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Community Structure of Gut Microbes in Busseola Fusca (Lepidoptera: Noctuidae)

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Community Structure of Gut Microbes in Busseola Fusca (Lepidoptera: Noctuidae) Community structure of gut microbes in Busseola fusca (Lepidoptera: Noctuidae) M Snyman 21155852 Dissertation submitted in fulfilment of the requirements for the degree Magister Scientiae in Environmental Sciences at the Potchefstroom Campus of the North-West University Supervisor: Prof CC Bezuidenhout Co-supervisor: Dr S Claassens Assistant Supervisor: Prof J van den Berg May 2015 ABSTRACT Bt-maize is engineered to express insecticidal toxins derived from the bacterium Bacillus thuringiensis and has been shown to be very effective against pests like Busseola fusca. However, resistance of this pest against Bt-maize has developed and spread throughout South Africa. This study was inspired by the lack of knowledge over the microorganisms associated with the gut of these insects as they play a vital role in insect growth and development. Microbial-derived enzymes may have a role during an insect‟s adaption in different environmental conditions and to new diets. Previous studies suggest (1) that gut bacteria are required for B. thuringiensis-induced mortality in most Lepidoptera species and (2) that the toxicity of B. thuringiensis depends on microbial community interactions within the gut. The aim of this study was to determine the microbial diversity present in the midgut of B. fusca larvae occurring in maize. Busseola fusca larvae were collected from 30 sites throughout South Africa and dissected to collect their midgut contents. Serial dilutions were made of the contents and spread plated onto nutrient agar after which morphotypes were identified. One-hundred and five morphotypes were identified; DNA were extracted from the selected morphotypes and subjected to PCR analysis followed by secquencing. Sequencing results revealed the dominance of Enterococcus spp., specifically Enterococcus casseliflavus and Enterococcus gallinarum, Klebsiella spp., espesially Klebsiella pneumoniae and Klebsiella oxytoca and Bacillus spp. such as .B. thuringiensis and B. subtilis. Other organisms isolated, included Achromobacter spp., Brevudimonas spp., Caulobacter spp., Enterobacter spp., Halomonas spp., Ochrobactrum spp., Pantoea spp., Pseudomonas spp., Serratia spp., Stenotrophomonas spp., Arthrobacter spp., Brevibacterium spp., Leucobacter spp., Microbacterium spp., Planomicrobium spp. and Staphylococcus spp. The microbial diversity of larvae collected at the respective sampling sites were determined with the Shannon diversity index. The data were compared to several factors regarding the sampling sites. No significant differences were observed between the microbial diversities isolated at the respective sites. This may imply that the microbial community within B. fusca larvae are relative consistent throughout the maize production area. It is important to understand the distribution and structure of gut microbial communities within insects and whether the gut community is influenced by the geographical distribution of the insects. A better understanding of the distribution of the insects and community structure of their gut microbiota may aid in the development of better insect control strategies. Keywords: Busseola fusca, microbial community, gut microbes, PCR, resistance, symbionts. i PREFACE The experimental work done and discussed in this dissertation for the degree Master of Science in Environmental Sciences was carried out in the School of Biological Sciences, North-West University, Potchefstroom Campus, South Africa. This study was conducted fulltime during the period of January 2013 - November 2014, under the supervision of Prof. Carlos Bezuidenhout, Dr. Sarina Claassens and Prof. Johnnie van den Berg. The research done and presented in this dissertation signifies original work undertaken by the author and has not been submitted for degree purposes to any other university. Appropriate acknowledgements in the text have been made where the use of work conducted by other researchers have been included. References were done according to: NWU. 2014. NWU Referencing Guide. Potchefstroom: Library Services of North- West University, Potchefstroom Campus. Maxi Snyman November 2014 ii ACKNOWLEDGEMENTS I would like to express my appreciation to the following persons at the North-West University, Potchefstroom Campus for their contributions towards the successful completion of this study. My supervisors Prof Carlos Bezuidenhout, Dr. Sarina Claassens and Prof. Johnnie Van den Berg for giving me this exceptional opportunity and for all of their support and guidance throughout this period. Dr. Jaco Bezuidenhout for assisting with the statistical aspects, and Dr. Arvind Gupta with the phylogenetic analysis of this study. Megan van Staden for the time spent sampling and assistance in the laboratory. Stephan Barnard for constructing the map that illustrates the respective sampling sites. Furthermore I would also like to thank my family and friends for their encouragement and support during my studies: Simone Booyens and my fellow colleagues My parents, Kenny and Marinda Snyman for supporting me since the beginning of my studies. My lifelong friends, Marizaan de Jong and Carla Pohl for their friendship and emotional support. Johan Smit, for the love and inspiration that kept me from giving up. Without you none of this would have been possible. iii TABLE OF CONTENTS ABSTRACT ..................................................................................................................... i PREFACE ...................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................. iii TABLE OF CONTENTS ................................................................................................ iv LIST OF ABBREVIATIONS ......................................................................................... vii LIST OF FIGURES ........................................................................................................ ix LIST OF TABLES ........................................................................................................ xii CHAPTER 1 ................................................................................................................... 1 INTRODUCTION ............................................................................................................ 1 1.1. General introduction and problem statement ............................................................ 1 1.2. Research aim and objectives ...................................................................................... 3 CHAPTER 2 ................................................................................................................... 4 LITERATURE STUDY ................................................................................................... 4 2.1. The importance of maize ............................................................................................. 4 2.2. Busseola fusca ............................................................................................................. 4 2.2.1 Distribution ............................................................................................................ 4 2.2.2. Behaviour and life-cycle ....................................................................................... 6 2.2.3. Pest status ........................................................................................................... 8 2.2.4. Management and control ...................................................................................... 9 2.3. Genetically modified crops ....................................................................................... 11 2.3.1. What are genetically modified crops? ................................................................. 11 2.3.2. Development of genetically modified crops......................................................... 12 2.4. Bt Maize ...................................................................................................................... 13 2.4.1. Bacillus thuringiensis .......................................................................................... 14 2.4.2. Mechanism of Bt ................................................................................................ 16 2.4.3. Advantages and disadvantages of Bt crops ........................................................ 18 2.5. Resistance development of Busseola fusca to Bt maize ........................................ 21 iv 2.6. Associations between insects and gut microbes .................................................... 23 2.6.1. Structure and physical conditions of the insect gut ............................................. 23 2.6.2. Function of gut microbes .................................................................................... 26 2.6.3. Lepidopteran gut community structure ................................................................ 29 2.6.4. Effect of geographical distribution of insects on gut microbe content .................. 32 2.7. Methods to identify gut community structure .......................................................... 33 2.8. Approaches to evaluate microbial community structure ........................................ 35 CHAPTER 3 ................................................................................................................. 37 MATERIALS AND METHODS ....................................................................................
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