approved by the Examining Committee on May 3, 2017 as satisfying the thesis requirement of the University Honors Program Examining Committee: ___________________________ Thesis Director: David John, Ph.D. Lecturer, College of Arts and Sciences ____________________________ Thesis Committee Member: Thomas Smith, Ph.D. Honors Program Director and Associate Professor of Political Science, College of Arts and Sciences ___________________________ Thesis Committee Member: Debby Cassill, Ph.D. Associate Chairperson and Associate Professor, College of Arts and Sciences Abstract The emerging field of epigenetics seeks to study the interactions of the environment and other factors on gene expression in organisms. Epigenetic mechanisms involve modification to the DNA and/or surrounding structures, resulting in the activation or repression of gene expression. One epigenetic mechanism, DNA methylation, involves the addition of a methyl group to carbon 5 of cytosines in CpG dinucleotide regions in vertebrates and possibly other phyla. Vertebrate genomes tend to be globally methylated, whereas genes tend to be the target of methylation in insects. Genomic analyses of the order Hymenoptera revealed the evolutionary persistence of DNA methylation in social insects. Fully functioning DNA methylation systems have been discovered in several bee and ant species, including Solenopsis invicta. DNA methylation in insects may play a role in caste determination, as ant embryos are capable of following different developmental pathways. Social insects prove a promising model for understanding methylation in developmental regulation due to the presence of phenotypic plasticity and the potential for genomic imprinting. This study aims to determine whether differential methylation is present among different castes in the fire ant species, Solenopsis invicta. Preforming bisulfite treatment on selected genes derived from whole body DNA extraction could provide data on the presence of differential methylation among castes. Further investigation may reveal whether DNA methylation is the mechanism by which environmental cues affect the developmental trajectory of ant embryos determining their caste. i Table of Contents Abstract………………………………………………………………………………………………………………..i TABLE OF CONTENTS………………………………………………………………………………………..…ii List of Tables………………………………………………………………………………………………………..v List of Figures……………………………………………………………………………………………………viii Chapter 1: Introduction………………………………………………………………………………..………1 1-1: Introduction to epigenetics…………………………………………………………………1 1-2: DNA methylation……………………………………………………………………….…….....2 1-2-1: DNA methylation mechanism………………………………………………...2 1-2-2: DNA methylation in vertebrates……………………………….……………4 1-2-3: DNA methylation in insects…………………………………………………...6 1-3: Identification of differentially methylated regions…….………………………..10 1-4: Phenotypic plasticity………………………………………………………………………...12 1-4-1: Genotype – phenotype mapping function..………………….…..……12 1-4-2: Apis mellifera: An example of phenotypic plasticity ……………...15 1-5: Caste differentiation in social insects…………………………………………………18 1-6: Solenopsis invicta………………………………………………………………………………20 Chapter 2: Materials and Methods……………………………………………………………...……….23 2-1: Determination of target genes...…………………………………………………………23 2-2: Primer design……………………………………………………………………………...……26 2-3: DNA extraction….……………………………………………………………………………...28 2-4: PCR amplification…………………………………………………………………………..…30 2-4-1: PCR overview………………………………………………………………..……30 ii Table of Contents 2-4-2: Primer reconstitution……………………………………………………..…..31 2-4-3: PCR reaction setup……………………………………………………………...31 2-4-4: Thermal cycler parameters………………………………………………….32 2-5: Gel electrophoresis…………………………………………………………………………...34 2-6: Cloning…………………………………………………………………………………………….35 2-6-1: Retailing reactions………………………………………………………………36 2-6-2: PCR cleanup……..…………………………………………………………………36 2-6-3: Cloning ligation…………………………………………………………………..37 2-6-4: Cloning transformation……………………………………………………….37 2-6-5: Cloning……………………………………………………………………………….38 2-7: Sequencing……...……………………………………………………………………………….39 2-8: PCR troubleshooting…………………………………………………………………………40 2-9: Bisulfite conversion…………………………………………………………………………..43 Chapter 3: Results………………………………………………………………………………………………45 3-1: Primer design…………………………………………………………………………………...45 3-2: DNA extraction…………………………………………………………………………………48 3-3: PCR amplification and gel electrophoresis…………………………………………49 3-3-1: Asunder Whole………………………………………………………………...…49 3-3-2: Asunder Front…………………………………………………………………….51 3-3-3: Asunder Rear and Rab11 Rear……………………………………………..53 3-4: Cloning…………………………………………………………………………………………….56 3-5: Sequencing……………………………………………………………………………………….58 iii Table of Contents 3-5-1: Asunder Front………………………………………………………………….…58 3-5-2: Asunder Rear……………………………………………………………………...60 3-5-3: Rab11 Rear…………………………………………………………………………61 3-6: PCR troubleshooting…………………………………………………………………………62 3-6-1: Asunder Front, Asunder Rear, and Rab11 Whole PCR and gel electrophoresis.…………………………………………………………………………….62 3-6-2: Asunder Front, Asunder Rear, and Rab11 Whole major and minor worker PCR and gel electrophoresis…………………………………….66 3-6-3: PCR troubleshooting analysis………………………………………………70 Chapter 4: Discussion…………………………………………………………………………………………77 Works Cited……………………………………………………………………………………………………….81 iv List of Tables Table 1.1 Conserved Differentially Methylated Genes Between Queens and Workers in Camponotus floridanus and Harpegnathos saltator...……………………………....………...24 Table 1.2 Gene Locus Numbers for Camponotus floridanus, Harpegnathos saltator, and Solenopsis invicta…………………………………………………………………………………………25 Table 1.3 Solenopsis invicta Gene Orthologs………………………………………………………..26 Table 2.1 Primer Design for Asunder and Rab11 Genes in Solenopsis invicta………..27 Table 2.2 Concentrations of Primers……………………………………………………………….….33 Table 3.1 Thermal Cycler Parameters…………………………………………………….…………..33 Table 3.2 Thermal Cycler Parameters for Retailing Reactions……………………………..36 Table 4 Colony Numbers for Asunder Front, Asunder Rear, and Rab11 Rear…….….39 Table 5 Vector Cloning Site Sequences…………………………………………………………....….40 Table 6.1a Asunder Whole Master Mix……………………………………………………………….49 Table 6.1b Asunder Whole Individual PCR Reactions………………………………………….50 Table 6.1c Asunder Whole Thermal Cycler Parameters………………………………………50 Table 6.2a Asunder Front Master Mix………………………………………………………………...52 Table 6.2b Asunder Front Individual PCR Reactions…………………………………………...52 Table 6.2c Asunder Front Thermal Cycler Parameters………………………………………..52 Table 6.3a Asunder Rear Master Mix………………………………………………………………….54 Table 6.3b Asunder Rear Individual PCR Reactions…………………………………………….54 Table 6.4a Rab11 Rear Master Mix………………………………………..…………………………...54 Table 6.4b Rab11 Rear Individual PCR Reactions……………………………………………….54 Table 6.4c Asunder Rear and Rab11 Rear Thermal Cycler Parameters………………..55 v List of Tables Table 7.1a Asunder Front Repeat Master Mix……………………………………………………..63 Table 7.1b Asunder Front Repeat Individual PCR Reactions………………………………..63 Table 7.1c Asunder Front Repeat Thermal Cycler Parameters…………………………….64 Table 7.2a Asunder Rear Repeat Master Mix………………………………………………………64 Table 7.2b Asunder Rear Repeat Individual PCR Reactions…………………………………64 Table 7.2c Asunder Rear Repeat Thermal Cycler Parameters……………………………...64 Table 7.3a Rab11 Whole Master Mix………………………………………………………………….65 Table 7.3b Rab11 Whole Individual PCR Reactions…………………………………………….65 Table 7.3c Rab11 Whole Thermal Cycler Parameters………………………………………….65 Table 7.4a Asunder Front Major and Minor Worker Master Mix………………………….67 Table 7.4b Asunder Front Major and Minor Worker Individual PCR Reactions…….67 Table 7.4c Asunder Front Major and Minor Worker Thermal Cycler Parameters…67 Table 7.5a Asunder Rear Major and Minor Worker Master Mix…………………………..67 Table 7.5b Asunder Rear Major and Minor Worker Individual PCR Reactions……..68 Table 7.5c Asunder Rear Major and Minor Worker Thermal Cycler Parameters…..68 Table 7.6a Rab11 Whole Major and Minor Worker Master Mix……………………………68 Table 7.6b Rab11 Whole Major and Minor Worker Individual PCR Reactions………68 Table 7.6c Rab11 Whole Major and Minor Worker Thermal Cycler Parameters…...69 Table 7.7a 0.2 μM Primer Asunder Front Master Mix………………………………………….71 Table 7.7b 0.2 μM Primer Asunder Front Individual PCR Reactions…………………….71 Table 7.8a 0.4 μM Primer Asunder Front Master Mix………………………………………….72 Table 7.8b 0.4 μM Primer Asunder Front Individual PCR Reactions………………...…..72 vi List of Tables Table 7.9a 0.8 μM Primer Asunder Front Master Mix…………………………………….……73 Table 7.9b 0.8 μM Primer Asunder Front Individual PCR Reactions…………………….73 Table 8 PCR Analyses of Various Conditions for Asunder Front…………………………...76 vii List of Figures Figure 1. Four Hypotheses for the Genotype–Phenotype Mapping Function……...…14 Figure 2.1 Graphical Representation of Primer Location on Asunder Gene in Solenopsis invicta………………………………………………………………………………………………..46 Figure 2.2
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