INTERACTIONS BETWEEN SCHISTOCERCA GREGARIA (FORSKAL) AND LOCUSTA MIGRATORIA MIGRATORIOIDES (REICH & FARMAIRE) IN RELATION TO PHASE POLYMORPHISM BY ABDOULAYE NIASSY INTERACTIONS BETWEEN SCHISTOCERCA GREGARIA (FORSKAL)AND LOCUSTA M1GRATORIA MXGRATORIOIDES (REICH & FARMAIRE) IN RELATION TO PHASE POLYMORPHISM A Thesis presented to the Department of Crop Science of the Faculty of Agriculture, University of Ghana, Legon in fulfilment of the requirements for the degree of "‘Doctor of Philosophy in Crop Science (Entomology) BY ABDOULAYE NIASSY B.Sc. (AGRICULTURE), M.Sc. (ENTOMOLOGY) OKLAHOMA STATE UNIVERSITY, STILLWATER OKLAHOMA, USA Crop Science Department, Faculty of Agriculture Univ. of Ghana Legon September, 1996 Q 3527*9 H 51 17-}£S'&S ^O o j v DECLARATION ii I hereby declare that the work herein now submitted as a thesis for the Doctor of Philosophy Degree in crop Science (Entomology) is the result of my own investigations and has not been submitted for a similar degree in any other University. Dr. ICIPE Professor Ahmed Hassanali ICIPE Supervisor Dr. ICIPE Supervisor Professor^jjll. Ayertey University Supervisor Dr. D. Obeng-Ofori University Supervisor DEDICATION To my father, Baboucar Niassy, my mother Fatoumata Badji, my adopted mother, Sire Badji, whose love, care, and encouragement have helped me strive to this level. They all never lived to see me through. I also dedicate this work to all my family members for their patience and endurance while I was away for these studies; to my step mother Seynabou Badji who took care of the family after mother passed away; to my elder brother Mamadou Lamine Niassy, my elder sister Hady Djikoher Niassy, my cousins and friends Daouda Diatta, Estella Djitome Kassa Mane (Diatta's wife), Augustin Niassy, and Saliou Djiba; to my uncles El-Hadj Ibrahima Badji, Djissambou Amanga Niassy, Vincent Niassy; and to all my aunts, especially to Famata Adjouberay Kangouye Niassy and Marie Aloumbeuteu Niassy, for their care and encouragement. iii ABSTRACT iv Cross pheromone-mediating releaser effects between the desert locust, Schistocerca gregaria gregaria, and the African migratory locust, Locusta migratoria migratorioides, were investigated in olfactometer bioassays. These were compared with responses of gregarious individuals of the two locust species to their own air-borne volatiles. Similar to previous reports, nympha1 and adult stages of S. gregaria responded strongly to their own volatiles; immature and mature adults responded to mature adult, but not to nymphal volatiles; nymphs did not respond to mature adult volatiles. The responses of both nymphal and adult stages to their respective volatiles were dose- dependent . In L. migratoria, nymphal and adult stages also responded strongly and in a dose-dependent fashion to their own volatiles. Immature adults responded to volatiles of mature adults, but both immature and mature adults did not respond sufficiently to nymphal volatiles. Nymphs also responded to volatiles of mature adults, but not to those of immature adults. The two locust species cross-responded to each other's volatiles in a dose-dependent fashion. Both nymphal and mature adult stages of S. gregaria were less responsive to the volatile emissions of the corresponding stages of L. migratoria. On the other hand, volatiles from nymphal and mature adults of S. gregaria evoked strong aggregation responses in corresponding nymphal and mature adult stages of L. migratoria. S. gregaria immature adults were more indifferent to volatiles from nymphs of L . migratoria, but immature adults of L. migratoria were actually repelled by conspecific nymphal volatiles; they further responded poorly to volatiles of nymphal S. gregaria. These results confirm previous findings that in S. gregaria, different pheromone systems mediate grouping behaviour in different stages of the locusts. They also suggest that there is an overlap in the pheromone systems mediating grouping behaviour in S. gregaria and L. migratoria. The changes in the phase characteristics (primer effects) of nymphal and adult solitarious desert locusts reared mixed with gregarious migratory locusts and the converse, were investigated. Body colour changes, the number of instars and stage duration, pheromone titres (as measured by the amounts of phenylacetonitrile produced by males), morphometries, and haemolymph pigments composition (as measured by the absorbance ratio at 460 and 680 nm) in test insects were determined. In cage bioassays, significant changes occurred in the phase V characteristics of solitarious nymphs and immature adults of S. gregaria which were reared with gregarious nymphs or immature adults of L. migratoria with respect to all parameters monitored (though at differerent rates). Similarly, solitarious immature adults of L. migratoria which were reared with immature adults of S. gregaria, changed significantly in their phase characteristics. Significant changes in phase characteristics also occurred in solitarious S. gregaria exposed to volatiles of L. migratoria. These findings confirm previous reports that interactions between certain groups of acridids are able to provide the necessary stimuli to initiate locust gregarization (shift from solitarious to gregarious phase). In another experiment, the effects of gregarious fift’n-instar nymphs and mature adults of L. migratoria on the sexual maturation of newly moulted gregarious immature males and females of the desert locust, S. gregaria, and vice-versa, were investigated by monitoring colour changes and copulation in males, and basal oocyte- length in females. Maturation in S. gregaria was signifcantly accelerated by gregarious fifth-instar nymphs of gregarious L. migratoria; mature adults did not produce consistent effects. Fifth-instar nymphs and mature adults of S. gregaria significantly delayed maturation of newly fledged L. migratoria. vi Gas-chromatographic (GC) and GC-mass spectrometric analyses of volatiles of similar stages of S. gregaria and L. migratoria showed quantitative and qualitative differences. In particular phenylacetonitrile was found to be present in the volatiles of nymphal and mature adult L. migratoria migratorioides. The implications of these results are discussed in relation to the behavioural ecologies of the two locust species. vii ACKNOWLEDGEMENTS I wish to acknowledge the following: a) My employer, the "Minister of Agriculture", Senegal, and the "Minister for State Modernisation" for granting me a study leave to concentrate on this project. b) The Director of the ICIPE, Dr. Hans Herren, and the Head of Capacity Building Unit, Dr. V.O. Musewe, for their efforts, in securing me from the International Fund for Agricultural Development (IFAD), a fellowship, thus offering me a valuable opportunity to conduct this important research project towards the scientific level of Ph.D, I have always been striving for. c) My supervisors at the ICIPE: Dr. B. Torto, major Supervisor, for his continuous effort in the follow-up, experimental designs, and in checking the validity of the results. I also thank him for training me in the techniques of analytical chemistry needed in my studies, and for his effort in reviewing my write up; Professor Ahmed Hassanali (Head of the Chemical Ecology Department, Coordinator of the Locust Semiochemical Project, and Deputy Director General of the ICIPE), for his continuous, prompt and pinpointed pieces of advice especially in the overall conceptualization of this research project, and for his pertinent and objective critique of the results; Dr. P.G.N. Njagi, at the viii Chemical Ecology Department, Locust Semiochemical Project, also for his assistance in many of the volatile analyses, and for his valuable critique and contributions in this write-up. d) My University Supervisors: Prof. J.N. Ayertey (former Head, Crop Science Department, Head of the Entomology Laboratory at the University of Ghana, Legon; and ARPPIS Regional Coordinator for West Africa) who followed my progress through valuable discussions, and encouragement; Dr. D .Obeng-Ofori who, when at the ICIPE got me started in the aggregation bioassay experiments, and while at the University followed my work with particular attention. e) Colleagues from the African Regional Postgraduate Programme in Insect Science(ARPPIS), and the technicians at the Chemical Ecology Department. Mr. David Munyinyi, Biomathematician at the ICIPE for his assistance in the massive data analysis. Prof. S. El-Bashir, former Supervisor, and Locust Research Programme Leader at the ICIPE, for his assistance and encouragement. f) My former work supervisor at the Dakar Training Center, Senegal, my friend, Dr. William A. Overholt (Project Leader ICIPE / WAU Project, ICIPE) for being the first to appreciate my qualifications and for his effort to get me into Oklahoma State University in 1980, and for having encouraged me to get into the ARPPIS. ix g) My former Oklahoma State University Supervisor, Prof. Don. C. Peters;|Dr. Walter Knaussenburger, USAID Washington; Prof. Ellis Huddleston, New Mexico State University; Dr. Mbaye Ndoye, Organisation of the African Unity; Dr.Ba Daoule, former Director of the IPM project, Coordinator of the UCTR/PV, and Permanent Secretary of the Sahelian Pesticide Committee (CSP) at the Permanent Inter State Committee for Drought Control in the Sahel (CILSS). All for their encouragement in my undertaking these studies. h) Colleagues from the Environmental Impacts of Locust Control(LOCUSTOX) Project: Dr. Eloi Dieme, Mrs. Jude Andreasen, Dr. James Everts, Mr. Harold Van Der Valk, Mr. Samba Thioub, and Mme Aminata Samb, for their encouragement and assistance; and Colleagues from the National Crop Protection Directorate: Mr. Faustin Diatta (Director), Mr. Seni Dieme(Head of the Quaranteen Division) , Abdoulaye Danfa, Alpha Omar Diallo, Aliou Badji, and Moussa Konate. i) The Senegalese Community in Nairobi: Mr. and Mrs Abdoulaye Sene, Mr. and Mrs. Ibrahima Coly, Dr. Amadou Diaite, Mr. Aladji Diack, Dr. and Mrs. Cheikh Mbacke. j) Dr. Melaku Girma, and all the Ethiopian Community in Nairobi. X xi COPYRIGHT All rights reserved. No part of this thesis may be reproduced, stored in any retrieval system or transmitted in any form or by any means: electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the author or University. TABLE OF CONTENTS TITLE PAGE Title page .................................................1 Declaration................................................ii Dedication................................................ii i Abstract................................................... iv Acknowledgements......................................... viii Copyright................................................... xi Table of contents......................................... xii List of tables.......................................... xviii List of figures........................................... xix List of plates........................................... xxiv CHAPTER ONE 1.1 General introduction................................... 1 1.2 Objectives of the study................................. 6 CHAPTER TWO 2.0 Literature review................................... 9 2.1 Interactions in insects........................... 9 2.1.1 Negative interactions..............................10 2.1.2 Positive interactions..............................15 2.1.3 Neutral interactions............................... 16 2.2 Phase transformation in locusts.................. 17 xii 2.2.1 Interactions with other locust and grasshoppers.19 2.2.2 Environmental conditions......................... 21 2.2.3 Density .......................................... 24 2.2.4 Chemical stimuli...................................2 8 2.2.5 Dietary factors.................................... 31 2.3 Methods of characterizing phase in locusts...... 33 2.3.1 Morphometries...................................... 33 2.3.2 Body colour.........................................35 2.3.3 Eye stripes and shading........................... 36 2.3.4 Haemolymph pigments............................... 37 2.3.5 Pheromones..........................................38 2.4 Developmental life cycle..........................38 2.4.1 Egg development.................................... 40 2.4.2 Nymphal development ............................. 41 2.4.3 Adult sexual maturation...........................42 2.4.4 Rainfall, humidity, and temperature complex 43 2.4.5 Chemical stimuli......... ......................... 44 2.4.6 Interactions between species......................46 2.4.7 Host and nonhost plants........................... 47 2.5 Methods of characterizing sexual maturation 48 2.5.1 Integumental colour......................... 48 2.5.2 Copulation..........................................49 2.5.3 Pheromone emission................................. 49 2.5.4 Length of basal oocytes and oviposition...........49 xiii CHAPTER THREE 3 General materials and methods..................... 52 3.1 Insects.............................................. 52 3.2. Bioassays............................................54 3.2.1 Single chamber olfactometer....................... 54 3.3.2 Cage assays..........................................56 3.3 Collections of volatiles........................... 58 3.4 Analysis of volatiles.............................. 60 3.5 Integumental colour grading........................61 3.6 Statistical analyses................................67 CHAPTER FOUR 4 Aggregation responses of S. gregaria and L. migratoria migratorioides to their airborne volatiles........................................... 68 4.1 Introduction........................................ 68 4.2 Materials and methods..............................69 4.2.1 Olfactometer assays................................ 69 4.3 Data analyses....................................... 72 4.4 Results..............................................74 4.4.1 Responses of 5. gregaria to volatiles of L. migratoria migratorioides......................... 74 4.4.2 Responses of L. m. migratorioides to volatiles of S. gregaria..................................... . xiv 4.4.3 Responses of S. gregaria or L. migratoria to choices of volatiles of both species............ 80 4.4.4. Sex responses to volatiles in L. migratoria migratorioides......................................82 4.4.5. Intra and inter-stage responses in L. migratoria migratorioides...................... 85 4.5 Discussion...........................................87 CHAPTER FIVE 5.0 Primer effects of gregarious population of one species on solitary individuals of the other........................... .................. 92 5.1 Introduction........ 92 5.2 Materials and methods.............................93 5.2.1 Effects of the presence of gregarious L.m migratorioides and two grasshopper species on the gregarization of solitarious S. gregaria........................................... 94 5.2.2 Effects of gregarious S. gregaria on the gregarization of solitarious L. migratoria........................................104 5.2.3 Effects of volatiles of gregarious L. migratoria on the gregarization of solitarious S. gregaria......................... 105 XV 5.3 Data analysis, 106 xvi 5.4 Results.......................................... ■*-07 5.4.1 Effects of the presence of gregarious L. m. migratorioides and two grasshopper species on the gregarization of solitarious S. gregaria .....................................107 5.4.2 Effects of gregarious S. gregaria on the gregarization of solitarious L. migratoria...134 5.4.3 Effects of volatiles of gregarious L. migratoria on the gregarization of solitarious S.gregaria.........................136 5.5 Discussion....................................... 144 CHAPTER SIX ?_* •I 6 Inter-and intra-specific effects of volatiles of S. gregaria and L. m. migratorioides on their sexual maturation times............... 149 6.1 Introduction......................................149 6.2 Materials and methods........................... 151 6.2.1 Effects of gregarious L. m. migratorioides on sexual maturation of gregarious S. gregaria.......................................... 151 6.2.2 Effects of gregarious S. gregaria on sexual maturation of gregarious L. m. migratorioides................................... 154 6.2.3 Data analysis..................................... jV vH O 6.4 Results........................................... 158 6.4.1 Effects of gregarious L. m. migratorioides on sexual maturation of gregarious S. gregaria....................................... 158 6.4.2 Effects of gregarious S. gregaria on sexual maturation of gregarious L.m. migratorioides....................................162 6.5 Discussion........................................ 169 CHAPTER SEVEN 7.1 General Discussion............................... 172 7.2 Recommendations.................................. 181 REFERENCES.............................................182 APPENDICES.............................................2 02 xvii xviii LIST OF TABLES Table 1, Table 2. The E/F and F/C ratios of S. gregaria and L. migratoria (Meinzingen, 1993).. .34 Aggregation responses of immature adults of S. gregaria (SG) and L. migratoria migratorioides (LM) to nymphal and mature adult conspecific and interspecific volatiles in the olfactometer............. .76 Table 3. Responses of S. gregaria (SG) and L. migratoria migratorioides (LM) to volatiles emanating simultaneously from 10 locusts placed in olfactometer choice tests...................................... . 83 Table 4. Sex responses of fifth-instar nymphs and mature adults of L. m. migratorioides to volatiles in the olfactometer.................84 Table 5. Cross-stage aggregation responses of L. m. migratorioides to volatiles in the ol f actometer.....................................8 6 Table 6. Time(days) taken by test S. gregaria(SG) and L. migratoria migratorioides(LM) to reach gregarious colour stage and the corresponding percent shift with respect to colour when reared mixed or exposed to volatiles.......................................Ill Table 7. Developmental time(days) of solitarious S. gregaria(SSG) reared with P. viridipes (PV)„ [ (PV/SSG)] and E. plorans(EP) , [(EP/SSG)] as compared to those from gregarious[(GSG)/GSG)] and solitarious [ (ISSG/cage) ] controls........................ 129 LIST OF FIGURES XXX Figure 1. Figure 2. Figure 3. Figure 4 . Figure 5, Figure 6. Figure 7. Figure 8. Breeding (recession) habitats and invasion areas of S. gregaria and L. migratoria migratorioides...................2 Life cycle of gregarious desert locust and migratory locust(Steedman, 1988) (solitary S. gregaria and sometimes L. migratoria have a sixth- instar) ......................................... 39 Diagrams of standard cages for rearing crowded (A) and isolated locusts (B)..........53 Diagram of the olfactometric aggregation bioassay system................................ 55 Dose-aggregation response curves for fifth-instar nymphs of S. gregaria (FSG) to volatiles of fifth-instar conspecifics[(FSG/FSG)] and to those of L. migratoria(FLM),[(FSG/FLM)] in the olfactometer (bars are SE).................... 75 Dose-aggregation response curves for mature adults of S. gregaria (ASG) to volatiles of mature adult conspecifics[(ASG/ASG)] and to those of L. migratoria(ALM),[(ASG/ALM)] in the olfactometer (bars are SE)............78 Dose-aggregation response curves for fifth-instar nymphs of L. migratoria (FLM) to volatiles of fifth-instar conspecifics[(FLM/FLM)] and to those of S. gregaria (FSG),[(FLM/FSG)] in the olfactometer(bars are SE).... .79 Dose-aggregation response curves for mature adults of L. migratoria (ALM) to volatiles of mature adult conspecifics[(ALM/ALM)] and to those of S. gregaria(ASG), [(ALM/ASG)] in the olfactometer (bars are SE).......................... Figure 9. Black colour pattern used in grading nymphal colour changes in test S. gregaria......................... 98 XX Figure 10. The standard morphometric chart for locust phase determination............... 101 Figure 11. Developmental times of solitarious test S. gregaria[SSG) reared with gregarious L. migratoria(GLM) , [ (SSG/GLM)] compared to gregarious [(GSG)] and solitarious[(SSG)] controls................................... 113 Figure 12. Nymphal instar and stage durations of solitarious test S. gregaria(SSG) reared with gregarious L. migratori a (GLM) , [(SSG/GLM)] and that of those which it was reared with [(GLM)]................... 114 Figure 13. Adult E/F and F/C ratios of solitarious S. gregaria(SSG) reared with gregarious L. migratoria{GLM) from nymphal stage [(SSG/GLM)] compared to gregarious[(GSG)] and solitarious controls [ (SSG) ]......... 116 Figure 14. Haemo lymph pigment ratios (at 460 over that at 680 nm wavelength) of test S. gregaria[(SSG/GLM)] compared to gregarious[(GSG)] and solitary[(SSG)] controls................................... 118 Figure 15. Gas chromatograms showing the production of phenylacetonitrile (PAN) in adult males of S. gregaria reared with gregarious L. migratoria at 10-12, 15-20, 25-30, and 35-40 days after fledging (30m, 0.2mm ID methyl silicone column) ........ 119 Figure 16. Gas chromatograms showing peaks of phenylacetonitrile (PAN) produced by gregarious locusts from control cages at 10-12, 15-20, 25-30, and 35-40 days after fledging (30m, 0.2mm ID methyl silicone column)........................... 120 Figure 17. Phenylacetonitrile titres (pg) produced by test S. gregaria(SSG) when reared with gregarious L. migratoria(GLM)at nymphal stage[(SSG/GLM)] and immature adult [(ISG/GLM)] as compared to titres of conspecifics from gregarious control [ (GSG) ]..............................122 xxi Figure Figure Figure Figure Figure Figure Figure 18. Gas chromatograms showing the production of phenylacetonitrile (PAN) in gregarious mature adults of L. migratoria at 10-12, 15-17, 20-22, and 25-27 days after fledging (30m, 0.2mm ID SPB-1 methyl silicone column)........................... 123 19. Gas chromatograms showing A) peaks of phenylacetonitrile (PAN) produced by S. gregaria reared from immature solitary adult stage with gregarious adults of L. migratoria ; B) produced by L. migratoria reared from immature solitary adult stage with gregarious adults of S. gregaria; and Oproduced by adults of S. gregaria reared from immature solitary adult stage with fifth-instar nymphs of L. migratoria(50m, 0.2mm ID Carbowax column)...................126 20. Adult E/F and F/C ratios of solitarious S. gregaria (SSG) reared with P. viridipes (PV) at nymphal stage[(SSG/PV)] compared to gregarious[(GSG)] and solitarious[(SSG)] controls......................................131 21. Adult E/F and F/C ratios of solitarious test S. gregaria (SSG) reared with E. plorans(EP) at nymphal stage[(SSG/EP)] compared to gregarious[(GSG)] and solitarious [ (SSG) ] controls................ 133 22. Production(in pg) of phenylacetonitrile by solitary immature L. migratoria(ILM) when reared with gregarious immature adults of S. gregaria(ISG) ,[(ILM/ISG)] compared to those reared with gregarious conspecific controls [ (ILM/ILM) ]............135 23. Nymphal instar and stage duration of test solitarious S. gregaria[(SSG)) exposed to volatiles of gregarious source L. migratoria[(GLM)] from nymphal stage stage [ (SSG:GLM) ]............. 137 24. Nymphal instar and stage durations of solitarious test S. gregaria(SSG) exposed to volatiles of gregarious L. migratoria (GLM),[(SSG:GLM)] from nymphal stage compared to source gregarious L. migratoria [(GLM)]..........................139 xxii Figure Figure Figure Figure Figure Figure 25. Nymphal instar and stage duration of test solitary S. gregaria(SSG) reared with gregarious L. migratoria (GLM),[(SSG/GLM)] or exposed to its volatiles [ (SSG: GLM) ]........................ 140 26. E/F and F/C ratios of solitary S. gregaria(SSG) exposed to volatiles of gregarious L. migratoria(GLM) from nymphal stage [ (SSG:GLM) ]....................141 27. Stage durations of solitarious S. gregaria(SSG) a) reared with[(SSG/GLM)] or b) exposed to volatiles of [(SSG:GLM)] gregarious L. migratoria(GLM) c)reared with P. viridipes (PV) ,[ (SSG/PV) ] and d) reared with E. plorans(EP) , [(SSG/EP)] compared to gregarious conspecifics from the control [ (GSG) ].....................143 28. Time (days) taken by maturing adult males of S. gregaria (ISG) to attain stage colour III when exposed to volatiles of live conspecific fift'n-instar nymphs and mature adults[(ISG/SG)]; conspecific immature adults[(ISG/ISG)] ; and to those of similar stages of L. migratoria [ (ISG/LM) ]................. 159 29. Amounts(pg) of phenylacetonitrile produced by maturing males of S. gregaria(ISG) exposed to volatiles from live conspecific fifth-instar nymphs(FSG) , [(ISG/FSG)]; immature adults[(ISG/ISG)] and mature adults (MSG) , [(ISG/MSG)] ; and to those of fifth-instar(FLM),[ (ISG/FLM)] and mature adults(MLM), [ (ISG/MLM)] of L. migratoria................................ 160 30. Time taken by maturing adult females of S. gregaria(ISG) to attain mature oocyte length when exposed to volatiles of conspecific(SG) fifth-instar nymphs and mature adults[ (ISG/SG)]; conspecific immature adults[(ISG/ISG)]; and to those of similar stages of L. migratoria(LM), [(ISG/LM) ].................................. 163 xxiii Figure 31. Time(days) taken by maturing adult males of L. migratoria(ILM) to attain stage colour III when exposed to volatiles of live conspecific(LM) fifth-instar nymphs and mature adults, [(ILM/LM)]; conspecific immature adults,[(ILM/ILM)]; and to those of similar stages of S. gregaria(SG) , [(ILM/SG)].............................. 164 Figure 32. Amounts(pg) of phenylacetonitrile produced by maturing males of L. migratoria(ILM) exposed to volatiles Of live conspecific fifth-instar nymphs(FLM),[(ILM/FLM)] immature adults [(ILM/ILM)], and mature adults(MLM) [(ILM/MLM)]; and to those of fifth-instar (FSG),[(ILM/FSG)] and mature adults(MSG), [(ILM/MSG)] of S. gregaria...................165 Figure 33. Time taken by maturing adult females of L. migratoria(ILM) to attain mature oocyte length when exposed to volatiles of conspecific(LM) fifth-instar and mature adults,[(ILM/LM)]; conspecific immature adults[(ILM/ILM)]; and to those of similar stages of S. gregaria (SG) , [(ILM/ISG)]....................................167 xxiv LIST OF PLATES A. Two chamber bioassay cages, a) source chamber, b) recipient (test) chamber................. 57 B. Volatile collection system(B-l) showing connected to it a)three-neck round bottomed flask containing mature S. gregaria b)quick- fit trapping chambers, and the medical air source cylinders; quickfit chambers(c) with connected charcoal traps(d) (B—2)....................59 C. Immature adults of S. gregaria(C-l) and L. migratoria(C-2)(note the pink and blackish body colours of the respective insects)............ 62 D. Gregarious(D—1) and solitarious(D-2) mature adults of S. gregaria................................ 63 E. Gregarious(E—1) and solitarious(E-2) adults of L. migratoria migratorioides.....................64 F. Gregarious fifth-instar(F-l) and solitarious fourth-instar(F-2) nymphs of L. migratoria migratorioides........................................ 65 G. Olfactometer tests showing locusts (1,2,..) orienting themselves towards or sitting on the treated side of the arena....................... 73 H. One chamber bioassay cage used for mixed rearing experiments and described in Chapter III, and showing a)glass door, b)egg laying tube insertion holes.......................................95 I. Gregarious fifth-instar and fourth-instar nymphs of S. gregaria from gregarious (I — 1) and solitarious(1-2) control cages respectively.......................................... 99 J. Solitarious S. gregaria{A) reared with gregarious L . migratoria migratorioides(B)....... 109 K. Adult male S. gregaria{A) reared with crowded L. migratoria(B) and showing yellow colour typical of mature gregarious locust............... n o L. Solitarious S. gregaria(A) reared with P. viridipes(B)(L-l), and E. plorans(B) (L-2) from nymphal stage............................ 128 CHAPTER ONE 1 General Introduction Locusts are short horned grasshoppers, a large group in the order Orthoptera. There are six major locust species in Africa: - the desert locust, Schistocerca gregaria gregaria (F), - the African migratory locust, Locusta migratoria migratorioides (R & F), - the Red locust, Nomadacris septemfasciata (Serville), - the Brown locust, Locvstana pardalina (Walker), - the Moroccan locust, Dociostaurus maroccanus (Thur.berg! , and - the Tree locust, Anacridium melanorhodon(Walker) The first four species are major agricultural pests in Africa, but the desert locust is regarded as the single most serious pest due to its polyphagous feeding behaviour, and its migratory habits (Popov et al. 1 9 9 1 ; Steedman, 1988; Meinzingen, 1993). While the desert locust is widely distributed in Africa, the Middle-East, and parts of Southwest Asia, the African migratory locust is restricted to Africa where it breeds in three ecological zones including the Sahelian, Sudanese and Guinean zones(Zolotarevsky, 1938; Showier, 1995) (Fig.1) . 1 2Figure 1. Breeding (recession) habitats and invasion areas of S. gregaria and L. migratoria migratorioides S. gregaria and L. migratoria both show density dependent phase polymorphism which refers to the ability of these insects to exist reversibly, depending on density, in two extreme forms or phases: solitaria and gregaria (Uvarov, 1921; 1923), with intermediate phases known as "transiens". The two locust phases differ in behavioural, morphological, and biochemical features, and in fecundity (Uvarov, 1921; 1923) . Between plagues, the two locust species live as solitary, scattered (less than 100 individuals/ha) , and harmless individuals in locust affected countries, in areas commonly known as "recession areas" (Fig. 1). When weather conditions(rainfall) become suitable, these individuals concentrate to breed successfully into large numbers, and become gregarious (Roffey and Popov, 1968) . Gregarious hoppers do not have developped wings to fly, but they march in dense groups. Gregarious adult stages, however are winged, and therefore, they can swarm over long distances (Descamps, 1961; Duranton and Lecoq, 1990; Steedman, 1988; and Meinzingen, 1993). A typical swarm of S. gregaria can be of 300 km2 in size containing about 40 million individuals, and can travel up to 1000 km a week (Steedman, 1988; Meinzingen, 1993). Swarms of the African migratory locusts are smaller, seldom of similar size, and are usually restricted to their breeding areas (Steedman, 1988). 3 Locust hopper bands and swarms are very voracious and are, therefore, very destructive. Swarms of the desert locust can cause losses of thousands of tons of crop such as cereals, and vegetables. Meinzingen(1993) reported that damage caused by the desert locust stems from the fact that a locust consumes 1.5 - 3 g of vegetation daily, the equivalent of its own weight. Thus a medium density swarm of 50 million individuals per km2 can consume 100 tons, and a swarm of 1000 Km; can eat 100.000 tons, enough to feed 500,000 people a year (Meinzingen, 1993). However, damage varies with the locust stage and as Meinzingen (1993) had indicated, 8% of the total damage by locusts is due to hoppers, 69% to immature and 23% to mature adults. Direct yield losses from damage due to the desert locust during the plagues of 1954 to 1958, were estimated to vary from 15 million US Dollars in Morocco, 55,000 tons of cereals in Sudan, 16.000 tons of millet in Senegal, and 167,000 tons of cereals in Ethiopia (Steedman, 1988; Popov et al., 1991; Meinzingen, 1993). About 76,000 to 900,000 tons of sorghum were lost in the Sahelian Africa during the desert locust plagues from 1986 to 1993, with an estimated maximum cost of control in 1988 of about 102.7 million US Dollars (Herok and Krall, 1995). The gregarious migratory locust is also polyphagous, but to a lesser extent compared to the desert locust, and 4 is restricted to graminaceous wild and cultivated plants such as millet, sorghum, maize, rice, sugar cane, and bamboo (Meinzingen, 1993) . No yield loss assessment exists for this species. Locust control mainly relies on the use of pesticides. Despite the fact that large scale locust control involves intensive surveys and mapping in order to optimize pesticide use and reduce environmental degradation, the risk of casualities and extensive contamination still exist in locust affected countries (Everts, 1990), because of the fragile ecosystems due to recurrent droughts. Alternative methods including monitoring locust breeding zones for rains and hatchings using remote sensing techniques, the development of gregarization models (Popov et al., 1991), and the use of biological control agents such as entomopathogenic fungi and protozoans (Lomer and Prior, 1992; Thomas et al., 1995), have been proposed. More recently, an integrated pest management approach (El-Bashir, 1991; Joffe, 1995) has been proposed using different, and environmentally sound, packages. It is in this context that the International Center of Insect Physiology and Ecology (ICIPE), launched the Desert Locust Semiochemical Project in 1990 to investigate the semiochemicals that mediate aggregation behaviour, sexual maturation, oviposition, and gregarization of this insect. By the end of 1993, 5 significant progress had been made by this project in the characterization of these pheromones, and the mechanisms underlying some aspects of phase transformation. However, the project did not include investigations on the role played by other locust species and grasshoppers on the phase dynamics in the desert locust. To fill this gap, the present study was designed to investigate the interactions between the desert locust, S. gregaria and the African migratory locust, L. migratoria migratotioides with regard to releaser effects of semiochemicals, and primer effects of all the phase mediating factors. 2 Objectives 2.1 General objectives The main objective of this work is to establish whether there are interactions between S. gregaria and L. migratoria, and to investigate releaser effects of semiochemicals and primer effects of the interactions (all phase mediationg factors) on both locust species. 6 2.2 Specific objectives To investigate : 1) phase associated releaser effects between and within stages of S. gregsria and L. migratoria; 2) phase associated primer effects of crowded populations of one species on isolated individuals of the other species in relation to: a) the rate of phase transformation in solitarious individuals of the two locust species; b) the influence of different stages of crowded locusts on phase change; c) the role played by volatiles in these interactions; and 3) cross-effects of crowded live locusts of one species on the rate of sexual maturation of the other. The procedures used to achieve these objectives are presented in this thesis which is divided into seven chapters. The first chapter contains the general introduction, the second, the review of literature. A general description of the methods and materials used in experimentation follows. Chapters four, five and six deal with studies on locust aggregation (releaser effects), gregarization (primer effects), and maturation 7 (primer effects), respectively. The last chapter contains two sections comprising a general discussion of the results, and recommendations for future studies. A list of references, and appendices are also provided at the end. 8 CHAPTER TWO 2 Literature review 2.1 Interactions in insects Insect species may exhibit diverse types of interactions (Odum, 1971; Price, 1984) . Odum (1971) reported that between any two species there could be as many as nine types of interactions including neutralism (neither of the interacting species is benefited), competition (direct interference, and resource use), amensalism (one is inhibited, the other not affected), parasitism, predation, commensalism, protocooperation (non obligatory, but beneficial for both species), and mutualism (both species benefit from their interactions and have become totally dependent on each other for their survival) . He added that these can be grouped into negative types of interactions such as predation, parasitism, antibiosis, and interspecific competition; and positive types of interactions including commensalism, cooperation and mutualism (Odum, 1971). To this could be added neutral interactions. Saini and Hassanali (1991) pointed out that in any multitrophic network, a large number of theoretical combinations are possible. For example, between any two levels, six 9 theoretical categories of interactions are possible depending upon the benefit or harm associated with the signal emitted by a member of interacting pairs (Saini and Hassanali, 1991). Due to the complexity of these ecological relationships, this review is focussed only on examples of recent studies on one and two stage negative, positive and neutral types of interactions. 10 interactions between two populations which result in negative effects of growth and survival of one of the interacting populations. Where the two populations have had a common evolutionary history in relatively stable ecosystem, the negative effects tend to be relatively small. Thus natural selection tends to ultimately reduce the detrimental effects and the interactions altogether (Odum, 1971). Severe negative interactions, when coupled with drastic changes in the ecosystem may lead to the extinction of one species and the resurgence of the other. This is probably how pest epidemics (i.e locust or grasshopper invasions) are born, especially when natural ennemies of a species are affected. Predator or parasitoid-prey relationships are the most widely studied interactions because they are of special interest and provide an understanding of the mechanisms of host location by pest and predators and/or parasitoids (Saini and Hassanali, 1991). According to Price (1984) , this type is the central issue in understanding both the adaptative strategies for getting food and for avoidance of becoming food. While a predator consumes more than one prey for its survival, a parasitoid survives on a single prey (Price, 1984). Models of predator and/or parasitoid-prey interactions which are developed independently by different authors are used in ecological studies especially in those related to biological control (Price, 1984) . There are as many as three levels of interactions of this type: between the natural enemy species and the host species (carnivore-herbivore); between species of natural enemies (carnivore-carnivore); and between species or host plants, natural enemies, and the herbivore species (plant-carnivore-herbivore). Carnivore-herbivore The example reported by Ngi-Song et al.(1995), in which they determined the suitability of the herbivore as food for the carnivore helps to illustrate this level of interaction. These workers found that the exotic Cotesia flavipes Cameron preferentially oviposited on larvae of the pyralid species Chilo partellus Strand, Chilo 11 orichalcociliellus Strand, Busseola fusca ('Fuller), and Sesamia calamistis (Hampson). However, the local endoparasitoid, Cotesia sesamiae Cameron, preferred S. calamistis, probably because of some evolutionary adaptation whereby this parasitoid has learned to use this lepidopteran species as a host. They also found that both Chilo species and S. calamistis were suitable for development of C. flavipes with Chilo being the preferred hosts, and that C. sesamiae preferred 5. calamistis to the two Chilo species. B. fusca was not a suitable host for the two parasitoids because of its ability to encapsulate parasitoid eggs in the haemolymph, a form of evolutionary resistance by B. fusca to parasitization by this wasp. Carnivore-carnivore This level of interaction is well illustrated by the work of Heinz and Nelson (1996) who studied the interactions between the natural enemies of the whitefly Bemisia argentifolii (Grenadius) (ex B. tabaci) , and found that there were no detrimental competitive interactions among the three species of natural enemies studied which would have negatively affected the level of predation and/or parasitism. 12 Herbivore-herbivore 13 Coexistence among insects involves spatial interactions which could be viewed to some extent as competition for space resource. The interacting species may learn to coexist for ever or will show competitive periodic displacement (Odum, 1971). This aspect had been studied by Brown et al.(1995) on two aphid species on apple, and found that in such an ecosystem, the Spirea aphid, Aphis spirascola Patch and the apple aphid, Aphis pomi De Geer, were similar in their distribution and abundance in late summer when apple is a less suitable host. No significant positive and negative interactions occurred between the two species. A. spirascola reproduced faster to yield more generations through spring to the point that, by the end of the summer, it was the only species present. PI ant-ca m i vore-herbi vore Recent work on this tritrophic level of interactions include the comprehensive studies by Wiedenmann et al. (1992) involving stemborers such as Diatrea saccharalis Dyar, their host plants, and parasitoids such as Cotesia flavipes. These studies have revealed some interesting aspects of these interactions especially with regard not only to the role played by the plant in host location by parasitoids, but also the suitability of the host for survival of the parasitoids. Abundance of the natural enemies may be favoured by host plant density. Brust et al.(1986) showed that the activity of four most important Carabid predator species of the genera Pterostichus, Amphasia, Pterosticus, Ambacidus, and Harpalus was more important in no tillage than in tillage corn ecosystem. Thus in no tillage system host plants are more abundant favouring rapid build-up of pest species and consequently, higher populations of their natural enemies. Perfecto et a1. (1996) studied the effects of plant diversity and density, on the migration of two ground beetles of the genera Harpalus and Evarthrus. They found that the emigration rate was higher at low plant density, with negative interactions between the insect migration, plant density, and plant diversity. Thus, emigration was higher in dense polyculture than in dense monoculture. Rupasas et al. (1996) studied in a 4-armed air flow olfactometer the chemical attraction of fourteen Oryza sativa Savannah cultivars and one wild Oryza species, to the mirid and coccinellid predators of the genera Cyrtorhinus, and Micraspis, respectively, of the brown planthopper, Nilaparvata sp. They found that odours from the plant attracted significantly Cyrtorhinus species. 14 The host plant can, in many forms, resist attack by herbivores, thus enhancing the actions of carnivores. The effects of plant resistance and natural enemies on sawfly were compared by Stein and Price (1995), who found a strong relationship between oviposition preference and larval performance of two tenthredinid sawflies, Euura sp., and Pontiana sp. Plant resistance to attacks, in addition to natural enemies, played an important role in the reduction of the sawfly populations. 2.1.2 Positive interactions Positive association between two species populations are widespread in nature. Positive interactions may be considered as having evolved from commensalism to protocooperation and to mutualism (Odum, 1971). Thus mutualism is the ultimate type of positive interaction, and is discussed in this Section. Symbiotic or mutualistic trophic interactions occur between various species of insects. For example, aphids excrete honeydew consisting mainly of excess ingested sap which is a favourite food for ants (Borror et al., 1976). The ants in turn protect the aphids against predation. Termites, Nasutitermes corniger (Motschulsky) obtain nitrogen-rich nutrients and additional nest defence from various species of ants which shelter in their nests during the 15 wet season (Jaffe et al., 1995). The ants also prey on some live and dead termite workers. Other forms of somewhat positive trophic interactions include facultative carnivory and/or cannibalism among species, e.g., orthopterans such as Chrotogonus spp., Aulacara spp., Eyprepocnemis spp., and S. gregaria (Duranton and Lecoq, 1990; Whitman et al., 1994). In S. gregaria, and L. migratoria migratorioides, cannibalism is common among fifth-instar nymphs and adults, in Cataloipus cymbiferus Krauss, and Aulacara spp., it is common among adults. It has been suggested that this provides a source of necessary proteins to insects that feed on protein-deficient plants (Whitman et al., 1994). This feeding habit has also been documented in many other insect species of the orders Isoptera and Heteroptera (Whitman et al., 1994). 2.1.3 Neutral interactions Neutral interactions occur when neither of the interacting species are benefited. However, there may never be total neutral interactions unless species are geographically far apart. There are situations in which non related species (in habit and niche) are forced to coexist and with time tend to share some characteristics of survival value such as colour mimicry or semiochemical 16 signals, without directly affecting each other's life. Such types of interactions are common in grasshoppers, and solitarious or gregarious locusts coexisting in certain breeding areas {Johnston and Buxton, 1949; Steedman, 1988; Showier, 1995). Johnston and Buxton (1949) reported that mixed bands of S. gregaria and L. migratoria migratorioides, in the Red Sea coastal breeding zones, exhibited marching behaviour characteristic of their gregarious phase. It was not known whether there was change in colour and morphometries associated with phase change, nor whethe they might share the same chemical stimuli. El-3ashir and Abdel-Rahman (1991) later reported some changes in colour and morphometries in mixed bands of the species These are discussed in more detail in the following Sections. 2.2 Phase transformation Phase transformation refers to the ability of locusts to exist reversibly in two extreme phases: solitarious and gregarious. The forms between the two extremes are known as transiens (Uvarov, 1921; 1923). Uvarov (1921; 1923) pointed out that all gregarious acridids belong to polymorphic species, and are true locusts. However, some grasshoppers, e.g., Oedaleus senegalensis (Krauss), and Gastrimargus africanus Saussure show colour polymorphism and marching behaviour like locusts, but do not swarm nor transform into different phases (Rowell, 1970; Launois and Launois- Luong, 1989). In the family Pyrgomorphidae, some species in the genera Zonocerus, Phymateus, and Poikylocerus, produce aggregation and defence pheromones (Whitman, 1990). However, they do not exhibit reversible phase change, nor form bands and adult swarms like locusts. Phase transformation is an important trait of locusts which facilitates their survival through migration and recolonization of old habitats and colonization of new areas. It is affected by many factors including, environmental (Steedman, 1988; Popov et al., 1991; Zolotarevsky, 1938), density (Pasquier, 1950; Duranton et Lecoq, 1990, Popov et al., 1991; Deng et al., 1996), chemical stimuli (Nolte, 1963; Gillett, 1975a; Obeng Ofori et al, 1993; Torto et al. 1994; Deng et al., 1996), diet (Jackson et al., 1978), and host and nonhost plants (Steedman, 1988) . Interactions with other locusts have also been implicated (Johnston and Buxton, 1949; El Bashir and Abdel-Rahman, 1991). 18 2.2.1 Interactions with other locusts and grasshoppers The coexistence of locusts and grasshoppers may be regarded as a neutral type of interaction whereas those between locusts are either mutual inhibitory competition or protocooperation (in accordance with the classification by Odum (1971) . Experiments under laboratory conditions have demonstrated that locusts affect phase polymorphism and development of one another when they are crowded in cages while grasshoppers do not (Gillett, 1968; Ba-Angood, 1976). Gillett (1968) studied the grouping behaviour of the desert locust, S. gregaria, reared singly with a non-swarming grasshopper species, Humbe tenuicornis (Sc’naum) . Isolated locusts and those reared crowded with H. tenuicornis were unable to form groups like gregarious populations. She concluded that the grasshopper had no significant effect on the phase status of the desert locust. In a similar experiment (Ba-Angood, 1976), crowded individuals of S. gregaria, and individuals of S. gregaria kept among groups of three grasshopper species, Kraussaria spp.r Oedaleus spp., and Aiolopus spp., had no significant effect on phase characters such as the number of eye stripes and morphometries. On the other hand, locust species have been reported to affect phase polymorphism and development of one another when they 19 occur crowded in the same ecological zones in the field (El-Bashir and Abdel-Rahman, 1991). Johnston and Buxton (1949) documented the occurrence of mixed populations of S. gregaria and L. migratoria migratorioides whose adults laid eggs together in some breeding areas in the Sudan. They further noted that, within the mixed hopper bands, hatchlings of the two locust species could also be differentiated by their host plant species, and in addition, hatchlings of L. m. migratorioides were better swimmers than those of S. gregaria. Although effect of the presence of either locust species on the phase characteristics of the other was not investigated, their observations suggest some mutual interactions in addition to overlap of the ecological niches of the two locust species. Gillett (1983) investigated the effects of the fecal volatiles of either L. migratoria and S. gregaria on each other's gregarization, and found that faeces of the latter affected the melanization and behaviour of the former lending support to Nolte's (1973) observations that these two species produce a common aggregation pheromone (locustol) . However, the faeces of L. migratoria did not seem to produce the same effects (Gillett, 1983). El-Bashir and Abdel-Rahman (1991) reported observations of mixed populations of the desert locust and the migratory locust in the Sudan. They observed 20 that solitarious hoppers of the desert locust which interacted with dense populations of gregarious stages of the migratory locust tended to change into transiens. There were increased encounters between hoppers of both species as they marched, roosted, and sheltered under Zygophyllum simplex L. They fed together near water courses where the vegetation was greener and soil moisture was conducive to egg-laying. They concluded that the prevailing edaphic factors appeared to have stimulated marching of mixed bands, colour and morphological changes of solitarious S. gregaria towards the transient phase. Such observations were confirmed by Torto in 1995 (personnal communication) in the locust breeding areas along the Red Sea coast in the Sudan. The observations by these authors did not involve analyses of parameters such as locust body colour, morphometries, marching behaviour, number of eye stripes, and pheromone production. Therefore, detailed studies on the interactions between the desert locust and the migratory locust are lacking. 2.2.2 Environmental conditions Environmental factors, especially rainfall play a significant role in the phase transformation of both the desert locust and the African migratory locust 21 (Zolotarevsky, 1938; Ellis and Carlisle, 1965; Carlisle and Ellis, 1965; Popov et al., 1991). Adequate rainfall supports the growth of annual plants and shrubs which when fed on by immature solitary adults hasten their sexual maturation. This is followed by congregation of individuals at suitable sites for mating and egg laying. Uvarov (1966), Rowell (1970), and Fuzeau-Braesch (1972) have shown that in locusts, environmentally regulated phenotypic colour polymorphism such as homochromy, green brown polymorphism, and density dependent polymorphism may be found. Faure (1942), Zolotarevsky (1938), and Albrecht (1964) showed that solitary L. migratoria were green under high humidity, but in low humidity they were near brown in colour. Furthermore, they observed that under low humidity, solitary hoppers adjusted their colour from whitish-cream, yellow, brown, grey, or black to match the colour of the background environment they were in. Zolotarevsky (1938) studied the effects of humidity on the ratios of lengths of the elytron to the femur (E/F), and the length of the femur to the width of the head capsule (F/C) of the migratory locust. He found that dry conditions (RH 5 45%) tended to favour gregarious characters as compared to wet conditions (RH £ 70%) . Increasing humidity coupled with increasing daylength and temperature favoured solitarious 22 morphometric ratios in L. migratoria (Albrecht and Lauga, 1978; 1979). Temperature has an effect on nymphal displacement behaviour in L. migratoria (Descamps, 1961). When temperatures are high (above 30”C) nymphs tend to find shelter by hiding under shrubs, trees or roost on top of grasses (Descamps, 1961: Steedman, 1988). At very low temperatures (below 26°C), they tend to find cover under dead leaves, and shrubs. This facilitates grouping of solitarious nymphs especially in patchy vegetation, and may lead to gregarization of nymphs if they are in contact with each other for sufficiently long periods Oouaichi et al., 1995). Colour patterns of locust cuticle also vary with temperature. Field and laboratory observations (Stower, 1959; Dudley, 1964), showed that at low temperatures the black patterns were predominant on locust hoppers while at high temperatures they were fewer. Morphometries of adults were found to change significantly with temperature. At a temperature of about 30CC and relative humidity of about 90%, locusts tended to shift toward gregarious E/F ratios. On the other hand, temperatures greater than 30°C, and at the same relative humidity of 90%, E/F ratios of individuals were characteristic of solitarious phase. F/C ratios only changed at relatively lower humidity (50%) . Stower (1959), and Meinzingen (1993) showed that at high 23 temperature (above 30°C) gregarious desert locusts showed morphometries which were similar to those of solitarious locusts reared at temperatures below 30°C. Thus there is ecological relative fitness of locusts to their environment as manifested by the changes in morphometric and colour, in relation to changing condtions. Habitat patchiness also plays an important role in phase dynamics, especially during the dry season when host plants are found in patches throughout the habitat. It is more evident in the case of the migratory locust in the breeding areas of the Niger (Lean, 1931; Zolotarevsky, 1938; Steedman, 1988) where individuals are forced to concentrate on green patches of vegetation resulting from previous floods. Grouping of solitary adults is then facilitated, and if conditions are conducive, synchronous egg-laying occurs leading to synchronous egg development and hatching (Roffey and Popov, 1968) . 2.2.3 Density Gregarization is mainly a density-driven process (Pasquier, 1950). Under favourable environmental conditions (e.g. sufficient rainfall and vegetation) adult locusts mature faster, copulate, and gravid females concentrate at suitable sites for egg-laying. The 24 attraction of such females to egg laying sites is favoured by adequate soil moisture and froth volatiles from deposited eggs (Saini et al., 1995). Gregarized females lay eggs which yield gregarious nymphs, a phenomenon known as "maternal inheritance" (Hunter-Jones 1958; Injeyan and Tobe, 1981; Bouaichi et al., 1995; Islam et al., 1995). Furthermore, grouping of nymphs that hatched together favoured prolonged contact and emission of aggregation pheromones (Deng et al., 1996), which could keep nymphal groups together. In addition, at high densities tactile, visual, olfactory, and acoustic interactions play a role in maintaining the gregarious phase. Nymphs hatching together form small cohesive groups at the early stage, feed from plant to plant, and keep merging with other small groups in their paths into larger groups (Steedman, 1988; Meinzingen, 1993). This progressive increase in density was referred to by Pasquier (1950) as "densation". Roffey and Popov (1968) and Duranton and Lecoq (1990) described three stages (multiplication, concentration, and gregarization! in the process leading to the build up of locust populations in the field, and which also influence phase shifts. They observed that in the Tamesna mountains breeding area in the Niger, the multiplication factor (ratio between parental and actual density) was estimated at 16 - 20 fold. Popov et al. (1991) suggested 25 that the actual multiplication factor in such situations could be about 100 - 200 fold. Population build up could also be facilitated by active immigration, e.g. through attraction (Pedgley, 1981), or passive immigration e.g. by random effects of wind (Roffey, 1969; Popov, 1969; Shaeffer, 1972). Hunter-Jones (1958) studied the inheritance of phase characters in hoppers and adults of L. migratoria, and S. gregaria. By monitoring hopper colour patterns, weight of hatchlings, number of nymphal instars, and adult colour and morphometries, he found that hoppers reared in isolation appeared green similar to solitarious locusts. Morphometric ratios (E/F and F/C) also corresponded to those of solitarious insects. He correlated weights of hoppers of S. gregaria with colour or adult morphometries and found that, the green hatchlings (solitary) had relatively higher morphometries and lower weight compared to their black and yellow counterparts (gregarious). Nolte (1973) showed that crowding at high densities in the field and in the laboratory increased melanin deposition, chiasma frequencies in L. pardalina, and L. migratoria while transient morphometries occurred in S. gregaria. Crowding of the grasshopper Paracinema tricolor Thunberg had no effect on chiasma frequency (Nolte, 1973). Deng et al. (1996) studied the effects of shifting S. gregaria from crowded to isolated conditions and vice- 26 versa on the emissions of adult aggregation pheromones in the laboratory, and compared this with changes in morphometries. They demonstrated the extreme sensitivity of the desert locust to crowding, such that even pairing two solitarious locusts provided each other with the necessary stimuli to cause their gregarization. Further, they showed that adult males of the parental generation resulting from shifting crowd-reared hoppers, immature or mature adults to isolated conditions did not produce phenylacetonitrile similar to solitary-reared adults. Conversely, adults of the parental generation resulting from shifting solitary-reared hoppers, fledglings, or mature adults to crowded conditions produced pheromone at levels which were similar to those of control adults from the crowd-reared colony. In contrast, they showed that morphometric changes were slow and required several generations to show significant variations, confirming earlier findings by Chapman (1979) . Also, the F/C ratio (length of the femur to the width of the head capsule) was shown to be more sensitive to treatment effects than the E/F ratio (length of the elytron to the length of the femur). 27 2.2,4 Chemical stimuli Nolte (1963) was the first researcher to demonstrate the mediation of a chemical stimulus in the gregarization of locusts. He reported the presence of a chemical factor in the air-borne volatiles of crowded locusts which acted as a "primer" pheromone facilitating the grouping of locusts reared in isolation. Gillett (1968) confirmed the existence of such a chemical stimulus in the air­ borne volatiles of crowded locusts. Nolte referred to this as a "gregarization pheromone" which was later identified as 5-ethyl guaiacol in the nymphal faeces and was called "Locustol" (Nolte, 1970). Locustol evoked such gregarious characteristics in crowded locust populations as increase in chiasma frequency, melanin pigmentation, and changes in adult morphometric ratios towards those of phase gregaria. More detailed studies by Nolte (1973) suggested that three locust species, S. gregaria, L. migratoria, and L. pardalina produced, and utilized locustol for gregarization. The red locust, Nomadacris septemfasciata (Serv), the Australian locust, Chortoicetes terminifera Walk., and a grasshopper Paracinema tricolor, did not demonstrate evidence of the production of this pheromone (Nolte, 1973) . Studies by Gillett (1975) showed that the aggregation pheromone mediated grouping behaviour in 28 gregarious nymphs and it was not produced by isolated nymphs in quantities that could alter each other's behaviour. She discussed two sets of pheromones, a gregarizing pheromone which emanated from faeces of crowded nymphs, and a solitarizing pheromone, only detectable in the faeces of crowded adults (Gillett and Phillips, 1977). A more detailed comparative study on the pheromonal systems of S. gregaria and L. migratoria migratorioides was conducted by Fuzeau-Braesch et al. (1988) . They detected four volatile aromatic compounds in the environment surrounding S. gregaria, L. migratoria migratorioides, and Locusta migratoria cinearensis (Fabricius) of which three were identified as phenol, guaiacol, and veratrole. Phenol, guaiacol, and the blend of the three compounds were found to act as cohesion pheromones for the first two locust species, while L. m. cinearensis did not produce detectable amounts of these pheromones. Recently, detailed work relating to releaser pheromones mediating aggregation behaviour in the desert locust revealed that a complex of pheromones produced by different locust stages and sexes, and in their faeces are involved (Obeng-Ofori et al. 1993; 1994a;1994b; Torto et al., 1994; 1996). The pheromone systems mediating aggregation behaviour in nymphal and adult stages of S. gregaria were charaterized by Torto et al.(1994; 1996) 29 using a more efficient volatile collection technique. They identified six electrophysiologically active compounds in the volatiles of adult males of the desert locust comprising anisole, benzaldehyde, veratrole, guaiacol, phenylacetonitrile, and phenol. Veratrole, guaiacol, and phenol which were previously detected by Fuzeau-Braesh et a1. (198 8) , were also present, but in minor amounts in the atmosphere surrounding fifth-instar nymphs and adults of S. gregaria. Of the six compounds, only four compounds phenylacetonitrile, benzaldehyde, guaiacol, and phenol were found to elicit aggregation responses (Torto et al., 1994). Phenylacetonitrile was found to be a major and key component since it elicited aggregation responses in adults similar to the crude extracts. Furthermore, it was shown that only gregarious mature adult male locusts produced the aggregation pheromone, and that solitary locusts responded to it (Torto et al., 1994; Njagi et al., 1996). It was suggested that, male aggregation pheromone may play a role in the 'arrestment' and recruitment of solitary individuals into gregarizing or gregarious groups in the early stages of locust outbreaks (Njagi et al., 1996). Subsequently, it has been shown that production of phenylacetonitrile by gregarious mature adult male desert locusts follows a sigmoidal pattern, starting in the first 10 days after fledging, peaking between 15 and 20 30 days, and leveling off by the 35th day after which it starts to drop to reach very low levels in very old adults (Torto et al., 1994; Deng et al., 1996; Assad et al., 1996). Deng et al.(1996) showed that pheromone titres (as measured by the amounts of phenylacetonitrile) are a more sensitive measure than morphometries in determining the onset of phase change in S. gregaria. The nymphal pheromone system of gregarious S. gregaria was characterized as consisting of three sets of corrpounds: aliphatic C.;,Ci-C:o aldehydes and their corresponding acids produced by the insects themselves, augmented by faecal phenols, guaiacol and phenol (Torto et al., 1996) . 2.2.5 Dietary factors Locusts use their host plants for food and/or shelter. The effects of diet from such plants as alfalfa, hedge mustard, Johnson grass, and the mixture of the three, on the lesser migratory grasshopper, Melanoplus mexicanus mexicanus (Saussure) were studied by Barnes (1955). He monitored many parameters of which nymphal development and weight, and adult body dimension ratios (morphometric) showed significant diet-dependent variations. Adults reared on hedge mustard and mixed diet showed traits towards the migratory phase, those 31 reared on Johnson grass were intermediate whereas those reared on alfalfa showed little or no development toward the migratory phase. Toye (1973) studied the effects of five different food plants on the development of the desert locust, S. gregaria and found that hoppers fed on Agropyron and Poa grasses showed the highest developmental rate of nymphs (29 - 32 days). The heaviest nymphs and adults had morphometries typical of the gregarious phase. When fed on lime, privet, and spinach they produced morphometrically smaller and less viable males which had some abnormalities in nymphal development (five instars with a long fourth and without the fifth instar). Jackson et al.(1978) investigated the effects of natural food plants on the phase of the desert locust, S. gregaria using cultivated plants Pennisetvm typhoides(Burm.f.) and Sorghum sp. and the desert host plant Dipterygium glaucum Oecn. Tribulus spp., Chrosozophora oblongifolia(Del.), Panicum turgidum Forsk. and Zygophyllum simplex L. They found that a diet of Pennisetum, and Sorghum tended to enhance gregarious traits while Dipterygium accentuated solitarious characteristics. This was also apparent on the progenies of parents fed on these plants. Mishra and Singh (1992), Langewald and Schmutterer (1992), Schmutterer et al. (1993), and Doumbia (1994) found that feeding on, or topical application of, neem (Azadirachta indica L.) 32 extracts to nymphal and adult locusts tended to reverse their phase from gregarious to solitarious. 2.3 Methods of characterizing phase in locusts The phase status of S. gregaria and L. migratoria has commonly been characterized on the basis of morphometric ratios (Uvarov, 1921; 1923), behaviour (Ellis, 1962), body colour (Stower, 1959), physiological characters such as eye colour (Stower, 1959), eye stripes (Uvarov, 1966), and fecundity as expressed by the number of ovarioles, egapods, and eggs per pod (Norris, 1950; Albrecht et al., 1953; Hunter-Jones, 1953; Papillon, 1960; Injeyan and Tobe, 1981a), haemolymph pigment composition (Mahamat et al., 1996), and pheromone emission (Torto et al., 1994). These phase charateristics are discussed below. 2.3.1 Morphometries Morphometric measurements were one of the first methods used to separate phases (Uvarov, 1921) . It was established that Locusta danicus Linnaeus was the same as L. m. migratorioides, and this was the basis of Uvarov's phase theory. Uvarov (1923) used the morphometric E/F ratio {ratio of the length of the elytron (E) to that of 33 the hind femur (F) }, to separate the solitarious and the gregarious phases of S. gregaria. Dirsh (1953) used uhe F/C ratio { ratio of the length of the hind femur (F), to the width of the head capsule (C) }, and found that it was a more reliable phase parameter than the E/F ratio. The E/F and F/C ratios of S. gregaria and L. migratoria are summarized in Table 1. 34 Table 1. E/F and F/C ratios of S. gregaria and L. migratoria ( Meinzingen, 1993) Locust species Phase E/F males F/C males E/F Females F/C Females S .gregaria solitaria <2 . 075 >3.75 <2.075 >3. 85 S.gregaria gregaria <2 .23 <3.15 <2 . 27 <3.15 L.migratoria solitaria <1 .83 3. 67 <1.83 3.46 L.migratoria gregaria <2 . 0 2 .96 <2 . 09 2.86 A method by which locust phase could be determined using both E/F and F/C ratios in a chart was proposed by Rungs (1954) and later modified by Duranton and Lecoq (1990) for more practical use in the field. It allows quick phase determination in a locust population. The proportion of any locust population of interest which undergoes phase shifts in the test population could be quickly determined. However, Chapman (1979) and Deng et al.(1996) suggested that morphometric parameters should be considered with caution for phase description since they change slowly over several generations. 35 2.3.2 Body colour Body colour patterns can be valuable for monitoring phase shift from the solitary to the gregarious phase, and vice-versa. Nymphal stages of the solitarious desert locust are often characterized by green straw colour and adult stage by greyish brown (Pener, 1991). In the gregarious phase, nymphs have a black pattern on a yellow background (Nickerson, 1956; Stower, 1959; Pener, 1991) while adults are pinkish when immature and bright yellow when mature (Norris, 1952, 1954). Stower (1959) and Duranton and Lecoq (1990) classified colour stages in nymphal desert locusts from 0-5, based on the extent of the black emaculation on the head and other body parts. A grade of 0 corresponded to no black markings on either the head, hind femora, pronotum, or the abdominal tergites, while grade five corresponded to full black markings on these body parts. Norris (1954) used a colour classification scheme made of five grades for adult S. gregaria. Grade I corresponded to pink immature adults, with no flush of yellow at all on abdominal tergites and hindwings, grade III denoted adults with a flush of yellow on abdominal tergites, wings, and thorax, and grade V corresponded to full bright yellow colour in fully mature adults. This classification was modified slightly by Loher (1960) into four stages to monitor the acceleration of maturation in the desert locust. In L. migratoria, the solitarious adult locusts are green or brown while gregarious nymphs are black with an orange ventrum (Faure, 1942; Gunn and Hunter-Jones 1952; Hunter-Jones, 1958). Hunter-Jones (1958) described six colour types in hatchlings of L. migratoria : type 1 corresponded to pale brown pattern on a yellow brown ground colour, type 3 a medium to dark brown covering on one third of the body surface, and type 6 to black hatchlings with little to no apparent ground colour. No such classification has been proposed for adults. The number of eye stripes in S. gregaria and N. septemfasciata has been correlated with the number of hopper instars in crowded and isolated hoppers (Uvarov, 1966). In S. gregaria, solitarious hoppers have seven stripes corresponding to six nymphal instars whereas the gregarious hoppers have six showing nymphal development through five instars (Uvarov, 1966) . Stower (1959) and Duranton and Lecoq (1990) showed that eye colouration could also be a phase marker in the desert locust. They 2.3.3 Eye stripes and shading found that while solitarious locusts had clear eyes with all the stripes visible, gregarious ones tended to have totally darkened eyes. Based on this, they graded the eye colouration from 0-5 as follows: 0, no shading of the eye; 1, one third of the eye shaded black; 2, one half shaded black; 3, one half shaded with additional black spots on the other half; 4, two thirds shaded with additional spots; and 5, eyes fully coloured black. 2.3.4 Haemolymph pigments Mahamat et al.(1996) studied the presence of the blue biliverdin pigments in the haemolymph of S. gregaria. They showed that these pigments are associated with all stages of and ages of the solitary-reared (solitarious) insects, irrespective of their origin or diet, but absent or present in very small amounts in the crowd-reared (gregarious) ones. The ratio of absorbance at 460 nm (max for the carotenoids) and 680 run (max for the biliverdin pigments) in a UV spectrophotometer was found to be a convenient and reliable phase marker. Such ratio falls in the range of 3.99 - 4.78 in the gregarious and from 0.64 - 1.67 in the solitarious locusts (Mahamat et al., 1996). 37 38 2.3.5 Pheromones The use of aggregation pheromones as a phase marker was recently demonstrated by Torto et al.(1994) and confirmed by Deng et al. (1996) by comparing pheromonal emissions of gregarious and solitarious adult desert locusts. Only adult mature male gregarious locusts were able to produce aggregation pheromone (Torto et al., 1994; Obeng-Ofori et al., 1994a). Phenylacetonitrile is the major and key compound in the pheromone system of gregarious adult S. gregaria. Deng et al.(1996) showed that pheromone titres (as measured by the amounts of phenylacetonitrile) represent a more sensitive measure than morphometries of the onset of phase change in S. gregaria. 2.4 Developmental life cycle The developmental cycle of both L. migratoria and S. gregaria comprises three stages: egg, nymph, and adult (Steedman, 1988; Meinzingen, 1993) (Fig. 2). Eggs are deposited by mature females in the soil where the whole embryonic development occurs. At the completion of the embryonic development, the eggs hatch and neonates crawl 39 Immatura ADULT Figure 2. Life cycle of gregarious desert locust and migratory locust (Steedman, 1988) (solitary S. gregaria and sometimes L. migratoria have a sixth-instar) 40 their way up to the ground surface. Nymphs develop through five to six instars depending on their phase and species before fledging into adults (Uvarov, 1966; Steedman, 1988; Meinzingen, 1993). The adult develops through two stages, a sexually immature stage corresponding to pink coloured adults and which do not copulate nor oviposit (Norris, 1954), and a mature stage corresponding to yellow coloured and sexually mature locusts which copulate and oviposit (Popov, 1954; Norris, 1954). This developmental life cycle is reviewed in more Eggs are laid in the soil where, under favourable conditions of soil humidity and temperature, they take, on the average, 15 days to incubate. Their development is affected by environmental conditions. For example, Ackowor and Vajime (1995) found that in the lake Chad breeding area of L. migratoria migratorioides, egg development is closely related to season, soil structure, and natural enemy complex in the area. The fastest egg development was recorded during the main rainy season in sandy soil at temperatures of around 29°C with an incubation period of 13.6 days while the slowest was recorded during the Harmattan (dry) season at 2.4.1 Egg development detail in the following sections. I temperatures ranging from 19°C to 26°C. Incubation time was 24 days in clayey soils. No distinct diapause has been observed in the egg development in both S. gregaria and L. m. migratorioides, even though Meinzingen (1993), and Akowor and Vajime (1995) showed that in the field, some slowing or temporary arrest of the egg development is possible in conditions of low soil humidity and temperature conditions. In L. migratoria, the incubation period ranges between 20 - 40 days in cold conditions, but only 10 - 20 days in warm conditions (Price and Brown, 1990; Meinzingen, 1993) . The longer incubation time is a quiescent period (quiescens) and development and hatching proceed normally as soon as conditions become favourable. 2.4.2 Nymphal development Nymphal developmental time varies from 24 - 57 days depending on such conditions as temperature and humidity, and on locust phase. Under conditions of optimum temperature and humidity, gregarious locusts develop faster than solitarious ones (Duranton and Lecoq, 1990). In S. gregaria, nymphal development goes through five instars over a period of 25 days in the gregarious phase, and through six nymphal instars in a period of 30 41 days in the solitarious phase. Under adverse conditions, development may take 50 days in the gregarious phase and up to 90 days in the solitarious phase (Duranton and Lecoq, 1990). In L. migratoria, nymphal development goes through five instars regardless of the phase, over a period of 24 to 35 days. However, under adverse conditions, solitarious individuals may go through six or seven instars and development could last as long as 60 days (Meinzingen, 1993) . 2.4.3 Adult sexual maturation At the end of the last instar, nymphs fledge into sexually immature adults. When water and food are adequate, the immature adults of S. gregaria become sexually mature, copulate and oviposit in about 18 to 30 days (Steedman, 1988; Duranton and Lecoq, 1990) . A mature adult female can lay 2 to 3 eggpods containing 30- 70 eggs each when gregarious, and more than 3 eggpods containing 55-140 eggs each, when solitary (Steedman, 1988; Duranton et Lecoq, 1990). In L. migratoria, this number varies from 55-140 with an average of 67 eggs/pod (Steedman, 1988; Meinzingen, 1993) in the solitarious phase, and an average of 39.4 eggs/pod in the gregarious phase (Meinzingen, 1993). The time from egg to first oviposition in s. gregaria can vary from 50 to 332 days, 42 depending on soil moisture, relative humidity and temperature conditions, and locust phase status (Duranton and Lecoq, 1990). In both species adult longevity varies from 75 to 150 days. On the average, S. gregaria may go through 3 generations (1—5 generations) a year (Steedman, 1988; Meinzingen, 1993) depending on how fast the immature adults find suitable breeding conditions. Also, L. migratoria can produce up to five generations per year in the main outbreak area, but only two elsewhere in Africa (Meinzingen, 1993). Adult sexual maturation is affected by such factors as rainfall, humidity, temperature (Norris, 1954; 1957; Steedman, 1988), chemical stimuli (Norris, 1954, 1968; Loher, 1960; Mahamat et al., 1993; Assad, 1995), host and nonhost plants (Jackson, 1978; Assad, 1995). 2.4.4 Rainfall, humidity, and temperature complex When rainfall, humidity, and temperature are not adequate, immature adult locusts undergo arrested sexual maturation known as quiescence or imaginal diapause (Meinzingen, 1993). In S. gregaria, this state can last as long as seven months (Duranton and Lecoq, 1990) . When conditions are favourable, sexual maturation proceeds. Rainfall leads to growth and germination of nutritious 43 vegetation that supports rapid development (Carlisle and Ellis, 1965) and sexual maturation in immature adults. This leads to mating and egg laying both in solitary and gregarious locusts (Steedman, 1988; Meinzingen, 1993) . Duranton and Lecoq (1990) and Meinzingen (1993) reported that at low temperatures (17CC and below), locusts tend to slow their maturation even if rainfall was adequate. Maturation is equally slowed down at high temperatures (above 30=C) in the absence of rainfall, but accelerated at temperatures between 27-30:C and adequate rainfall 20- lOCrr.m (Steedman, 1988; Duranton and Lecoq, 1990) . 2.4.5 Chemical stimuli Pheromones play an important role in locust maturation and is particularly effective in inducing yellowing in males and females, testicular and ovarial development in male and female maturing locusts, respectively (Loher, 1960). Norris (1964) studied the accelerating and inhibiting effects of crowding on sexual maturation in two locust species, L. m. migratorioides and S. gregaria, and found that crowding accelerated maturation in the latter, but delayed it in the former. She further showed that young adults exerted inhibiting effects whereas older adults exerted accelerating effects on maturing adults. She concluded that the depression of 44 activity is a response to some non-mechanical, non­ visual, and presumably, a chemical stimulus which accelerated maturation in males and stimulated ovarial development and readiness to copulate in females of S. gregaria. When immature adults of the desert locust, S. gregaria, were exposed to themselves, they tended to copulate after 28 days, however when they were exposed to mature males most of them copulated after 17 days (Loher, 1990). In the migratory locust, L. migratoria migratorioides the acceleration was from 17-25 days to 13-14 days (Loher, 1990). In the absence of mature adult locusts maturation is delayed in S, gregaria (up to 28 days), but in L. migratoria, it is accelerated to only 7 days after final molt (Loher, 1990). Norris (1964) observed that young adults of L. migratoria in crowded conditions tended to inhibit each other from maturing over a certain period. The nature of the inhibition was not identified. In S. gregaria, it was the opposite, isolation preventing pheromone production (Njagi et al., 1996; Deng et al., 1996). In a recent study, Mahamat et al.(1993) applied the colour grading scale of Norris (1954) and pheromone release in gregarious adults described by Torto et al. ( 1994 ) to study maturation- acceleration in immature S. gregaria. They observed that the compounds identified by Torto et al.(1994), 45 especially phenylacetonitrile, were the ones responsible for hastening maturation of immature males and females. Assad et al. (1997) investigated the effects of the nymphal stage of 5. gregaria on the maturation of immature adults. Sexual maturation was significantly delayed in adults that were exposed to volatiles of fifth-instar nymphs. They further showed that live male and female nymphs placed in an upper compartment of two chamber cages were equally effective in inducing this delay. However, their faeces were ineffective, suggesting the mediation of a volatile signal from the nymphs themselves. Nymphal volatiles containing Z-, C;-C:; aldehydes and acids, phenol, and guaiacol which are nymphal aggregants (Torto et al., 1996), also act as maturation retardants for young adults of S. gregaria (Assad et al., 1997). 2.4.6 Interactions between species Norris (1964) studied sexual maturation in S. gregaria and L. migratoria and reported that mature adult S. gregaria accelerated sexual maturation of young adults of L. migratoria. It was not clear whether mature adults of L. migratoria had the same effects on immature adults of S. gregaria. 46 2.4.7 Host and nonhost plants Carlisle and Ellis (1967) studied the synchronisation of sexual maturation in desert locust swarms and found that locusts fledging at the beginning of the dry season and feeding on senescent vegetation failed to mature. This was attributed to the low concentrations of giberrellin and the monoterpenoid eugenol in their diet confirming their earlier findings (Ellis and Carlisle, 1965) . Maturation occurred in young adults which fed on green leaves containing these substances at the onset of the rainy season. Sexual maturation was also affected by odours of essential oils from certain desert shrubs (Carlisle and Ellis, 1967). Jackson et al.(1978) showed that different food plants variably affected many aspects of locust growth and development. For example, Pennisetum, Diptarygium, Tribulus, and Chrozophora supported rapid growth and synchronized moulting. On the other hand, hoppers reared partly on a pure diet of sorghum completed their nymphal development, but many were retarded with poor moulting synchronization and low body weight. Sexual maturation was adversely affected. Price and Brown (1992) found that migratory locusts had a high reproductive performance in maize monoculture during summer and in wheat monoculture during spring 47 seasons. They suggested that green food was essential for continuous reproduction, and that drying out of food plants could stop reproduction even at the oviposition stage, forcing locusts to enter into reproductive quiescence or delayed maturation. Effects of a desert plant of the genus Commiphora on the sexual maturation of the immature males and females of the desert locust were investigated by Assad et al. {1997) . They found that extracts (containing 55 detectable compounds mainly terpenoids) from this plant, when collected before winter rains accelerated sexual maturation of immature locusts. However, when collected after the winter rains no such acceleration was observed. Thus, volatiles from the host plant affect sexual maturation in locusts, and the effects vary with seasons (Carlisle and Ellis, 1967; Assad et al., 1997). 2.5 Methods of characterizing sexual maturation 2.5.1 Integumental colour Maturing adults of S. gregaria become progressively yellow according to Norris (1954) and Loher (1960) colour classifications (Section 2.3.2 of this thesis). Such a scheme has not been applied in L. migratoria even though 48 in both locusts, yellowing is a sign of sexual maturation (Norris, 1950; 1964; Loher, 1990; Whitman, 1990). 49 2.5.2 Copulation Norris (1950; 1954) found that mature males of S. gregaria, and L. m. migratorioides copulated as soon as they became sexually mature. This was applied later by Mahamat et al. (1993), and Assad (1995) and Assad et al. (1997) to study maturation acceleration and retardation, respectively, in the desert locust, S. gregaria. 2.5.3 Pheromone emission Emission of the aggregation pheromone was used by Mahamat et al. (1993) and Assad (1995) to study sexual maturation of the desert locust, S. gregaria. They showed that, pheromone emission is also a sensitive indicator of sexual maturation since maximum pheromone production in males coincides with age at full maturity. 2.5.4 Length of basal oocytes and oviposition The length of basal oocyte was used as an indicator of sexual maturity in studying the rate of maturation of immature adult females of S. gregaria, since this indicates the rate of ovulation (Norris, 1954). Norris (1954) showed that oocyte length between 6 and 8mm corresponded to sexual maturity in females. Descamps and Wintrebert (1961) defined four maturity stages in female migratory locusts, L. m. migratorioides. Locusts in stage III maturity or reproductive stage had oocyte lengths of 3-7 mm. Highnam and Haskell (1964) studied the relationship between the endocrine system, flying activity and oocyte lengths in both S. gregaria and L. m. migratorioides. They showed that in S. gregaria, the oocyte lengths varied from 0.7 mm in fledglings to 8.0 mm in fully mature adults. In L . m. migratorioides the lengths varied from 0.7 mm in fledglings to 6.5 mm in fully mature adults. They further showed that the variations in oocyte lengths were also influenced by the locust phase as in isolated S. gregaria, and crowded L. m. migratorioides, oocyte growth was slow, but in crowded S. gregaria and isolated L. m. migratorioides, oocyte growth was faster. There was a strong correlation between the variations in the lengths of oocytes and the size of the corpora cardiaca, on one hand, and the amount of neurosecretory material released by these glands in the pars intercerebralis of the brain, on the other (Highnam and Haskell, 1964). 50 The time to first oviposition is also a useful parameter in determining sexual maturation of the desert locust (Norris, 1950; 1954; Mahamat et al., 1993). 51 CHAPTER THREE 3 General materials and methods 3.1 Insects Crowded desert locusts, S. gregaria were obtained from a colony maintained at the ICIPE. The colony was propagated (for seventeen generations by the beginning of this work), from an original stock which was obtained from the Desert Locust Control Organisation for East Africa (DLCO-EA) in Addis Ababa, Ethiopia (Ochieng-Odero et al., 1991). Crowded migratory locusts were obtained from a stock maintained (for 16 to 17 generations at the beginning of the experiments) at the University of Nairobi, Kenya, and propagated for the experiments. To keep the colony gregarious, locusts were reared crowded (100-200 nymphs or 60-100 adults) in aluminium cages of 50 x 50 x 50 cm (Fig. 3A; Plate H) whereas for a solitarious stock, locusts were reared individually in 10 x 10 x 15 cm aluminium cages (Fig.3 B)(Ochieng-Odero et al., 1991). The rearing room (dimension: 2.5 x 2 m) was maintained at 29 ± 2°C., 58 ± 1 % RH, and 12:12 H light:dark period. The room was aerated by a duct system which maintained a negative pressure that facilitated a 52 53 Electric cab le Wire m esn G la ss w m ccw N^iCer C a g e ho icer - ■- — Cage comcanmem aise Hoor seal 2c.*n {Observation) ^ewing/feeding ! window | ' L 5cm OviDosid'on tube holder Figure 3. Diagrams of standard cages for rearing crowded (A) and isolated locusts (B) flow of fresh air at 10-15 air changes per hour (Ochieng- Odero et al., 1991). Fresh shoots of wheat (variety 'Nyangumi') and wheat bran obtained from Nakuru (Kenya) were provided daily to the colony. 3.2 Bioassays 3.2.1 Single chamber olfactometer The olfactometer (Fig. 4) was a 60 x 30 x 30 cm single glass chamber with a two choice arena whose top was covered with a removable wire gauze (Obeng-Ofori et al., 1993). An aluminium metal plate drilled with 2 mm-diameter holes (1cm apart) was fitted at the bottom of the chamber. Attached to each half of the arena and from underneath was a 28 cm (base length) pyramidal aluminium funnel. Each of the two funnels was connected by teflon tubing to a 2-litre round bottomed flask. Insects were placed in one of the flasks to serve as the source of volatiles; the other flask was left empty to act as a control. The olfactometer was placed under an extraction hood which maintained a continuous flow of air through the chamber, and prevented accumulation of volatiles. Two 60 cm fluorescent light tubes, each 60 W, were fitted 54 55 Figure 4. Diagram of the olfactometric aggregation bioassay system over the olfactometer arena to provide uniform illumination. Temperature in the experimental room was maintained at 26-29°C. 3.2.2 Cage assays Bioassays to investigate the gregarization of solitarious desert locusts in the presence of gregarious colony of migratory locusts were conducted in standard 50 x 50 x 50 cm aluminium cages (Ochieng-Odero, 1991) which were easy to clean and had no risk of adsorbing volatiles (Fig. 3A) . The front side of the cage had a sliding plexiglass door to allow observations on the locusts. One of the lateral sides had an opening with a sliding door to enable placement of food. The floor had a wiremesh (5 mm mesh) which allowed the faeces to drop out of the cages. The large cages had an outlet in the back of their ceiling for a 40 W bulb to provide light and heat to the insects inside. For bioassays where only the effects of volatiles on phase shift or maturation were tested, double storey aluminium cages (15 x 15 x 30 cm), described by Mahamat et al. (1993) were used (Plate A). Wire gauze partition 56 57 A. Two chamber bioassay cages, a) source chamber, b) recipient (test) chamber. between the two chambers allowed flow of volatiles from the top chamber to the lower one. 58 3.3 Collection of volatiles Volatiles were adsorbed onto charcoal traps employing the method described by Torto et al.(1994) (Plate B-l). The traps were 8mm-diameter and 8cm long glass tubes inside which, 60 mg of activated charcoal (80 - 100 mesh Chrompack, Middlesburg, The Netherlands) was packed between two glass wool plugs (Plate B-2d) . Before use, the charcoal was cleaned thoroughly by Soxhlet extraction with dichloromethane (Merck, Germany) for 4 8 hours followed by activation under nitrogen (250 ml/min) at 250:C for one hour. Air from a compressed air cylinder was cleaned by passing it through a charcoal filter before passing it over locusts contained in 0.5, 1.0, or 2 1 round-bottomed flask (Plate B-la) depending on the number of locusts. When trapping was from smaller numbers of locusts ( 1 - 2 adults or 30 - 50 early instar nymphs), quick-fit glass tubes (male 2.8 cm ID, female 3.4 cm ID, and length 9 cm) (Plate B-lb; B-2c) were used. All joints were sealed with teflon tape to avoid air leakage during trapping sessions. Each trapping session lasted overnight (16 hours) after which, traps were B 59 1 2 B. Volatile collection system (B-l) showing connected to it a) three-neck round bottomed flask containing mature S. gregaria b) quick- fit trapping chambers, and the medical air source cylinders; quickfit chambers (c) with connected charcoal traps (d) (B-2) eluted into a vial placed in ice with 5 ml HPLC grade (Aldrich Ltd, UK) dichloromethane. Volatile extracts were stored at -15°C, and concentrated under a stream of nitrogen at 0°C to approximately 300 ^1 prior to analysis. 3.4 Analyses of volatiles Analyses of volatiles were carried out by capillary gas chromatography (GC) on a Hewlett-Packard (HP) 5390 Series II gas chromatograph equipped with a flame ionization detector (FID) and a HP capillary column methyl silicone SPB-1 (30m x 0.2 mm ID x 0.2 /^ m film thickness), or Carbowax (50m x 0.2 mm ID x 0.2 ^m film thickness). When the Carbowax column was used, the analyses were run at initial temperature of 60-C isothermal for 10 min, then programmed to 180"C at a rate of 5'C/min and maintained there for 5 min, then to 220:C at a rate of 10DC/min and maintained isothermal for 15 min. When the methyl silicone column was used, the analyses were run at an initial temperature of 45°C for 5 min, then programmed to 150°C at the rate 5°C/min, then to 250°C at a rate of 10°C/ min and maintained there for 13 min. Chromatographic peaks were integrated using a HP 3396 Series II integrator. Samples from L. migratoria were analyzed by GC-MS on a VG 12-250 mass spectrometer (El, 70 eV) coupled to a HP5790 gas chromatograph. The 60 GC conditions were the same as for analyses using Carbowax. 3.5 Integumental colour grading Test locusts, S. gregaria were monitored for visible colour changes of the cuticle as an indication of gregarization (Plate C-E) or sexual maturity, in accordance with the colour classification of Norris (1954) for adult locusts as follows : I : insect totally deep pink ; II : insect clear pink; III : insect with slight pink and yellow on the abdomen, thorax, hindwings, and frons. This is obvious in male Schistocerca and Locusta (frons); IV : Yellow colour generalized over body and hindwings; V : Insect totally yellow even females showing frons and hindwings yellow; Pink immature adults (Plate C—1) or stage III yellow mature adults (Plate D-l) were considered to have reached the transient or gregarious phase. Immature gregarious adult L. m. migratorioides (Plate C-2), also show some faint pink colour but usually pale to creamy white frons. The hind wings are hyaline at soft immature adult stage but become yellow after 61 C. Immature adults of S. gregaria{C-l) and L. migratoria(C-2) (note the pink and blackish body colours of the respective insects)... 62 D. Gregarious (D-l) and solitarious (D-2) mature adults of S. gregaria. Gregarious (E-l) and solitarious (E-2) adults of L. migratoria migratorioides 65 1 F. Gregarious fifth-instar (F-l) and solitarious fourth- instar (F-2) nymphs of L. migratoria migratorioides. cuticular hardening. The frons turns progressively yellow, especially in males, followed by the pronotum and legs as the locust matures. Solitarious adults (Plate E- 2) have a green pronotum head and legs while the hind wings' hyaline colour turns yellow as the insect matures. The front wings are golden greyish. Mature solitarious males although green in general appearance can show yellow frons at maturity. Thus, in the adult migratory locust the yellow colouration on frons was a good indicator of sexual maturity. The colour grading was limited to the extent of the yellow colouration on frons of both gregarious and solitarious adult males, and the green colouration on the pronotum and legs for both male and female solitarious adults. The following scale was adopted: Grade 1 : No yellow apparent, frons pale with the brown colour fading out. Hind femora with no yellow; Grade 2 : Frons are pale with some faint yellow, less than 20% appearing at the base of the frons. Hind femora not yellow; Grade 3 : About 50% of frons yellow with some yellow appearing on the pronotum and hind femora; Grade 4 : More than 50 % of frons yellow and hind femora with more pronounced yellow; Grade 5 : Almost whole frons yellow, hind legs and pronotum with more pronounced yellow colouration. 66 Grade 3 colouration corresponded to sexual maturation of the locusts as males stridulated, copulated, and produced pheromone whereas adult females started ovipositing, 3.6 Statistical analyses Statistical analyses were carried out by SAS General Linear Model Procedure (GLM) in which simple or multiple analysis of variance (ANOVA, MANOVA) were run, with the Least Significant Difference test (LSD test at P < 0.05) for mean comparisons (SAS, 1988). Counts in the olfactometer, morphometric ratios, and haemolymph absorbance ratios, were transformed into arcsine, square roots, log(x), or log(x+l), prior to analysis. 67 CHAPTER FOUR 68 4 Aggregation responses of S. gregaria and L. migratoria migratorioides to their airborne volatiles. 4.1 Introduction Fuzeau-Braesh et al. (1988) identified guaiacol, phenol, and veratrole as three of the four major volatile constituents in the air surrounding fifth-instar nymphs and older adults of S. gregaria, and L. m. migratorioides. However, they did not study the responses of the species to the crude volatile blend of the other. A more detailed study on the aggregation responses of different stages of S. gregaria has been recently carried out by Obeng-Ofori et al. (1993). They found that, aggregation in the desert locust S. gregaria is mediated by two sets of releaser pheromones: a juvenile aggregation pheromone produced by nymphal stages to which only nymphs respond, and an adult aggregation pheromone which is specific to adult stages only. Aggregation responses in L. m. migratorioides are unknown and neither are cross-aggregation responses between the two locust species. In this chapter, comparative responses of the two locust species to their own and to each other's volatiles are described. In L. m. migratorioides, it was further investigated whether there are stage and sex differences as reported for S. gregaria (Obeng-Ofori et al., 1994a) 4.2 Materials and Methods 4.2.1 Olfactometer assays Aggregation responses of 5. gregaria and L. m. migratorioides to air-borne volatiles were conducted in olfactometric bioassays using the single chamber olfactometer (described in Chapter III, page 51 of this thesis), which eliminates visual and contact stimuli between source and test locusts. Air from a compressed air cylinder was cleaned by passing it through a charcoal filter, then split into two streams, each passing into a round-bottomed flask, and then into either of the sides of the arena at a flow rate of 120 ml/rain. Locusts placed in one of the flasks provided volatiles for the assays. Sets of five, each of mixed or separate sexes of 3-4 day old fifth-instar nymphs, immature, and 25-40 days old mature adults of L. m. migratorioides and S. gregaria were tested against their own, and to each other's volatiles at five doses 3, 7, 10, 20, and 40 Locust equivalents (LEQ). One LEQ represents the emission of 1 locust from 2L flask for 10 69 rain. The standard dose in this work was set at 10 LEQ, and the dose responses were studied in the range from 0 to 10, and from 10 to 50. Thus in the lower range, doses 7 and 3 {decrease by 3 units) were tested while in the upper range, doses 20 and 40 (increase by a factor of 2) were so. Test insects were introduced into the olfactometer via the entry box, and after 10 minutes, the number of insects on each side of the arena was recorded (Plate G). The aggregation index (Al) for each test was calculated using the equation: Al = 100 ( T - C ) / N ; where T= number of locusts found on the treated side, C= number of locusts found on the control side, and N= total number of locusts tested. Experiments involved different stages of S. gregaria and L. m. migratorioides (L. migratoria) as mixed (M) and separate (S) sexes and the controls (*) in the following combinations: 70 71 EXPT SOURCE RECIPIENT (SEX) Fifth-instar Fi fth-instar 1 L. migratoria L. migratoria (M, S)* 2 S. gregaria S. gregaria (M) * 3 L. migratoria S. gregaria (M) 4 S. gregaria L. migratoria (M) Mature adults Mature adults 5 L. migratoria L. migratoria (M, S)* 6 S. gregaria S. gregaria (M) * 7 L. migratoria S. gregaria (M) 8 S. gregaria L. migratoria (M) Fifth-instar Immature adult 9 L. migratoria L. migratoria (M) * 10 S. gregaria S. gregaria (M) * 11 L. migratoria S. gregaria (M) 12 S. gregaria L. migratoria (M) Mature adults Immature adults 13 L. migratoria L. migratoria (M) - 14 S. gregaria 5. gregaria (M) ' 15 L. migratoria S. gregaria (M) 16 S. gregaria L. migratoria (M) Between experiments, the olfactometer was carefully cleaned with acetone. Air was also flushed through the tubes for 2-3 hours to remove any volatile residues. The source and the control flasks were systematically switched in between experiments to minimize bias. Combinations 1 to 8 were also repeated and distribution of locusts to their volatiles after 10 and 30 min of exposure at the standard dose of 10 LEQ was noted. Additional tests were conducted with L. migratoria for stage and sex-specific responses to volatiles. These involved combinations 1, 5, 9, and 13 in the treatment combination list. Cross responses to eight to ten grams of 80% wet faeces of nymphs and immature adults of the respective locust species were also studied. 4.3 Data analysis The data were analyzed using the SAS (1988) package. Tests for independence were run using the Chi-square test. For separation of means by treatment, the data were transformed into arcsine or square roots prior to analysis of variance using LSD test at P < 0.05. 72 73 G. olfactometric tests showing locusts (1,2,..) orienting themselves towards or sitting on the treated side of the arena 74 4.4 Results 4.4.1 Responses of S. gregaria to volatiles of L. migratoria migratorioides Aggregation reponses of fifth-instar S. gregaria nymphs to their own volatiles and to those of fifth- instar L. migratoria nymphs are shown in Fig. 5. Responses were dose-dependent and fifth-instar S. gregaria nymphs responded maximally to volatiles produced by S. gregaria and L. migratoria at 10 and 20 LEQ, respectively. Fifth-instar S. gregaria nymphs had similar aggregation responses to their own volatiles and to those produced by fifth-instar L. migratoria nymphs. They also responded to faecal volatiles of fifth-instar nymphs of the latter species (Table 2). Immature adults of the two species had significant aggregation responses to conspecific volatiles. S. gregaria immature adults were indifferent to volatiles from nymphs of L. migratoria while immature adults of L. migratoria were actually repelled by volatiles from conspecific nymphs and responded weakly and insignificantly to those of nymphal S. gregaria. On the other hand, immature adults of both species responded equally to volatiles produced by mature adults of the two species, but significantly more to those from their Ag gr eg at io n In de x (% ) 75 Doses (LEQ ) Figure 5. Dose-aggregation response curves for fifth-instar nymphs of S. gregaria (FSG) to volatiles of fifth-instar conspecifics[(FSG/FSG)] and to those of L. migratoria(FLM),[(FSG/FLM)] in the olfactometer (bars are SE). 76 Table 2. Aggregation responses of immature adults of S. gregaria (SG) and L. m. migratorioides (LM) to nymphal and adult conspecific and interspecific volatiles in the olfactometer. Source Test Aggregation Index (% ± SE) Fifth-instar Immature adult LM LM -34 ± 10 c SG LM 16 ± 8 a LM SG -2 ± 8 b SG SG 18 ± 8 a Mature adult Immature adult LM LM 60 ± 11 a SG LM 22 ± 12 b LM SG 2 4 + 1 1 b SG SG 40 i 6 b NvmDhal faeces Fifth-instar LM SG 23 ± 8 a SG LM 24 ± 7 a NvmDhal faeces Immature adult LM SG 32 ± 13 a SG LM 32 ± 5 a Mean indices in each group followed by the same letter are not significantly different(at 5% level, LSD- test) . conspecifics (Table 2) . They also responded significantly higher than the untreated control (P < 0.05), to faecal volatiles of fifth-instar nymphs of L. migratoria similar to the responses to conspecific immature adult fecal volatiles (Table 2). Responses of mature adults of S. gregaria to their own volatiles and those of L. migratoria were dose- dependent. Adults of S. gregaria aggregated strongly (P< 0.05) and significantly more to their own volatiles than to those produced by L. migratoria especially at doses of 10 and 20 LEQ (Fig. 6). 4.4.2 Responses of L. migratoria to volatiles of S. gregaria. The reponses of fifth-instar nymphs of L. migratoria to their own volatiles and to those of fifth-instar nymphs of S. gregaria are shown in Fig. 7. Responses to volatiles of nymphal S. gregaria were not dose-dependent and there were no significant differences in responses of nymphal L. migratoria to their own volatiles and to those produced by fifth-instar S. gregaria over the range of doses tested (P <0.05) (Fig. 7) . However with regard to their own volatiles, nymphs of L. migratoria had significantly higher (P <0.05) aggregation responses to volatiles from pecifics at 20 LEQ than at other doses. They also 77 Ag gr eg at io n In de x (% ) 78 Doses (LEQ ) Figure 6. Dose-aggregation response curves for mature adults of S. gregaria(ASG) to volatiles of mature adult conspecifics[ (ASG/ASG)] and to those of L. migratoria (ALM),[(ASG/ALM)] in the olfactometer(bars are SE). Ag gr eg at io n In de x (% ) 79 Doses ( LEQ ) Figure 7. Dose-aggregation response curves for fifth-instar nymphs of L. migratoria (FLM) to volatiles of fifth-instar conspecifics[(FLM/FLM)] and to those of S. gregaria(FSG),[(FLM/FSG)] in the olfactometer(bars are SE). responded significantly (P <-05 ) compared to untreated control, to faecal volatiles of fifth-instar nymphs of S. gregaria (Table 2). Immature adults of the two species aggregated weakly to volatiles of fifth-instar nymphs. They responded less to volatiles produced by mature adults of S. gregaria, compared to responses to their own volatiles. Responses were significantly greater within than between species. They also responded to faecal volatiles of fifth-instar nymphs of S. gregaria (Table 2). Responses of mature adults of L . migratoria to their own volatiles and to those of S. gregaria were dose- dependent and there were no significant differences over the range of doses tested (Fig. 8). 4.4.3 Responses of S. gregaria or L. migratoria to choices of volatiles of both species Nymphs of S. gregaria responded to volatiles of nymphs of L. migratoria similar to their own. Similarly, there were no significant differences (P > 0.05)in aggregation responses of nymphs of L. migratoria to volatiles of S. gregaria and to those of their conspecifics during 10 or 30 min exposure (Table 3) . Mature adults of L. migratoria showed a significantly stronger response to their own volatiles 80 Agg reg atio n Ind ex (%) 81 Doses ( LEQ ) Figure 8. Dose-aggregation response curves for mature adults of L. migratoria (AIM) to volatiles of mature adult conspecif ics [ (ALM/ALM) ] and to those of S. gregaria(ASG), [ (ALM/ASG) ] in the olfactometer (bars are SE) . than to those of S. gregaria after 10 min of exposure (P < 0.05). However, after 30 min, mature adults of L. migratoria responded significantly more to volatiles of S. gregaria than to their own (P £ 0.05) . Mature adults of S. gregaria responded to volatiles of mature adults of L. migratoria similar to their own at the two time (10 and 30 min) intervals. Aggregation responses to volatiles of S. gregaria of the two locust species after 30 min of exposure to volatiles were not significantly different (P > 0.05) (Table 3) . 4.4.4 Sex responses to volatiles in L.m. migratorioides. Male and female nymphs of L. migratoria migratorioides did not show any preference for their respective volatiles (Table 4) . Mature adult females were more strongly attracted (P< 0.05) to volatiles from mature males than males to those of females. However, mature females showed significantly (P < 0.05) lower aggregation responses to their own volatiles compared to the aggregation of males to their own, and this was not significantly different (P > 0.05) from the control (Table 4). 82 83 Table 3. Responses of S. gregaria (SG) and L. m. migratorioides (LM) to volatiles emanating simultaneously from 10 locusts of each species placed in olfactometer choice tests. Test (stages) Time (min) Aggregation index (% ±SE) to LM volatiles Aggregation index (% ±SE) to SG volatiles Fifth-instar LM 10 42 ± 6 a 52 ± 7 a SG 10 42 ± 9 a 60 ± 8 a LM 30 44 ± 9 a 50 ± 12 a SG 30 46 ± 6 a 54 ± 6 a Mature adult LM 10 56 ± 8 a 32 ± 5 b SG 10 44 ± 4 a 54 ± 4 a LM 30 32 ± 5 b 66 ± 5 a SG 30 44 ± 8 a 56 ± 9 a All means in each column(3 and 4), followed by the same letter are not significantly different (at 5% level, LSD test) . 84 Table 4 Sex responses of nymphs and mature adults of L. m. migratorioides to volatiles in the olfactometer. Source Test Aggregation Index (% ± SE) Fifth-instar Fi fth-instar Males Females 40 ± 9 a Males Males 42 + 11 a Females Males 42 ± 9 a Females Females 46 ± 13 a Mature adult Mature adult Males Females 56 ± 6 a Males Males 40 ± 8 b Females Males 38 ± 9 b Females Females 18 ± 8 c Means in last column and by stage, followed by the same letter are not significantly different (at 5% level, LSD test) . 85 4.4.5 Intra and inter-stage aggregation responses in L. a.migratorioides. Male and female fifth-instar nymphs responded highly to their volatiles which produced a repellent effect on immature adults (Table 5) . On the other hand mature adults were indifferent to nymphal volatiles, but responded highly to their own volatiles to which fifth- instar, young, and older adults were also responsive (Table 5). 86 Table 5 Cross-stage aggregation responses of L. m. migratorioides in the olfactometer. Source Test Aggregation Index (% ± SE) Fifth-instars Fifth-instars Fifth-instars Fifth-instars Immature adults Mature adults 57 ± 8 a -34 ± 10 c 6 ± 10 b Mature adults Mature adults Mature adults Fifth-instars Immature adults Mature adults 40 ± 10 b 60 ± 11 ab 7 6 ± 7 a In each group, means followed by the same letter in the last column are not significantly different (at 5% level, LSD test). 4.5 Discussion 87 The present results show some similarity in the aggregation response patterns of different stages of S. gregaria and L.m. migratorioides. Particularly noteworthy is the extent of cross-reactivity between the nymphal and mature adults stages respectively of the two species. Thus, the aggregation responses of fifth-instar nymphs to their respective volatiles as well as to those of one another were remarkably similar (Figs. 5 and 7). As in the case of S. gregaria (Obeng-Ofori et al., 1994a), no sexual differences in the emissions of, or responses to, the nymphal pheromone was observed in L. migratoria. Nymphs, and immature adults cross-responded to their fecal volatiles. These results suggest the presence of either similar compounds or pheromone blends with similar releaser effects on each other's nymphs in the nymphal emissions of these species. This may account for the occurrence of mixed hopper bands of S. gregaria and L. m. migratorioides often observed in the field (Johnston and Buxton, 1949; El-Bashir and Abdel-Rahman, 1991; Torto, pers. comm.). Similarly, mixed populations of immature adults and fifth-instar nymphs could be explained by the fact that immature adults of the two locust species are responsive to each other's nymphal faecal volatiles. The responses of mature adults to their respective volatiles as well as to those of one another were also quite similar (Figs. 6 and 8). However, in this case, S. gregaria responses to volatiles of conspecifics was significantly higher at some doses (10 and 20 LEQ) compared to its responses to volatiles of mature adults of L. m. migratorioides (Fig. 6) suggesting some differences in the composition of the two pheromone systems or possibly in the amounts of the active compounds. On the other hand, significant differences were observed between the two species when the aggregation responses of one stage of the insect to volatiles of another were compared. Thus, although immature adults of S. gregaria were indifferent or aggregated weakly to volatiles of conspecific fifth-instar nymphs as previously reported (Obeng-Ofori et al., 1993), those of L.m. migratorioides were significantly repelled by conspecific nymphs of similar stage (Table 2). Interestingly, immature adults of L. m. migratorioides were stimulated to aggregate, albeit weakly, in response to volatiles of fifth-instar nymphs of S. gregaria, whereas S. gregaria immature adults were indifferent to volatiles of nymphal L. m. migratorioides. These results clearly point toward compositional differences in the nymphal volatile blends of the two species. Comparison 88 of the aggregation responses of immature adults to volatiles of conspecific and interspecific mature adults also shows compositional differences in the adult pheromone blends (Table 2) . Thus, although immature adults of both species aggregated significantly to volatiles of conspecific mature adults, their cross responses were weak and statistically insignificant. Differences in pheromonal responses between the two locusts both at nymphal and adult stages may also be quantitative, inferring then that the two locusts have different treshold responses to the pheromones, and probably produce different amounts of the necessary compounds. This implies that in the ecological point of view these two locusts still maintain their specificity as two different species with distinct niches. Cross responses between mature adults and nymphal stages also show differences between the two species (Tables 2 and 5) . Thus, whereas in S. gregaria mature adults and nymphs are indifferent to each other's volatiles, in L. m. migratorioides, although mature adults are also indifferent to nymphal volatiles, the nymphs respond positively to those of mature adults. A major difference between the adults of the two locusts is that in S. gregaria males do not respond significantly to volatiles from females (Obeng-Ofori et al., 1994), while in L. m. migratorioides both males and 89 females cross responded to each other's volatiles. In the latter species (Table 4), females were strongly attracted to male volatiles, and, conversely males were significantly strongly attracted to female volatiles. The attraction of females to female volatiles was very weak and statistically insignificant. These results suggest the mediation of two sets of volatile pheromones in mature adults of L.m. migratorioides: an aggregation pheromone produced by males that elicits aggregation from conspecifics of both sexes, and a sex pheromone that is produced by mature females that attracts/arrest male conspecifics. In summary, the present study has demonstrated some similarities as well as differences in the pheromone- mediated aggregation behaviour of S. gregaria and L. migratoria migratorioides. The nymphs, as illustrated by the fifth stadium, were remarkably similar and indeed, the two species cross-responded to each other's pheromones; but S. gregaria showed some specificity for its own pheromone. Major differences between the two species occurred in cross- stage responses and in sex differentiation in the pheromone systems of mature adults. It is concluded that despite cross-activity between the aggregation pheromones of the two species both the nymphal and adult aggregation pheromones have 90 significantly different compositions possibly with some overlapping compounds. In addition, L. migratoria has a volatile sex-recognition signal produced by females. 91 CHAPTER FIVE 5. Primer effects of a gregarious population of one species on solitary individuals of the other. 5.1 Introduction Locusts, as well as other living creatures, interact with their environment in general, and particularly with other insects sharing the same habitat. The behaviour and maturation of the desert locust and the migratory locusts have been reported to be affected by the presence of other locusts (Gillett, 1963; Loher, 1990) and host plants(Jackson et al., 1978; Assad, 1995). Nolte (1977), Ba-Angood (1976), and Gillett (1968) showed that when solitarious locusts were reared with certain grasshoppers their morphometries, colour, and eye stripes did not change, but when reared with other locust species, they acquired some gregarious characteristics. Studies described in Chapter IV show that there are strong pheromone-mediated interactions between the two locust species. The long term (primer) effects resulting from interactions between the two locust species, S. gregaria and L. migratoria migratorioides, are the subject of this Chapter. Primer effects were by more sensitive phase markers such as pheromone production 92 ( Deng et al. 1996 ) and haemolymph pigment composition (Mahamat et al., 1996) in addition to previously known characters based on body colour changes and morphometries. 5.2 Materials and methods This section addresses three different objectives. The first one is on the investigation of the effects of gregarious migratory locusts and two grasshopper species on the gregarization of solitarious desert locust in mixed population; the second is on the investigation of the effects of gregarious desert locusts on the gregarization of solitarious migratory locusts; and the third is on the effects of the volatiles of gregarious individuals of one species on the gregarization of solitarious ones of the other, with S. gregaria responses to volatiles of L. migratoria taken as a case study. 93 94 5.2.1 Effects of the presence of gregarious L.m. migratorioides and two grasshopper species on the gregarization of solitarious S. gregaria Insects and experiments Preliminary rearing tests at random densities of 30, 60, 70, 80, 110, 120, 130, 180, 200, 270, 400 nymphs and adults of L. migratoria migratorioides were conducted to determine the appropriate locust density to use as stimulus source on S. gregaria in subsequent assays. Integumental colour changes were used to monitor the degree of gregarization in test locusts. All locusts tested shifted to gregarious colour (grades 4 and 5, see page 59-52) at all densities tested. A standard density of 80 locusts was used in subsequent assays. The size of the cage (50 x 50 x 50 cm) allowed free movement of the locusts. One solitary desert locust, at nymphal or adult stages, was placed in the cage already described in Chapter III (Plate H) containing specific stages of either 80 two-day-old nymphs of gregarious L. migratoria, Phymateus viridipes Stal, Pyrgomorphidae, or Eyprepocnemis plorans (Charpentier), Eyprepocnemidinae. The latter two are grasshopper species collected as first instars behind the ICIPE fence. 95 H H. One chamber bioassay cage used for mixed rearing experiments and described in Chapter III, and showing a)glass door, b)egg laying tube insertion holes. 96 T r e a t m e n t s (TRT) w e r e d e s i g n e d a s f o l l o w s : RATIO 80 : ] TRT STIMULUS qnT.TTARIOUS TEST 1 . First-instar First-instar L. migratoria S. gregaria 2. Two-day-old Two-day-old immature immature adult L. migratoria adult S. gregaria 3. Fifth-instar Two-day-old immature L. migratoria adult S. gregaria 4 . First-instar First-instar P. viridipes S. gregaria 5. First-instar First-instar E. plorans S. gregaria 6. First-instar First-instar 5. gregaria S. gregaria 7 . First-instar First-instar S. gregaria S. gregaria 80 80 19 10 19 19 Treatments 6 and 7 were referred to in the text as control gregarious or solitarious respectively. Test nymphal locusts were monitored for changes in integumental colour, and durations of instars (and stage) while adult locusts were monitored for changes in integumental colour, eye shading, morphometries (E/F and F/C ratios), eye colour, haemolymph pigment composition, and titres of the male-produced aggregation pheromone. Integumental colour Nymphal colour changes were recorded daily, following Stower (1959) nymphal colour grading as follows (Fig. 9): 0 : no black maculation on head, abdomen and legs. 1 : a few black spots on the head; less than 1/4 of the abdomen is black; a black line appearing in the mid horizontal axis of the femur. 2 : 1/3 of head including mouthparts black; less than 1/4 of the abdomen is black; black line on the femur conspicuous. 3 : 1/2 of head and mouthparts black; less than 1/4 of the abdomen is black; femur over 1/3 black. 4 : 2/3 of head and mouthparts are black; 1/4 of the abdomen is black; 1/2 femur is black. 5 : over 2/3 of head and 1/2 of abdomen and femur black ( Plate 1-1; 1-2 ). 97 Adult colour changes from immature pink stage to mature yellow stage were also recorded daily until maturation colour stage III was attained as described in Chapter III. Number of nymphal instars, nymphal and total developmental (to mature adult stage) times. The nymphal developmental time in the desert locust was determined by monitoring the number of instars. This was done by recording each nymphal moult, and by counting Head 0 Abdomen J U S s ^ ^ * Femur Eyes Grade I I I I Figure 9. Black colour pattern used in grading nymphal colour changes in test s. gregaria 98 99 I Gregarious fifth—instar and fourth-instar nymphs of s. gregaria from gregarious (1-1) and solitarious(1-2) control cages respectively. the eye stripes of adult locusts. The duration of each instar was the time between consecutive moults whereas the nymphal developmental time was computed as the total of all instar durations. The total developmental time to end of immature stage was computed as the total nymphal developmental time added to the time the adult lived as immature. Morphometric measurements The length in millimeters (mm) of the elytron (E), the hind femur (F) , and the width of the head capsule (C), were measured on mature adult males and females of both species using an electronic Sylvac (T) caliper with a range of 0-150 mm and an accuracy of ± 0.03 mm. Morphometric changes were determined by calculating E/F and F/C ratios (Fig. 10). Average morphometric ratios were calculated and compared to those of control locusts to determine the phase status of all test locusts. To determine the individual phase status and the proportion of transformed individuals of the desert locust, a standard morphometric chart (Duranton and Lecoq, 1990) was used (Fig. 10) . 100 cF/C i GREGARIOUS i i i i i i 1 !o i i V i i ! i I 1 TRANSIENS i 1 1 i i 1 1 J 1 1 I11 u I | 1 111 ! , . i ' i ; iT i i i 1 i i i i SOLITARIOUS 1 i 14 1-6 1-8 2-0 2-2 lk 2-6 2-8 E/F Figure io. The standard morphometric chart for locust phase determination Adult eye shading 102 The eye dark shading was recorded following Duranton and Lecoq (1990) grading scheme as follows: 0 : no shading of the eye, 1 : 1/3 of the eye shaded black, 2 : 1/2 shaded black, 3 : 1/2 shaded with additional black spots on the other 1/2, 4 : 2/3 shaded with additional spots, and 5 : eyes fully black coloured. Grade 0 corresponds to the solitarious phase, grade 3 to transiens congregans, and grade 5, fully gregarious desert locusts (Fig. 9). Collection and analysis of haemolymph Samples of locust haemolymph were collected from 33 day-old male and female adults of S. gregaria. This was done by puncturing the membrane at the precoxal cavity with a sterilized needle, and sucking the fluid up to 15 ^1 using microcapillary pipets (Sigma Chemical Co. St Louis Missouri). The sample fluid was diluted to 300 n 1 with phosphate buffer saline (PBS) pH 7.4 in Eppendorf tubes and kept at - 20°C until use (Mahamat et al. 1996). The collected samples were analyzed for their uv- visible absorption spectra from 460 to 680 nm using a Beckman DU-50 UV Spectrophotometer with phosphate buffer saline as the reference (Mahamat et al., 1996) . Absorbance ratios, R, were calculated as Al / A2 where Al was the absorbance reading at 4 60nm, and A2 the absorbance reading at 680nm. Collection and analysis of volatiles Air-borne volatiles were collected following the procedure described in Chapter III. Air from a compressed air cylinder was cleaned through a charcoal filter and passed over locusts placed in a 2 liter three­ necked round bottomed flask. The volatiles released by the locusts in the air stream were adsorbed onto traps containing 60 mg of activated charcoal. After 16 hours (overnight), the traps were removed and eluted with 4 ml of cichloromethane (Aldrich Ltd., UK). The extract was then concentrated under a stream of nitrogen at 0:C to approximately 100 /ul and stored at - 15:C prior to analysis. Collections were done on adult locusts at age 5-7, 10 - 12, 15 - 17, 20 - 22, 25 - 27, 30 - 32, and 35 37 days after fledging. Four microliters were analyzed by capillary gas- chromatography (GC) and by GC-Mass spectrometry (MS). The columns used were methyl silicone SPB-1 (30m, 0.2 ^m, and 0.2 mm ID), and Carbowax (50m, 0.2 /urn, and 0.2 mm ID) (Chapter III, page 59) . Nitrogen was used as the carrier gas at a flow rate of 0.35 ml/min. 103 5.2.2 Effects of gregarious S. gregaria on the gregarization of solitarious L. migratoria 104 Insects and experiments •it ' '* , Solitarious individuals of L.m. migratorioides •we.^e*^ reared under conditions as described in Chapter III of this thesis. For this experiment only immature adults from the 3rd generation of isolated locusts were used. Locust isolated rearing was required, at least, for three generations, to make sure that they were solitary enough to be used in such experiments. It was impossible (because of low hatchability of eggs) to obtain a sufficient number of nymphs from mature adults of this generation to run this experiment at the nymphal stage. Two-day-old immature adult solitarious locusts were placed in the cages as described above (Plate H) and reared with same age gregarious immature adults of S. gregaria to the end of the adult stage. Three treatments (TRT) were set up as follows: 2E1 GREGARIOUS STIMULUS SOLITARIOUS TEST RATIO REPS 1. Two-day-old immature Two-day-old immature 80 : 1 9 adult S. gregaria adult L. migratoria 2. Two-day-old immature Two-day-old immature 80 : 1 9 adult L. migratoria adult L. migratoria 3. Two-day-old immature Two-day-old immature 0 : 1 9 L. migratoria L. migratoria Parameters monitored were integumental colour changes (on pronotum and frons) in immature and mature adults, and the amounts of phenylacetonitrile produced by maturing adult male locusts (Chapter III and Section 5.2.1) . 5.2.3 Effects of volatiles of gregarious L. migratoria on the gregarization of solitarious S. gregaria Insects and experiments One two-day-old first instar solitarious desert locust was exposed to the volatile emissions from crowded (15) first-instar migratory locusts of similar age in a 15 x 15 x 30 cm bic'namber cage (Plate A) . Within each cage gregarious locusts placed in the top chamber, were visually separated from the solitary test locust by a black cloth placed at the bottom of the top chamber. Between cages, solitary locusts in the lower chamber were visually separated from each other by a black cloth taped over the window facing the neighbouring cage. Locusts were reared from nymphal to the adult stages. The following treatments (TRT) were set up: 105 TRT nRKGARinns STIMULUS S&Lt t a r tW $ TEST RAXIG REPS 1. First-instar L. migratoria 2. First-instar S. gregaria 3. First-instar S. gregaria Test locusts were monitored for nymphal integumental colour, number and duration of instars (nymphal developmental time), and adult integumental colour, morphometries, duration of immature stage, and total developmental time (to the end of immature adult stage). The colours of nymphs and adults were graded daily as described for nymphs (page 97) and adults (Chapter III, see page 62). The number and duration of nymphal instars were obtained from recording moulting periods, and counting adult eye stripes. Adult morphometries were taken at age 12-15 days (Chapter III, ) after fledging when locust integument was expected to have fully hardened. 5.3 Data analysis Data were analyzed using SAS (1988) . The data were transformed into log(x) or square roots, and means were separated, after analyses of variance, using LSD-test at P <; 0.05. 106 F i r s t - i n s t a r 15 : 1 6 S. gregaria F i r s t - i n s t a r 15 : 1 6 S. gregaria First-instar 0 : 1 6 S. gregaria 107 5.4.1 Effects of the presence of gregarious L.m migratorioides, and two grasshopper species on the gregarization of solitarious S. gregaria. Experiment 1. Effects of the presence of nymphal L. migratoria on the gregarization of nymphal solitarious S. gregaria Integumantal colour Integumental colour patterns in all nymphal desert locusts varied from green (grade 0 to 1) to yellow and black (grades 4 and 5) when reared mixed (singly) with gregarious migratory locusts. Nymphs changed to gregarious colour by the third instar, about 15 days after rearing of the two locust species together (Plate J; Table 6) . About 13 (9 males and 4 females) out of the 19 desert locusts reared with each other reached grade 4 colour pattern and above by the fifth instar, representing a shift of 69% (75% in males, and 57% in females)( Table 6). These nymphs monitored to the pink fledglings attained stage III yellow colour at 29 ± 2 days (Plates C-l; D-l). The appearance of the yellow colour (Plate K) was, however, somewhat delayed by 7 days compared to gregarious controls. 5.4 Results Number of nymphal instars, nymphal and total developmental times (to mature adult stage) times. Solitarious S. gregaria reared with gregarious L. migratoria went through five or six nymphal instars. Only 50% (44% in males and 6% in females) of them went through five nymphal instars similar to the gregarious control while the remaining 50% (56% in males and 94% females went through six instars similar to the solitarious control. These results show that males were more responsive to the treatments and, therefore, transformed faster than females. 108 109 J. Solitarious S. gregaria{A) reared with gregarious L. migratoria migratorioides (B). 110 K. Adult male S. gregaria[A) reared with crowded L. migratoria(B) and showing yellow colour typical of mature gregarious locust Ill Table 6. Time (days) taken by test S. gregaria (SG) and L. migratoria (LM) to attain gregarious colour, and the corresponding percent shift when reared mixed with or exposed to volatiles Source Test locust Rearing Days(± SE) in Test locusts Days (± SE) in control locusts % shift in test locusts Nymphs LM Nymph SG mixed 15.0 + 2 a 4.0 ± 2 a 69 b Nymphs PV Nymph SG mixed 17.0 ± 2 a 4.0 ± 2 b 63 b Nymphs EP Nymph SG mixed - 4.0 ± 2 0 c Nymphs LM Adult SG mixed 20.0 ± 0.6 b 22.4 ± 0.6 a 90 a adult LM Adult SG mixed 15.3 ± 0.3 b 22.4 ± 0.6 a 89 a adult SG Adult LM mixed 10.3 ± 0.3 b 11.8 ± 0.4 a 100 a Nymphs LM Nymph SG exposure 25.0 ± 3.0 a 22 . 4 ± 0 . 6 b 100 a Treatment and control means in each experiment followed by the same letter are not significantly different (at 5 % level; Student T-test). Percent (°o) shifts(to gregarious colour) in last column were compared between experiments. PV refers to the grasshopper P. viridipes and EP to E . plorans. Fifth and sixth instars were the longest while those of the other instars were quite variable compared to gregarious and solitarious controls. Solitarious S. gregaria reared with gregarious L. migratoria took 13 to 19 days longer than the gregarious and solitarious controls, respectively, to complete their nymphal stage. There was, however, significant similarity (P<0.05) in development between nymphs from the test and those from the control colonies, especially at second, and from the fourth, fifth, and sixth-instars (Fig. 11). There significant synchrony in developmental time between the test desert locusts and the source migratory locusts (Fig. 12) The total nymphal developmental time of the test locust was 46 days and immature adult took 23 days to reach maturation making it a total developmental time to mature adult of about 79 days. 112 CO >N 50-- o ov—y CD 40-- E __ 30-- O-4—' c CD E 20- Cl _o reared with (L*?i 1= 1P&s (B ) (L-l) , and E. plorans(B) > fr« nymphal stage, P^°rans(B) 129 Table 7. Developmental time (days) of isolated S. gregaria(SSG) reared with P. viridipes (PV),[(PV/SSG)] and E. plorans(EP), [(EP/SSG)] as compared to those from gregarious[(GSG/GSG)] and solitarious (1 SSG/cage) controls. Source/Test 1st- instar 2nd- instar 3rd- instar 4 th­ ins tar 5th- instar 6th- instar Total stage Young adul t stage Total time to mature PV/SSG 15±la 4±lbc 7±lb 9± 1 a 9+ lb ll±2a 45+3a 54±10a 99±10a EP/SSG 5±lcd 6±la 9±0ab 6±2b 12 + la 9± la 47±3a 8±lc GSG/SSG 5±lcd 5±lab 5±lc 5±lb 8± lb - 27±2c 22± lb 55±3c 1SSG/cage - 4±ld 5±lab 4±lc 3±lc 7±lb 12±la 32±lb 74±2b In columns, means followed by the same letter are not significantly different(at 5% level; LSD-test). days. The remaining 50% stayed pink by the conclusion of the experiment (55-60 days after fledging). 130 Morphometries E/F ratios of males of test S. gregaria were closer to solitarious controls while females were intermediate between solitarious and gregarious controls. F/C ratios of males were more were more like solitarious and females were transients (Fig. 20). Comparison with the standard morphometric chart (Duranton and Lecoq, 1990) showed that test locusts were more like transiens, a shift of about 67% (75% in males and 50% in females). Volatile emissions Mature adult males of S. gregaria which fledged from nymphs reared with P. viridipes, produced trace amounts of phenylacetonitrile in their volatile emissions 15 and 25 days after fledging. Analysis of the volatiles from P. viridipes showed the presence of benzaldehyde, phenol, and guaiacol three of the aggregation pheromone components of mature desert locust. M or ph om et ri c ra ti os 131 ^ GSG VZZA SSG' b a Males Females Males Females E /F F/C Figure 20. Adult E/F and F/C ratios of solitarious S. gregaria (SSG) reared with P. viridipes (PV) at nymphal stage[(SSG/FV)] compared to gregarious[(GSG)] and solitarious[(SSG)] controls. These compounds are also present in the volatile emissions of the migratory locust (Appendix VII) 132 Eye shading Five out of the six monitored desert locusts reared with P. viridipes showed significant dark shading of the eye of grade 3 and above, similar to those reared with L. migratoria and the control gregarious colony. This represents a shift of 84% (all males and 75% of females). Experiment 5. Effects of the presence of nymphs of E. plorans on the gregarization of nymphal solitarious S. gregaria Nymphs of solitarious S. gregaria reared with E. plorans, maintained the colour characteristics of the solitarious controls (nymphs green and adults grey colour). Six nymphal instars were recorded and the total nymphal developmental time was up to 47 days (Table 7). Adults (which live for 8 days as immature) had eye shading and morphometries typical of solitarious locusts (Fig. 21), ancj did not produce phenylacetonitrile. 133 cn.2 '■4—'o o *L_ -J—' , o O CD 0) > CD O 50- 40- 10 - eza gsg ggg ssg H SSG : GLM ° 30J- c " "-Ii -cies as a maturation accelerant im accordance to what was observed in S. gregaria by Mahamat et al.(unpublished). Thus, pheromones may also contribute to synchrony of maturation within and between populations of the respective species. Whitman (1990) pointed out that if stimulatory and inhibitory pheromonal effects operate in the field, the demographic consequences would be great especially in the synchronisation locust maturation and in phase transformation. It was observed in these studies that L. migratoria matures relatively faster than S. gregaria. This is an important difference between the two species which in the field suggests their separation even if they coexisted as mixed bands, or up to the immature adult stage. The desert locust and the migratory locust appear to differ in maturation such that, within similar stages, they tend to inhibit each other's sexual maturation whereas between stages they tend to either inhibit or accelerate each other's maturation. Thus the cross­ effects on maturation may be affected by conditions in which they coexist. The inhibitive interactions between the two locusts were also apparent in the primer effect studies on phase shift and development. The antagonistic primer effects between S. gregaria and L. migratoria have ecological significance in that they lead to species divergence in the field, thus to separation in space and time. Therefore, these locusts may not be seen in mixed swarms, unless by accident and for a limited period. CHAPTER SEVEN 7.1 General discussion The results of the present study confirm that the interactions between L. migratoria migratorioides and S. gregaria observed in the field are not fortuitous. Interspecific and intraspecific assays have shown that the signals from the two locust species can elicit aggregation responses from each other, and affect each other's phase shifts, development, and maturation. An important finding of these studies is the fact that the two species share some similar components in their volatile pheromone systems. It is pertinent to compare the chemical communication system of the two species and to discuss the implications of interactions in relation to their possible biological role. General comparisons between the two species The present study revealed several similarities and differences in the pheromone systems of S. gregaria and L. migratoria. In both species, there was no sex differentiation in either the production or responses to nymphal aggregation pheromone. In the adult stages, the older maturing males produce the adult aggregation pheromones in both species. Also, in both species, the 172 young adults use their fecal volatiles and those of nymphal conspecifics to aggregate. However, there are major sexual differences in the adults of the two species. First, whereas young adults of S. gregaria are indifferent to the conspecific nymphal pheromone, those of L. migratoria are actually repelled by pheromones of conspecific nymphs. Among mature adults of S. gregaria, there was no evidence of a volatile sex pheromone in females. However, in L. migratoria, the female attracted male conspecifics but not the females, indicating the mediation of a volatile sex pheromone in the gregarious phase of this species. Differences between the two species are also apparent in the maturation process. L. migratoria matures relatively faster (7-13 days) than S. gregaria (18-30 days). Interestingly, whereas S. gregaria nymphal pheromone retards the maturation of conspecific young adults, that of L. migratoria accelerates it. The adult S. gregaria pheromone accelerates the maturation of conspecific young adults. The sequential retardation by the nymphal pheromone and acceleration by that of adults ensure maturation synchrony in the adults of this species. In L. migratoria, the male-produced adult pheromone also accelerates the maturation of young adults albeit to a lesser extent than that emitted by conspecific nymphs. Synchrony possibly arises from the 173 augmentative accelerated effects of the nymphal and mature adult pheromone systems. The present study did not include the oviposition behaviour of L. migratoria nor the aggregation behaviour of first to fourth-instars nymphs of this species. These are, by themselves, whole areas of research, and difficult ones. However, future behavioural studies in such aspects will help throw light on the extent of similarities in the pheromone systems mediating behaviour in the two species. Interactions between the two locusts The present study has shown remarkable interspecific aggregation responses to pheromone emissions of the two species at both nymphal and adult stages. In particular, the nymphs responded to one another's pheromone without any discernable specificity. Likewise the young adults, which do not produce any significant amount of the adult pheromones, also responded to each other’s fecal volatiles. Among mature adults, S. gregaria was less responsive to volatiles of L. migratoria. However, the latter was less discriminatory. This suggests the existence of a specific factor in the volatile blend of L. migratoria not present in that of S. gregaria. The existence of similarities as well as differences in the pheromone blends of the two species is also 174 suggested by the cross-primer gregarization and maturation effects of the two species. Solitarious individuals from both locust species gregarized when reared with gregarious groups of the other species. However, mixed rearing led to longer developmental cycles compared to rearing with conspecific gregarious locusts in both species. Thus though intra and interspecific locust pheromone systems contributed to gregarization, only conspecific pheromone leads to normal development. Cross-maturation effects also reflect some differences in the pheromone systems of the two species. Thus, whereas the maturation of adult S. gregaria is retarded by conspecific nymphal pheromone, on the other hand, it is accelerated by that of L. migratoria. The maturation of adult L. migratoria is retarded by S. gregaria nymphal pheromone. Likewise, mature S. gregaria pheromone retards the maturation of young L. migratoria; but that of mature L . migratoria had no consistent effects on young adult S. gregaria. Pheromone composition The similarities or differences between locust pheromonal systems is of special interest. One of the key findings in these studies is that the two locust species share some components in their aggregation Pheromone systems. However, in contrast to what was 175 suggested in the past by other authors (Fuzeau-Braesh] 1988; Whitman, 1990} there are important qualitative and quantitative differences between the pheromone systems of the two species. Analyses of volatiles had shown that both species produce phenylacetonitrile. It is however not produced by nymphs of S. gregaria while mature adult males produce it in relatively high amounts (Torto et al., 1994). In contrast to nymphal S. gregaria, both male and female nymphal L. migratoria produce it in substantial titres in their volatiles (Torto unpublished) whereas mature adult males produce it in very small amounts, nearly 100 times less than by mature male S. gregaria. Other aggregation compounds i.e. guaiacol, and phenol, are present in the volatiles from the respective locust species. However a detailed chemical study of L. migratoria needs to be conducted. The presence of substantial amounts of phenylacetonitrile in nymphal L. migratoria explains why fifth-instar nymphs of this species accelerated sexual maturation of immature males and females of S. gregaria. Pheromone-mediated interspecific interactions: biological role Some of the key findings described in this thesis revolve around the volatile (pheromonal) factors in these interactions. Therefore, it is pertinent to discuss briefly the importance of pheromones in locust biology 176 and in particular interspecific interactions that may be mediated by pheromones. Pheromone communication is probably the most important mode of communication in insects (Whitman, 1990). Many insects use pheromones for either simple communication within species or for interspecific defense, as in pyrgomorphids, against natural enemies. In most social insects e.g. termites, ants, bees, and locusts, the survival of the colony is dependent on these chemical messengers which have various functions: aggregation, dispersion, alarm, and defense. The role played by pheromones is primarily to ensure communication within species as a way of keeping populations either together or separated when necessary, and for species and mate recognition during courtship. However, in locusts, pheromones are the most important mediators of key attributes of the gregarious phase of the insect. In grasshoppers, chemical communication is less well known probably because of lack of research in this area. These insects also make extensive use of acoustics and vision besides olfactory signals within their habitats (Whitman, 1990). Interactions between species occur when species become receptive to each other's semiochemicals, due to some ecological relationship or coevolution, such as in predator/prey, symbiotic, and mutualistic relationship (Jaffe et al., 1995). A list of compounds shared by grasshoppers and locusts was summarized by Whitman (1990). For example, phenol, guaiacol and benzaldehyde, 177 which are components of the male produced aggregation pheromone in S. gregaria (Torto et al., 1994) are also present in the defense secretions of Romalea sp., a very aposematic pyrgomorphid grasshopper. They are also found in the volatiles of P. viridipes. Interestingly, solitary S. gregaria reared with P. viridipes turned gregarious similar to those reared with L. migratoria. The results on the cross reactivity of the aggregation pheromone systems of L. migratoria and S. gregaria account for the occurrence of mixed bands and mixed populations of immature adults and 5th-instar nymphs of the two locust species often observed in the field (El— Bashir and Abdel-Rahman, 1991; Johnston and Buxton, 1949; Torto, pers. comm.). I can only speculate on an adaptative value of these pheromone mediated interactions to both species. They share common ecological niches and outbreak areas. Solitarious S. gregaria and L. migratoria occupy arid to semi-arid zones where food source is scarce. They are in the constant need of finding new areas where they can develop and reproduce. To achieve this, both species have developed phase polymorphism which allows them to transform their behaviour into migratory phase with long flight characteristics. These are important for recolonizing lost (formerly colonized) habitats and the discovery of new ones. Gregarization in both species is density- dependent. In .«? gregaria and L. migratoria, but 178 particularly in S. gregaria, populations occurs in very patchy vegetation with limited resources. Rapid gregarization and the resulting migratory capability of the gregarious phase are common to both species ano aj-low the insects access to a wide profile of food plants, and thus a rapid increase in numbers. Cross-facilitation of gregarization in the early stages when the densities of each species is low can thus be adaptatively advantageous to both species. However, the question which arises is "how long would this situation persist in the field"? In breeding areas where green vegetation and moisture are found in restricted areas such as water courses, locusts are also forced to encounter each other and interact. This also occurs during hot or cold temperatures in the habitats when insects have to seek for refuge under such plants as Zygophyllum simplex L. or other shrubs or straw (Descamps, 1961a; Steedman, 1988; El-Bashir and Abdel-Rahman, 1991). Nymphs are more subject to these sorts of interactions because they cannot move over long distances. Long contact between the two species, however, may lead to mutual inhibition between different stages such that development and maturation between the two species are not synchronized, leading to divergence in developmental rates. Our studies on mixed rearing have shown that L. migratoria slows down nymphal development of S. gregaria; therefore, in mixed nymphal populations the former species tends to develop faster, 179 fledge into immature adult stage and swarm earlier than the latter. Thus, the occurrence of mixed bands is a temporary phenomenon which may last until locusts reach the soft immature stage, not beyond. Under optimal conditions, immature L. migratoria matures relatively faster than immature S. gregaria. If immature adults of the latter are in the presence of fifth-instar nymphs of the former, their sexual maturation is accelerated; if immature adults of the former are in the presence of fifth-instar nymphs of the latter, their maturation is delayed. Thus divergence also occurs in mixed populations that may persist to the fledgling stages. Another limiting factor to prolonged coexistence (interactions) of the two species is their food habits. S. gregaria is a general feeder while L. migratoria is restricted to graminaceous plants, thus the two are likely to diverge into different habitats even in the nymphal stage. Whether speculations outlined here represent a valid description of real events in the field must await more detailed ecological studies of mixed locust populations. 180 7.2 Recommendations 181 The preceeding discussions suggest the following lines for future research: 1) - Studies of the same scale as those by Obeng-Ofori et al. (1993, 1994a;b), and Torto et al. (1994) need to be done for L. migratoria, L. pardalina and other locusts and grasshoppers, in order to have a better understanding of the possible cross influence between chemical communication systems in these species, and where they co-occur, locust phase dynamics and swarm formations; 2) - Detailed chemical studies involving the isolation and characterization of aggregation pheromones of the migratory locusts, and their similarity or differences with those of the other grasshoppers and locusts, in order to have a more comprehensive understanding of the general effects of pheromones on the ecology of acridids in general. 3) - Similar to work done in S. gregaria (Hansson et al., 1996; Anton and Hansson, 1996), physiological studies may be conducted to elucidate the detection of the aggregation pheromones in the central nervous system i_2— — — —— ' ratorioides. REFERENCES cited 182 Ackowor, J.B., andVajime, C.K., 1995. 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COMBINATIONS Doses of source (LEQ) 3 7 10 20 40 Source / Recipient Fifth -instar nymphs LM / SG 58 + 8 a 62 ± 6 a 66 + 7 a 74 + 6 a 60 + 8 a SG / SG 66 + 9 a 72 ± 5 a 74 + 7 a 68 + 7 a 68 + 7 a Blank / SG 36 + 7 b 36 ± 7 b 34 + 7 b 24 + 3 b 36 + 7 b SG / LM 72 + 6 a 78 ± 5 a 74 + 9 a 74 + 5 a 78 + 7 a LM / LM 56 + 7 b 68 ± 5 a 77 + 5 a 80 + 4 a 66 + 7 a blank / LM 22 + 4 c 20 ± 4 b 20 + 7 b 16 + 4 b 20 + 7 b Mature adults LM / SG 52 + 8 a 70 ± 7 a 72 + 4 a 66 + 7 b 62 + 11 a SG / SG 64 + 7 a 76 ± 7 a 82 + 5 a 88 + 4 a 62 + 7 a Blank / SG 36 + 9 b 22 ± 6 b 24 + 5 b 22 + 6 c 36 + 10 b SG / LM 66 + 7 a 74 ± 6 a 78 + 5 a 86 + 4 a 66 + 7 a LM / LM 56 + 5 a 72 ± 7 a 82 + 6 a 76 + 6 a 72 + 4 a Blank / LM 22 + 6 b 20 ± 4 b 18 + 5 b 8 + 4 b 28 + 6 b 204 Appendix II. Average responses (% ± SE) of nymphs and mature adults of S. gregaria (SGR) and L. m . migratorioides (LMI) to each other's volatiles in the olfactometer. Stage Treatment (Source / Test) combinations Fifth-instar Mature adults LMI / LMI SGR / LMI SGR / SGR LMI / SGR 46 ± 7 ab 53 ± 8 a 56 ± 8 a 52 ± 8 a 40 ± 10 ab 37 ± 10 b 31 ± 10 b 55 ± 8 a In lines and columns, means followed by the same letter are not significantly different (P < 0.05 LSD test). Treatments are compared horizontally, and stages vertically (80 replicates of 10 min each). Appendix III. Dose responses of nymphal and mature adult locusts of species S. gregaria and L . migratoria in a two choice olfactometer. Locust Stage Doses (LEQ) 3 7 10 20 40 Nymphs Mature adults 33 ± 10 be 31 ± 10 c 43 ± 7 ab 51 ± 8 b 48 ± 9 ab 64 ± 8 ab 54 ± 7 a 66 ± 7 a 39 ± 10 ab 36 ± 10 C Within each line and column, means followed by the same letter are not significantly different ( P < 0.05 LSD test). 205 Appendix IV. Densities of crowded L. m. migratorioides (LM) in relation to the gregarization of each isolated S. gregaria (SG) under study. NYMPHAL STAGE ADULT STAGE Density Density of Density Eyes of test SGR Phase of test No Sexe of LM test SG of adult SG LM stripes shading 1 Female 273 5 154 7 Transiens 2* Male 117 5 117 6 3 Transiens 3 Female 71 5 66 7 - ii 4 Female 120 5 33 7 - ii 5 Male 122 5 57 7 3 ii 6 Male 200 5 185 6 5 gregarious 7 Male 400 5 244 6 3 ii 8 Female 200 - 200 7 - 9 Male 131 5 108 6 1 transiens 10 Male 200 5 154 7 3 ii 11 Male 400 5 200 6 3 gregarious 12 Male 80 2 120 7 1 Solitary 13 Male 80 3 . 5 120 7 1 Solitary 14 Male 80 3 . 5 80 7 1 Solitary 15 Male 80 2 80 7 1 Solitary 16 Male 80 5 80 6 3 gregarious 17 Female 80 5 80 6 3 II 18 Female 80 5 80 6 3 II 1 19 Female 80 2 80 7 1 Solitary Mean h SE 152 ± 24 4 ± 0 . 3 118 + 13 6.6 ± .1 2.2 ± 0.4 Transiens 206 Appendix V. Developmental time of s. gregaria (SG) reared with L. migratoria migratorioides (LM), P. viridipes (PV) and E . plorans (EP), exposed (:) to volatiles of L. migratoria, and in controls. Trts Nymphal instars Im. ad Total S/C 1 2 3 4 5 6 Total SG/LM 6 + lc 5 + lab 8 ± lab 8 ± lab 9 + 2b 10 + 2a 46 + 3a 29 + 2b 75 + 6.4 b SG/PV 15 + 0a 4 + lbc 7 ± lb 9 ± la 9 + lb 11 + 2a 45 + 3a 54 + 10a 99 + 10 a* SG/EP 5 + 0c 6 + la 9 ± Oab 6 ± 2b 12 + la 9 + la 47 + 3a 8 + Id 55 + 3 c** SG: LM 10 + lb 6 + la 10 ± la 7 ± 2ab 8 + 2b 9 + 2a 50 + 4a 29 + 6c 79 + 8 b GLM 6 + lc 4 + lbc 7 ± lb 6 ± lb 10 + lb . 33 + 2b 12 + Id 45 + 3 c GSG 5 + led 5 + lab 5 ± lc 5 ± lb 8 + lb . 27 + 2c 22 + lc 49 + 2 c SSG 4 + Od 5 + lab 4 + lc 3 ± lc 7 + lb 12 + la 32 ± lb . . SLM 4 + Od 3 ± 0c 4 ± 0c 8 ± 2ab 12 + la ■ 31 + lb • • SSG: solitary control; Trts = treatment combinations; S: solitarious test locust; / : over. C : gregarious source. In columns, means followed by the same letter are not significantly different (P < 0.05, LSD-test). Im. ad = immature adult; * : 50% reached maturation colour in 34 ± 3 days. The other 50% never did by the end of the experiment. ** : died 8 days after fledging 207 Time (min) Appendix VI. Chromatograms showing phenylacetonitrile (PAN) produced by gregarious fifth- instar nymphal male (A) and female (B) L. migratoria 3 - 4 days after molt (50m, 0.2mm ID Carbowax column). 208 Appendix VII : Compounds identified from the volatiles of adult P. viridipes Staal, 1873 used in the studies of primer effects of pheromones ( + means that the compound is found in that species ). Compound P. viridipes L. migratoria S . gregaria Benzaldehyde + + + Pentanoic acid + Hexanoic acid + Guaiacol + + + Heptanoic acid + Phenol + + + Octanoic acid + Nonanoic acid + Decanoic acid + 209 Appendix VIII. Percent (%) of S. gregaria showing phase change after rearing with different stages of L. migratoria Parameters Nymph/Nymph Immat. /Immat. Immat./Nymph Nymph : Nymph M F Tot. M F Tot. M F Tot. M F Tot. Body colour 75 57 69 100 32 89 80 90 75 100 100 100 Morphometries 27 38 33 • . . . . . 50 50 50 Haemolymph 80 100 81 • . . . . . . • PAN * * * * * * * * * * Eye stripes 44 6 50 . . . . . . 50 0 25 Eye shading 100 13 56 80 75 78 100 100 100 • • . / = mixed rearing; : = exposed to volatiles of L.m. migratorioides. M = males F = Females. PAN = phenylacetonitrile. * : males were grouped for pheromone collection; they produced the pheromone; . : parameter not monitored for this experiment. Tot. : total immat. = immature adult 210 Appendix IX. Mean basal oocyte lengths in maturing females of S. gregaria (ISG) and L. m . migratorioides exposed to volatiles of fifth-instar nymphs (FSG or FLM), conspecific immature, and mature adult (MSG or MLM) volatiles. Combinations Days after fledging (range of ± 1 day) Source / Test 6 11 16 21 26 31 ISG / ISG 2.4 ± 0.2 4.1 ± 0.3 4.9 ± 0.2 5.3 ± 0.3 5.6 ± 0.1 MSG / ILM 1.7 ± 0.6 2.4 ± 0.3 5.0 ± 0.5 4.3 ± 0.3 4.8 ± 0.5 3.0 ± 0.2 MLM / ISG 1.8 ± 0.2 2.8 ± 0.5 3.8 ± 0.9 5.0 ± 0.9 3.2 ± 0.5 . FSG / ISG . 2.7 ± 0.9 4.9 ± 0.8 5.9 ± 0.4 5.6 ± 1.0 4.1 ± 0.8 FSG / ILM 1.0 ± .01 1.8 ± 0.3 5.5 ± 0.0 4.2 ± 0.2 4.9 ± 0.5 3.9 ± 0.5 FLM / ISG . 2.2 ± 1.0 6.5 ± 0.3 5.0 ± 0.6 6.0 + 0.8 5.6 ± 0.1 MSG / ISG . 2.0 ± 0.5 5.2 ± 0.5 6.4 ± 0.7 3.3 ± 0.7 5.4 ± 0.6 MLM / ILM 1.9 ± 0.1 4.9 ± 0.3 5.5 ± 0.0 4.4 ± 0.4 4.7 ± 0.2 4.3 ± 0.1 ILM / ILM 1.0 ± 0.4 3.2 ± 0.0 4.1 ± 0.1 4.9 ± 0.8 4.9 ± 0.2 3.6 ± 0.1 ILM / ILM 2.7 ± 0.8 5.8 ± 0.7 3.0 ± 0.6 4.9 ± 0.5 5.1 ± .04 5.5 ± 0.1 A total of 25 female locusts in clusters (blocks) of 5 locusts were tested for each experiment or combinations. 211 Appendix X. Summarized effects of L. migratoria and S. gregaria on each other's sexual maturation with respect to integumental colour, copulation, oocyte length, using two conspecific effects as references. Combinations Parameters Effects Source Test Colour On ISG On ILM ISG FSG MSG ILM FLM MLM FLM ISG + + . + + + Acceleration FSG ILM . . . - - . Delay MLM ISG - . + + + - intermediate MSG ILM - • • - • + Delay Copulation FLM ISG + + . + + + Accelerate FSG ILM . . . + - . delay MLM ISG - . - + + - Delay MSG ILM - • • - • — Delay Pheromone FLM ISG + + . + + + Accelerate FSG ILM + - . Intermediate MLM ISG + . + + + - Accelerate MSG ILM - • ■ + • + Accelerate Oocyte lengths FLM ISG + + + + + Accelerate FSG ILM . . . + - . Intermediate MLM ISG + . - + + - Delay MSG ILM • • • + • + Accelerate FLM : fifth-instar L. migratoria; FSG : fifth-instar S. gregaria MLM : mature adults L. migratoria; MSG : mature adult S. gregaria.