çhrvIFU$ kþ.n "rW d "Gfri;iyf$t$ A COMPARISON BETWEEN THE SARIVARY PHYSIOLOGY OF THE CRUSADER Míctis profana Fabricius (COREIDA CrË AND THE GREEN LUCERNE MIRID, Creontíades dilutus (Stål) BT GARY STE\ryART TAYLOR A thesis submitted for the Degree of Doctor of Philosophy in the Faculty of Agricultural and Natural Resource Sciences at The University of Adelaide. Depar:tment of Crop Protection V/aite Campus The University of Adelaide May 1995 \ ,.' . t \'t,.' ì \ . r, ll TABLE OF CONTENTS CHAPTER ONE A REVIEW OF SALIVARY FI.]NCTION IN THE PHYTOPHAGOUS HETEROPTERA, WITH SPECIAL REFERENCE TO THE INVERTASE AND PECTINASE OF THE FAMILIES SALIVARY 1 COREIDAE AND MIRIDAE' RESPECTWELY I 1.1. SALIVARY FUNCTION IN ÍIETEROPTERA 1.1.1. Morphology of the salivary apparatus and mouthpafis of 1 3 3 4 5 5 6 6 8 t.2. ETIOLOGY OF COREID AND MIRID LESIONS 1.2.1. Australian Coreidae and Miridae considered in this discussion 9 10 1.3. ENZYME zuNCTIONS 1.3.1 Invertase 10 10 1.3.1.1. t.3.1.2. and invertase and phloem transport ...... t2 1.3.1.3. Intervention of Hemipteran saliva in nutrient transPort in I4 Plants 16 1.3.2. Pectinase.......... 1.3.2.1. Properties 16 1.3.2.2. The role in the saliva of 20 t.3.3. salivary physiologY of coreids and 20 mirids: 111 CHAPTER TWO THE BIOLOGY ANT) BEHAVIOUR OF Mictis .ONVBIOPMENTnrofana Fabricius OBSERVATIONS ON THE (coRErDAE) WITH 2L OF FEEDING LESIONS 2l 2.1. ABSTRACT......... 2l 2.2. INTRODUCTION 22 General biologY ..... 2.2.I. 23 2.2.2. Host Plant association 2.3. MATERIALS AND METI{ODS............... """""23 2.3.1. Fe€dúg b"h*io*....:."",""""' """"""'23 2.3.2. O¡seruãtiónJ on feeding Èsions of M' profarul""""""' """"2/+ Z.Z.Z.l.--Olservationã on rhe rate of stem abscission by ,\^ M.Profana """' L+ 23.2.í. Moistote aphylla fed upon bY M.ProÍ4na.-...."onte """'24 2.3.3. Hiroiógi*f ðiu.i rnsof M. proÍ4tt4"""""""'24 stn¡ctufe of.the stylet sheath............................. ..7! 2.3.4. O¡se*ítiãnson the z) 2.3.5. Measurements of stylet penetration ""' ZS.{t-. Maximúm èxsertion of the stylets based on -r"r*å-ttttt of --ip"rãiJ mouttrpartl """""""' """"""" 25 2.3.5.2. Measurem"ntt oiìtyfa þne'tration in plant tissue " " " " " " " " " " 25 25 2.4. RESULTS.. - proÍano 25 2.4.r. Feeding behaviour and rate of lesion formation bY M 25 2.4.1.1. Field observations 29 2.4.2. Histological examination of feeding lesions of M.profara Structure of the stylet sheath and feeding behaviour on agaf 2.4.3. 29 gels., 30 2.4.4. Measurements of stylet penetration 30 2.5. DISCUSSION...... CHAPTER THREE 34 34 34 3.2. INTRODUCTION 35 3.3. MATERIALS AND METHODS Dissection of salivarY glands of M. and C. dilutus 35 3.3.1. 35 3.3.2. Dissection of digestive tract ofM 38 RESULTS...... 3.4. 38 Salivary glands of M. proÍana 3.4.1. 39 3.4.2. Salivary glands of C. dilutus 39 3.4.3. The digestive tract of M. profaru 4T 3.5. DISCUSSION lv CHAPTER FOUR 43 43 43 4.2. INTRODUCTION M 4.3. MATERIALS AND METHODS............... their use 4 4.3.L. Livç materials and M 4.3.2. Collection of salivary preparations """""""' ¡""""""""""""' 4.3.2.1. Collection of watery saliva"""" 4 45 45 4.3.3. material and sulohvdrvl groups 45 +.i.12.'Peterniination of PH of saliva and contents of lobes 45 of the salivary glands....... 4.3.3.3. Obsérvations on turbidity and surfactant properties of 45 4.3.4. Preparation and use of gland extracts for qualitative enzYme 45 tests -......... --- i.s.+.t. Ireparation of buffered salivary gland extracts..... 45 4.3.4.2. M 46 4.3.4.3. 4.3.4.4. and cellulase""""""""' 47 4.3.4.5. Test for 47 4.3.4.6. Test for 49 4.3.4.7. Oxidase feacüons of salivary glands and saliva. 49 49 4.3.5. Determination of free amino acids......... 50 4.3.6. Uèié.tion of enzymes in the digestive tact of M ' proÍ4t14 50 4.3.6.1. Test for Proteinase 4-.1:.6.i. Test fór additional invertase/sucrase input from digestive tract.......... 50 50 4.4. RESULTS............ """"" EA 4.4.1. Properties of watery saliva """"""""" ru ¿.+.r.ï.-ôórbCtioñof watery saliva."""' """""""50 4.4.L.r. betermination of pH of contents of lobes of the salivarY gl """""""51 4.4.1.i. b ProPerties of saliva....... """"""""' 51 4.4.2. """"""""' 51 ........:::::::::::::::::::::::::::::::::::3i <1 4.4.3.4.4.4. """"' JJ 4.4.5. tive tract of M ' profana ""' 53 59 4.5. DISCUSSION 4.5J. Origins of the stylet sheath....... 59 4.5.2. Detection of salivary enzymes 60 4.5.3. Detection of salivary amino nitrogen compounds"""""""" 61 v CHAPTER FIVE THE CHARACTERISATION, SOURCE AryD SECRETORY ß¡,rv¡.ÍrY SUCRASE OF Míctis profana Ãðiwrif or inn 63 Fabricius (COREIDAE) .. 63 5.1. ABSTRACT......... 63 5.2. INTRODUCTION g 5.3. MATERIALS AND METHODS -. & 5.3.1. Quantitaúve determinations of salivary sucrase.......... 5.3.2. Sucrase activitY and pH of secreted saliva & Determination of soluble protein and specific activity of 5.3.3. 65 sucrase 65 5.3.4. Electrophoretic seParation of denatured 5.3.5 Identification of sucrase and invertase 65 separated proteins .... 6 66 7l 7l 7L 75 5.5. DISCUSSION CHAPTER SIX THE POSSIBLE FUNCTION OF A SALIVARY SUCRASE IN THE ñúinir1õñ Or- ulù¡i profana Fabricius (C9REIDAE) IN rHE noL'n ör suc¡,Rs IN PLANI PHLoEM äÈiÃnioñ ro 77 TRANSPORT ............ 77 6.1. ABSTRACT......... 6.2. INTRODUCTION .............. 77 MATERIALS AND MET{ODS 79 6.3. 79 6.3.1. 79 6.3.2. 79 6.3.3. 80 6.3.4. 80 6.3.5 80 6.3.6. Pressure bomb experiments............." 6.4. RESULTS............ """"" 81 6.4.1. """"""' 81 6.4.2. yeast invertase"""""" """""' 81 6.4.3. n stem sections """ 81 6.4.4. infilration and pressure bomb õ*p.ri."nts.............. """""""""' 82 6.5. DISCUSSION 85 vt CHAPTER SEVEN ON FEEDING BEHAVIOUR AND THE OBSERVATIONS (MIRTDAE) 88 SALIVARY PECTINASE O'F Creontitde s dilutus (stål) 88 7.1. ABSTRACT.......... 88 7.2. INTRODUCTION 88 General biologY 7.2.I. 89 Pectinase in the saliva of Hemiptera 7.2.2. 90 7.2.3. Feeding lesions of mirids 90 7.3. 90 90 teins from individual lobes......... 90 -l¿."tification of pectinase in electrophoretically 7.1i.ã. 91 separated Proteins 9T 7.3.2. OËsè*atioïs on the structurc of th""""""""""""":""""' ï.1.1. óbservations on feeding on lucerne 91 7.3.3.1. Feeding lesions tlssue""""' 91 7.3.3.2. Simulation of p ase"""""" 9r 92 RESULTS ............... 7.4. 92 7.4.1. grectróprroretic separation of salivary prote ins o1 C. dilutus Identifiäation of pectinase in electrophoretically 7.4.1.i 92 92 7.4.2. 92 7.4.3 agar 7.4.4. viour on 95 gels 95 7.5. DISCUSSION CHAPTER EIGHT FUNCTION OF SALIVARY PECTOLYTIC ENZYMES IN THE 98 LESIONS CAUSED BY Creontíades dílutus (sråt) (MTRTDAE) 98 8.1. ABSTRACT 98 99 8.3. MATERIALS AND METHODS..... 99 8.3.1. 99 8.3.2. 100 8.3.3. by pectinase 100 8.3.3.2. Elution of amino nitrogen from bean sections bY Dectinase (with maceration)...... 100 'S.t.i.t. Èiution of amino nitrogen from bean sections by 100 Dectinase vs time...... '8:t.3.¡. Elution of amino nitrogen from bean sections by salivary gland extract from C. dilutw vs time'.." 101 vll of other plant chemicals and oxidation products from 8.3.4. Elution ..... 101 bean sections bY Pectinase.. .....101 8.4. .....101 .....toz 102 102 roz t02 8.5. DISCUSSION CHAPTER NINE 113 GENERAL DISCUSSION 113 9.1. The salivary glands insect saliva"""" é:i: to insect feeding tt4 -Ä'õã-pî¡io"Pit;tñ"fidut"tt strategies of coreids and é.ã. tr4 APPENDIX ONE The Madhusudhan, v.v., Taylor, G.S. and Miles, P.w._(1994). A;;".tt";- oi sativaiy of phylophagous lremiotera: a "ity-.s Api;iiäà líi;l;il-tzt, 4o3-4r2""""""""' 117 compiration of methäã '.-Âi*äit APPENDIX TWO by Miles, P.V/. and Taylor, G.S. (1994)' 'Osmotic plryP'-Iegglng 7 3: 163 - 17 3 t26 coreids. n ntå^ob eío-Eip er:ime'ntalis et Applic ata " " APPENDIX THREE Taylor, G.S. and Miles, P.W. ( the saliva of coreids in relation of the salivary 138 et 73l. 277 r52 REFERENCES I viii LIST OF FIGURES Fig. 2.1. Exsertion of the stylets of M ' prolana"" ""'26 Fig.2.2. Axial row of stylet sheath flanges on stem of A' iteaphy11a""""""""""""""27 feeding by an Fig. 2.3. Stem of A. ite¿ hylla 2 hr from commencement of adult M. profana.... n Fis. 2.4. Terminal wilting on stem of A' iteaphylla 4 hr from .óä."nt"ment of feeding by ao adult M' profara' n Fig. 2.5. Terminal wilting and leaf senescence on stem of A. iteaphyllø8hr frõn commencement of feeding bY an adult M. profana- n of M. proþna nea¡ vascular bundles of Fig. 2.6. Branching.of stylet track 3l broad bean stem sectlon. Transverse sections of broad bean stem showing oxidative Fis.. 2.7. proÍono. 32 me"lanisation of v ascular bundles following 2 hr feedingbY M 36 Fig. 3.1. Digestive tract of M. profaø-"' """"""""" 37 Fig.3.2. Salivary glands of M.proÍana"" 40 Fig. 3.3. Salivary glands of C- diluttu 48 Fig. 4.1.