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Thesis Rests with Its Author University of Bath PHD Paedogenetic reproduction in Mycophila speyeri and Heteropeza pygmaea (Cecidomyiidae, Diptera) Hunt, Justine L. Award date: 1996 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 11. Oct. 2021 Paedogenetic Reproduction in Mycophila speyeri and Heteropeza pygmaea (Ceeidomyiidae, Diptera). Submitted by Justine L. Hunt for the degree of PhD. of the University of Bath, 1996. Copyright Attention is drawn to the fact that the copyright of this thesis rests with its author. This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without the prior written consent of the author. This thesis may be made available for consultation within the University Library and may be photocopied or lent to other libraries for the purpose of consultation. UMI Number: U092157 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U092157 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 LIBRARY Contents Abstract 1 Chapter 1 Introduction: The Paedogenetic Cecidomyiidae 1.1 The Cecidomyiidae 3 1.2 External morphology of the larvae 3 1.3 Gall midge biology 4 1.4.1 Mushroom production 6 1.4.2 Pests of mushroom crops 8 1.4.3 Cecids as pests of mushrooms 9 1.5 Paedogenesis 12 1.5.1 Paedogenetic reproductive system 13 1.5.2 Oogenesis 15 1.5.3 Nurse chamber formation 16 1.5.4 Oocyte-nurse chamber complex 17 1.5.5 Follicular epithelium 18 1.5.6 Embryogenesis 19 1.5.7 Chromosomes 21 1.6 Spermatogenesis 24 1.7 Lifecycles 24 1.8 Environmental control of life cycles 25 1.9 Hormonal control of life cycles 25 1.10 Larval nutrition 27 Chapter 2 Observations onMycophila speyeri and Heteropeza pygmaea 2.1 Introduction 28 2.1.1 Life cycles 28 2.1.2 Mycophila speyeri 28 2.1.3 Heteropeza pygmaea 32 2.1.4 Other species 33 2.1.5 Description of different sexes 34 2.1.6 Culture techniques 35 2.1.7 History of in vitro culture 36 2.2 Materials and methods 36 2.2.1 Culture of Mycophila speyeri and Heteropeza pygmaea 38 2.2.2 Fixing and sectioning for light microscopy 38 2.2.3 Culture of ovaries, follicles and embryos 39 2.3 Results 42 2.3.1 Lifecycles 44 2.3.2 Mycophila speyeri 44 2.3.3 Heteropeza pygmaea 50 2.3.4 Follicle and ovarian development 52 2.3.5 In vitro culture of ovaries, follicles and embryos 54 2.4 Discussion 62 Chapter 3 Environmental Control of Development and Reproduction 3.1 Introduction 68 3.1.2 Sex determination 68 3.1.3 Factors influencing sex determination 70 3.1.4 Developmental fate 72 3.1.5 Introduction to experiments 73 3.2 Materials and methods ' 74 3.2.1 The influence of nutrition on sex determination in Mycophila speyeri 74 3.2.2 The culture of Mycophila speyeri on defined medium 74 3.2.3 The effect of temperature on the growth of Mycophila speyeri 75 3.2.4 The effect of fungal species on the growth of Mycophila speyeri 75 3.2.5 The effect of crowding on the growth of Mycophila speyeri 76 3.3 Results 78 3.3.1 The influence of nutrition on sex determination in Mycophila speyeri 78 3.3.2 The culture of Mycophila speyeri on defmed medium 79 3.3.3 The effect of temperature on the growth of Mycophila speyeri 82 3.3.4 The effect of fungal species on the growth of Mycophila speyeri 83 3.3.5 The effect of crowding on the growth of Mycophila speyeri 86 3.4 Discussion 89 iii Chapter 4 Endocrine Control of Reproduction 4.1 Introduction 92 4.1.1 Insect developmental hormones 92 4.1.2 Production of hormones 92 4.1.3 Juveniie hormone 96 4.1.4 Ecdy steroids 97 4.1.5 Hormonal control of reproduction 98 4.1.6 Insect growth regulators 99 4.1.7 Role of hormones in cecid development 102 4.1.8 Alternative ecdysteroids 105 4.1.9 Aims 108 4.2 Materials and methods 110 4.2.1 Treatment of insects with hormones and insect growth regulators 110 4.2.2 Identification of dietary sterol 111 4.2.3 Sterol and ecdysteroid extraction from insect tissue 112 4.2.4 Identification of sterols using GC-MS 114 4.2.5 Identification of insect ecdysteroid using HPLC-RIA 115 4.3 Results 118 4.3.1 Treatment of cecids with hormone analogues 118 4.3.2 Identification of dietary sterol from GC-MS - 121 4.3.3 Identification of insect sterols from GC-MS 122 4.3.4 Identification of ecdysteroid from insect tissue 123 4.4 Discussion 142 Chapter 5 Larval Nutrition 5.1 Introduction 146 5.1.1 Viviparity 146 5.1.2 Larval nutrition in the Cecidomyiidae 147 5.1.3 Vitellogenesis 150 5.1.4 Fat body 154 5.1.5 Fat bodies in the Cecidomyiidae 160 5.1.6 Role of the fat body in vitellogenesis 162 5.2 Materials and methods 165 5.2.1 Ultrastructural study of the fat bodies of Mycophila speyeri 165 5.2.2 Insect tissue preparation and protein content determination 166 5.2.3 SDS-PAGE of insect tissue samples 167 5.2.4 Observations of fat bodies in vitro 168 5.3 Results 170 5.3.1 Fixation 170 5.3.2 Ultrastructural study of the fat body tissue of Mycophila speyeri 170 5.3.2.1 Observations of cytoplasmic structure at different stages 172 5.3.2.2 General observations 175 5.3.3 Insect tissue preparation and protein content determination 179 5.3.4 Minigels of all parts of the insect 180 5.3.5 Observations of fat bodies in vitro 182 5.4 Discussion 203 Chapter 6 General Discussion 210 Appendix 1 Leatham’s medium 216 Appendix 2 Solutions for electron microscopy preparation 218 Appendix 3 Lowry protein assay for microplates ' 219 Appendix 4 SDS-PAGE 220 Appendix 5 RIA of free ecdysteroids 221 References 222 v List of Figures. Figure Page number 1.1 Cultivated mushroom infested with Mycophila speyeri 11 2.1 Life cycle of Mycophila speyeri 29 2.2 Life cycle of Heteropeza pygmaea 31 2.3 Petri dish containing 2% malt-extractagar, inoculated with Chondrostereum purpureum and Mycophila speyeri 43 2.4 Diagramatic representation of the life cycle of Mycophila speyeri and Heteropeza pygmaea at Bath. 45 2.5 Day 1, paedogenetic, Mycophila speyeri larva 47 2.6 Day 3, paedogenetic, Mycophila speyeri larva 47 2.7 Day 5, paedogenetic, Mycophila speyeri larva 47 2.8 Mother larva with offspring emerging 48 2.9 Day 3, paedogenetic, Heteropeza pygmaea larva 48 2.10 Pre-pupal Mycophila speyeri larva 49 2.11 Mycophila speyeri pupa 49 2.12 Mycophila speyeri adult 49 2.13 Longitudinal section through day 3 Mycophila speyeri larva 55 2.14 Longitudinal section through day 3 Mycophila speyeri larva , 55 2.15 Transverse section through a day 2 Mycophila speyeri larva 56 2.16 Transverse section through a day 4 Mycophila speyeri larva 56 2.18 Mycophila speyeri embryo at 24 hours 57 2.19 Mycophila speyeri fat body cells at day 5 57 2.20 Egg follicles from a newly emergedMycophila speyeri larva 59 2.21 Individual eggs from 24 hour old Mycophila speyeri larva 59 2.22 Embryos from a day 2 Mycophila speyeri larva 60 2.23 Embryos from a day 2 Mycophila speyeri larva after 24 hours in culture 60 2.24 Embryos from a day 3 Mycophila speyeri larva 61 2.25 Embryos from a day 5 Mycophila speyeri larva 61 4.1 A generalised scheme for side chain dealkylation of phytosterols in insects 106 vi 4.2 Graph showing, fractions from cecid ecdysteroid sample exhibiting ecdysone-like immunoreactivity 124 4.2b Total ion current for sterol sample extracted from C. purpureum 132 4.3 Mass spectrum for peak 1; ergosta-5,7,22-trien-3(3-ol 133 4.4 Mass spectrum for peak 2; ergosta-7.22-dien-3P-ol 134 4.5 Mass spectrum for peak 3; ergosta-5.7-dien-P-ol 135 4.6 Mass spectrum for peak 4; ergosta-7-en-3p-ol 136 4.7 Total ion current for Heteropeza pygmaea sterol sample 137 4.8 Reconstructed ion current Heteropeza pygmaea sterol sample 138 4.9 Mass spectrum for ergosterol from Heteropeza pygmaea sterol sample 139 4.10 Mass spectrum for sterol from Heteropeza pygmaea sterol sample: cholesta-5,7,22-3P-ol 140 4.11 HPLC trace from Heteropeza pygmaea ecdysteroid sample 141 5.1 Mycophila speyeri embryo fat body cells 184 5.2 Mycophila speyeri embryo fat body cells 184 5.3 Mycophila speyeri embryo fat body cells 185 5.4 Mycophila speyeri embryo fat body cells 185 5.5 Day 1, Mycophila speyeri fat body cells 186 5.6 Day 1, Mycophila
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