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Microbial Colonization of the Gut Environment
The insect gut is an extreme environment, with unstable conditions making colonisation by microbial organisms difficult. Firstly, microorganisms need to persist against harsh physiochemical conditions, digestive processes and host immune defence (Douglas, 2015). Secondly, insects undergo several events in which the gut is shed, affecting the in situ microbial communities (Broderick and Lemaitre, 2012, Engel and Moran, 2013, Douglas, 2015). In insect groups producing a PM, microorganisms are unavoidably expelled during regular shedding of this lining (Engel and Moran, 2013). Likewise, larvae moult several times during development, at which time the lining of the fore- and hindgut is shed, along with it any inhabiting microorganisms (Douglas, 2011,Broderick and Lemaitre, 2012, Engel and Moran, 2013). This results in variable gut microbiomes across the different life stages in holometabolous insects (Engel and Moran, 2013). However, moulting events stop once the insect is an adult, providing a more stable environment for colonisation of the fore- and hindgut (Engel and Moran, 2013). Microorganisms found in the insect gut environment are typically either resident (naturally occurring), successfully inhabiting the gut environment, or transient, acquired through contact with the environment or ingested with the food (De Vries et al., 2001, Bright and Bulgheresi, 2010). While a small number of transient microorganisms may be harmful (pathogenic) to the host, the majority have little influence on the host, typically being removed from the gut (Douglas, 2009). Some ingested microorganisms are beneficial to the host, as a food-source, assisting with digestion of the ingested food and supplementing the nutritional
needs of the host (Douglas, 2009). Transient microorganisms may be prevented from colonising the insect gut by the presence of the resident microflora (Veivers et al., 1982). However a small number of bacteria are capable of persisting in the gut (Vallet-Gely et al., 2008).
Metagenomic DNA Extraction for Amplicon Pyrosequencing
Gut mDNA extraction was performed using a modified version of the protocols previously described by Calderón-Cortés et al. (2010) and Shi et al. (2012). Gut sections were weighed and crushed in liquid nitrogen using a sterilised epi-crusher. For 10mg of gut tissue, 100μl of a preheated (60°C) 2% Cetyl trimethylammonium bromide (CTAB) solution was added. The mixtures were incubated for 30min at 60°C before centrifugation for 5min at 10000rpm. The supernatant was transferred to a clean collection tube and enzymatic digestion of the gut samples was carried out with the addition of 2μl lysozyme (5mg/ml) per 100μl CTAB solution for 30min at 37°C under continuous shaking (120 rpm). 0.5μl Proteinase K (20mg/ml) per 100μl CTAB solution was then added (Priya et al., 2012), followed by an overnight incubation at 55°C with continuous shaking. One volume phenol:chloroform:isoamyl alcohol (25:24:1) solution was added. Tubes were inverted and centrifuged at 13000rpm at 4°C for 4min. One volume chloroform:isoamyl alcohol (24:1) solution was added to the top aqueous phase and the mixtures were inverted before centrifugation at 13000rpm at 4°C for 15min. This step was repeated until no protein contamination was observed (Priya et al., 2012). DNA was precipitated with 3M NH4Ac (Lagisz et al., 2010) and ice cold 99.9% EtOH followed by overnight incubation at -20°C. Mixtures were centrifuged for 60min at 14000rpm at 4°C. The DNA pellet was washed twice with ice cold 70% EtOH and allowed to dry completely for 2 hours. The DNA pellet was resuspended in 50μl filter-sterilized nanopure H2O overnight at
4°C (Lagisz et al., 2010), and stored at -20°C for downstream analysis.
Chapter 1: Literature Review
1.1 Desert Environments
1.2 Desert Insect Communities: A Brief Overview
1.3 Diets of Insects
1.4 Structure of the Insect Gut
1.5 Insect Gut Microbial Communities
1.6 Sequence-Based Methods in Microbial Ecology
1.7 Aims and Objectives
1.8 References
Chapter 2: Materials and Methods
2.1 Chemical Reagents
2.2 General Methodologies
2.3 454 Amplicon Pyrosequencing Methods – Chapter 3
2.4 Hiseq Shotgun Sequencing Methods – Chapter 4
2.5 References
Chapter 3: Amplicon Sequencing of Bacterial and Fungal Gut Communities of Pachysoma Species
3.1 Introduction
3.2 Results and Discussion
3.3 Conclusion
3.4 References
Chapter 4: Shotgun Metagenomic Sequencing of the Gut Microbiomes of Pachysoma endroedyi and Pachysoma striatum
4.1 Introduction
4.2 Results and Discussion
4.3 Conclusion
4.4 References
Chapter 5: Concluding Remarks
5.1 Studying the Pachysoma gut microbiome
5.2 Limitations of this study
5.3 Future study prospects
5.4 References
