Genetic variability of the whitefly Bemisia tabaci and its secondary endosymbionts in the Arabian

62 Pages·2013·5.52 MB·English

by  Alaa I Ragab

Genetic variability of the whitefly Bemisia tabaci and its secondary endosymbionts
in the Arabian Peninsula

Thesis by
Ala'a Ragab

In Partial Fulfillment of the Requirements
For the Degree of
Master of Science

King Abdullah University of Science and Technology
Thuwal, Kingdom of Saudi Arabia

May 2013
2

EXAMINATION COMMITTEE APPROVALS FORM

The thesis of Ala'a Ragab is approved by the examination committee.

Committee Chairperson Prof. Dr. Nina Fedoroff
Committee Co-Chair Dr. Ali Idris

Committee Member Prof. Dr. Arnab Pain
3

© 2013
Ala'a Ragab
All Rights Reserved
4

ABSTRACT
Genetic variability of the whitefly Bemisia tabaci and its secondary endosymbionts
in the Arabian Peninsula
Ala'a Ragab

The whitefly Bemisia tabaci species complex has been well documented as one of the
most economically important emergent plant virus vectors, through serious feeding
damage to its broad range of plant hosts and transmission of plant viruses to important
agricultural crops. It has been shown to have associations with endosymbionts which
have significant effects on the insect fitness. The purpose of this study was to provide
information for the biotype and secondary endosymbiont distribution for B. tabaci
populations in the relatively unstudied Arabian peninsula. The geographical localization
and variation in endosymbiont populations across the region were identified using a
sequence-driven analysis of the population genetics of the secondary endosymbiont. Live
field specimens were collected from 22 different locations in the region and preserved in
70% ethanol for genetic studies. Previously established procedures were used to extract
and purify total insect DNA from 24-30 individual whiteflies for each location (Frohlich
et al., 1999; Chiel et al., 2007). Specimens were subjected to PCR amplification using the
respective 16S rDNAprimers for the Rickettsia, Hamiltonella, and Wolbachia to amplify
endosymbiont DNA. PCR was run with primers for the highly conserved whitefly
mitochondrial cytochrome oxidase subunit I (COI) gene for biotyping. Samples were
sequenced using the Sanger method and the data analyzed to correlate the presence,
prevalence and geographical distribution of endosymbionts in B. tabaci. Phylogenies
5

were constructed to track evolutionary differences amongst the endosymbionts and
insects and how they have influenced the evolution of the regional populations. Samples
were characterized by differences in the genomes and endosymbionts of common
whitefly ‘biotypes’ that have different host plant preferences, vector capacities and
insecticide resistance characteristics. It was found that the B biotype is the predominant
haplotype, with no evidence of the Q biotype. All endosymbionts were detected, with
Hamiltonella as the most predominant. Several instances of co-infection by two or more
endosymbionts were observed. Samples from the geographically isolated and
mountainous region of Fayfa demonstrated higher genetic variability compared to the
other locations, leading to the possible identification of a new haplotype, as well as the
first time identification of the A biotype in the region.
6

ACKNOWLEDGEMENTS
I would like to thank my committee chair, Professor Nina Fedoroff, and my committee
members, Dr. Ali Idris and Professor Arnab Pain, for their guidance and support
throughout the course of this research.
My appreciation also goes out to my friends for making my time at King Abdullah
University of Science and Technology a great experience. I would especially like to thank
the friends who helped and supported me throughout: Sarah Al Mahdali, Alan Itakura,
William Bass, Jesse Cochran, Octavio Salazar, Alex Barthel and Mariam Awlia.
Finally, my heartfelt gratitude is extended to my parents for their encouragement and
support.
7

TABLE OF CONTENTS
Page
EXAMINATION COMMITTEE APPROVALS FORM .......................................... 2
COPYRIGHT PAGE ................................................................................................ 3
ABSTRACT ............................................................................................................... 4
ACKNOWLEDGEMENTS ...................................................................................... 6
TABLE OF CONTENTS ........................................................................................... 7
LIST OF ABBREVIATIONS .................................................................................... 8
LIST OF ILLUSTRATIONS ..................................................................................... 9
LIST OF TABLES ..................................................................................................... 10
1. INTRODUCTION ……………………………………………………………….. 11
1. 1. Biotypes…………………………………………………………… ..... 11
1.2. Virus Transmission………………………………………………….. ... 14
1.3. Endosymbionts ……………………………………………………….. . 14
1.4. Pest Management Strategies………………………………………... .... 19
1.5. Current Research…………………………………………………….. ... 20
2. MATERIALS AND METHODS…………………………………………….. ..... 24
2.1. Sample Collection…………………………………………………… ... 24
2.2. DNA Extraction……………………………………………………...... 25
2.3. Primer Design………………………………………………… ............. 25
2.4. Biotype Identification………………………………………………… . 25
2.5. Endosymbiont characterization……………………………………….. . 26
2.6. Gel Electrophoresis………………………………………………….. ... 26
2.7. Cloning………………………………………………………….. .......... 27
2.8. PCR Product Cleaning……………………………………………….. .. 27
2.9. Sequencing, Sequence Assembly and Data Analysis………………… . 28
3. RESULTS ………………………………………………….. ............................... 29
3.1. Identification of B. tabaci biotypes and genetic subgroups………… .... 29
3.2. Detection and identification of the secondary endosymbiotic bacteria .. 29
3.3. Phylogenetic Analyses……………………………………………….. .. 35
4. DISCUSSION………………………………………………………………….. .. 40
5. CONCLUSION & FUTURE WORK………………………………………….. .. 45
REFERENCES…………..................…………………………………………..…... 47
APPENDICES………………………………………………………………….…... 57
8

LIST OF ABBREVIATIONS

mtCOI Mitochondrial cytochrome oxidase subunit I
SS Secondary endosymbiont
ddH O Double distilled water
2
PCR Polymerase chain reaction
dNTP Deoxyribonucleotide triphosphate
DMSO Dimethylsulfoxide
rDNA Ribosomal DNA
CI Cytoplasmic incompatibility
PI Parthenogenesis induction
TYLCV Tomato yellow leaf curl virus
CLCuV Cotton leaf curl virus
IRM Insecticide-resistance management
CAPS Cleaved amplified polymorphic sequences
bp Base pair
PAUP Phylogenetic anaylsis using parsimony
9

LIST OF ILLUSTRATIONS

Figure 1. Map of Sample Locations ................................................................................. 24
Figure 2. Gel of Polymerase Comparison......................................................................... 29
Figure 3. Gel of Wolbachia Clone Digest......................................................................... 30
Figure 4. Secondary Endosymbiont (SS) Infection Frequency For All Locations........... 33
Figure 5. Secondary Endosymbiont (SS) Infection Frequency in the East and South..... 34
Figure 6. Maximum Likelihood Tree For Mitochondrial Cytochrome Oxidase I (mtCOI)
..................................................................................................... ..................................... 37
Figure 7. Maximum Likelihood Tree For Wolbachia............................ .......................... 38
Figure 8. Maximum Likelihood Tree For Hamiltonella......................... ......................... 39
Figure 9. Histogram Of Endosymbiont Infection Frequency........................ .................. 62
10

LIST OF TABLES
Table 1. PCR Primers Used in the Study...................................................................... 27
Table 2. Sample Locations with Endosymbiont Distribution........................................ 31
Table 3. Sample Locations with Endosymbiont (SS) Infection and Co-infection
Frequency....................................................................................................................... 32
Table 4. PCR Conditions............................................................................................... 57
Table 5. PCR Reaction Components for Each Primer................................................... 57
Table 6. Ligation Components...................................................................................... 58