Biology and Biological Control Potential of Bethylid Wasps Sahand Kamal Khidr (MSc) Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy May 2012 Table of Contents Table of Contents .................................................................... II ••• ii List of Figures .. II. 11"1" II ••••• II •••••••••• 1 III' II •• II ••••••• 1 •• 1 •• II' '1""'" II •••••• ,.viii List of Tables ...............................................................................x Abstract ............ I." I ••••••• I •••• I •• I •• I I I ••••••••• I II •••••• 1.1 •••••••••• ,. I I ••• I I ••••••• xi Impact statement"I'I"" ............................ " ................................. xi Acknowledgements. I •••••••••••• I I ••••• I I ••••••••••••••••••••• I" I •••••• I ••••••••••••••• xiii Chapter 1: General introduction .................................................. 1 1.1 The aim of biological control .................................................... 1 1.1.1 Most selected organisms ...................................................................... 2 1.1.2 Risks and requirements in biocontrol agents ........................................... 2 1.2 Parasitoids and evolutionary studies ................................... · .. · .. 4 1.2.1 Clutch size .......................................................................................... 6 1.2.2 Sex ratio ...................................... 111 •• '1 ••••••••••••••••••••••••••••••••••••••••••••••• 7 1.3 Parasitoid aggressive behaviour ............................................. 10 1.4 Bethylid parasitoids .............................. I •••••••••••••••• I •••••••••••••• 13 1.4.1 Biology of Goniozus nephantidis .......................................................... 15 1.4.2 Biology of Goniozus legneri ................................................................. 16 1.5 Contest behaviour ................................................................ 16 1.5.1 Contest behaviour in Goniozus legneri and G. nephantidis ...................... 19 1.6 Proton Nuclear Magnetic Reasonance (NMR) and data analysis .. 21 1. 7 Microsatellite markers and parasitoids .................................... 22 ii 1.8 From behavioural ecology to population biology and biocontrol .. 24, 1.9 Summary of thesis structure ................................................. 25 1. 9.1 Part one: Resource value, host quality and parasitoid age ...................... 25 1.9.2 Part two: Kin recognition, aggressive behaviour and chemical cues ......... 26 1.9.3 Part three: Molecular genetics and sex ratios ........................................ 26 Chapter 2: Post-paralysis declines in host quality assessed by parasitoid behaviour, development and metabolomics ............. 28 2.1 Abstract .... I I ••••••••••••••• I I I •••••••••••••••••••• I • I • I • I • I •••••••••••••••• I •••••• I' 28 2.2 Introduction ............................................................... , ........ 29 2.2.1 Biology of the host-parasitoid study system .......................................... 30 2.3 Material & Methods ...................................................... 11 •••••• 31 2.3.1 Part 1: Longevity of paralysed hosts .................................................... 31 2.3.2 Part 2: Effects of host age on host weight, parasitoid reproductive behaviour and life-history ......... , .................................................... , .......... " ............... 31 2.3.3 Part 3: Effects of host age on parasitoid contest behaviour ..................... 32 2.3.4 Part 4: Effects of host age on metabolomic state ................................... 33 2.3.5 Data analysis .................................................................................... 34 2.4 Results ......... 1 •••••••••••••••••••••••••••••••••••••••••••••••• , •••••••••••••••••••• 36 2.4.1 Part 1: Longevity of paralysed hosts .................................................... 36 2.4.2 Part 2: Effects of host age on host weight, parasitoid reproductive behaviour and life-history ..... , ....................................................... , ........... " ........ , ...... 37 2.4.2.1 Host weight change ..................................................................... 37 iii 2.4.2.2 Host acceptance .......................................................................... 38 2.4.2.3 Clutch size .................................................................................. 39 2.4.2.4 Developmental mortality .............................................................. 40 2.4.2.5 Sex ratio .................................................................................... 41 2.4.2.6 Offspring size .............................................................................. 41 2.4.3 Part 3: Effects of host age on parasitoid contest behaviour ..................... 42 2.4.4 Part 4: Effects of host age on metabolomic state ................................... 44 2.5 Discussion ............. 11.1 •••• 11' •• 1'11.1 ••••••••••••••••••• I'" •••••••••• II •••••••• so 2.6 Conclusions ............ s ••••••••••••••••••••••••••••••••••••••••••• ,11 •••••••••••••• 54 Chapter 3: Two components of kin recognition influence parasitoid aggression in resource competition .................. I "" II. I S6 3.1 Abstract ....................................................... 1 •••••••••••••••••••••• 56 3.2 Introduction ......................................... ".............................. 57 3.3 Material & Methods ........................ 11 •••••••••••••••••••••••••••••••••••• 59 3.3.1 Parasitoids ....................................................................................... 59 3.3.2 Experiment 1: Identifying determinants of aggression ............................ 59 3.3.3 Experiment 2: Focussing on kin recognition determinants of aggression ... 61 3.3.4 Statistical analysis ............................................................................. 62 3.4 Results I •••••••••••••••••••••••••••••••••••• , ••••••••••••••••••••••••••••••••••••••••• 62 3.4.1 Experiment 1: Identifying determinants of aggression ............................ 62 3.4.2 Experiment 2: Focussing on kin recognition determinants of aggression ... 66 3.5 Discussion ........................................................................... 68 iv 3.6 Conclusion ............................................................................ 72 Chapter 4: Cuticular hydrocarbon profiles of Goniozus species and of hosts influence wasp aggressiveness ............................. 73 4.1 Abstract .............................................................................. 73 4.2 Introduction ........................................................................ 74 4.3 Material & Methods ................................................................ 76 4.3.1 Parasitoids ....................................................................................... 76 4.3.2 Culturing .......................................................................................... 77 4.3.3 Sampling programme ........................................................................ 77 4.3.4 Sample extraction ............................................................................. 78 4.3.5 Cuticular hydrocarbons analysis .......................................................... 78 4.3.6 Statistical analysis ............................................................................. 79 4.4 Results ............................................................................................. 79 4.5 Discussion ........................................................................... 88 4,6 Conclusion. II ........ 1.11 ••••• I ......................................................... 90 Chapter 5: The development of molecular genetic markers in lJE!tl1"licl '"'CI!tJ)!S •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 92 5.1 Abstract .................................................................................. 92 5.2 Introduction ........................................................................ 93 5.2.1 Biology of bethylids ................•......... , ...................................•............ 94 5.3 Material & Methods .............................................................. 95 v 5.3.1 Parasitoid origins and cultures ............................................................ 95 5.3.2 Design and preparation of the primers ................................................. 95 5.3.3 DNA extraction .................................................................................. 99 5.3.4 Polymerase Chain Reaction (PCR) ....................................................... 100 5.3.5 Agarose Gel Electrophoresis ............................................................... 101 5.3.6 Quantitation test of DNA templates and Goniozu5 individuals ................. 101 5.3.7 Capillary sequencing: Preparing fragment samples for analysis .............. 101 5.4 Results ....................................... 11 ••••••••••••••••••••••••••••••••••••• 102 5.4.1 Primer design in the Microsatellite library ............................................ 102 5.4.2 Annealing gradient tests ................................................................... ·l02 5.4.3 DNA quality test ...................................................................... · .... · .. ·103 5.4.4 Second round of PCR runs ................................................................. 104 5.4.5 Capillary sequencing ........................................................................