TAXONOMY, ECOBIOLOGY AND MANAGEMENT OF ON COTTON IN PAKISTAN

BY

GHULAM ABBAS M.Sc. (Hons) Agri.Entomology

A thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY IN AGRICULTURAL ENTOMOLOGY

FACULTY OF AGRICULTURE UNIVERSITY OF AGRICULTURE FAISALABAD, PAKISTAN

2010 To :

The Controller of Examinations,

University of Agriculture,

Faisalabad.

We, the Supervisory Committee, certify that the contents and form of thesis submitted by Mr. Ghulam Abbas, Regd. No. 83-ag-700 have been found satisfactory and recommend that it be processed for evaluation by the External Examiner (s) for the award of degree.

SUPERVISORY COMMITTEE:

1. CHAIRMAN : ------(Dr. Muhammad Jalal Arif)

2. MEMBER : ------(Dr. Muhammad Ashfaq (TI))

3. MEMBER : ------(Dr. Muhammad Aslam Khan)

4. SPECIAL MEMBER : ------(Dr. Shafqat Saeed)

ii

DEDICATED

To My father,

Mahr Sharif Muhammad (May his soul rest in heaven)

Who had to face the bitter realities of the life in his early childhood. He was unborn when his father died. He was only nine months old when his mother also passed away. As an orphan he was brought up by his grandfather Maher Isa (Jesus). He was an illiterate and self-made person but he educated all his six sons in spite of the hardships of his life as a poor farmer. He is no longer in this world to be congratulated on the success of his son but his greatness is still alive and smiling on the fruit of his ambitions and efforts.

iii CONTENTS

CHAPTER TITLE PAGE NO.

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 7

3 MATERIALS AND METHODS 25

4 RESULTS AND DISCUSSION 42

5 SUMMARY 125

REFERENCES 129

APPENDIX 140

iv

TABLE OF CONTENTS

TABLE OF CONTENTS ...... v LIST OF TABLES ...... ix LIST OF FIGURES ...... xii LIST OF APPENDICES ...... xiv LIST OF ANNEXURES ...... xv LIST OF ABBREVIATIONS ...... xvi ACKNOWLEDGEMENTS ...... xvii Abstract……………………………………………………………………………….xviii Chapter No. 1 INTRODUCTION 1.1 Background……………………………………………………………………………1 1.1.1 Agriculture in Pakistan ...... 1 1.1.2 The Importance of ...... 1 1.1.3 The cotton belt of Pakistan ...... 3 1.2 The Problem ...... 4 1.3 Objectives of This Work ...... 6 Chapter No.2 REVIEW OF LITERATURE ...... 7 2.1 The Taxonomic Literature on ...... 7 2.1.1 Mealybug Classification ...... 7 2.1.2 The Identity of Pest Mealybugs in Pakistan ...... 8 2.1.3 The Mealybug Genus Phenacoccus ...... 9 2.2 The Economic Importance of Mealybugs ...... 10 2.2.1 Pest Behavior of Phenacoccus Species Other than CMB ...... 12 2.2.2 The Economic Importance and Spread of Phenacoccus solenopsis ...... 13 2.2.3 The Global Distribution of P. solenopsis ...... 15 2.3 The Biology and Ecology of P. solenopsis ...... 17 2.3.1 The Biology of P. solenopsis in Pakistan ...... 17 2.3.2 Host Plants of P. solenopsis ...... 18 2.3.3 Associated with Mealybugs ...... 19 2.3.4 Natural Enemies of P. solenopsis ...... 19 2.4 The Management of Pest Mealybugs on Cotton in Pakistan ...... 20 2.4.1 Principles of Pest Management ...... 20

v 2.4.2 Chemical Control of P. solenopsis ...... 21 2.4.3 Pesticide Hazards and the Loss of Biodiversity ...... 22 2.4.4 Biological Control of Mealybugs ...... 22 Chapter No.3 MATERIALS AND METHODS ...... 25 3.1 Methods Used in Taxonomic Studies ...... 25 3.1.1 Preparation of Mealybugs for Authoritative Identification ...... 25 Table of contents (continued) 3.1.1.1 Collection and Labeling ...... 26 3.1.1.2 Preservation ...... 27 3.1.1.3 Maceration ...... 27 3.1.1.4 Staining ...... 27 3.1.1.5 Dehydration ...... 28 3.1.1.6 De-waxing and Clearing ...... 28 3.1.1.7 Slide-mounting Adult Female Mealybugs ...... 29 3.1.1.8 Drying Canada Balsam Slide Mounts ...... 30 3.1.2 Observation and Description ...... 30 3.1.3 Identification ...... 30 3.2 Methods Used in Ecobiology Studies ...... 30 3.2.1 Life Span and Life Cycle ...... 31 3.2.2 Sex Ratio Study ...... 31 3.2.3 Reproduction and Developmental Stages ...... 32 3.2.4 Fecundity...... 33 3.2.5 Effect of Host-Plant Species on Fecundity ...... 34 3.2.6 Alternate Host Plants ...... 35 3.2.7 Distribution and Dispersal ...... 37 3.2.8 Overwintering and Carry Over of the Pest ...... 37 3.2.9 Recording Natural Enemies of CMB ...... 38 3.3 Methods Used in CMB Management Studies ...... 38 3.3.1 Host-Plant Resistance to CMB ...... 38 3.3.2 The Impact of Narrow-Spectrum Pesticides and IGRs ...... 39 3.3.3 The Optimum Volume of Sprayable Material ...... 40 3.3.4 The Effects of Additives in Sprayable Material ...... 40 Chapter No.4 RESULTS AND DISCUSSION: 4.1 ……………………………………………………………………...42 4.1.1 The Identification Problem ...... 42 4.1.2. Diagnosis of the Family Pseudococcidae ...... 43 4.1.3 Records of Phenacoccus Species on Malvaceae ...... 43 4.1.4 Morphological Description of the Cotton Mealybug in Pakistan ...... 44 4.1.5 Morphological Variation Between Samples of P. solenopsis ...... 45

vi 4.1.6 Biological Differences Between P. gossypiphilus and P. solenopsis .. 47 Taxonomic Discussion ...... 47 4.2 ECOBIOLOGY...... 49 4.2.1 Biology ...... 49 4.2.1.1 The Life Cycle of CMB ...... 49 4.2.1.2 The Reproduction of CMB ...... 50 4.2.1.3 The Crawler and Wax Secretions ...... 51 4.2.1.4 Mortality of CMB ...... 52 4.2.1.5 The Instar Durations of CMB ...... 53 4.2.1.6 The Life Span of CMB ...... 54 4.2.1.7 The Sex Ratio of CMB ...... 54 Table of contents (continued) 4.2.1.8 The Effect of Host-Plant Species on Fecundity ...... 55 4.2.1.9 Alternate Host-Plants of CMB ...... 60 4.2.2 Ecology ...... 69 4.2.2.1 Study of CMB Developmental Stages at Different Seasons ...... 69 4.2.2.2 Population Dynamics of CMB ...... 70 4.2.2.1.1 Intensive Survey of CMB on Shoe Flower ...... 70 4.2.2.1.2 Intensive Survey of CMB on Cotton ...... 75 4.2.2.3 Distribution of CMB in the Cotton Field ...... 80 4.2.2.4 Distribution of CMB in Pakistan ...... 81 4.2.2.5 Overwintering and Carry Over of CMB in Pakistan ...... 85 4.2.2.6 Natural Enemies of CMB in Pakistan ...... 86 4.2.3 Ecobiology Discussion...... 88 4.2.3.1 Life Cycle Study ...... 88 4.2.3.2 The Number of Instars in CMB ...... 89 4.2.3.3 Sexual Dimorphism in CMB ...... 89 4.2.3.4 Time and Duration of Mating in CMB ...... 89 4.2.3.5 Mode of Reproduction of CMB ...... 90 4.2.3.6 CMB Crawler Emergence and Movement ...... 90 4.2.3.7 Deposition of Wax on CMB Crawlers ...... 91 4.2.3.8 Mortality in CMB ...... 91 4.2.3.9 The Average Duration of one CMB Instar ...... 92 4.2.3.10 The Total Lifespan of CMB ...... 92 4.2.3.11 The Sex Ratio of CMB ...... 92 4.2.3.12 The Effect of Host-Plant Species on CMB Fecundity ...... 93 4.2.3.13 Alternate Host Plants of CMB ...... 93 4.2.3.14 Study of Developmental Stages in Different Seasons ...... 97 4.2.3.15 Population Dynamics of CMB: Intensive Surveys ...... 97 4.2.3.16 Distribution of CMB in Pakistan ...... 99 4.2.3.17 Overwintering and Carry Over of CMB in Pakistan ...... 100 4.2.3.18 Recording the Natural Enemies of CMB ...... 101 RESULTS AND DISCUSSION: 4.3 PEST MANAGEMENT ...... 103

vii 4.3.1 Relative Resistance of Various Cotton Cultivars to CMB Infestation ...... 103 4.3.2 Narrow Spectrum Pesticides and IGR Impacts on CMB and Beneficials . 105 4.3.3 Optimum Volume of Sprayable Material ...... 111 4.3.4 The Effect of Additives in Sprayable Material ...... 112 4.3.5 Pest Management Discussion ...... 113 4.3.5.1 Relative Resistance of various Cotton Cultivars to CMB Infestation 113 4.3.5.2 Narrow-Spectrum Pesticides and IGR Impacts on CMB and Beneficials...... 113 4.3.5.3 Optimum Volume of Sprayable Material ...... 115 4.3.5.4 The Effects of Additives in Sprayable Material ...... 115

Table of contents (continued) 4.3.6 Future Work ...... 116 4.3.6.1 The Origin of CMB ...... 116 4.3.6.2 Factors determining CMB Infestation Levels ...... 117 4.3.6.3 Factors Facilitating Dispersal of CMB ...... 118 4.3.6.4 Factors Facilitating Carry Over of CMB ...... 118 4.3.6.5 Appropriate Pesticide Use...... 119 4.3.6.6 Proposal for an Experiment in Sustainable Control of CMB on Cotton ...... 120 4.3.6.7 The Role of Ants ...... 121 4.3.6.8 Other Associations of CMB ...... 121 4.3.6.9 A Possible Impact of a Change in Microclimate ...... 122 4.3.6.10 Tools for CMB Management ...... 124 Chapter No.5

Summary ……………………………………………………….. 125 REFERENCES ...... 129 APPENDIX: Additional data and details of analyses ...... 140

viii LIST OF TABLES

S. no. Title Page 1. Cotton Area and Yield of the top Five cotton-producing Countries worldwide, 2005-2008 ...... 2 2. Cotton and Textile Sector Growth in Pakistan ...... 3 3. Proportional Area and Production of Cotton in various Provinces of Pakistan for the Year 2006-07 ...... 3 4. Biological Control Agent releases globally against various Pest Orders ...... 24 5. Pesticides tested for Control of CMB on Cotton and the Dose used for each ... 39 6. Summary of the Life Cycle of Cotton Mealybug in Laboratory Conditions ...... 49 7. Mode of Reproduction of CMB Females isolated on Cotton Leaves in vitro ..... 50 8. Instar Durations of CMB on Cotton in the Field, Faisalabad, 6 March to 11 April 2007 ...... 54 9 Sex Ratio determination of CMB on various Hosts in Field and Laboratory Conditions ...... 55 10. Analysis of Variance and Comparison of Means for the Number of Eggs developing inside a dissected, mature Adult CMB Female collected from various Hosts on different Dates ...... 55 11. Average Number of developing Eggs visible in the Body of a dissected, full-sized, field-collected Adult Female in August 2007 ...... 56 12. Average Number of CMB Crawlers in one Batch, in different Months and on various Hosts ...... 58 13. Environmental Data for the Weeks of Study for the Host-related Fecundity Experiment with ten Host-plants at UAF...... 59 14. A List of Alternate Host Plants of CMB, confirmed in the Laboratory ...... 60-62 15. A List of Host Plants of CMB observed in the Field, not confirmed in the Laboratory ...... 62 16. CMB Host Plants listed in Order of Percentage Infestation Level observed during survey of CMB on various Host Plants, 2005-2008 ...... 63-64 17. CMB Host Plants listed in Order of maximum Intensity of Infestation observed during survey of CMB on various Host Plants, 2005-2008 ...... 64-66

ix List of Tables (continued) 18. Year-wise Summary of the Maximum CMB Population Intensity on 20 grams of Fresh Biomass on various Host Plants, 2005-2008 ...... 67 19. Maximum CMB Population Intensity on various Host Plants, observed during Field Surveys of CMB in Pakistan in different Months, 2005-2008 ...... 68-69 20. Average Number of different Stages of the Pest damaging Cotton in three different Months, on the top three inches of a Cotton Twig ...... 70 21. Linear Multiple Regression Models between the CMB Population on Shoe Flower and Ecological Factors, along with Coefficient of Determination Values ...... 74 22. Interactions between various Biotic and Abiotic Factors and CMB Population Growth ...... 79 23. Comparison of Means of the Weekly Population Growth Rate of CMB through the Cotton Season ...... 80 24. Maximum CMB Population Intensities recorded at different Locations in Pakistan on various Host Plants during field surveys during 2005-2008 ...... 83-84 25. Occurrence and average Population of CMB in various Agro-ecological Zones of Pakistan ...... 86 26. List of Beneficial Fauna associated with CMB in the Field in Pakistan ...... 87 27. Comparison of Studies on Features of the Life Cycle of P. solenopsis ...... 88 28. List of reported Host Plants of CMB, not verified personally or in the Laboratory ...... 97 29. Analysis Of Variance of CMB Population on each of ten Cotton Cultivars ..... 103 30. Comparison of Means of CMB Population on each of ten Cotton Cultivars, as an Indicator of Relative Resistance against CMB ...... 104 31. Analysis of Variance for the Percentage Mortality of CMB, recorded 24, 72 and 168 hours after Application of various Pesticides...... 105 32. Comparison of Means for the Percentage Mortality of CMB, recorded 24, 72 and 168 Hours after Application of various Pesticides ...... 106 33. Comparison of Means for the Population of Beneficial Fauna present on Cotton before and after Application of various Insecticides ...... 108 34. Comparison of Orthogonal Contrasts of Different Groups of Pesticides and their Relative Efficacy against CMB ...... 109

x

List of Tables (continued) 35. Comparison of Orthogonal Contrasts of Different Groups of Pesticides and their Relative Effects on the Population of Beneficial Fauna, 168 hours after the pesticide application ...... 110 36. Comparison of Means for the CMB Population present after Application of various Volumes of Insecticide Spray ...... 111 37. Analysis of Variance for the Effects of various Additives in Population Reduction of CMB on Cotton ...... 112 38. Comparison of Means of CMB Population Reduction on Cotton by various Additives to Insecticide Treatments...... 112 39. Average Meteorological Conditions in Cotton Fields at Faisalabad from 1996 to 2008, in three Distinct Periods ...... 122 40. Comparison of Means Analysis of average Relative Humidity in Cotton Fields at Faisalabad from 1996 to 2008, for three Distinct Periods...... 123 41. Comparison of Means Analysis of Monthly Mean Relative Humidity for the period 1996-2008 ...... 123

xi LIST OF FIGURES

S. no. Title Page 1. Map of Pakistan, showing its Position in Asia ...... 1 2. The Global Distribution of Phenacoccus solenopsis ...... 15 3. CMB Infestation on Aerial Parts of a Cotton Plant in Pakistan ...... 46 4. Adult Female CMB on a Cotton Leaf underside, slightly displaced to show the Crawler Sac ...... 51 5. Graph to show in vitro Mortality of CMB ...... 52 6. In vitro Mortality of CMB Immatures, shown in four Sectors based on the Number of Days after hatching ...... 52 7. Number of Development Days in different Instars of P. solenopsis, under controlled Conditions ...... 53 8. Box Plot showing the Range and Mean Number of developing Eggs per dissected Adult Female of CMB, on various Host Plants at UAF in 2007 .... 57 9. Total Number of Crawlers per Female of CMB, on various Hosts in July 2007 ...... 57 10. Average Number of Crawlers per Batch per CMB Female, in different Months, observed on three different Host-plant Species (Cotton, Itsit and Hazardani) ...... 59 11. Average Number of Crawlers per Batch per CMB Female, in different Months, observed on three different Host-plant Species (Cotton, Itsit and Hazardani) in July and December 2006 and May 2007 ...... 60 12. Maximum CMB Population on 20 grams of fresh Biomass of Host Plant observed during Surveys during 2005- 2008 ...... 66 13. Year-wise Summary of the Percentage CMB Infestation of various Host Plants, 2005-2008 ...... 67 14. Averaged Total damage-inducing CMB Population on a three-inch Cotton Twig in May, July and October 2007 at Faisalabad, Pakistan ...... 70 15. Graph to show Population Dynamics of CMB and various Features of the Host Plant, Shoe Flower (Hibiscus rosa-sinensis) ...... 71 16. Graph to show Properties of the Data of Residuals of Population Dynamics of CMB on Shoe Flower (Hibiscus rosa-sinensis) at UAF in 2007, against a Regression Model Fitted Line...... 72

xii List of Figures (continued) 17. Graph of the Residual of the CMB Population per Twig (variable) versus Population of Beneficials (predictor) for CMB on Shoe Flower, against a Regression Model Fitted Line...... 75 18. Graph of the Residual of the CMB Population per Twig (variable) versus percent Relative Humidity (predictor) for CMB on Shoe Flower, against a Regression Model Fitted Line...... 75 19. Population Dynamics of CMB on Cotton (Gossypium hirsutum ) ...... 76 20. Population Dynamics of CMB as compared with cumulative Population Growth, and Growth of the Host-Plant (Cotton) ...... 77 21. Weekly Growth Rate of CMB on ten Cotton Cultivars in 2007 ...... 77 22. Weekly Population Growth Rate of CMB through the Cotton Season, averaged from ten Cotton Cultivars in 2007 ...... 78 23. Different features of the Weekly CMB Population Data through the Cotton Season, averaged from ten Cotton Cultivars ...... 79 24. Graph to show various Features of Cotton Plants infested by CMB ...... 81 25. Graph to show the CMB Infestation Levels at Survey Sites in Pakistan, based on summarized Survey Data for 2005- 2008 ...... 82 26. Distribution of CMB-infested Areas on a Map of Pakistan ...... 84 27. Agro-ecological Zones of Pakistan ...... 85 28. Graph to show the Population Dynamics of CMB on ten Cotton Cultivars ...... 104 29. Graph to show the Effect of different Pesticides on the Population of CMB on Cotton ...... 107 30. Graph to show the Population Dynamics of CMB after various Pesticide Treatments...... 107 31. Graph to show the Population Dynamics of the Beneficial Fauna after various Pesticide Treatments ...... 108 32. Average Population Reduction of CMB on Cotton after spraying different Volumes of Water with Pesticides ...... 111

xiii LIST OF APPENDICES

S. no. Title Page 1. Additional data and details of analyses ...... 136

xiv LIST OF ANNEXURES

S. no. Title 1. Abbas, G., M.J. Arif, S. Saeed and H. Karar, 2009. A new invasive species of genus Phenacoccus Cockerell infesting cotton in Pakistan. Int. J. Agri. Biol. 11:54-58. 2. Hodgson, C.J., G. Abbas, M.J. Arif, S. Saeed and H. Karar, 2008. Phenacoccus solenopsis Tinsley (: Coccoidea: Pseudococcidae), a new invasive species attacking cotton in Pakistan and India, with a discussion on seasonal morphological variation. Zootaxa 1913:1-33.

xv LIST OF ABBREVIATIONS ave. average BMNH British Museum of Natural History BZU Baha ud din Zakariya University, Multan °C degrees Celsius (= degrees centigrade) cm centimeter CMB the pest mealybug on cotton in Pakistan, Phenacoccus solani Tinsley DAS days after sowing Fig. Figure g gram ha hectare IGR insect growth regulator IPM integrated pest management kg kilogram Lab. Laboratory Lbs pounds LAI leaf area index m meter ml milliliter mln. ha million hectares mln. bales million bales mln. ha million hectares mln. kg million kilograms mln. m2 million square meters mm Millimeter misc. miscellaneous NA Not applicable RH Relative humidity UAF University of Agriculture, Faisalabad UCA University College of Agriculture, BZU, Multan

xvi ACKNOWLEDGMENTS

This work was made possible by a scholarship and funding from the Higher Education Commission of Pakistan. I am extremely grateful to my supervisor, Dr. Muhammad Jalal Arif, Associate Professor, Department of Agri. Entomology, University of Agriculture, Faisalabad (UAF), for providing me with facilities, guidance and advice. I am most obliged to my Committee for their guidance and encouragement in spite of other commitments: Dr. Muhammad Ashfaq, (Tamgha e Imtiaz), Professor and Chairman, Dept of Agri. Entomology, and Dr. Muhammad Aslam Khan, Professor and Chairman, Dept of Plant Pathology, UAF; and Dr. Shafqat Saeed, Assistant Professor, University College of Agriculture, Baha ud Din Zakariya University, Multan. Special thanks are due to Dr. Christopher J. Hodgson, National Museum of Wales, Cardiff, UK for exhaustive assistance with description of the pest mealybug in the global context; also to Dr Gillian W. Watson, California Department of Food and Agriculture, Sacramento, California, USA for her encouragement, advice and invaluable assistance in refinement of this thesis to its present form. Dr. Ijaz Pervez, Director General, Pest Warning & Quality Control of Pesticides, Punjab, Lahore kindly provided unrestricted access to official reports. I am grateful to those who sent me specimens, particularly Dr. S. Suresh, Department of Agricultural Entomology, Centre for Plant Protection Studies, Tamil Nadu, and Prof. A.K. Dhawan and Mrs S. Saini, Department of Agriculture, Agricultural University, Ludhiana, Punjab, India.; also to Dr. Douglass R. Miller, ex-USDA, Maryland, USA; Dr. Douglas J. Williams, ex-BMNH, London, UK; and Dr. Yair Ben-Dov, Bet Dagan, Israel, for their invaluable opinions. Friends and colleagues provided technical support and field assistance, particularly Bashir Ahmad Kirio, Malik Najm-ul-hassan, Jamshed Khalid Sindhu, Afzal Naeem Cheema, and my student fellows Atif Shuja, Qaisar Abbas, Muhammad Rafiq Shahid, Sabir Hussain, Abdul Majeed, Haider Karar, Muhammad Arshad, Muhammad Dildar Gogi, and Unsar Naeemullah. The moral support of my family was essential, particularly that of my mother, Alam Khatoon, who always prayed for my success, my brothers and sisters, my wife, my parents-in-law and my children, Atteya, Madiha, Muneera, Ali, Saliha and Maad, who had to suffer the scarcity of the care and affection they deserved during my absence from home. May Allah Almighty be caretaker of us all!

GHULAM ABBAS IPM Lab., UAF, 8 September 2008

xvii ABSTRACT

A fairly exhaustive survey of morphological characters on the material from Pakistan, India and from several other sites in Asia, have revealed that the morphological variability of the species in Pakistan falls within that of Phenacoccus solenopsis Tinsley, and it is recommended that, until the DNA studies currently being undertaken in the United States are completed, the name P. solenopsis Tinsley(Sternorryncha: Pseudococcidae)should be used for this pest. It is an aerial pest and passes all of its life cycle on aerial parts of the host plats, on tender shoots, leaves, flower buds and even on stem. It has been noted to reproduce sexually. Its mode of reproduction is ovoviviparous ie. it retains the eggs in the body until they are ready to hatch. Number of crawlers is variable and depends upon source of food and environmental conditions. Its life cycle is variable with response to changing environmental conditions, availability of preferred host and its physical health. It is dimorphic insect having a winged male and wingless female. The crawlers can be identified for their sex with a very careful examination under microscope but after second instar the male can be identified with naked eye as the female moults into 3 rd instar whereas, males transforms into prepupa. It is most active earlier instars and most of the dispersal occurs through initial instars. The number of eggs developing in one female is variable depending upon the type of the host plant. Newly emerged crawlers are capable of moving and feeding freely. The newly crawler are tiny (0.5 mm) and relatively transparent, therefore they can hardly be observed with an overview except a careful observation. In 1-2 days size is increased and wax is deposited on the body which increases its visibility. It has been recorded on 55 host plants in 18 families. In addition to cotton tract it has also been recorded in other districts. It has been observed in 20 districts of Punjab, 14 districts of Sindh, one district each from NWFP and Baluchistan, in 6 out of 10 agro ecological zones of Pakistan. These districts have been confirmed by the author, still there are some districts and localities which are prone to the occurrence of this pest. This pest can find a large number of alternate host plants in agro ecological conditions of Pakistan. A number of beneficial insects and spiders have been observed feeding on the pest but these are

xviii wiped out by the indiscriminate spraying process adopted to protect the crops. Relative resistance of the present 10 cotton cultivars shows that they are nearly equal in their response towards infestation of cotton mealy bug Psolenopsis none of them is resistant to this pest. The relative efficacy of the insecticides shows that the pesticides used fall in the following sequence after 72 hours of the application; Methidathion> Profenophos > Methomyl > Imidacloprid > Carbosulfon > Bifenthrin > Acetameprid > Fenpropathrin >Buprofezin > Control. Any how for safety to benificials the sequence was reverse ie., Control> buprofezin> Imidacloprid> Methomyl> Fenpropathrin > Bifenthrin> Acetameprid> Profenophos> Methidathion. The research trial for optimum quantity of spray volume showed that 100 & 120 liters water used in one acre (43560 sq ft) was the optimum volume, more than this was also good but less than this volume resulted in low control as there was no proper coverage of the spray material on the target pest and the pest escaped and resulted in build up of population again. Similarly,it was revealed that there is no additional effect of the additives like detergent, vegetable oil and mineral oil in the spray material, which were recommended as hit and trial from various agencies and persons, rather it affected the plant health so it should be avoided.

xix Chapter No.1 INTRODUCTION

1.1 Background 1.1.1 Agriculture in Pakistan Pakistan is situated in southern Asia, between the latitudes 23° 35’ to 37° 05’ North and longitudes 60° 50’ to 77° 50’ East (GOVPK, 2008) (Fig. 1 below). It is among the most important agricultural countries of the world.

Figure 1. Map of Pakistan, showing its Position in Asia (World Atlas, 2007)

Agriculture is the single largest economic sector in the economy of Pakistan, and a dominant driving force for growth and poverty reduction (FBS, 2008). It contributes about 25% to the national economy. Over 44% of the labor force is employed in the production, processing and distribution of agricultural products, for example cotton, wheat, edible oil, sugar, milk and meat, so agriculture is the main source of income for the rural population and the main source of livelihood for 66% of the population (FBS, 2008). Agriculture contributes to growth as a supplier of raw materials to industry, to a market for indigenous industrial products, and as a substantial source of foreign export earnings. 1.1.2 The Importance of Cotton to Pakistan Pakistan is the fourth largest grower and third largest exporter of cotton (Gossypium hirsutum L., Dicotyledones: Malvaceae) globally. Cotton is an important non-food cash crop in Pakistan and a significant source of foreign exchange earnings. It contributes 22.11% of the value added in agriculture (Naqvi and Nosheen, 2008) and contributed 11 percent to the economy’s gross domestic products (GDP) in 2004-05 (Altaf, 2008). The value added in major crops account for 37.1% of the overall agriculture earnings (GOP, 2005). Pakistan has a reputation for producing high quality fiber exporting raw and value-added products of cotton (Table 1 below).

Table 1. Cotton area and yield of the top five cotton-producing countries worldwide, 2005-2008

Country 2005-06 2006- 07 2007-08 2005-06 2006-07 2007-08 Last name mln. ha mln. ha prel. mln. mln. prel. three mln. ha bales bales mln. years (480 lbs) (480 lbs) bales ave. yield (480 lbs) kg/ha USA 5586 5152 4246 23890 21588 19400 946 China 5500 6000 6100 29500 35500 35500 1235 India 8873 9166 9500 19050 21800 25000 519 Pakistan 3101 3250 32 50 10165 9900 9000 660 Uzbakistan 1432 1430 1450 5550 5350 5500 828 Source: USDA (2008).

Cotton occupies a unique position in the agrarian economy of Pakistan. It plays a vital role in boosting the country’s economy. An increment of one million bales in cotton production translates into a 0.5% increase in the GDP, so this crop is believed to be the lifeline of the economy. In 2004-5, the gross export receipts for Pakistan were estimated at US $14.41 billion, of which cotton and textiles accounted for $8.68 billion or 60% of the total (GOP, 2005). About 70.6% of households in Pakistan are classified as rural and approximately 40.7% are engaged in farming. About 25% of the farmers produce cotton and almost all of them also produce wheat. Nearly 70% of cotton farmers are landowners; the remaining 30% are sharecroppers or have other tenancy arrangements (Altaf, 2008). Cotton provides critical rural income. It constitutes 46% of the entire manufacturing sector, 38% of industrial employment and 31% of the investment sector (Altaf, 2008). Progress made in the cotton sector over the past 57 years to 2007-08 (since the creation of Pakistan in 1947) is presented in Table 2 below, which illustrates the past and present status of cotton in Pakistan.

2 Table 2. Cotton and Textile Sector Growth in Pakistan

Detail 1947-48 2007-08 Growth (%) Area (mln. ha) 1.23 3.54 287.82 Production (mln. bales) 1.10 11.66 1059.55 Yield (kg/ha) 160.00 649.00 45.63 Ginneries 31 .00 1200 .00 3870.97 Textile mills 2.00 458 .00 22900 .00 Mill consumption (mln. bales) 0.04 12.40 31000.00 Yarn production (mln. kg) 6.20 1939.00* 31274.00 Cloth production (mln. m2) 29.50 683.00* 2315.30 Sources: Naqvi and Nausheen (2008); Chang and Sultan (2007). * = estimated.

1.1.3 The Cotton Belt of Pakistan The area in Pakistan where cotton thrives best is called the cotton belt. It lies at about 26-33° North 67-73° East, comprising the central and southern areas of Punjab to Northern Sind (the two cotton-producing provinces of the country). Of the two provinces, Punjab is the main contributor towards national cotton production, growing 87% of the total production of Pakistan. It enjoys four seasons a year: summer is from May to August , autumn is September and October, winter is from November to February, and spring is in March and April. The cotton belt has the most fertile alluvial soils from the River Indus and other rivers (that is, the Jhelum, Chenab, Ravi and Sutluj rivers). In the cotton belt, the general cropping pattern is an alternation between cotton and wheat. Cotton was sown in lines in the 1990s; however, today more than 50% of cotton is cultivated in beds or ridges (Yasin, 2005). Punjab is the largest province of Pakistan and the largest agricultural producer of cotton and other crops. The figures for 2006-07 regarding cotton sowing and production in Pakistan shows the importance of the Punjab among the cotton-producing areas of Pakistan (Table 3 below).

Table 3. Proportional Area and Production of Cotton in various Provinces of Pakistan for the Year 2006-07

Province Area (mln. ha) % Production in mln. bales % (of 375 lbs. each) Punjab 2.4629 80.10 10.350 80.51 Sindh 0.5701 18.54 2.398 18.65 NWFP 0.0003 0.01 0.001 0.01 Baluchistan 0.0416 1.35 0.107 0.83 Total 3.0749 100.00 12.856 100.00 Source: FBS (2008).

3 1.2 The Problem In the decade 1995-2004, while agricultural growth in Pakistan averaged around 4.5% per annum, the rate of growth fluctuated mainly because of adverse weather, crop pest outbreaks, shortage of inputs and lack of attention to sub-sectors other than crop farming (FAO, 2007). In 2004-07, Pakistan’s overall economic growth was rapid (7.0%) (Naqvi and Nausheen, 2008). Between 2002 and 2008, the average agricultural growth rate was 4.1% annually, but the production of cotton declined for three successive growing seasons (2005-6, 2006-7 and 2007-8), by -8.7%, -1.2% and -9.% respectively (Naqvi and Nausheen, 2008). Cotton in Pakistan is attacked by an insect pest complex that is controlled using various management strategies. Successful pest control is one of the major factors determining cotton yield. During 2005-06, the performance of the agricultural sector was weak because the crops sector, particularly major crops, did not perform up to expectations. In 2005, mealybug was first noted as a serious pest of cotton in both the cotton-growing provinces, the Punjab and the Sindh (CCRI, 2006; Parvez, 2008b; Zaka et al., 2006). It was found in 11 of the 18 cotton-growing districts of Punjab province simultaneously (Hodgson et al., 2008), affecting nearly 15000 square kilometers of cotton (Abbas et al., 2006; Saeed et al., 2007) reducing yield by 0.2 million bales {1 bale = 170 kg of lint (Muhammad, 2007)}. Patches of cotton plants heavily infested by the mealybug showed considerable reduction in yield. Heavily infested plants dried out completely, as though they had been sprayed with a defoliator (Abbas et al., 2007a, b; Arif et al., 2006; Arif et al., 2007a, b; Arif & Abbas 2007). Cotton yield in 2005-06 was lower than expected, partly because of the mealybug infestation (CCRI, 2006; USDA, 2008). The pest mealybug overwintered successfully on a number of host plants (perennials, crops and weeds), so an early infestation of cotton was initiated in 2006. It spread rapidly through the cotton-growing areas (Parvez, 2008b; GOS, 2008) and the level of infestation and severity of mealybug damage increased (Khaskheli, 2006). In 2006-07 the agriculture sector registered a sharp recovery, growing by 5.0% in contrast to the preceding year’s growth of 1.6%. Major crops posted a strong recovery from -4.1% in the previous year to +7.6%, mainly because of increased production of wheat and sugarcane. However, cotton production in 2006-07 was 13 million bales, slightly below the 13.02 million bales produced the previous year. Among many causative factors, infestation by cotton mealybug (henceforth abbreviated as CMB) was an important one (Johnson et al., 2008). The presence of the mealybug was noticed even

4 earlier in 2007 than in 2006 (Khaskheli, 2007) and the infestation was particularly damaging (Parvez, 2008b; Khaskheli, 2007). CMB attained the status of a regular pest posing a threat to the entire cotton industry (FBS, 2008). Similar reports have also been received from neighboring countries where it has been recorded as serious pest of cotton (Nagrare et al ., 2009). In 2007-08, excessive rain combined with even more widespread damage by CMB caused cotton yield to fall below the preceding three-year average by nearly 20%. Infestation occurred in patches, causing considerable reduction in yield and even premature dehydration and defoliation. Heavily infested plants dried out as if they had been sprayed with a defoliator (Arif et al ., 2007a, b; Arif and Abbas, 2007). Problems were experienced in the management of CMB using conventional control measures, because little was known regarding its identity, origin, biology or management. Desperate growers responded to the problem with a large number of hit-or- miss, untested chemical control methods. For example, they sprayed the crop with pesticides mixed with additives like surfactants (like washing powders), or mineral or vegetable oils, or sweet attractants of ants for example gur or sugar to pesticide applications; they used mixtures of more than one pesticide used against sucking pests, or mixtures of pyrethroids and other pesticide groups with reported synergism or potentiation; they resorted to overdosing with pesticides; and they used restricted, broad spectrum pesticides. Such practices are expensive, wasteful of resources, and put the health of farm workers and the environment at risk. The Government of Pakistan convened a group of cotton stakeholders, the Central Cotton Management Group (CCMG), comprising researchers, botanists, entomologists, Heads of Pest Warning and Quality Control of Pesticides, Extension wings, representatives of the Irrigation Department, the Seed Corporation, progressive growers, administrative representatives of Provincial and Federal Agriculture Departments and cotton ginners and textile mill owners. This group meets once or twice per month to review the situation of the cotton crop. The pest mealybug problem caused great concern in all the CCMG meetings throughout 2005-2008. The unidentified mealybug was a matter of great concern not only for the Agriculture Departments but also for researchers, extension workers, farmers, ginners, exporters, and all commercial organizations involved with cotton. There was great need to address this issue. So far, no well-defined, environmentally friendly Integrated Pest Management (IPM) strategy has been developed against CMB.

5

1.3 Objectives of This Work The rapid spread of CMB, and escalation of severe damage caused by this pest to the most important cash crop in Pakistan, called for immediate action. Authoritative identification was required, to discover whether CMB was a known species with effective control strategies available. This study is the first attempt to identify and characterize the pest mealybug and to explore possible methods for short-term management of the pest. The biology and ecology of the insect in Pakistan needs to be studied to fine-tune control methods to local conditions and assess their impact on its natural enemies. The aims of this work are to:- (1) identify the pest mealybug attacking cotton in Pakistan (2) document the basic biology and ecology of CMB in Pakistan (3) assess the threat CMB presents to crops in Pakistan and neighboring countries (4) explore the short-term management of CMB using available pesticides (5) document any limitations encountered, that might need to be taken in to account for the effective management of CMB using IPM methods. The overall goal of this study was to provide a factual foundation from which further research could develop effective IPM against the mealybug pest on cotton in Pakistan.

6 Chapter No.2 REVIEW OF LITERATURE

The subject material in this work on the cotton mealybug pest (CMB) in Pakistan falls into three categories: taxonomy, ecobiology and pest management. The review of literature below is presented, therefore, in three sections: 2.1 on the taxonomic literature on mealybugs , 2.2 on the economic importance of mealybugs, 2.3 on the biology and ecology of mealybugs in Pakistan, and 2.4 on the management of pest mealybugs on cotton in Pakistan.

2.1 The Taxonomic Literature on Mealybugs Mealybugs are soft-bodied, sap-feeding insects with mouthparts adapted to piercing and sucking: they secrete a powdery, white wax covering over the body (Osborne, 1994). Mealybugs are of ever-increasing importance in economic entomology. Some species are notorious crop pests and several have caused immense economic damage. In the last thirty years, four major outbreaks of mealybugs have occurred globally due to species being accidentally introduced to countries outside their area of origin, without the natural enemies that normally keep them in check (Williams, 2004). Phenacoccus manihoti Matile-Ferrero is an example; this neotropical species was accidentally introduced to Africa and became a very serious and rapidly spreading pest on cassava (Williams, 2004). Another example is Rastrococcus invadens Williams, which was accidentally introduced from southern Asia to West Africa, where it devastated vast areas of fruit trees (Williams, 2004). 2.1.1 Mealybug Classification Traditionally, mealybugs were placed in the order , suborder Homoptera. However, the term 'Homoptera' is no longer accepted as an order or suborder by taxonomists (Carver et al., 1991; Delabie, 2001; Gullan, 2001), because it is a paraphyletic group. Instead, three suborders have been erected within the Hemiptera and are accepted worldwide (Carver et al., 1991): i. Suborder Heteroptera (true bugs), ii. Suborder Auchenorrhyncha (cicadas, leafhoppers, planthoppers), iii. Suborder Sternorrhyncha (scale insects, aphids, whiteflies), sister group to the rest of the Hemiptera. The suborder Sternorrhyncha contains four superfamilies: Psylloidea (jumping plant lice); Aleyrodoidea (whiteflies); Aphidoidea (aphids) and Coccoidea (scale insects

7 and mealybugs) (Carver et al., 1991). Ben-Dov et al. (2006) established a very useful website, “ScaleNet”, which provided computer-searchable catalogues of 7355 species of scale insects (Coccoidea) described from all over the world, based on the world literature. It included morphological characteristics of all the families including mealybugs, their biology, distribution, host plants recorded, synonymy etc. Within the Coccoidea, the true mealybugs fall in the family Pseudococcidae (Ben-Dov et al., 2006). After the family Diaspididae, which comprises of 32% of the total described species, Pseudococcidae is the second largest family in the Coccoidea, comprising about 28% of species (Miller et al., 2005). Ferris (1950, 1953) documented the taxonomic history of the Coccoidea. He recognized the family Pseudococcidae, which previously had been treated as a subfamily of the family Coccidae (=Coccoidea). He gave a detailed account of the then-recognized families, and illustrated numerous species in 21 genera of the family Pseudococcidae. Williams (2004) reviewed the Pseudococcidae of southern Asia (Bangladesh, Bhutan, Brunei, Burma, Cambodia, India, Indonesia, Laos, Malaysia, Maldives, Nepal, Pakistan, Philippines, Singapore, Sri Lanka, Thailand and Vietnam), comprising 354 species in 62 genera. He gave identification keys to genera and species and detailed morphological descriptions of the species found so far, including 147 species and six genera new to science. Brief accounts of the economic importance and major breakouts of the invasive species were also given. Coverage of each species included the distribution, host plants, and notes on the biology and economic importance when known. 2.1.2 The Identity of Pest Mealybugs in Pakistan The term mealybug is familiar in Pakistan, mostly with regard to a pest of mango and other orchard crops, known as mango mealybug or giant mealybug. Previously identified as stebbingii (Stebbing), the orchard pest is now known to be Drosicha mangiferae Green (Sternorrhyncha: Coccoidea: Monophlebidae). It is a giant mealybug (family Monophlebidae, until recently part of family sensu lato). The mealybug pest damaging cotton in Pakistan (CMB) belongs to the family Pseudococcidae, different from the giant mealybug pest on mango and other orchard crops in Pakistan (Abbas et al., 2006). Tanwar et al. (2007) reported on recent mealybug infestations on various economic crops in India, and provided a short field identification key to the nine major pest mealybug species they found. Out of the nine reported species, eight belong to the Pseudococcidae and one to the Monophlebidae.

8 During this study, CMB was recognized as a species belonging to the mealybug genus Phenacoccus (Abbas et al., 2006) . A detailed morphological study of CMB by Hodgson et al. (2008) led to the conclusion that CMB is a known mealybug species, Phenacoccus solenopsis Tinsley (Tinsley, 1898), native to the New World. Workers in India, which is affected by CMB as well, also concluded that its identity was P. solenopsis , an introduced species of neotropical origin (Nagrare et al., 2009). However, not all were in agreement in India, as Bambawale (2008) thought that CMB might not be an introduced species – although he referred to it by the name P. solenopsis, which is a species of neotropical origin. Williams and Granara de Willink (1992) noted that in microscopic morphological details, P. solenopsis is very similar to P. solani Ferris and P. defectus Ferris (also native to the New World). However, live adult females of P. solenopsis generally have paired dark spots or stripes dorsally, whereas the other two species appear to be uniformly white dorsally (Miller et al., 2005). During this study, small morphological differences were found between the description of P. solenopsis in Williams and Granara de Willink (1992) (in which multilocular pores are limited to the mesal areas of the abdomen) and specimens of CMB collected and reared in this work (which often had multilocular pores on the ventral submargins of the abdomen). A description of CMB as a species new to science, P. gossypiphilus , was therefore submitted for publication but took a long time to appear in print, as Abbas et al. (2009) (Annexure 1). Meanwhile a detailed morphological study subsequently failed to find any consistent morphological differences between CMB and New World specimens of P. solenopsis [Hodgson et al., 2008 (Annexure 2)], leading to the decision to use the name P. solenopsis for CMB. Hodgson et al. (2008) was the later work, but it was published very quickly in an electronic journal and therefore became available before the description of P. gossipiphilus was published . 2.1.3 The Mealybug Genus Phenacoccus The genus Phenacoccus was erected by Cockerell (1893) to accommodate 17 mealybug species. Ferris (1950) provided a diagnosis, history and synonymy of the genus, and additional coverage of Phenacoccus was given in Ferris (1953), in which 18 species were mentioned but only 11 were illustrated. According to Ben-Dov et al. (2007) there are currently 186 described mealybug species placed in the genus Phenacoccus worldwide (or 183 species according to BayScience Foundation, 2008).

9 Cox (1987) recorded 114 species of Pseudococcidae in 28 genera from New Zealand, including two species in the genus Phenacoccus that were believed to be exotic introductions. She provided identification keys to the genera and species, and brief descriptions and illustrations of the species covered. Little had been reported on the mealybugs found in Pakistan either because of the scarcity of research on this subject, or a failure to share any such knowledge with the international community. Williams (2004) described and illustrated 14 species of Phenacoccus from southern Asia, including two species of Phenacoccus from Pakistan new to science: P. divaricatus Williams (collected from Ghari Dopatta on Olea cuspidate) , and P. puncticulatus Williams (collected from Kohala on Arundo donax).

2.2 The Economic Importance of Mealybugs Mealybugs are sap-feeding insect pests that inflict losses to their host-plants in several ways (Osborne et al., 1994; Gullan and Kosztarab, 1997; Oetting, 2004; Williams, 2004; Watson and Kubiriba, 2005; Abbas et al., 2008). They: i. Suck sap from the host-plant phloem tissue, removing biomass and water ii. Egest sugary honeydew that fouls plant surfaces, blocking stomata, so impeding gas exchange, respiration and photosynthesis, and hence yield. iii. The honeydew forms a medium for the growth of sooty mold, blocking light from the leaves, so impeding photosynthesis. iv. Some mealybug species transmit plant virus diseases while feeding v. The feeding punctures facilitate infection by secondary diseases vi. Mealybugs on live plant material in trade present a quarantine threat that may prevent the export, or cause the rejection of, fresh produce vii. Waxy mealybugs impair the aesthetic value of ornamental plants, presenting a serious threat to interior landscaping and greenhouse crops. Many mealybug species have been reported attacking vegetables, fruit trees, glasshouse- and field-crops around the world (Cox, 1987; Daane et al., 2008; Osborne et al., 1994; Oetting, 2004; Rothwangle et al., 2004; Williams, 2004; Watson and Kubiriba, 2005; Culick and Gullan, 2005; Moghaddam, 2006; Zaka et al., 2006; Tanwar et al., 2007). Economic losses from mealybug damage to crops can be very substantial (Zeddies et al., 2001). A number of polyphagous mealybug species in various genera were reported from southern Asia by Williams (2004); the most important of these species are mentioned below.

10 • Antonina maritima Ayyar, reported from India on the nodes of Cynodon dactylon (khabbal grass). • Dysmicoccus brevipes Cockerell, common on pineapples worldwide and reported from Malaysia. • Ferrisia virgata (Cockerell), a widespread and polyphagous species known as a serious pest of cotton in India. • (Green), the most economically important of the polyphagous mealybugs of southern Asia, especially in India. • Nipaecoccus viridis (Newstead), a polyphagous mealybug common throughout southern Asia. • Phenacoccus madeirensis Green, a common polyphagous mealybug, was recorded from southern Asia for the first time by Williams (2004). • Phenacoccus saccharifolii (Green), the sugarcane mealybug, is known from India, Nepal and Pakistan. • Phenacoccus solani : most records of this pest in Williams (2004) were from quarantine interceptions, which showed that this species had been introduced to southern Asia and had become established there. • Planococcus citri (Risso), a cosmopolitan species, was one of the first species to have been recorded as pest in southern Asia. • Pseudococcus longispinus (Targioni Tozzetti) was one of the first mealybugs ever reported from southern Asia; severe infestations have been recorded on black pepper in India. • Saccharicoccus sacchari (Cockerell), which probably originated from New Guinea, is probably distributed wherever sugarcane is grown. • Trabutina serpentina (Green) is confined to Tamarix spp., and has potential for use in the biological control of Tamarix spp. in countries where it has attained weed status. Gullan and Kosztarab (1997) reviewed the occurrence, pest behaviour and distribution of scale insects on various plants around the world and concluded that in spite of being wingless, widespread introduction of mealybugs between countries is possible through various means. Miller et al. (2005) listed all the mealybug species accidentally introduced to the USA; they reported on 356 species.

11 Major state universities in the U.S. states of Florida, Georgia, California etc. provide on-line information about the more notorious pest mealybug species in those states, including information on invasive species of Phenacoccus in the U.S.A. 2.2.1 Pest Behavior of Phenacoccus Species Other than CMB There are about 186 species in the genus Phenacoccus (Ben-Dov et al., 2006) , quite a few of which are polyphagous. The majority of the species are native to the neotropics (Ben-Dov et al., 2006), although some are native to other parts of the world including southern Asia (Williams, 2004). Some, like P. madeirensis and P. parvus Morrison, have spread outside their natural ranges and have become cosmopolitan pests (Williams, 2004). Others have achieved pest status on a single continent, like P. manihoti Matile-Ferrero, a very important invasive species damaging cassava in Africa, which originated from the Neotropical Region. Its introduction outside its native region, in the absence of its natural enemies, resulted in its unchecked multiplication and the devastation of cassava in equatorial Africa (Williams and Granara de Willink, 1992; Watson and Kubiriba, 2005). P. manihoti was also reported as a cassava pest in Argentina (Granara de Willink, 2003). Apparently it is moderately polyphagous, being found on hosts in at least 14 plant families (Ben-Dov et al., 2006) and has been taken at quarantine inspection in the USA from Cuba (on Sida ), Dominican Republic (on Euphorbia ), Ecuador (on Cucurbita ) and Mexico (on many hosts) (Williams, 2004). Miller et al. (2005) listed all the mealybug species introduced to the USA accidentally, including four species of Phenacoccus: P. aceris (Signoret), P. parvus Morrison, P. dearnessi King and P. graminicola Leonardi. The first two of these species are polyphagous. Several Phenacoccus species have been intercepted on imported plant material at US ports-of-entry, including: P. avenae Borchsenius (from Turkey on bulbs), P. azaleae Kuwana (from Japan on Azalea ), P. manihoti (from Central Africa and South America on Manihot ) and P. pergandei Cockerell (from Japan and Korea on Diospyros , Magnolia , Malus , Prunus , Punica , and Rhododendron ) (Williams and Granara de Willink, 1992; Miller et al., 2005; Culik and Gullan, 2005; Ben-Dov et al., 2008). In his list of economically important mealybugs in southern Asia, Williams (2004) mentioned three species of Phenacoccus: P. madeirensis, P. saccharifolii and P. solani (see the list above). Of these species, P. madeirensis and P. solani are introductions from the New World; while P. saccharifolii is native to the Indian subcontinent (Ben-Dov et al., 2006).

12 A number of coccidologists worldwide are involved in ongoing research on Phenacooccus species due to their economic importance and pest potential (G.W. Watson, 2009, personal communication). 2.2.1 The Economic Importance and Spread of Phenacoccus solenopsis Phenacoccus solenopsis was described originally from the U.S.A. (New Mexico) by Tinsley (1898) and remained known only in the U.S.A. (where it is widespread in the southern and eastern states) until 1992. This suggests that the species is native there. It was not reported as a serious pest of any economic crop until 1990 (Ben-Dov, 2008), when Fuchs et al. (1991) reported it damaging cultivated cotton in Texas, USA and infesting 29 other host species in 13 plant families. In 1992, P. solenopsis was reported from Central America, the Caribbean, and Ecuador (Williams and Granara de Willink, 1992). It was also reported from the Caribbean region (Barbados, Cuba, Dominican Republic, Jamaica and Panama) by Watson and Chandler (2000). Subsequently, P. solenopsis was reported from a state park in Florida, USA, on caesar weed [Urena lobata L. (Malvaceae)], where a severe infestation was found on 80% of several hundred plants (Halbert, 2000). Then it was reported from Chile in 1995-1997 as a pest on Papino (Solanum muricatum Aiton, Solanaceae), where it was controlled using pesticides (Patricia, 2002; Larraín, 2002). Similarly invasive behaviour of P. solenopsis was observed when it was reported from Argentina (Granara de Willink, 2003). P. solenopsis was reported for the first time from Brazil (Espírito Santo state) by Culik and Gullan (2005) as a pest of tomato (Lycopersicon esculentum Miller, Solanaceae) and as being very common on host plants in the plant families Amaranthaceae and Caricaceae. In 2005, P. solenopsis was found Asia for the first time, in Pakistan (GOVPK, 2005) . Heavy infestations on cotton were found in the cotton-growing areas of Pakistan (Punjab and Sindh) (GOVPK, 2005; GOS, 2008; Parvez, 2008b; Zaka et al., 2006). It was found in 11 of the 18 cotton-growing districts of Punjab province simultaneously - Faisalabad, Rahim Yar Khan, Bahawal Pur, Lodhran, Multan, Muzaffar Garh, Rajan Pur, Dera Ghazi Khan, Layyah, Vehari and Khanewal (Hodgson et al., 2008), affecting nearly 15,000 square kilometers of cotton (Abbas et al., 2006; Saeed et al., 2007). The infestation was widespread (Khaskheli, 2006) and devastating (Khushk and Mal, 2006a and b). Cotton yield in 2005-06 was lower than expected, partly due to the mealybug infestation (CCRI, 2006; USDA, 2008). In 2006, after overwintering successfully on various perennials, crops and weeds, an early infestation was initiated and the mealybug spread rapidly through the cotton-growing areas of Pakistan (Parvez, 2008b; GOS, 2008)

13 and the level of infestation and severity of mealybug damage increased (Khaskheli, 2006). The mealybug was noticed even earlier in 2007 than in 2006 (Khaskheli, 2007), and the infestation was particularly damaging (Parvez, 2008b; Khaskheli, 2007). In 2006- 7 cotton production fell slightly below that of the previous year (from 13.02 to 13 million bales) (Johnson et al., 2008). CMB had become a regular pest, threatening the entire cotton industry in Pakistan (FBS, 2008). In 2007-08, excessive rain and even more widespread damage by CMB caused cotton yield to fall below the preceding three-year average by nearly 20%. Patchy infestations caused considerable reduction in yield and even premature dehydration and defoliation. Heavily infested plants dried out as if they had been sprayed with a defoliator (Arif et al., 2006; Abbas et al., 2007a and b; Arif et al., 2007a and b; Arif and Abbas 2007). Then CMB was reported from India, where it was initially considered a minor pest but subsequently emerged as a major pest posing a severe threat to their cotton crop. The mealybug has been reported from almost all the cotton-growing centres of India (NCIPM, 2008), particularly from the Indian states of Punjab (Yousuf et al., 2007; Siani and Ram, 2008) and Haryana (Monga et al., 2008). The incidence and severity of the CMB outbreak in the Indian Punjab was worst in villages adjoining the Pakistan boundary, where the infestation consisted of a mixture of species, mostly P. solenopsis and M. hirsutus (Monga et al., 2008). P. solenopsis has had a devastating impact on cotton production in India (NCIPM, 2008; Nagrare et al., 2009). Similar reports of damage by P. solenopsis were received from some vegetable- growing areas of Thailand (USDA, 2008; Bambawale, 2008; Hodgson et al., 2008; Tanwar et al., 2007). In Iran (Shiraz), another mealybug closely related to CMB (identified as P. solani ) was found on the leaves and roots of Chrysanthemum morifolium (Mughaddam, 2006); it has been suggested that this record should be treated as a morphological variant of P. solenopsis [Hodgson et al. (2008), Annexure 2]. The UK Plant Health Interception and Outbreak Chart, 13 May – 02 June 2007, documented the interception of two live specimens of P. solenopsis from Ghana (UK, 2007). P. solenopsis has also been reported from Nigeria on Hibiscus rosa-sinensis L. (Dicotyledones: Malvaceae) (Akintola and Ande, 2008). An up-to-date list of mealybugs occurring in Colombia was published that documented 78 species; 11 of these were new records for Colombia, including P. solenopsis (Kondo et al., 2008). Most recently P. solenopsis was reported to be a potential pest in 17 provinces of the Peoples’ Republic of China (Wang et al., 2009).

14 2.2.3 Global Distribution of P. solenopsis Phenacoccus solenopsis (as presently understood) has been reported from other countries. Its global distribution is mapped in Fig. 2 below, followed by a list of the literature records on which the map is based. Locality records in the list are listed to state or province level only, although some of the references provide more locality details.

Adapted from MapQuest (2008).

Figure 2. The Global Distribution of Phenacoccus solenopsis

Asia China : Anhui; Fujian; Guangdong; Guangxi; Guizhou; Hainan; Henan; Hubei; Hunan; Inner Mongolia; Jiangsu; Jiangxi; Shandong; Yunnan; Zhejiang. Possibly also present in: Beijing; Chongqing; Gansu; Hebei; Liaoning; Ningxia; Shaanxi; Shanxi; Shanghai; Sichuan; Tianjin and Xinjiang Uygur (Ben-Dov et al., 2009, based on Wang et al., 2009). India : Andhra Pradesh; Gujarat; Haryana; Karnataka; Madhya Pradesh; Maharashtra; Punjab; Rajasthan and Tamil Nadu (Nagrare et al., 2009). Pakistan : Punjab and Sindh (GOVPK, 2005; GOS, 2008; Hodgson et al., 2008; Parvez, 2008b; Zaka et al., 2006). Thailand : (Hodgson et al., 2008; UK, 2007). Taiwan : (Hodgson et al., 2008).

15 Pacific Region Galapagos Islands (part of Ecuador): (Hodgson et al., 2008) New Caledonia : (Hodgson et al., 2008). Africa Nigeria : (Akintola and Ande, 2008; Hodgson et al., 2008). Benin : (Hodgson et al., 2008). Cameroon : (Hodgson et al., 2008). North America USA : Arizona; California; Idaho; New Jersey; New Mexico; Texas (Hodgson et al., 2008); District of Colombia; Michigan; Mississippi (Ben-Dov, 1994); Illinois; Maryland; New York; Ohio; Virginia (Ben-Dov et al., 2009, based on Kosztarab, 1996); and Florida (Halbert, 1999). Mexico : Veracruz; Hidalgo; Zacatecas; Tamaulipas; Guanajuato (Hodgson et al., 2008). Central America Guatemala : (Hodgson et al., 2008). Panama : (Williams and Granara de Willink, 1992; Watson and Chandler, 2000). Caribbean Region Barbados : (Watson and Chandler, 2000). Cayman Islands : Grand Cayman (Hodgson et al., 2008). Cuba : (Williams and Granara de Willink, 1992). Dominican Republic : (Hodgson et al., 2008; Watson and Chandler, 2000). Guadeloupe : (Ben-Dov et al., 2009, based on Matile-Ferrero and Etienne, 2006). Haiti : (Ben-Dov et al., 2009, based on Perez-Gelabert, 2008). Jamaica : intercepted at Miami, USA (Hodgson et al., 2008; Watson and Chandler, 2000). Martinique : (Ben-Dov et al., 2009, based on Matile-Ferrero and Etienne, 2006). St Martin and St Barthelemy : (Ben-Dov et al., 2009, based on Matile-Ferrero and Etienne, 2006). South America Argentina : (Granara de Willink, 2003). Brazil : Rio de Janeiro (Hodgson et al., 2008); Espírito Santo (Culik and Gullan, 2005). Chile : (Patricia, 2002; Larraín, 2002). Colombia : (Kondo et al., 2008) Ecuador : (Williams and Granara de Willink, 1992).

16 2.3 The Biology and Ecology of P. solenopsis Remarkably little has been published about mealybug biology in Pakistan. This is particularly true of CMB (P. solenopsis) because it only became a pest recently, although American cotton (Gossypium hirsutum L., Dicotyledones: Malvaceae) has been grown in Pakistan since the 1960s. A recent review article (ICAC Recorder, 2008) commented on the biology of P. solenopsis on cotton, on some of its important host plants, and on symptoms of damage caused by the pest. Akintola and Ande (2008) described the morphological features and life cycle of P. solenopsis on another host plant (shoe flower, H. rosa-sinensis) in Nigeria. 2.3.1 The Biology of P. solenopsis and other species in Pakistan Mohyuddin and Mehmood (1993) studied the life cycle of the giant mealybug on mango (Drosicha mangiferae, family Monophlebidae) and reported that the diapausing eggs which remain in the soil for six months are the major weak point in the life cycle of the pest for control purposes. However, giant mealybugs are not true mealybugs (family Pseudococcidae) and their biology is not the same (G.W. Watson, 2009, personal communication). Khushk and Mal (2006a and b) provided information on the biology of mealybugs on cotton in Pakistan, but they had confused CMB with another mealybug, Maconellicoccus hirsutus . Subsequent articles discussed the genera Phenacoccus and Maconellicoccus in detail, clarifying that the new mealybug pest on cotton was a Phenacoccus species, not M. hirsutus (Abbas et al., 2006; Arif et al. , 2007a and b). Phenacoccus solenopsis has been shown to be sexually dimorphic, having short- lived, winged males and longer-lived, wingless, larviform females (Abbas et al., 2008). It was found to reproduce sexually, producing live young instead of laying eggs. The eggs are retained in the body until they are ready to hatch, a phenomenon known as ovoviviparity (Arif et al., 2007a and b; Yousuf et al., 2007, Abbas et al., 2008). Nakahira and Arakawa (2006) studied variation in the life cycle of P. solani (an invasive species closely resembling P. solenopsis) at 20°, 25° and 30°C in a photoperiod of 16 hours light : 8 hours of darkness at Kochi University, Japan. They found that the total developmental periods of the immature stages and the pre-reproductive period of the adults decreased significantly with increased temperatures, and that the survival rates of the immature stages were high at all temperatures. However, the adults lived significantly longer at 20°C than at higher temperatures, and the number of offspring produced per female was significantly lower at 30°C than at lower temperatures. The net reproductive rate and the

17 intrinsic rate of natural increase of P. solani were highest at 25°C (Nikahara and Arakawa, 2006). Research by Calalatyud et al. (2002) on the feeding of the cassava mealybugs P. herreni Cox and Williams (present in South America) and P. manihoti (the invasive pest in Africa), using the electrical penetration technique, showed that the mealybugs feed primarily on phloem sap. The principal nitrogenous compounds in phloem are free amino acids, often in an exceptionally unbalanced composition. Glutamine and or asparagine are the main amino acids in the phloem sap of various plant species (Calatayud, 2000). In cassava, the amino acids in the phloem sap were so unbalanced that glutamine and asparagine together accounted for 55% of the total. Consequently, amino acid metabolism is crucial in these mealybugs (Calalatyud et al, 2002). Phagostimulants play a very important role in phloem-feeding insects. Sucrose was found to be a strong phagostimulant (Calalatyud et al, 2002), which was one of the factors that led to the successful rearing of the mealybugs. Certain amino acids, either alone or in combination, act synergistically with sucrose as phagostimulants. The studies concluded that some essential amino acids played a vital role in the growth and development of the pests (Calatayud et al., 2002). 2.3.2 Host Plants of P. solenopsis Phenacoccus solenopsis has been recorded on members of 31 major plant genera in 13 families (Ben-Dov et al., 2008). The detailed host list includes the following genera: Atriplex, Suaeda (Chenopodiaceae); Achillea, Ambrosia, Encelia, Enceliopsis, Eriophyllum, Franseria, Helianthus (Compositae); Cucurbita (Cucurbitaceae); Euphorbia (Euphorbiaceae); Lupinus (Fubaceae); Cevallia (Loasaceae); Althaea, Gossypium, Hibiscus, Sida (Malvaceae); Boerhavia (Nyctaginaceae); Orobanche (Orobanchaceae); Rubiaceae; Physalis, Solanum (Solanaceae); Lantana (Verbanaceae); and Kallstroaemia (Zygophyllaceae) (Ben-Dov et al., 2008). Study of P. solenopsis in Pakistan found some important alternate dicotyledonous host-plants. This information was shared in popular articles for the awareness of all concerned, for example, records on shoe flower (Hibiscus rosa-sinensis), okra (Abelmoschus esculentus (L.) , Malvaceae), sunflower (Helianthus annuus L. , Compositae), brinjal (Solanum melongena L. , Solancaeae), tomato (Lycopersicon esculentum ) and some weeds (Abbas et al., 2007a; Arif et al., 2007a and b). Another study documented all the hosts (154 species) on which CMB was found incidentally or in

18 low, medium or high intensity in the agro-ecological conditions of Multan, Pakistan (Arif et al., 2009). 2.3.3 Insects Associated with Mealybugs A number of workers have found a mutualistic relationship, known as trophobiosis, between ants and members of Hemiptera: suborders Sternorrhyncha and Auchenorrhyncha (formerly 'Homoptera') (Delabie, 2001). In the most archaic form of the relationship, ant foragers collect the honeydew casually expelled onto foliage by sap- sucking 'Homopterous' insects; thus this commonest of trophobiotic relationships is facultative. This form of mutualism is extremely diverse and has led to development of a range of physiological, morphological and behavioral adaptations in sap-sucking insects of the ‘Homoptera’ (mainly members of the Sternorrhyncha). The more differentiated trophobioses are symbioses where extreme morphological changes can be observed in the phytophagous insects, whereas the ants show mainly behavioral adaptations, resulting from a long co-evolutionary process (Delabie, 2001). Some ants closely attend mealybugs for the honeydew they egest, and protect the mealybugs from nocturnal predation by spiders (Buckley, 1990). The more aggressive the ants are, the more protection they provide to the sap-feeding insects from predators and hymenopteran parasitoids (Buckley and Gullan, 1991). This relationship has some IPM implications (McKenzie, 1967). Phenacoccus solenopsis was described from the roots of a weed associated with ants of the genus Solenopsis (Tinsley, 1898), which is why it is commonly known as solenopsis mealybug in the USA (Hodges and Hodges; 2005; Ben-Dov et al., 2008). 2.3.4 Natural Enemies of P. solenopsis Tanwar et al. (2007) mentioned some coccinellid such as Cheilomenes sexmaculata (Fabricius), Rodolia fumida (Mulsant), Scymnus coccivora Aiyar and Nephus regularis (Sicard) (Coleoptera: ) as general and important predators of mealybug nymphs. An exotic ladybird , Cryptolaemus montrouzieri (Mulsant) (Coleoptera: Coccinellidae) has been reported to feed voraciously on P. solenopsis Tinsley in India (Nagrare, 2008) and in Pakistan (I. Parvez, 2008). A parasitoid wasp, Aenasius sp. (Hymenoptera: Encyrtidae) has been reported from coastal and sub-coastal areas of Sindh, identified by Dr. John Noyes (BMNH) (Dr. R. Mahmood, CABI Bioscience Pakistan, 2008, personal communication via G.W. Watson, California, USA).

19 2.4 The Management of Pest Mealybugs on Cotton in Pakistan Achievement of effective pest management is the ultimate objective of all basic studies of a new agricultural pest. Integrated Pest Management (IPM), of all the management strategies available, is the most desirable because it uses a system approach to reduce pest damage to tolerable levels through a variety of techniques, and only uses chemical pesticides when necessary and appropriate (van Emden, 2002). This concept has further developed into bio-intensive IPM, in which living organisms such as predators, parasites, fungi, resistant varieties, trap crops etc. are the main components of the IPM program (Dufour, 2001; van Emden, 2002). The biological control of vine mealybug [Planococcus ficus (Signoret)] was discussed by Daane et al. (2008). 2.4.1 Principles of Pest Management Insects are the most abundant and diverse on the planet. Insects that compete with human beings for food, causing economic losses, are termed as pests. Global crop losses due to phytophagous insects, plant diseases and weeds, increased from 34.9% of yield in 1965 to 42.1% in 1988-1990, despite the intensification of pest control measures (Lewis et al., 1997). There are four major problems encountered with conventional pesticides: toxic residues, development of pest resistance, emergence of secondary pests because their natural enemies have been killed, and pest resurgence. The latter three of these are consequences of reliance on interventions that disrupt the ecology, and are of diminishing value because of countermoves of the pest organisms. A switch to targeted, non-toxic pest controls, such as microbial organisms or inundative releases of natural enemies, although helpful in reducing environmental contamination and safety problems, still does not truly address the ecological weakness of conventional pest control using pesticides (Lewis et al., 1997). For the major crops, Pedigo (2003) developed methods to monitor the populations of various pests; to evolve and evaluate various sampling techniques; and to assess the threshold population of the pest which actually inflicts an economic loss. To minimize disruption of the ecological system, a number of measures have been developed to deal with pest problems in an eco-friendly manner (Azam et al., 2002), such as working out the economic threshold level (ETL) of each pest using various sampling methods. In the development of IPM, the ETLs of important pests have been worked out. Biological control has been reported to be a successful, long-term method of management for mealybugs (Williams, 2004) that is costly to develop initially but incurs no further running costs once established. It requires authoritative identification of the

20 pest as well as the parasitoid (Oetting, 2004). Four major outbreaks of mealybugs have been reported globally in last thirty years, all of which have been controlled biologically using natural enemies (insects in most cases, and mites in few cases) (Williams, 2004). The best-known example is the biological control of cassava mealybug, P. manihoti, which had become a severe problem in equatorial Africa; it was successfully controlled by an exotic parasitoid introduced from South America (Zeddies et al., 2001). 2.4.2 Chemical Control of P. solenopsis An early chemical control effort against P. solenopsis in the USA proved unsatisfactory (Fuchs et al., 1991). Although the problem of P. solenopsis on cotton in Pakistan is recent, some work on its chemical control has been done. Zaka et al . (2006) worked on the relative efficacy of different pesticides against CMB in Pakistan. They concluded that Methomyl, Triazophos and Methamidophos were the most effective pesticides against CMB, followed by Imidacloprid. Tanwar et al. (2007) reported on recent mealybug infestations on various economic crops in India, and found nine major pest mealybug species (eight Pseudococcidae and one Monophlebidae). A number of pesticides were tested against these pests: Lamdacyhalothrin (Boxer, 2.5 EC), Bifenthrin (Talstar, 10 EC), Profenophos (Craker, 50 EC), Imidacloprid (Crown, 200 SL), (Alarm, 1.8 EC), (Proclaim, 19 EC), Chlorpyriphos (Lorsban, 40 EC), Mathidathion (Supracide, 40 EC), (Advantage, 20 EC), Acetameprid (Rani, 20 EC) were tested in a laboratory bioassay and then in the field. After 72 hours Profenophos was most effective, followed by Supracide and Talstar (with mortality rates of 68.34%, 65.83% and 48.23% respectively). However, after 168 hours Supracide superceded Profenophos, causing 94.7 % and 92.87% mortality rate respectively (Arif et al ., 2008). Similar results were reported by Saeed et al. (2007), who tested insecticides of different groups against CMB in the laboratory as well as in the field in Pakistan. In the laboratory, using the leaf dip method, Bifenthrin, Profenofos and Chlorpyrifos proved to be the best insecticides for mealybug control, based on the LC 50 . In field conditions, the recommended application rates of Methomyl, Profenofos and Chlorpyrifos provided the best control; lethal time studies proved their efficiency for timely control of this sporadic pest. Profenofos, Chlorpyrifos, Methomyl and Bifenthrin, provided satisfactory control of CMB (Saeed et al., 2007) while more than 90% mortality of the pest resulted from application of Acetameprid and Imidacloprid at double the recommended dose, that is, 250 g/acre and 500 ml/acre respectively (Rasheed Ahmad, 2007, personal communication), where an acre is 43560 sq. ft. Dhawan et al. (2008) found Emmamectin Benzoate most effective

21 against CMB in a bioassay test, followed by chlorantroniliprole, then pyridalyl, nuvaluron, quinalphos, thiodicarb, flubendiamide, acephate and chlorpyriphos, with endosulfan being the least effective. Profenophos, Thiodicarb, Quinalphos, and Acephate gave more than 90% kill of the pest using the recommended doses and 200 litres of water as spray material (Saini and Ram, 2008). 2.4.3 Pesticide Hazards and the Loss of Biodiversity The growth of pest-susceptible, high-yielding varieties of major crops in Pakistan involves widespread use of chemical pesticides. Widespread pesticide use has resulted in contamination of the surface water and soil, threatening sustainable production through health hazards to farm workers and loss of biodiversity (ecological damage) (Suhail and Ahmad, 2006) . In a chemical analysis of surface water samples (rivers, lakes, canals, ponds and tanks), 75% of the water samples taken showed pesticide contamination, and 58% of the tested samples contained pesticide residues above the maximum residue limit identified by the World Health Organization (Chiller and Kumari, 2008). 2.4.4 Biological Control of Mealybugs Many mealybugs are attacked by relatively host-specific parasitoid wasps, making them particularly suitable for biological control (Watson, 2008, Per. Com.). For example, the cassava mealybug (P. manihoti), a neotropical species, was accidentally introduced to Africa in the early 1970s (Alene et al ., 2004), reportedly on infested planting material from South America. The infestation was so serious that within ten years, it threatened to wipe out cassava in sub-Saharan Africa (SSA), the staple food for more than 200 million people. IITA launched a biological control project against the pest. IITA's successful experience with the biological control of the cassava mealybug was later employed to address various other pest problems in SSA. Zeddies et al . (2001) worked out the economics of biological control of P. manihoti in SSA. The data were derived from field, socio-economic impact and financial reports by some international institutions for a period of forty years (1973-2013). They found that, with a reasonable calculation and taking into consideration compound interest, biological control by the parasitoid wasp Epidinocarsis (Apoanagyrus) lopezi (De Santis) (Hymenoptera: Encyrtidae) resulted in a benefit : cost ratio of about 1: 200 when cassava was valued at world market prices, and of between 1 : 370 and 1 : 740 when inter-African country prices were considered. Frank and McCoy (2007) listed a number of biological agents that were introduced deliberately into Florida to control insect pests. They discussed 59

22 invertebrate species, of which at least three had become important biological agents against mealybug pests including a species of Phenacoccus . Of these three important agents, one was Cryptolaemus montrouzieri from Australia, which was released in Florida in 1930 against citrus mealybug, Planococcus citri (Risso) (Pseudococcidae), a mealybug native to Asia. The second biological control agent was the parasitoid wasp, Leptomastix dactylopii Howard (Hymenoptera: Encyrtidae) from the Neotropical Region, which was released in 1940 against P. citri ; however, they suspected that this wasp might have been accidentally introduced earlier, before the deliberate release. L. dactylopii includes some species of Phenacoccus in its host range. The third biological agent reported on was the parasitoid wasp, Pseudaphycus mundus Gahan (Hymenoptera: Encyrtidae) from Louisiana, which was released in Florida in 1932 against gray sugarcane mealybug, Dysmicoccus boninsis (Kuwana) and pink sugarcane mealybug, Saccharicoccus sacchari (Cockerell) (both Pseudococcidae) - although S. sacchari was not found in Florida until 1944. This parasitoid is not monophagous, but includes mealybug species of various genera (including Phenacoccus) among its hosts. Tanwar et al . (2007) provided detailed information about invasive mealybugs found in India. They described the biology, mode of damage, and precautions and practices for invasive mealybug management. They were of the opinion that biological control was the most effective long-term solution to mealybug infestations because the parasitoids and predators are self perpetuating, persisting even when the host mealybug is at only low population densities. As general feeders, they mentioned coccinellid beetles such as Cheilomenes sexmaculata (Fabricius), Rodolia fumida Musant, Scymnus coccivora Aiyar and Nephus regularis (Sicard) as important predators of mealybug nymphs. They said that biological control by release of natural enemies has proved very successful. Among the biological control agents, introduction of the predatory Australian ladybird, Cryptolaemus montrouzieri ; the parasitoid wasps Anagyrus pseudococci (Girault) and Leptomastix dactylopii (Hymenoptera: Encyrtidae) ; the predatory mite Hypoaspis sp., and the entomophagous fungi Lecanicillium lecanii (Zimmerman) and Beauveria bassiana (Bals.-Criv.) Vuill. have been effective in managing mealybug infestations. Hypoaspis sp . is a small, predatory mite that feeds on mealybug crawlers. They concluded that for effective management of mealybugs an integrated approach is best, involving conservation and augmentation of predatory coccinellids together with other control tactics.

23 Zehnder et al . (2007) described in detail various means of arthropod management. Generally they found that with use of different organic management practices, there is an increase in the level of biodiversity within the agricultural production system. Such increases in biodiversity may be at the first or higher trophic levels, and generally are probably compatible with, and supported by, organic agriculture. The increase in plant biodiversity may lead directly to reduced pest densities via the resource concentration hypothesis or trap crop effects. Botanical diversity may also enhance the third trophic level (natural enemies of pests), leading to top-downwards suppression of herbivores. Zehnder et al. (2007) also reported the abundance of natural enemies in several studies of organic systems. On European farms, organic agriculture was associated with a 62% increase in spider density compared with conventional farms. A recent meta-analysis has shown such effects to be robust for species richness and abundance, and that the abundance of predatory insects, particularly carabid beetles, increased under organic conditions while pest populations declined. Dhaliwal (2008) reported 5634 recorded releases of 2119 species of entomophagous to control 597 insect pest species globally (Table 4 below). These resulted in substantial control in 419 (70.2%) cases, whereas full control was obtained in only 212 (35.5%) cases. The primary control agents in the biological control programmes belonged to Hymenoptera (3887), Coleoptera (1168) and Diptera (457), for which successful control was achieved in 12.3%, 9.2% and 8.1% of cases respectively.

Table 4. Biological control agent releases globally against various pest orders

S. Pest order against which No. releases of Percentage of total releases no. releases were recorded natural enemies against the pest category 1. Sternorrhyncha and 2420 42.95 Auchenorrhyncha (“Homoptera”) 2. Lepidoptera 1772 31.45 3. Coleoptera 596 10.58 4. Diptera 533 9.46 5. Hymenoptera 137 2.43 6. Heteroptera 96 1.70 7. Others 80 1.43 Total 5634 100.00 Source: Dhailwal (2008).

24 Chapter No.3 MATERIALS AND METHODS

The broad range of topics studied in this work fall into three categories: taxonomy, ecobiology and pest management. Accordingly, the methods used in this study are presented in three sections below: 3.1 on taxonomic studies , 3.2 on ecobiological studies and 3.3 on pest management studies.

3.1 Methods Used in Taxonomic Studies Authoritative identification of mealybugs requires preparation of the stained cuticle of adult females as microscope slide mounts for detailed examination under high magnification. This was necessary to determine the species identity and to prepare a morphological description of the pest mealybug on cotton (CMB). Samples of CMB from the field and from screen-house cultures were collected, preserved and prepared as microscope slide mounts for morphological study and identification. 3.1.1 Preparation of Mealybugs for Authoritative Identification The following materials were used in making permanent slide mounts of adult female mealybugs, and for their identification: 1. Ethanol of good quality, at 70%, 95% and 100% concentrations 2. Insect collection kit containing: 10x magnifying glass with folding metallic body; Swiss army knife; polythene bags with closing edges; a sharp blade; forceps; a mounted needle with plastic handle; and a fine camel-hair brush 3. Distilled water 4. Fine camel-hair brushes, size no. 1 size 5. Cylindrical glass vials, each 20 ml capacity, 2 cm in diameter with a tight, screw- on lid 6. Large magnifying glass with handle 7. 10% potassium hydroxide (KOH) 8. Bulb Pasteur pipette 9. Acid Fuchsin stain (commercially available) 10. Anhydrous, high quality clove oil 11. Canada balsam thinned with xylene to a consistency of runny honey 12. Xylene 13. Micro-spatula

25 14. Watch glass 15. Slides and cover slips 16. Slide labels 17. Forceps and mounted needles 18. Binocular dissection microscope (Wild M30 with 6x and 16x objectives) 19. Micro-spatula, hand-made from a metallic handle 20. Micro-scissors 21. Syringe (used for few specimens only) A good specimen suitable for authoritative identification and morphological study should have completely clean cuticle; the body should be dorso-ventrally flattened with the cerarii positioned at the margins; and the stain should be well differentiated to make small structures easier to see. The method used for preparing permanent slide mounts was adapted from those used by coccidologists worldwide (Cox, 1987; Hodgson and Henderson, 2000; Hodges and Hodges, 2005; Watson and Kubiriba, 2005). The steps involved were: collection and labeling; preservation; maceration; staining; dehydration; de-waxing and clearing; slide-mount preparation and drying. 3.1.1.1 Collection and Labeling For each CMB sample, mature females, immature stages and males wherever available, were collected. A camel-hair brush was used to gently transfer the specimens into a 20 ml glass vial of 70% alcohol, carried in an insect collection kit for this purpose. Each sample was labeled on the spot with at least the following information: locality, host plant, date of collection and the collector’s name. Collected samples were stored in the shade in the insect collection kit during the field visit before transfer to the laboratory for further processing. At the laboratory, samples were stored in a refrigerator to minimize deterioration before further processing. At each sampling site, the prevailing population of CMB on each host-plant species was noted. The surroundings were also examined for alternate host plants. Photographs were taken and the host-plant, if unidentified, was taken to the laboratory for accurate identification by the botanist. Samples from unknown plants were kept in a notebook with the leaves and flowers intact, even after the leaves dried. More then one sample of each unknown host-plant was collected in this way, to maintain clear host records. Whether the infestation was primary or secondary, damage symptoms and other field characters were noted on the spot also.

26 During the cotton-growing season, CMB was sampled from infested cotton in the field. Infestation hot spots declared by the Pest Warning and Quality Control of Pesticides (PWQCP), Department of Agriculture, Govt. of the Punjab were preferred collection sites. At each site, samples of CMB from alternate host plants were collected outside the cotton-growing season. In cotton-growing districts of the Punjab, surveys were conducted in each ecological zone, as described in 4.2.10 below. To ensure easy access to laboratory facilities, samples collected from cotton zone Punjab were taken to the laboratory of Bahauddin Zakariya University (BZU), Multan. Samples from others parts of the country were taken to the University of Agriculture, Faisalabad (UAF). 3.1.1.2 Preservation Specimens were killed and preserved in alcohol. Samples sent abroad for authoritative identification were collected into 100% ethanol (in case they were used for molecular studies), but specimens to be processed in Pakistan were collected into 70% ethanol (described in 3.2.1 above). Similarly, portions of host-plants were also preserved in this way for study in the laboratory when necessary. 3.1.1.3 Maceration Identification of mealybugs is based on morphological details of the insoluble, chitinous integument. The mealy wax powder secreted onto the body surface obscures morphological characters of taxonomic importance. This wax powder partly dissolved in the alcohol used for preservation, which was removed with a pipette. A small hole was made in the dorsum of each insect and wax, fats, proteins and carbohydrates were hydrolyzed and removed by soaking the adult females in 10% KOH for 6-18 hours at room temperature until the bodies were completely translucent. Sometimes specimens that had been collected in hot summer conditions required gentle heating in KOH. Eggs were pushed out of each body using a blunt mini-spatula, leaving the integument dorsoventrally flattened and completely empty. Then the specimens were removed from KOH and soaked in distilled water to rinse out the KOH and any remaining ionic solutes. 3.1.1.4 Staining Acid Fuchsin was used to stain the chitinous integument, to make tiny pores and other details more visible. This stain turns red in acid conditions but is colourless in alkaline conditions. Specimens were transferred from distilled water into a mixture of distilled water and acidified stain and soaked until all the chitin turned red (15-30 minutes to overnight, depending on the specimens). After staining the specimens were rinsed

27 briefly in distilled water to remove excess stain, leaving some structures darker red than others (a process known as differentiation). Some coccidologists recommend using triple stain (an aqueous mixture of acid Fuchsin, lignin pink and erythrosin); however, erythrosin was not available. Double stain, made using acid Fuchsin with lignin pink, did not work well so it was used in only a limited number (less than 10) of slides in this work. Details for the preparation and use of triple stain [used by FDACS-DPI (Hodges and Hodges, 2005)] are given for general information below. It is a combination of three stains, mixed as follows: i. Aqueous lignin pink (2%), 10 ml (40 drops) ii. Aqueous acid Fuchsin (4%), 5 ml (20 drops) iii. Aqueous erythrocin (2%), 10 ml (40 drops) The mixture of stains (above) should be added to 100 ml Essigs Aphid Fluid (EAF) to form a stock staining solution. Essigs aphid fluid is made as follows (modified from Wilkey, 1962): i. Lactic acid 85%, 20 parts ii. Liquified phenol, 20 parts iii. Glacial acetic acid, 4 parts iv. Distilled water, 1 part One or two drops of stock solution should be added to half a mini-casserole of EAF to stain specimens overnight or even for several days if necessary.

28 3.1.1.5 Dehydration Ethanol was used as a dehydrating agent. Dehydration was completed gradually by soaking the specimen in (i) 70% ethanol for 1-2 minutes and then (ii) 95% ethanol for 15 minutes. This helped to remove any excess stain and resulted in gradual dehydration by slowly and evenly removing water, so reducing the risk of distortion of the integument. In this process alcohol replaced the water inside the specimen, fixing the stain in the cuticle and ensuring the permanence of the archival slide mounts. Specimens were never left overnight in alcohol, as it would evaporate and leave the insects full of air. In a few mounts, the 95% ethanol step was skipped to save time; but the residual water in the resulting Canada balsam mounts turned the mountant milky and made the specimens difficult to study. 3.1.1.6 De-waxing and Clearing Cuticular wax and residual fats in the body were dissolved and rinsed out when fully dehydrated specimens were soaked in xylene. Xylene is the solvent of the mountant, Canada balsam. However, xylene dries up quickly and makes the cuticle brittle. The specimens were therefore transferred to clove oil, which is less volatile than xylene, to rinse out any remaining wax and leave the cuticle relatively flexible. Specimens could be left in clove oil for 10 minutes to several days, but usually they were mounted onto microscope slides soon as possible. After completion of this process only the chitinous cuticle of the specimen remained; all the other body contents had been dissolved and rinsed away. Clove oil, which has an optical density similar to that of Canada balsam and is miscible with it, penetrated the cuticle preparatory to Canada balsam replacing it – a process called clearing. Once the cuticle has been mounted in Canada balsam (see 3.1.1.7 below), the xylene and clove oil gradually diffuse out of the cuticle and are replaced by Canada balsam as the slide-mount dries. This ensures there are no optical discontinuities in the mount. 3.1.1.7 Slide-mounting Adult Female Mealybugs Permanent slide mounts were prepared in very clean surroundings to avoid inclusion of foreign contaminants in the mounts. Canada balsam (clear, transparent, purified tree resin) was used as the mounting medium. If it was too thick it was diluted with xylene to the consistency of runny honey, to ensure that no air bubbles got trapped in the slide preparation.

29 A drop of Canada balsam about 9 mm in diameter was placed in the center of the slide. While observing the process through the binocular microscope, a specimen was removed from the clove oil using a micro-spatula or a mounted needle held horizontally, and transferred into the drop of mountant using a micro-spatula. It was placed with the dorsum upwards and was pressed gently down onto the surface of the slide. If the specimen was left suspended in the mountant it was sometimes difficult to identify. After a few moments to allow any air bubbles to escape, the cover slip was slowly lowered onto the wet Canada balsam, using forceps to grip one corner; the cover slip was allowed to settle under its own weight (not pressed on with any tool). The slide was labeled immediately to reduce any risk of confusion or loss of identity of the sample. The data recorded at the time of collection (the locality, host plant identity, date of collection and collector’s name) and any other relevant information were placed on one label, leaving the other label available for the identification to be noted. Any Canada balsam leaking from the sides of the cover slip was very carefully removed using a swab dipped in xylene, held with forceps. 3.1.1.8 Drying Canada Balsam Slide Mounts Freshly made slides were stored horizontally in the laboratory for drying, covered to protect them from dust and light. The mountant took at least 12 weeks to dry, even if the slides were kept in a thermostatically controlled incubator at a temperature of 35 ˚C. 3.1.2 Observation and description The specimens were ready for examination once the Canada balsam had dried. Each slide was examined at high magnification under a compound light microscope (Kyowa with 4x, 10x, 40x, 100x light ground objectives). The characters observed were noted and an illustration was made using a grid. The taxonomic illustrations in Hodgson et al. (2008) were kindly prepared by Dr. C.J. Hodgson (details in 3.1.3 below) using a camera lucida. 3.1.3 Identification Identifications were made using conventional taxonomic techniques and the most recent literature available. The general morphology of an adult female mealybug was illustrated clearly by Williams (2004). Identification to family level was made using ScaleNet (Miller et al., 2001), which was most useful. For genus-level identification, the most helpful publications were Cox (1987) and Williams (2004). For species-level identification, the most helpful literature was Williams (2004). For further study and

30 expert opinions on our initial findings, batches of specimens were sent to internationally known coccidologists: 1. Dr. Douglas R Miller (retired), Systematic Entomological Laboratory, USDA, Beltsville, Maryland, USA 2. Prof. Yair Ben-Dov, Bet Dagan, Israel 3. Dr. Gillian W. Watson, Insect Biosystematist, CDFA, California, USA 4. Dr. Christopher J. Hodgson, National Museum of Wales, Cardiff, UK. These specimens were also intended to be deposited in various international depositories after authoritative identification. The identification keys used to identify CMB are cited above and detailed identification keys and taxonomic illustrations of CMB (Phenacoccus solenopsis) were provided in Hodgson et al. (2008) (see Annexure 2).

3.2 Methods used in Ecobiology Studies The following aspects of the biology of CMB were studied: life span and life cycle, fecundity and the impact of host-plant identity and season, sex ratio, reproduction and developmental stages, developmental rate in different seasons, reproduction and developmental stages, the impact of host-plant species on fecundity, alternate host-plants, distribution and dispersal, overwintering, and natural enemies. 3.2.1 Life Span and Life Cycle Five fully mature adult CMB females of the same age, each with a cluster of crawlers, were field-collected from cotton and taken to the laboratory. Each female was placed in a cage made of a transparent plastic Petri dish, 5.5 cm in diameter with a reasonably tight-fitting lid. Each female was kept at 25 ±2°C temperature and 65±5% relative humidity (RH). The insect was fed with a fresh cotton leaf (or a leaf of shoe flower (Hibiscus rosa-sinensis ) when cotton leaves were not available). The leaf was changed daily and data was recorded before the change. Any alteration in the shape or morphology of the female was noted daily and the total number of days taken to complete the life process and reproduce again was recorded. The data recorded daily, following the guidelines in Nakahira and Arakawa (2006) were: date of observation; fed on; name of observer; no. of females with crawler sacs; no. of crawlers; no. dead; waxed; remarks. The data was then transformed on a Microsoft Excel spreadsheet and the life history of each parent female was studied for the parameters listed below. The averages and other statistical values were calculated using Minitab 15 software (Zgonc, 2007). 1. Length of life after crawler sac production

31 2. No. of crawler sacs produced in the lifespan of one female 3. Total generation time from hatching to hatching 4. Time taken from hatching to full-sized adult 5. Time taken from hatching to become a reproductive female 6. Maximum no. of crawlers per batch in lab.-reared progeny 3.2.2 Sex Ratio Study A CMB-infested twig (the top 3 cm of a tender, growing cotton shoot) was selected as a source of 100 second-instar CMB. The mealybugs were gently dislodged from the host in the field, using a camel-hair brush, and were kept in a 5.5 cm-diameter transparent plastic Petri dish, feeding on cut leaves, in laboratory conditions. Within 1-7 days the mealybugs moulted - to the third instar in the case of females, or to a prepupa in the case of males. The number of each sex was noted for calculation of the sex ratio. The number that died in the second instar was recorded and the sex ratio was calculated from the remaining population. The host plants were taken from three localities (Mailsi, Alipur and Jatoi) to ensure against the bias of genetic similarity. The host plants at each locality were sunflower (Helianthus annuus L., Compositae), cotton (G. hirsutum) and Aksun [Withania somnifera (L.), Solanaceae] respectively. Three repetitions were made for each host-plant species. The following data were recorded for sex ratio determination of CMB in different seasons: Sr. month 2007; locality; total specimens, no. that died; percentage mortality; no. specimens remaining; male, % M; female, % F. 3.2.3 Reproduction and Developmental Stages Observations were made during the conduct of experiments for the study of alternate hosts and dispersal and occurrence of the pest in the field. The morphological changes that took place in the mealybug during the course of its development were noted in the field and then confirmed in the laboratory. For in vivo studies, each cage was made from a 5.5 cm-diameter Petri dish with the flat surface of each half replaced by a fine net mesh, to allow air exchange. A leaf on a cotton plant in the field was inspected to ensure no insects were present on it. An adult female mealybug was placed on the leaf. Then the bottom and top halves of the cage were placed on either side of the leaf and were held together using metal wire. In this way the insect under study was caged and protected on the leaf under semi-natural conditions. The mortality and changes in morphology of the specimens, including the number of males and female progeny produced, were noted daily until the last specimen in the last Petri dish died. The number of days taken by each individual to complete the instar and to develop its wax covering after moulting was

32 noted. The number and duration of developmental stages, the mortality rate in each instar, and the ratio of males to females produced, were worked out from the history sheet of each Petri dish in the study. The data derived from the history sheets in this way were transformed using Minitab 15 software (Zgonc, 2007) for further statistical analysis. The data were used to work out the following: a. Mode of reproduction and production of offspring b. Time taken for the crawlers to develop a covering of mealy wax c. Movement of the newly emerged crawlers d. No. of developmental instars e. Average time taken in one immature instar f. Sexual dimorphism (occurrence of a winged male and wingless females) g. Mode and preferred time of mating h. Total lifespan of the pest on average and at the maximum. The in vitro study was conducted twice: in 2006 at University College of Agriculture (UCA), Bahauddin Zakariya University (BZU), Multan; and again in 2007 at Integrated Pest Management (IPM) Laboratory, University of Agriculture, Faisalabad (UAF). The conditions maintained in each laboratory were: temperature 25 ±2°C and relative humidity (RH) 65 ±5%, with natural day length. These conditions were subject periodically to scheduled or unscheduled power failures because of load-shedding of electricity. Ten mature adult females, each with a crawler sac, were collected from the field and taken to the laboratory. Each was placed in a 5.5 cm-diameter Petri dish, lined with a water-soaked piece of tissue paper to maintain the relative humidity. The adult and crawlers were fed on a detached cotton leaf cut to fit into the Petri dish; the moist tissue prevented the cut leaf from drying out. The cut leaves were changed daily; the crawlers were transferred gently from the old leaf to the new one using a camel-hair brush. Daily observations were made, as described above for the in vivo experiment in the field. To test the hypothesis that CMB only reproduces sexually, 30 immature female mealybugs were reared individually in Petri-dish cages in laboratory conditions. Out of the 19 females that survived to the adult stage, 14 were not provided with males and the remaining 5 females were provided with males for mating 3-4 times. The number of offspring produced by each female through her lifetime was recorded. (Data was tested under t test using two treatments, male provided versus no male provided.) 3.2.4 Fecundity

33 Five crawler sacs from each of the three host-plant species tested were opened and the number of crawlers in each sac was counted. Repetitions were made on the three hosts, in different seasons and at different localities. The results were statistically analyzed to assess the range of variation and average number of progeny, and to assess the interaction between these factors. The three host-plant species used in this study were: cotton (Gossypium hirsutum L., Malvaceae), Itsit (Trianthema partulacastrum L., Aïzoaceae) and Hazardani (Euphorbia granulata Forssk., Euphobiaceae). The study was carried out at: Alipur: in the cotton-growing area of the cotton zone Multan: in the mixed-crop area of the cotton zone Faisalabad: in the mixed-crop area of the non-cotton zone Data was collected in three different months: July, 2006 the middle of the cotton-growing season October, 2006 the end of cotton-growing season May, 2007 the start of cotton-growing season The data were processed for statistical analysis under factorial design using Statistica 6.0 software. 3.2.5 Effect of Host-Plant Species on Fecundity The effect of host-plant species on CMB fecundity was studied on ten host-plant species collected from Faisalabad (Pakistan). The plant species were not pre-determined initially because at that stage the host plants of CMB were not known. The ten host species found to be infested most frequently and heavily by CMB were selected. The following host-plant species were included in this study: itsit (Trianthema patulacastrum, Aïzoacaeae); tandla [Digera muricata (L.), Amaranthaceae); hazadani (Euphorbia granulata), lady’s finger [Abelmoschus esculentus (L.), Euphorbiaceae]; cotton (G. hirsutum), shoe flower (Hibiscus rosa-sinensis ); gule dupehri (Portulaca grandiflora Hook, Portulacaceae); aksun (Withania somnifera), chilli (Capsicum annuum L., Solanaceae); and lantana (Lantana camara L., Verbenaceae). For each host species tested, a mature adult female was collected from each of five plants. All the females selected were of the same size. Each female was dissected under a binocular dissection microscope to determine the number of eggs developing inside. The total number of eggs was counted and recorded. The number of developing eggs was taken as an indicator of the quality of that host-plant as a food source for CMB to support reproduction and development.

34 In addition, for each of the same ten plant species, a mature adult female was collected from each of ten plants in the field. All the females selected were of the same size. Each female was transferred to a Petri dish cage (described above) on a clean leaf of the same host-plant in the field. These females were caged individually to discover the total number of offspring produced by a single female in semi-natural conditions. The crawlers produced by each female were counted, recorded and destroyed, leaving the reproductive female alone again until she died in the cage. Rearing in this manner provided natural food, air and light, but there was no inter- or intra-specific competition, nor any interaction with natural enemies. The data obtained were analyzed statistically using Minitab 15.0 software for Anova followed by a Tucky HSD test for significant differences between the means. 3.2.6 Alternate Host Plants Various ecological aspects of the alternate host plants were studied in the field. Unidentified host plants were taken to the botanist for authoritative identification. The host-plant species studied were identified, listed, and the infested parts of each host were photographed. More than one locality was studied for each host species recorded, to show that the same findings occurred more than once. If CMB was recorded on the same host at more than five different localities, in each case with the host species harboring all the stages of the pest along with the breeding female, it was included in the host list. The host-plant species were listed in alphabetical order of the botanical families, using an Excel spreadsheet. The S. no. allotted to each host species in the list (Table 14 in 5.1.9 below) was used as the reference number of that host-plant species in subsequent analyses and graphs. The data on CMB infestation levels were standardized in the following ways. For host species recorded at five or more localities (and therefore included in Table 14), the maximum CMB population on 20 grams of fresh biomass of the host plant was recorded. The remaining host plants, found infested by CMB fewer than five times, were allotted a score of 0. Standardization was also necessary because the host-plant species varied in size from tiny plants like hazardani (Euphorbia prostrate Ait.) to large shrubs like shoe flower (Hibiscus rosa-sinensis L.), and because seasonal growth meant that observations on a growing weed having 3-5 leaves in January could not be compared properly with the same weed in April, when it had increased in size by 10-15 times. In order to make the data comparable, values taken per small plant or per upper six inches of the plant or per twig were

35 converted to number of CMB per 20 gram fresh biomass of the host plant. The following conversion formula was used:

Maximum population recorded per sample unit = X Average fresh biomass weight (grams) of unit = Y Conversion factor = X (20/Y) The maximum CMB population per 20 grams of fresh biomass for each host-plant species was compared statistically using Minitab 15 statistical software. The data for different years, different months of observation and different districts visited were summarized with descriptive statistics to facilitate viewing the results. In some cases where CMB infestation could not be confirmed in the field, the host plants were taken to the laboratory. Then the host-pest relationship could be confirmed by rearing an adult female CMB on cut pieces of the subject host plant in the laboratory using Petri dish cages. The growing tip of an un-infested plant was placed in a Petri dish daily, or on alternate days, under laboratory conditions at 25 ±2°C and RH 65 ±5%, and the mortality or establishment was observed daily, as in 3.2.3 above. Three replicate tests were made for each host-plant species. If the pest completed its life cycle and produced a crawler sac again on the host plant, it was recognized a host of the pest mealybug. If the crawler died or failed to mature and breed it was declared a non-host plant. Some host reports were received from reliable sources like Pest Warning or Quality Control of Pesticides or research and extension personnel working in the field, but could not be confirmed in the laboratory. These records were incorporated in the list of hosts. In this way three lists of CMB host-plant species were compiled: a. Confirmed host plants: host plants that were found in 5 or more different localities, or were confirmed by the author in laboratory rearing studies. b. Observed host plants: host plants that were observed in the field but were not confirmed by laboratory rearing, or were observed at fewer than 5 localities, since these might be transitory hosts. The botanical names and families were determined, as far as possible, by the botanist (Department of Botany, UAF). c. Reported host plants: this list was based on the observations of other technical persons (entomologists) where the author was unable to confirm the record himself because of shortage of time or resources or lack of access. These hosts were listed together with the source of the information (reporting agency or

36 person) and locality, so that future researchers can confirm the records if necessary. 3.2.7 Distribution and Dispersal The distribution and dispersal of CMB was studied in two different ways: (a) intensive surveys and (b) extensive surveys. a. Intensive surveys Intensive surveys were conducted in the experimental field at the Research Area, Department of Agri. Entomology, University of Agriculture, Faisalabad, using five plants of shoe flower (Hibiscus rosa-sinensis) in order to make five repeats. Shoe flower is a perennial plant, so the study was carried out throughout the year. Observations were made weekly over 50 weeks (from 7 January to 30 December 2007). In May and again in June, one observation was missed in order to complete other field activities. The following parameters were studied in each observation: average height of the plant in inches; number of major branches (of one inch diameter) on each plant; number of twigs on branches 1 inch in diameter; number of twigs capped in white wax because of infestation of CMB; average population of mealybugs per twig; and average population of beneficial insects per twig. For each plant, in order to assess the populations of mealybugs and beneficial insects, three twigs were taken at random from each of three randomly selected major branches. In situ counts of mealybugs were made and then averaged. The data recorded were compiled in a Microsoft Excel spreadsheet. The averages and other statistical values were calculated using Minitab 15 software (Zgonc, 2007). The aggregate average of the 5 selected plants under study was presented graphically using Microsoft Excel. b. Extensive surveys These surveys were conducted routinely throughout the cotton-growing tracts of the Punjab (by the Directorate General of Pest Warning and Quality Control of Pesticides, Punjab) and the Sindh (by the Govt. of Sindh Agriculture Department). The findings were analyzed to determine the dispersal and extent of infestation of the pest in these areas. The reports were checked personally by the author twice in each cotton- growing season (June to November) to ensure validity. Similar, personal confirmation was made of 20% of any uncertain or extraordinary situations reported. In case extraordinary situations were not reported, the fields were checked at random also.

37 3.2.8 Overwintering and Carry Over of the Pest Field surveillance of the pest was carried out throughout the year in the intensive and extensive surveys (see 3.2.7 above). Where overwintering was confirmed, this information was shared with the stakeholders; as a result, campaigns were launched by the departments concerned, to minimize the carry over of the pest. Overwintering was observed in the different various cropping systems used in the different agro-ecological zones of Pakistan. 3.2.9 Recording Natural Enemies of CMB During the course of surveillance of the pest in cotton-growing areas (for example, in 3.2.7 above), the observed and associated fauna, parasites and predators (insects, mites, spiders, fungi etc) were recorded, together with brief observation notes. The unidentified natural enemies were deposited at the Department of Agri. Entomology for isolation and identification by later researchers if required.

3.3 Methods Used in CMB Management Studies This project was a baseline study, so the work on CMB management was confined to investigation of possible methods of short-term management only. Long-term management - which is vital, unavoidable and of immense importance for sustainable and environmentally friendly agriculture - was beyond the scope of this project. The development of sustainable long-term CMB control will fall to later researchers. The following aspects of CMB short-term management were studied: host-plant resistance to CMB; the impact of narrow-spectrum pesticides and IGRs on CMB and its natural enemies; the optimum volume of sprayable material; and the role of additives in sprayable material. 3.3.1 Host-Plant Resistance to CMB Ten common cultivars of cotton (Gossypium hirsutum) , mostly those approved by the Pakistan Seed Certification Department, were tested for their innate resistance against infestation by CMB. The following 10 cultivars were tested using a complete randomized block design with three replications, in the research area at PARS (Punjab Agricultural Research Station ), under the management of the University of Agriculture, Faisalabad: 1. BT 121 2. FH 901 3. FH 1000 4. BH 160 5. FDH 170 6. FDH 228 7. MNH 786 8. CIM 541

38 9. CIM 554 10. CIM 496 The seed was de-linted with sulfuric acid, washed thoroughly with water rinses and dried in the shade. The lay-out and sowing of the cotton varieties was made on 25 May 2006, using a hand-driven, single-row cotton drill at the depth of approximately 4-6 inches. When the cotton plants attained a height of 1.2 ± 0.4 ft. (41 days after sowing), they were artificially infested with CMB at a rate of 5 mature adult female mealybugs per plant, on 5 plants of each variety, with three replicates of each treatment. This was to ensure homogeneous levels of infestation. All the treatments were observed for trends in the development of the pest in interaction with the innate resistance of these varieties. The CMB population-per-plant data were recorded weekly until harvesting and were analyzed using Minitab 15 software. The results obtained were tested statistically for significance (after Steel et al., 1996). 3.3.2 The Impact of Narrow-Spectrum Pesticides and IGRs Trials of commercially available pesticides with relatively narrow spectra of mortality were made in CMB-infested cotton fields to assess their relative efficacy against the mealybug under field conditions. These trials were made in the hope of identifying a possible short-term control for CMB. The mortalities of CMB and its natural enemies were noted. The nine insecticides tested in the field for their efficacy against CMB, and for their suitability for use in IPM regarding their impact on natural enemies, are listed in Table 5 below. The trial was conducted at the Government farm in Multan behind the office of the District Officer Agriculture, using a complete randomized block design with three replications. The outcome of this trial was shared with the stakeholders for onward transmission to cotton growers, and was published as popular articles.

Table 5. Pesticides tested for Control of CMB on Cotton and the Dose used for each

S. no. Active ingredient Product name Dose per acre 1. Buprofezin @ Buprofezin 500 g 2. Fenpropathrin # Fenpropathrin 300 ml 3. Bifenthrin # Talstar 250 ml 4. Acetamiprid N Rani 125 g 5. Imidacloprid N Confidor 250 ml 6. Carbosulphan * Advantage 500 ml 7. Methomyl * Lannate 500 g

39 8. Methidathion ° Supracide 150 ml 9. Profenophos ° Curacron 1000 ml 10. Control Water only Water only @ = chitin synthesis inhibitor (an IGR); # = pyrethroid; N = neonicotinoid; * = carbamate; ° = organophosphate

Solutions of insecticide were made at the dose per acre (43560 sq ft) recommended by the manufacturer (see Table 5 above). The plots were measured using proper calibration and the test pesticide solutions were made up in the appropriate quantities of water according to plot size, taking 120 liters of water per acre as a standard. Pesticide applications were made between 8:00 and 11:00 am using a manual knapsack sprayer with a hollow cone nozzle. The population density of mealybugs and associated beneficial fauna (beetles, lacewings, spiders, predator bugs) on the top three inches of each infested plant were noted before spray application. The percentage population decreases in CMB and associated beneficial fauna (beetles, lacewings, spiders, predator bugs) were recorded 24, 72 and 168 hours after spraying. The data were subjected to statistical analysis by SPSS 15.0 software at the 0.05 level of significance. 3.3.3 The Optimum Volume of Sprayable Material Another trial was conducted to evaluate the optimum volume of water to be used in pesticide applications for the control of CMB. This trial was laid out at the research area owned by the University College of Agriculture, (UCA), Baha ud Din Zakariya University (BZU), Multan, under the management of Welcon Pesticides Pvt. Ltd. (on lease). Five different spray volumes were tested using the manufacturer’s recommended dose for the pesticide. A single well-performing insecticide, Curacron (Profenophos) 40 EC marketed by Syngenta Pakistan Ltd. at 1000 ml per acre was used to determine the optimum volume of water used for total sprayable material. Five different volumes of water, that is, 80, 100, 120, 140 and 160 liters per acre sprayable solution were used in the trial after proper calibration for the plot size. The population density of mealybugs and associated beneficial fauna (beetles, lacewings, spiders, predator bugs) were recorded from the upper 6 inches of the cotton plant before spraying and again 24 and 72 hours afterwards, and one week after the spray. The results obtained were analyzed statistically at the 0.05 level of significance.

40 3.3.4 The Effects of Additives in Sprayable Material The additives tested were those that were used or recommended by field workers or experts in the public or private sectors as hit-or-miss control methods against CMB. The effectiveness of the additives was to be verified by scientific experimentation. A complete randomized block design trial was conducted in the research area at UCA, BZU, with 4 treatments and three replications. Imidacloprid at 330 grams per acre was used. The following treatments were tested: T1: 330 grams Imidacloprid + 50 gram detergent T2: 330 grams Imidacloprid + 50 ml mineral oil T3: 330 grams Imidacloprid + 50 ml vegetable oil T4: 330 grams Imidacloprid (control) The pre-spray CMB population on the top six inches of the plants was noted before the pesticide was applied, and again 72 hours after and seven days (168 hours) after application, to observe the mortality levels associated with the different additives. The resulting data were subjected to ANOVA and comparison of means analysis for significance at the 0.05% significance level.

41 Chapter No.4 RESULTS AND DISCUSSION: 4.1 TAXONOMY

4.1.1 The Identification Problem When CMB was first noticed on cotton in Pakistan in 2005, its identity, even at the family level, was unknown. The only known common pest mealybug in Pakistan was the mango mealybug, Drosicha magiferae (Qayyum, 1961, 1972; Karar et al., 2009) which is not a true mealybug but belongs to the family Monophlebidae. Initially it was thought that the mango mealybug might have shifted hosts from mango to cotton (Buriro, Khushk, 2005, personal communications; newspapers with reference to some other scientists). At first the new pest was noticed on cotton but as scouting of the pest was intensified, it was found on economic crops, vegetables, weeds and ornamental plants. The situation was complicated by discovery of another, unidentified monophlebid pest in cotton fields in Pakistan in 2006 (from Hasilpur, Bahawalpur, and Khanewal in Punjab). This monophlebid species normally lives on wild woody plants like ber (Ziziphus muritiana Lam.), kandi (Acacia spp .) and lasoori [Cordia gharaf (Forssk.) Ehrenb. ex Asch.]; it made localized attacks on cotton and had a passive carry over on tender plants. Unlike D. mangiferae, Drosicha sp. does not overwinter as cysts in the soil . This monophlebid was tentatively identified as Drosicha sp. and was sent to Dr. C. J. Hodgson, National Museum of Wales, UK, a scale insect expert, for identification to species level; his opinion is yet awaited. The pest mealybug on cotton was then misidentified or confused with another damaging mealybug, Maconellicoccus hirsutus (Hemiptera: Sternorrhyncha: Coccoidea: Pseudococcidae) which is also a known pest of cotton and 200 other plants in other parts of the world (Khaskheli, 2006; Khushk and Mal, 2006a and b; Khaskheli, 2007). These workers made a superficial or unscientific opinion that CMB was M. hirsutus and used M. hirsutus ’ biology and other features in popular articles. Meanwhile CMB was reported as Phenacoccus solenopsis Tinsley due to its close similarity to that species (Zaka et al, 2006). Then it was identified as Phenacoccus solani (Naveed, 2008; ICAC Recorder, 2008), but this was disputed by some entomologists (D. Miller, Y. Ben-Dov, 2007-08, personal communications). It was, therefore, necessary to identify the new pest mealybug on cotton at the family as well as genus and species level, using the most recent literature. A key to some families of Coccoidea was available (Cox, 1987) but it discussed only the few families

42 found in New Zealand. Another identification key available on-line was that of Ben-Dov et al. (2006), which covered all the extant scale insect families and contained much other helpful information; but this identification aid was not accessible to the author as it requires the program JAVA, which was not available. Therefore, the species was identified with the help of salient diagnostic characters mentioned for all the families in the recent literature (Williams, 2004; Ben-Dov et al., 2006). 4.1.2 Diagnosis of the Family Pseudococcidae The family of CMB was identified as Pseudococcidae by the presence of the following characters: body of the adult female normally elongate- to broadly oval, usually membranous, often with a pair of anal lobes, each terminating in an apical seta. Antennae each normally 6–9-segmented, but sometimes reduced. Legs normally present, each with a single tarsal segment and a single claw. Claw with a pair of digitules at base and sometimes with a denticle on plantar surface. Translucent pores frequently present on hind legs. A pair of dorsal ostioles normally present towards each end of the body. Circulus present or absent. Anal ring present, normally with at least 2 rows of cells and bearing 6 setae. Paired cerarii present around margins, but sometimes absent entirely. Swirled trilocular pores usually present on dorsum and venter, often abundant. Multilocular disc pores often present, at least on venter of abdomen. Quinquelocular pores present in some genera, usually on venter, rarely on dorsum. Oral collar tubular ducts normally present on venter, less frequently on dorsum. Oral rim tubular ducts sometimes present, at least on dorsum, rarely present on venter only (Williams and Granara de Willink, 1992). The Pseudococcidae is the second largest family in the Coccoidea and contains about 288 genera and 1947 species and subspecies (Ben-Dov et al., 2006). Williams (2004) recorded 62 genera of mealybugs from southern Asia, and provided a comprehensive key to genera. This key was used to identify CMB as belonging to the genus Phenacoccus Cockerell. 4.1.3 Records of Phenacoccus Species on Malvaceae Eleven species of Phenacoccus have been recorded on Malvaceae but only 5 have so far been found on Gossypium (Ben-Dov et al., 2007): P. gossypii Townsend and Cockerell, which is mainly known from the USA, Mexico and the Bahamas, but has fairly recently been recorded from the Western Palaearctic and Japan; P. madeirensis , which is now known to be cosmopolitan and has recently been recorded from Pakistan, the Philippines and Vietnam in southern Asia (Williams, 2004); P. parvus , also rather

43 cosmopolitan and now known in southern Asia from Indonesia, India, the Maldives, Singapore and Thailand (Williams, 2004); P. similis Granara de Willink, only known from the Neotropics; and P. solenopsis , which was originally restricted to the Nearctic and Neotropics but is now known from parts of West Africa and southern Asia. Apart from P. solenopsis (discussed above), these species should all be easily separable from P. gossypiphilus as they all have quinquelocular pores on the venter; in addition, P. gossypii and P. madeirensis also have quinquelocular pores and multilocular pores present on the dorsum. 4.1.4 Morphological Description of the Cotton Mealybug in Pakistan Phenacoccus Cockerell: Cockerell, 1893: 318. Type species: Phenacoccus aceris Signoret, by subsequent designation by Fernald (1903). The genus Phenacoccus currently contains about 180 species and is one of the largest genera in the Pseudococcidae (Ben-Dov, 1994). Several Phenacoccus species are known to be important plant pests and potentially (if not actually) invasive (for example, P. aceris , P. madeirensis Green and P. solani ). The genus Phenacoccus can usually be diagnosed by the adult female possessing a combination of some (but not necessarily all) the following characters: antennae usually each 9-segmented; legs well developed, claw usually with a well-developed denticle; translucent pores absent from hind coxae but sometimes present on hind femur and tibia; cerarii numbering 1-18 pairs, each containing lanceolate setae and trilocular pores, usually situated on membranous areas; dorsal setae usually short and lanceolate, sometimes with trilocular pores situated close to setal sockets; circulus usually present on venter between abdominal segments III and IV; multilocular disc pores usually present, often distributed in rows across venter and sometimes on dorsum; quinquelocular pores usually present on venter and occasionally on dorsum; oral collar ducts often present on dorsum and venter, either all one size or often with dorsal ducts larger than ventral; oral rim ducts absent; eyes without associated discoidal pores (modified slightly from Williams, 2004). The adult female CMB was identified as a species closely resembling P. solenopsis as described by Williams and Granara de Willink (1992), but it differed from it by some key characters, for example, the number and location of ventral multilocular disc pores and oral collar tubular ducts. These are important characters for separating other Phenacocccus species such as P. solani and P. aceris Ferris. Therefore, CMB was initially considered to be an undescribed species by several coccidologists (D.R. Miller and Y. Ben-Dov, personal

44 communications). On this basis, the species was described and named P. gossypiphilus Abbas and Arif (Abbas et al., 2008). However, it was then found that specimens collected at the end of the cotton season (March 2007) and kept in the screen house as a culture had many fewer multilocular pores and oral collar tubular ducts, so that these specimens were inseparable from P. solenopsis [for details, see Annexure 2, Hodgson et al. (2008)]. It is now considered that P. gossypiphilus is morphologically inseparable from P. solenopsis . Whether CMB in Pakistan is actually a separate species new to science remains to be clarified. The DNA analysis being conducted by Dr Alessandra Rung (CDFA, Sacramento, USA) should provide conclusive evidence for this dilemma (C.J. Hodgson and G.W. Watson, 2008, personal communications). At the present time, it is being recommended (see Hodgson et al., 2008 in Annexure 2) that CMB be referred to as P. solenopsis until such time as it is shown to actually be a different species. An identification key is provided in Annexure 2 to separate the species currently attacking cotton in the Indian subcontinent from other Phenacoccus species found in southern Asia. A full-length paper with a description and taxonomic illustrations is attached as Annexure 1 (Abbas et al., 2009). This research paper on the pest cotton mealybug in Pakistan designated as P. gossypiphilus Abbas and Arif was accepted on 26 June 2008 but was not published until 2009. Meanwhile, after a thorough morphological analysis of many samples of this species and comparison of its morphological range with specimens of P. solenopsis from all around the world, it was decided to regard it as P. solenopsis unless a molecular analysis proves them to be genetically different species; this was because off- season specimens of the cotton pest were morphologically indistinguishable from specimens referred to as P. solenopsis in the New World. The detailed paper on this thorough comparison with P. solenopsis (Hodgson et al., 2008) was published on 24 October 2008, and is attached as Annexure 2. 4.1.5 Morphological Variation Between Samples of P. solenopsis Multilocular pores are unknown along the abdominal sub-margins of P. defectus and most specimens of P. solani . They are also absent from the sub-margin of the type series of P. solenopsis (as illustrated by Williams and Granara de Willink, 1992, p. 389). As indicated above, some P. gossypiphilus specimens are distinguishable from the type series of P. solenopsis in lacking multilocular pores on the abdominal sub-margins. However, many other (non-type) collections also considered to be P. solenopsis have been made available from North and South America, and from around the Gulf of Mexico, and these often have multilocular pores on the abdominal sub-margins. Some of this material is

45 almost certainly the material referred to by Williams and Granara de Willink (1992, p. 390). It is clear that, if all of this material does represent P. solenopsis , then P. gossypiphilus and P. solenopsis (based on this wider understanding of the latter species) are very similar - indeed, no distinguishing character could be discovered to separate the screenhouse form of P. gossypiphilus from many P. solenopsis, despite an exhaustive search of all growth stages. To make matters more complicated, several samples of material from Asia (India, Thailand, Taiwan and New Caledonia) show a similar frequency of multilocular pores on the sub-margin to that noted for P. solenopsis and P. gossypiphilus and have the same distribution of oral collar ducts. Therefore, it would appear that, on the basis of the present understanding of P. gossypiphilus and P. solenopsis , specimens of these two populations can only be distinguished when the former species is collected from the field during the hot, cotton-growing season (C.J. Hodgson, 2008, personal communication).

Figure 3. CMB Infestation on Aerial Parts of a Cotton Plant in Pakistan

46 4.1.6 Biological Differences Between P. gossypiphilus and P. solenopsis Phenacoccus gossypiphilus (Abbas et al., 2009) closely resembles P. solenopsis , and some specimens of P. gossypiphilus (collected in the winter from a culture on cotton) cannot be distinguished from P. solenopsis morphologically (see Hodgson et al., 2008, Annexure 2). However, they differ as follows: i) P. gossypiphilus is an aerial species, spending its entire life on the upper parts of the host plants (Fig. 2 above) and no part of its life cycle in the soil (in North America, P. solenopsis is found almost exclusively on the roots of its many host plants). ii) P. gossypiphilus is visited by a number of ant species, which feed on the honeydew, but not by Solenopsis ants [ P. solenopsis was originally collected with Solenopsis ants (Tinsley, 1898).] iii) P. gossypiphilus is an invasive species on 55 host plants in 18 families [ P. solenopsis was not reported as being invasive in the Neotropics, its native region, between 1898 and 1990 (Fuchs et al., 1991). However, Williams and Granara de Willink (1992) and Culik and Gullan (2005) did report P. solenopsis as a pest.] iv) P. gossypiphilus is ovoviviparous, with the eggs being retained within the body until they are ready to hatch. It is not known how P. solenopsis produces its progeny. v) P. gossypiphilus can tolerate high temperatures and is active at 35ºC-50ºC although the rate of population growth is negatively affected. Population decreases have been reported in P. solenopsis at temperatures below this range on tomato; whether this was because of host conditions or rising temperatures was not clear (Culik and Gullan, 2005). If DNA analysis proves that CMB in Pakistan is a distinct species, then it is very similar to P. solenopsis . If it proves to be the same species as P. solenopsis then it has a very wide range of variation in its morphology and biology, which change in different environmental conditions. The recent invasions of this species on a large number of host- plant species including economic crops, vegetables, ornamentals and weeds have ranked it as a most important economic threat. Because of the extreme importance of this invasive species, great caution should be exercised regarding infested plant material arriving from Pakistan (and possibly other southern Asian countries) at ports of entry around the world.

4.1.7 Taxonomic Discussion Accurate identification of a pest is the primary requirement for a successful IPM program. The identity of the pest mealybug on cotton in Pakistan has been a hot issue.

47 Initially it was thought that the mango mealybug (Drosicha mangiferae) might have shifted from mango to cotton for some reason. CMB was then misidentified as, or confused with, Maconellicoccus hirsutus, a known pest of cotton and many other plants in other parts of the world. Then our studies identified it to genus level, as Phenacoccus spp. (Abbas et al, 2006). Various published sources referred to it as Phenacoccus solenopsis Tinsley (Zaka et al, 2006), Phenacoccus solani Ferris (Naveed, 2008; ICAC Recorder, 2008) and Phenacoccus gossypiphilous nom. nud. (Abbas et. al., 2007a). A research paper was submitted for publication in May, 2008 in which CMB was described as a species new to science, Phenacoccus gossypiphilus Abbas and Arif (Abbas et al., 2009); publication of this paper was relatively slow. A detailed morphological study subsequently failed to find any consistent morphological differences between CMB and New World material of P. solenopsis (Hodgson et al., 2008), leading to the decision to use the name P. solenopsis for CMB. Hodgson et al. (2008) was the later work, but it was published electronically and therefore was published before the description of P. gossipiphilus. The cotton mealybug pest has been reported from India, China and Nigeria as P. solenopsis Tinsley (Akintola and Ande, 2008; Arif et al, 2009; Nagrare et al, 2008; NCIPM, 2008; Tanwar et al., 2008; Wang et al., 2009). Since the publication of Hodgson et al. (2008) in October 2008, there is a consensus amongst coccidologists to refer to CMB as P. solenopsis until a molecular analysis shows it to be a distinct taxon.

48 RESULTS AND DISCUSSION: 4.2 ECOBIOLOGY

4.2.1 Biology 4.2.1.1 The Life Cycle of CMB The in vitro and in vivo experiments described in 3.2.1 to 3.2.5 above provided the data on which this section is based, which are summarized in Table 6 below. The in vitro studies used cut cotton leaves from the field as a food source for the mealybugs.

Table 6. Summary of the Life Cycle of Cotton Mealybug in Laboratory Conditions

S. no. Features of life cycle Units Freq. Mean ± S.D. Median 1. Number of ovisacs produced no./ female 10 2.6 ± 1.0 2.0 2. Maximum number of crawlers no./ female 10 39.1 ±12.0 40.5 in one crawler sac 3. Additional crawlers produced no./ female 10 22.5 ± 10. 7 23.0 4. Total crawlers per female no./ female 10 77.9 ± 22.2 80.50 5. Entire female life span to death days 10 25.1 ± 13.7 18.0 6. Duration of 1st instar I days 8 6.8 ± 1.8 7.0 7. Duration of 2nd instar II days 8 6.4 ± 0.7 6.5 8. Duration of 3rd instar female days 6 7.3 ± 2.5 7.0 9. Adult female maturation up to days 6 18.6 ± 2.9 18.5 progeny production 10. Life of female after days 10 16.4 ± 4.2 15.50 crawler production 11. Span of male prepupa + pupa days 4 5.0 ± 1.0 5.0 12. Full life span of male to death days 4 16.0 ± 2.5 16.5 13. Duration of winged adult male days 4 2.2 ± 1.8 2.5 14. Duration of mating minutes 3 5±1 5.0 15. Percentage of males in progeny % 3 42.0 ± 7.2 40.0 of lab-reared females 16. Number of crawler-sacs no./ female 10 2.6 ± 1.0 2.0 produced by one female 17. Offspring by unmated females no./ female >100 0 0 18. Deposition of wax on crawlers no. days 9 2.7 ± 0.7 3.0 to whitening

Observation of the in vitro studies found that there were 4 immature instars in the winged male and three in the wingless, larviform female. The developmental stages can be summarized as follows: Male : First instar, second instar, “prepupa”, “pupa”, winged adult Female : First instar, second instar, third instar, wingless adult

49 The sexes were identical in the first-instar (crawler) stage (duration 6.8 ± 1.8 days) and morphologically similar except for microscopic details in the second instar (6.4 ± 0.7 days), as discussed in Hodgson et al. (2008, Annexure 2). The female became morphologically recognizable when it moulted to the third instar (immature) whose body became covered in mealy white wax; after feeding for 7.3 ± 2.5 days (Table 6 above, s. no. 8) it then moulted to the fourth instar (the adult female), and mating took place soon after. In contrast to the female, the second instar male secreted a cocoon of fine wax filaments around its body, and moulted into an inactive “prepupa” inside the coccoon. By the time the coccoon was fully developed, the male had moulted again within it to the “pupal” stage. The male’s final moult also occurred within the cocoon before he emerged as a small, winged adult. Full morphological descriptions of all the immature stages and the adult male were given in Annexure 2. 4.2.1.2 The Reproduction of CMB When the male emerged from the cocoon he slowly spread his wings and then took flight to find a female. The male clung to the posterior end of the female’s abdomen with his wings vibrating, and curled his abdomen under the tip of hers. The aedeagus was inserted into the vulva of the female from the venter (not visible from above). Mating lasted up to 5 minutes in laboratory conditions (Table 6 above, s. no. 14). In the field, mating usually occurred in the early morning on hot summer days, but in months with mild temperatures (25-30 ºC), mating was observed to occur at any time of the day. In the in vitro experiments described in 3.2.1 above, it was found that an adult female was capable of reproducing only if she mated with a male. Unmated females failed to reproduce (Table 6 above, s. no. 16; Table 7 below), which supported the hypothesis that CMB only reproduces sexually (see also Appendix Table 9.0, p = 0.000).

Table 7. Mode of Reproduction of CMB Females isolated on Cotton Leaves in vitro

Population Total Died (%) No. survived (%) No. reproducing (%) Total population of females 30 11 (36.7%) 19 (63.3%) 4 (21.1%) Mated females 5 1 (20.0%) 4 (80.0%) 4 (100.0%) Unmated females 14 2 (14.3 %) 12 (85.7 %) 0 (0.0 %)

After mating, the fertilized adult female mealybug became relatively inactive. It anchored itself by its mouthparts to a single feeding site while the fertilized eggs

50 developed within the body, causing it to continue to enlarge. When the embryos were near to hatching, the female mealybug secreted a flexible, cottony sac of white wax filaments from the posterior of the abdomen. Initially the sac could hardly be seen from above but as more wax filaments were secreted, a white cottony sac became visible behind the rear end of the body when viewed from above (see Fig. 4 below). The sac extended up to half of the body length of the adult female.

Photograph by Sabir Hussain

Figure 4. Adult Female CMB on a Cotton Leaf underside, slightly displaced to show the Crawler Sac

Observations made on more than 300 occasions, in different seasons and on different hosts, always found that the female produced crawlers in the sac, not eggs. It was therefore concluded that CMB was ovoviviparous, retaining the eggs within the female’s body until they were ready to hatch. As soon as the crawler sac was secreted, the crawlers were produced. 4.2.1.3 The Crawler and Wax Secretions When the crawler sac ruptured or was detached from the female, the crawlers (20- 53 in laboratory conditions) were seen crawling about in search for a feeding site. Crawlers were each 0.2-0.5 mm long, with a yellowish white, transparent body. The following is based on observation of more than 300 specimens in summer conditions (at 28-48˚C): during the first 2-4 days after leaving the crawler sac, white powdery wax

51 secretions gradually accumulated on the crawler’s body, giving it a whitish appearance (Table 6 above, s. no. 18). On moulting, this powdery layer was lost with the old cuticle but was secreted again. The time taken to form a new white wax layer decreased with the increasing age (and size) of the mealybug, so in subsequent instars it only took few minutes to hours for the wax coating to be secreted. Wax deposits were thickest on the oldest adult females. The thicker the mealy wax layer was, the more prominent the wax pencils projecting from the body margins became. 4.2.1.4 Mortality of CMB Figurs 5 and 6 (below) show that, in vitro, 75% of the mortality occurred in the first 1-8 days of life (the crawler stage), followed by 20% in days 9-16 (the second instar). Only 1% of the mortality occurred after the twenty-fifth day.

Figure 5. Graph to show in vitro Mortality of CMB

25-32 days, 7, 1% 17-24 days, 33, 4%

9-16 days, 148, 20% 1-8 days, 554, 75%

Figure 6. In vitro Mortality of CMB Immatures, shown in four Sectors based on the Number of Days after hatching

52 Out of the 30 female mealybugs reared in isolation in the laboratory experiment described in 3.2.1 above, 11 (36.7%) died. The mortality in the first two instars was 36.7%, whereas in the third and fourth instars the mortality was only 15.8% resulting in cumulative mortality of 52.5%. This level of mortality was very different from the 66.3% recorded in the group-rearing in vitro study described in 3.2.1. The latter figure was based on 100 crawlers kept together in an over-populated environment (a Petri dish 5.5 cm in diameter). In both the laboratory and the field the life cycle of CMB, like all other insects, was highly dependent upon the environmental conditions. Increasing the temperature shortened the life span. Similarly, high humidity favored the reproduction and survival of the pest mealybug. 4.2.1.5 The Instar Durations of CMB The different developmental stages had different durations (Table 6 above; the data are from controlled laboratory conditions), as shown in Fig. 7 below.

30 25 20 15 10 5 Male 0 Female

a r a r f e I n s t r s t A d u l t l i t o t a l l i f e f i second inst

third /prepupa& pupa

Stages of the CMB life cycle

Figure 7. Number of Development Days in different Instars of P. solenopsis, under controlled Conditions

The duration of the instars in the field differed from that in laboratory conditions (Table 8 below). In addition, the duration of each instar varied according to the environmental conditions, becoming much shorter in high temperatures. Over the period

53 of field observations, environmental conditions spanned the ranges of 9.3°-34.5°C and 33.8-74.8% relative humidity.

Table 8. Instar durations of CMB on cotton in the field, Faisalabad, 6 March to 11 April 2007

Life cycle stage No. observationsAverage duration in days (± standard deviation) First instar 25 4.04 ± 0.73 Second instar 21 4.57 ± 1.69 Third instar female 14 4.07 ± 0.62 Fourth instar female 12 7.25 ± 5.79

4.2.1.6 The Life Span of CMB In laboratory conditions, the maximum female lifespan (from hatching to mortality) was 52 days (rough data, Appendix 1). However, this was an exceptional outlier in the normal distribution; normally she survived up to 38 days (mode of the number of days in the life of a mature female). The normal life span range was 16-43 days with an average of 25.1 ± 13.7 days (Table 6 above, s. no. 5). This average excluded immature mortality. In contrast, the median lifespan was only 18.0 days because most mealybugs die in the crawler stage. Once a female mealybug reached maturity, the chances of longevity increased by 42.9% compared to the average lifespan of a member of the population as a whole. Total lifespan data, if observed only in mature females, fell in a normal distribution (a bell shape), but over the whole developmental process it was asymmetric because of the high mortality early in life (Fig. 5 above). The mean life span of the male, based on data in Table 6 above, was 20.4 ± 5.3 days, out of which the winged adult lived only 2.2 ± 1.8 days. 4.2.1.7 The Sex Ratio of CMB The experiment described in 3.2.2 above provided the data in Table 9 below. The overall percentage of males in the population was 37.5 ± 2.7% and that of females was 48.5 ± 5.4%, giving a male : female ratio of 1 : 1.29. The proportion of the population that was female and the degree of dominance of this sex varied considerably (p value 0.29, Appendix Table 8.0). There was no significant difference in the sex ratio obtained from in vitro or in vivo conditions (p = 0.084, Appendix Table 8.0). The effect of host- plant species on the sex ratio also had a statistically non-significant effect (p = 0.721,

54 Appendix Table 8.1). The immatures that died before their sex could be determined morphologically were excluded from the sex ratio calculation . Table 9. Sex ratio determination of CMB on various hosts in field and laboratory conditions

S. Host plant Conditions % male % female % died no. 1. Shoe flower, Hibiscus rosa-sinensis In vitro 38.0 41.0 21.0 2. Cotton, Gossypium hirsutum In vitro 42.0 45.0 13.0 3. Sunflower, Helianthus annuus In vitro 36.0 48.0 16.0 4. Shoe flower, Hibiscus rosa-sinensis In vivo 34.0 57.0 9.0 5. Cotton, Gossypium hirsutum In vivo 38.0 49.0 13.0 6. Sunflower, Helianthus annuus In vivo 37.0 51.0 12.0 Average percentages (± SD) Overall 37.5 ± 2.7 48.5 ± 5.4 14.0 ± 4.1 Sex ratio (male: female) Overall 1.0 1.29 -

4.2.1.8 The Effect of Host-Plant Species on CMB Fecundity A female produced 2.6 ± 1.0 crawler sacs in her life (Table 6 above, s. no. 15). The initial experiment (described in 3.2.5 above) investigated the effects of various factors on the number of progeny produced by CMB fecundity on three different host- plants in the field. The three host-plants studied were cotton (G. hirsutum) , itsit (Trianthema portulacastrum) and hazardani (Euphorbia prostrate) . The comparisons of means (Table 10 below) revealed that the host-plant species had a significant effect on CMB fecundity. Locality and month, singly and in interaction with each other and with the host-plant species had no significant effects on the number of progeny.

Table 10. Analysis of Variance and comparison of means for the number of eggs developing inside a dissected, mature adult CMB female collected from various hosts on different dates

SOV SS df MS F ratio p value Host 67294.71 2.00 33647.36 283.06 0.00 Month 325.37 2.00 162.69 1.37 0.26 Locality 493.45 2.00 246.72 2.08 0.14 Host*Month 756.17 4.00 189.04 1.59 0.19 Host*Locality 504.90 4.00 126.23 1.06 0.38 Month*Locality 694.13 4.00 173.53 1.46 0.23 Host*Month*Locality 1073.05 8.00 134.13 1.13 0.36 Error 6300.15 53.00 118.87 Total 77441.94 79.00 34798.57 291.74 1.56

55 These results indicated that cotton and Hibiscus spp. were the preferred hosts, because it was such a good food source that it enabled the pest to reproduce vigorously. The population of CMB crawlers on cotton was significantly larger than that on either of the other two hosts [Itsit ( T. portulacastrum) and hazardani (E. prostrate)]. There was no significant difference between the latter two hosts in the number of crawlers produced. In view of the results above, more host-plant species were taken into observation at UAF in 2007 and the effect of ten host-plant species on fecundity was determined (described in 3.2.5 above). The data obtained are summarized in Table 11 below. Analysis of this data showed that there were significant differences in the numbers of developing eggs per adult female between the host-plant species studied (see Appendix Table 4, p = 0.000). The comparison of means analysis is shown in Appendix Table 5.

Table 11. Average number of developing eggs visible in the body of a dissected, full-sized, field-collected adult female in August 2007

S. Host plant Eggs/female no. Local or English name Latin name 1. Cotton Gossypium hirsutum 92.4 ± 6.9a 2. Lady’s finger Hibiscus esculentus 81.7 ± 5.8ab 3. Shoe flower Hibiscus rosa-sinensis 90.4 ± 7.1a 4. Itsit Trianthema patulacastrum 63.9 ± 6.3 bc 5. Hazadani Euphorbia granulate 52.8 ± 5.9 cd 6. Tandla Digera muricata 46.8 ± 7.2 de 7. Aksun Withania somnifera 68.6 ± 6.8bc 8. Gule dupehri Portulaca grandiflora 51.6 ± 7.3cd 9. Lantana Lantana camera 48.4 ± 6.8de 10. Chillies Capsicum annuum 46.7 ± 5.7de The values having same letters were statistically similar, α = 0.05 P = 0.000, pooled standard deviation = 6.60

Figs. 8 and 9 below illustrate the effect of different host-plant species on the number of eggs and crawlers produced by one female CMB, respectively. In both figures, cotton (G. hirsutum) and Hibiscus spp. were the most preferred food source, enabling the pest to produce the maximum number of offspring, followed (in order of decreasing mealybug fecundity) by shoe flower (H. rosa-sinensis), lady’s finger (H. esculentus), aksun (W. somnifera), itsit (T. patulacastrum), hazardani (E. granulate), gule dupehri (P. grandiflora), lantana (L. camara), tandla (D. muricata), and lastly chillies (C. annuum).

56

Boxplot of Number

100

90

80

70 The number of crawlers obtained in the field from females feeding on each of the Number hosts listed60 in Table 10 (above) showed the same host-related trend. The average total number of crawlers produced per female on cotton in the field was significantly greater 50 than that produced on chillies in the same conditions (Fig. 7 below). 40 1 2 3 4 5 6 7 8 9 10 Host

For host plant identities, see S. no. in Table 11 above

Figure 8. Box Plot showing the range and mean number of developing eggs per dissected adult female of CMB, on various host plants at UAF in 2007

140 122.5 120.7 120 111.9 94 98.7 100 83 77.1 81.8 78.7 76.8 80 60 40 20 0 number of crawlers /female crawlers of number r i e e n la n a s u ies tton g Itsit tan ll fin n i Co y Tand Aks a d L Ch a nese ro Hazada L i ule Dupehri Ch G

The figure above each column is the mean number of crawlers per female

Figure 9. Total number of crawlers per female of CMB, on various hosts in July 2007

Compared to the initial study on three hosts in three months (July, August and September 2006), the second experiment included some different months of the year that have different meteorological conditions, to test whether varying environmental

57 conditions had any effect on CMB fecundity. The observations were undertaken in July 2006 (cotton season), December 2006 (end of the cotton season) and May 2007 (start of the cotton season) on the same host-plant species. Table 12 (below) shows the average number of crawlers produced per batch on different host-plant species in the field. Analysis of means of the data (Appendix Table 6) revealed that the average number of CMB crawlers in one batch varied significantly between the host-plant species studied (p = 0.002); and the average number of crawlers per female also varied significantly between different months (p = 0.002). The interaction of host-plant species and month did not cause significant variation in the average population of CMB crawlers per female (p = 0.454). In overall comparisons, CMB fecundity was highest on cotton in July, when the temperature range was 28-41°C and relative humidity was 41.3±6.7%. In laboratory conditions the average number of crawlers per batch averaged 20-53; a female might produce up to four batches of crawlers at 2-5 day intervals. A trend in decreasing numbers of crawlers in successive batches was observed (Table 6 above), with the lowest number in the last batch.

Table 12. Average number of CMB crawlers in one batch, in different months and on various hosts

Month/Host Cotton Itsit Hazardani Monthly ave. July 62.4 ± 5.3 42.3 ± 4.7 36.7 ± 6.3 47.1 ± 12.6a December 42.6 ± 5.3 12.5 ± 4.1 9.4 ± 5.2 21.5 ± 16.4b May 58.7 ± 8.5 38.9 ± 8.6 37.8 ± 7.6 45.1 ± 12.4a Host-wise ave. 54.6 ± 10.7a 31.2 ± 15.1b 28.0 ± 15.0b 37.9 ± 17.9 Values sharing same letters are not significantly different

Cotton was the best quality food source (optimal host) as it enabled the mealybugs to reproduce prolifically. The number of crawlers produced on cotton was significantly higher (p = 0.000) than that on two other hosts [itsit (T. portulacastrum) and hazardani (E. prostrate)]. The difference between the number of crawlers produced on each of the latter two hosts was not significant (p = 0.534). The cumulative average of the number of crawlers produced differed significantly between the three different months (p = 0.002). The number of crawlers per female in July 2006 and May 2007 was not significantly different (p = 0.000), but differed significantly from the average population of crawlers per female produced in December, 2006 (p = 0.786). (For details of the analysis, see Appendix Tables 6.1 to 6.5). The

58 prevailing environmental conditions for the corresponding weeks of the months in the study are given in Table 13 below.

Table 13. Environmental Data for the Weeks of Study for the Host-related Fecundity Experiment with ten Host-plants at UAF

Date Period Temp. max. Temp. min. RH Rainfall °C °C % mm July 2006 3rd week 40.0 ± 1.3 29.4 ± 1.4 41.3 ± 6.7 0.0 Dec. 2006 1st week 21.8 ± 3.2 10.8 ± 3.2 60.6 ± 13.8 6.1 ± 16.3 May 2007 1st week 41.3 ± 3.2 24.7 ± 1.3 23.3 ± 6.2 2.1 ± 5.7

Both the month of observation and the host-plant species significantly affected CMB fecundity (p = 0.002 for both factors, see Appendix Table 6). These relationships are illustrated in Figs. 10 and 11 respectively, below.

70

60

50

40

30

20

10 Av.number of crwlers/female/batch

0 Jul06 Dec06 May07 Month

July = cotton-growing season; December = outside cotton-growing season; May = season for early sown cotton

Figure 10. Average number of crawlers per batch per CMB female, in different months, observed on three different host-plant Species (cotton, Itsit and hazardani)

59 70

60

50

40

30

20

10 Av. number of Av. crawlers/female/batch number

0 Cotton Itsit Hazardani Host

Figure 11. Average number of Crawlers per Batch per CMB Female, in different Months, observed on three different Host-plant Species (Cotton, Itsit and Hazardani) in July and December 2006 and May 2007

4.2.1.9 Alternate Host-Plants of CMB The plant species listed in Table 14 below were confirmed as alternate hosts of CMB, where it could complete its entire life cycle and bred successfully. These plants were found infested by the pest in more than five different localities, or were confirmed as being able to sustain more than one generation of CMB by rearing in the laboratory. Plant species where all the stages of the pest were present in a ratio similar to that observed on cotton usually proved to be viable hosts.

Table 14. A list of alternate host plants of CMB, confirmed in the laboratory (continued on the next two pages)

S. Plant family Latin name Vernacular English name no. Name 1 Aizoaceae Trianthema portulacastrum L. Itsit Horse purslane 2 Amaranthaceae Achyranthes aspera L. Puth kanda Prickly chafflower 3 Amaranthaceae Amaranthus spinosus L. Chulai Spiny amaranth 4 Amaranthaceae Amaranthus paniculatus L. Billi booti Scarlet 5 Amaranthaceae Amaranthus viridis L. Jangli chulai Pigweed 6 Amaranthaceae Digera muricata Mart. Tandla Digera 7 Asteraceae Carthamus oxyacantha Pohli Wild safflower M. Bieb. 8 Asteraceae Cirsium arvense (L.) Scop. Leh Canadian thistle

60 S. Plant family Latin name Vernacular English name no. Name 9 Asteraceae Conyza ambigua DC. Lusan booti Fleabane 10 Asteraceae Conyza bonariensis (L.) Lusan booti Hairy fleabane Cronquist 11 Asteraceae Eclipta prostrate (L.) L. Daryai booti 12 Asteraceae Helianthus annuus L. Suraj mukhi Sunflower 13 Asteraceae Launea nudicaulis Hook. f. Peeli dodhak 14 Asteraceae Parthenium hysterophorus L. Gajar booti 15 Asteraceae Xanthium strumarium L. Muhabbat Cocklebur Booti 16 Boraginaceae Heliotropium europeaum L. Namkeen Booti 17 Boraginaceae Heliotropium indicum L. Oont chra Wild heliotrope 18 Brassicaceae Coronopus didimus L. Sm . Jangli haloon Swine cress 19 Brassicace ae Lepidium sativum L. Haloon 20 Cannabinaceae Cannabis sativa L. Bhang 21 Chenopodiaceae Atriplex crassifolia C.A. Mey. Lani 22 Chenopodiaceae Chenopodium album L. Bathu Lambs quarters 23 Chenopodiaceae Chenopodium morale L. Krund Fathen 24 Convulvulaceae Convolvulus arvensis L. Lehli Field bindweed 25 Cucurbitaceae Cucumis melo L. Kharboza Musk melon 26 Cucurbitaceae Cucumis sativus L. Khera Cucurbits 27 Cucurbitaceae Cucurbita moschata Duchesne Kaddu Pumpkin 28 Euphorbiaceae Euphorbia prostr ate Ait . Hazardani 29 Euphorbiaceae Euphorbia granulate Forssk. Hazardani Trailing spurge Dodhak 30 Euphorbiaceae Euphorbia hirta L. Lal dhodhak Red garden spurge 31 Fabaceae Medicago alba E.H.L. Krause Do Honey clover 32 Fabaceae Medicago polymorpha L. Maina Black clover 33 Fabaceae Melilotus indicus (L.) All. Seinji Indian clover 34 Fumariaceae Fumaria indica Pugsley Shahtra 35 Malvaceae Abutilon indicum (L.) Sweet Kangi booti 36 Malvaceae Gossypium hirsutum L. Kapah Cotton 37 Malvaceae Abelmoschus esculentus (L.) Bhindi Lady’s finger Moensch 38 Malvaceae Hibiscus mutabilis L. - Cotton rose 39 Malvaceae Hibiscus rosa-sinensis L. Gudhal Shoe flower 40 Nyctaginaceae Boerhavia diffusa L. Jangli itsit 41 Nyctaginaceae Bougainvillea spectabilis Willd. Boganbilla Bougainvillea 42 Portulacaceae Portulaca oleracea L. Kulfa, lunak Common purslane 43 Portulaceae Portulaca grandiflora Hook. Gule dupehri 44 Solanaceae Capsicum annuum L. Mirch Chillies 45 Solanaceae Datura alba Rumph. Ex Nees Dhatura 46 Solanaceae Lycopersicon esculentum Mill. Tamater Tomato 47 Solanaceae Nicotiana tabacum L. Tamakho Tobacco 48 Solanaceae Solanum melongena L. Bengun Brinjal 49 Solanaceae Solanum nigrum L. Mako Black nightshade 50 Solanaceae Solanum tuberosum L. Aaloo Potato 51 Solanaceae Withania somnifera (L.) Dunal Aksun 52 Verbenaceae Clerodendron inerme Gaertn. Gardenia

61 S. Plant family Latin name Vernacular English name no. Name 53 Verbenaceae Duranta repens L. Duranta 54 Verbenaceae Lantana camara L. Lantana 55 Zygophyllaceae Tribulus terrestris L. Bhakra Puncture clover End of Table 14.

Some plant species were observed to be infested in the field at fewer than five different localities but the host record not be confirmed by laboratory rearing due to non- availability of the plant near the laboratory. These observed hosts, which may be transitory hosts only, have been listed in Table 15 below. Botanical names and families have been given as far as possible, as identified by the botanist (Dept. of Botany, UAF).

Table 15. A list of host plants of CMB observed in the field, not confirmed in the laboratory

S. Plant family Latin name Vernacular No. of observations no. Name & intensity 1. Anacardiaceae Mangifera indica L. Mango 3 : 2 Is. ad.* 2. Cucurbitaceae Cucumis melo Long melon 2 : Cl.>10 var. utilissimus (Roxb.) Duthie & Fuller 3. Cyperaceae Cyperus difformis L. Ghoin 3 : Cl >25 4. Cyperaceae Cyprus rotundus L. Deela 3 : Cl.<5 5. Cyperaceae Cyperus subgenus Iria sp. Bhoin 3 : Cl.>10 6. Fabaceae Dalbergia sissoo Roxb. Shesham 2 : Cl.>20 DC. seedlings 7. Poaceae Echinocloa colona (L.) Swanki 3 : Is. <5 Link 8. Polygonaceae Rum ex dentatus L. Jangli palak 2 : Is. cr. >4 9. Salvadoraceae Salvadora oleoides Jal, Peelu 2 : Is. ad.< 2 Note: Number = number of times observed; Is. = isolated; Cr. = crawler; Cl. = cluster; > = more than; < = less than; Is. ad. = isolated adult female; Is. cr. >4 = crawlers numbering more than four have been seen; isolated means that in the remaining population of the same plants, only one was observed harboring CMB.

In Table 14 above, the CMB host-plant species were listed in the order of botanical classification on the basis of plant families. In Table 16 below, the CMB hosts are listed in order of the CMB infestation levels found during the surveys of CMB during 2005-2008.

62 Table 16. CMB host plants listed in order of percentage infestation level observed during survey of CMB on various host plants, 2005-2008 (continued on the next page)

S. Host Vernacular No. No. Latin name name n Mean SD Median 1. 39 Hibiscus rosa-sinensis Gudhal 14 96.4 7.5 100.0 2. 47 Nicotiana tabacum Tamakho 5 44.8 35.0 50.0 3. 51 Withania somnifera Aksun 5 41.3 42.1 25.0 Bougainvillea 4. 41 spectabilis Boganbilla 5 40.2 37.0 33.3 5. 54 Lantana camara Lantana 7 38.0 39.3 20.0 6. 53 Duranta repens Duranta 6 35.3 34.1 29.0 7. 36 Gossypium hirsutum Kapah 25 29.3 33.5 16.0 8. 42 Portulaca grandiflora Gule dupehri 10 23.3 26.9 10.0 9. 37 Abelmoschus esculentus Bhindi 10 18.9 23.2 9.0 Muhabbat 10. 15 Xanthium strumarium booti 7 17.9 18.9 12.0 11. 48 Solanum melongena Bengun 7 16.1 20.1 12.0 12. 28 Euphorbia prostrate Hazardani 16 14.9 28.0 4.9 13. 52 Clerodendron inerme Gardenia 9 14.3 15.1 6.3 14. 49 Solanum nigrum Mako 11 13.7 15.6 5.0 15. 38 Hibiscus mutabilis Bhindi phool 10 13.3 18.0 10.0 16. 34 Fumaria indica Shahtra 7 13. 1 16.0 8.0 17. 24 Convolvulus arvensis Lehli 15 11.2 16.0 4.0 18. 2 Achyranthes aspera Puth kanda 8 10.9 5.2 11.0 19. 40 Boerhavia diffusa Jangli itsit 5 8.9 12.7 6.7 20. 23 Chenopodium morale Krund 12 8.7 16.7 0.0 Parthenium 21. 14 hysterophorus Gajar booti 8 8.2 6.0 10.0 22. 45 Datura alba Dhatura 6 7.8 3.2 7.3 23. 35 Abutilon indicum Kangi booti 7 7.6 3.3 8.0 24. 7 Carthamus oxyacantha Pohli 8 7.5 6.6 9.0 25. 31 Medicago alba Jangli methi 5 6.5 8.4 2.0 Lycopersicon 26. 46 esculentum Tamater 17 6.2 8.3 2.0 27. 34 Melilotus indicus Seinji 11 5.9 8.1 4.0 28. 30 Euphorbia hirta Lal dhodhak 12 5.7 5.7 4.2 29. 50 Solanum tuberosum Aaloo 6 5.0 5.7 3.0 Trianthema 30. 1 portulacastrum Itsit 15 4.9 5.8 4.0 31. 25 Cucumis melo Kharboza 8 4.6 3.2 5.0 32. 11 Eclipta prostrate Daryai booti 8 4.0 2.5 4.0 33. 13 Launea nudicaulis Peeli dodhak 6 4.0 6.9 1.5 34. 19 Lepidium sativum Haloon 5 4.0 2.5 4.0 35. 27 Cucurbita moschata Kaddu 9 4.0 5.2 2.0 36. 4 Amaranthus paniculatus Billi booti 7 3.9 4.0 4.0 Hazardani 37. 29 Euphorbia granulate dodhak 7 3.9 3.4 4.0

63 S. Host Vernacular No. No. Latin name name n Mean SD Median 38. 43 Portulaca oleracea Kulfa, lunak 10 3.8 3.3 3.5 49. 44 Capsicum annuum Mirch 5 3.8 2.0 4.0 40. 55 Tribulus terrestris Bhakra 6 3.8 2.2 3.1 41. 18 Coronopus didimus Jangli haloon 6 3.7 2.3 3.0 42. 8 Cirsium arvense Leh 6 3.6 3.8 3.0 43. 9 Conyza ambigua Lusan booti 7 3.6 4.1 3.0 44. 5 Amaranthus viridis Jangli chulai 16 3.5 2.6 4.0 45. 32 Medicago polymorpha Maina 5 3.3 2.5 2.0 46. 6 Digera muricata Tandla 8 3.1 2.5 47. 21 Atriplex crassifolia Lani 7 3.1 2.6 4.0 48. 26 Cucumis sativus Khera 7 3.1 2.9 2.0 49. 22 Chenopodium album Bathu 20 2.9 3.8 0.0 50. 17 Heliotropium indicum Oont chra 6 2.7 4.8 0.0 51. 20 Cannabis sativa Bhang 8 2.7 1.8 2.0 52. 10 Conyza bonariensis Lusan booti 7 2.4 3.6 4.0 53. 12 Helianthus annuus Suraj mukhi 9 2.2 3.3 1.0 54. 3 Amaranthus spinosus Chulai 9 2.1 2.7 2.0 Heliotropium 55. 16 europeaum Namkeen 9 1.8 2.0 1.0 End of Table 16. n = number of observations

Statistical analysis found that the level of CMB infestation on the observed host plants in the natural conditions of the different localities of Pakistan in different habitats differed significantly (p = 0.000, Appendix Table 3.3). The statistical difference between various means (see Appendix Table 3.3) and the order of the host plants with respect to the maximum CMB population intensity observed is given in Table 17 below.

Table 17. CMB host plants listed in order of maximum intensity of infestation observed during survey of CMB on various host plants, 2005-2008 (continued on the next page)

S. Host Vernacular no. no. Latin name name n Mean SD Median 1. 47 Nicotiana tabacum Tamakho 5 141.6 116.1 42.0 2. 36 Gossypium hirsutum Kapah 24 114.5 107.1 76.5 3. 39 Hibiscus rosa-sinensis Gudhal 14 105.7 62.0 88.5 4. 51 Withania somnifera Aksun 5 73.6 116.2 42.0 5. 37 Abelmoschus esculentus Bhindi 10 41.5 44.3 39.0 6. 2 Achyranthes aspera Puth kanda 8 36.9 31.6 22.5 7. 15 Xanthium strumarium Muhabbat b. 7 34.7 28.5 43.0 8. 48 Solanum melongena Bengun 7 31.0 24.8 32.0 9. 35 Abutilon indicum Kangi booti 7 28.4 22.3 34.0 10. 14 Parthenium hysterophorus Gajar booti 8 26.4 29.3 13.5 11. 54 Lantana camara Lantana 7 23.4 21.3 16.0

64 S. Host Vernacular no. no. Latin name name n Mean SD Median 12. 53 Duranta repens Duranta 6 17.7 13.4 18.5 13. 45 Datura alba Dhatura 6 17.5 14.7 11.5 14. 42 Portulaca grandiflora Gule dupehri 10 16.2 18.1 9.5 15. 46 Lycopersicon esculentum Tamater 17 15.1 21.8 2.0 Trianthema 16. 1 portulacastrum Itsit 15 14.9 20.6 0.0 17. 20 Cannabis sativa Bhang 8 13.6 10.0 11.0 18. 6 Digera muricata Tandla 8 12.4 9.8 13.0 19. 52 Clerodendron inerme Gardenia 9 11.9 11.7 12.0 20. 38 Hibiscus mutabilis Bhindi phool 9 11.8 9.8 12.0 21. 50 Solanum tuberosum Aaloo 6 11.0 9.6 10.5 22. 49 Solanum nigrum Mako 11 9.9 9.0 12.0 23. 19 Lepidium sativum Haloon 5 9.2 4.4 11.0 24. 55 Tribulus terrestris Bhakra 6 9.2 7.5 6.5 25. 25 Cucumis melo Kharboza 8 9 7.6 8.0 26. 18 Coronopus didimus Jangli haloon 6 8.5 5.7 6.0 27. 30 Euphorbia hirta Lal dhodhak 12 8.5 10.9 5.0 28. 41 Bougainvillea spectabilis Boganbilla 5 8 9.3 5.0 29. 7 Carthamus oxyacantha Pohli 8 7.6 6.1 7.0 30. 13 Launea nudicaulis Peeli dodhak 6 7.3 10.0 2.5 31. 26 Cucumis sativus Khera 7 7.1 4.9 7.0 Hazardani 32. 29 Euphorbia granulate dodhak 7 6.9 8.1 5.0 33. 4 Amaranthus paniculatus Billi booti 7 6.6 5.1 6.0 34. 32 Medicago polymorpha Maina 5 6.6 4.4 5.0 35. 10 Conyza bonariensis Lusan booti 7 6.3 5.3 6.0 36. 21 Atriplex crassifolia Lani 7 5.7 4.6 7.0 37. 40 Boerhavia diffusa Jangli itsit 5 5.6 5.9 6.0 38. 12 Helianthus annuus Suraj mukhi 8 5.5 7.7 2.5 39. 44 Capsicum annuum Mirch 5 5.2 4.8 4.0 40. 9 Conyza ambigua Lusan booti 7 5.1 5.4 4.0 41. 11 Eclipta prostrate Daryai booti 8 5 4.1 4.0 42. 43 Portulaca oleracea Kulfa, lunak 10 5 4.9 4.0 43. 5 Amaranthus viridis Jangli chulai 8 4.8 3.7 5.5 44. 3 Amaranthus spinosus Chulai 9 4.3 4.9 5.0 45. 27 Cucurbita moschata Kaddu 9 4.3 4.2 4.0 46. 16 Heliotropium europeaum Namkeen 8 4.1 5.4 1.0 47. 28 Euphorbia prostrate Hazardani 16 4.1 3.4 4.0 48. 23 Chenopodium morale Krund 12 3.7 6.9 1.0 49. 24 Convolvulus arve nsis Lehli 15 3.4 2.5 2.0 50. 17 Heliotropium indicum Oont chra 6 3.2 5.0 0.0 51. 34 Fumaria indica Shahtra 7 3.1 2.3 2.0 52. 8 Cirsium arvense Leh 6 2.7 2.3 3.0 53. 31 Medicago alba Jangli methi 5 2.4 2.1 2.0 54. 33 Melilotus indicus Seinji 11 2.4 2.0 2.0 55. 22 Chenopodium album Bathu 20 1.8 1.7 2.0

65 S. Host Vernacular no. no. Latin name name n Mean SD Median 56. 0 Hosts with <5 records (misc.) 23 1.7 2.9 0.0 End of Table 17. n = number of observations

The data from the CMB surveys 2005-2008 are illustrated in Fig. 12 below. This graph summarizes data from 506 sites in 44 districts of Pakistan, containing 55 listed CMB host-plant species. Host species that were observed at fewer than 5 sites were assigned the common number ‘0’ .

400

300

200

100 Max. pop of CMB/ 20 biomassgram of host plant

0

0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 Host plant No.

Lower and upper lines from both ends of the box (where applicable) show the first and fourth quartiles. The host plant no. denotes the S. no. of the host in Table 14 above. * indicates an outlying observation.

Figure 12. Maximum CMB population on 20 grams of fresh biomass of host plant observed during surveys during 2005- 2008

The CMB maximum population data (illustrated in Fig. 12 above) indicated that the most favoured host of CMB was cotton (host number 36); whereas the percentage infestation data in Table 17 (above) indicated that shoe flower was the most favoured. The year-wise CMB infestation intensity on various host-plants is given in Table 18 below. This indicated the way CMB was carried over on different plants in different years. Since there was a predetermined purpose for the survey (surveillance of CMB) the results might be biased, but even so, the results in Table 18 below show a trend of

66 infestation intensity on various host plants. The mean value for overall CMB infestation intensity in 2005 was high because there were fewer observations (only taken on cotton, itsit and hazardani, with a standardized CMB intensity of 254, 12 and 8 CMB on 20 gram fresh biomass of host plant respectively) which elevated the mean value for that year. The means for the remaining years (2006-2008) were all comparable and similar.

Table 18 . Year-wise summary of the maximum CMB population intensity on 20 grams of fresh biomass on various host plants, 2005-2008

Year n Mean SD Median Min. Max. 2005 3 91.3 140.9 12 8 254 2006 87 19.1 38.4 7 0 246 2007 387 19.6 45.4 5 0 368 2008 33 16.2 21.9 8 2 79 n = number of spots (1host plant species at 1 locality) recorded for infestation of CMB

The summarized annual data for the percentage of CMB infestation on various host-plants during 2005-2008 is shown in Fig. 13 below.

100

80

60

40

20 % infestation of CMB on observed host plants

0

2005 2006 2007 2008 Year of observations

Figure 13. Year-wise Summary of the Percentage CMB Infestation of various Host Plants, 2005-2008

67 Fig. 13 shows that, in spite of some outlier observations, the average and magnitude of the percentage infestation on different host plants was similar between the years, reflecting the consistency of the behavior of CMB on the host-plant flora. A month-wise summary of the intensity of CMB infestation data recorded from various host-plant species in the surveys during 2005-2008 is given in Table 19 below.

Table 19. Maximum CMB population intensity on various host plants, observed during field surveys of CMB in Pakistan in different months, 2005-2008

Month n Mean SD Median Min Max Host-plant species no. Jan 2 40.5 54.4 40.5 2 79 47,50 1, 3, 4, 5, 6, 10, 13, 19, 22, 32, 34, 40, 42, 43, 47, 50, Feb 26 15.4 29.3 9.0 3 156 52, 53, 54, 55 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37, 39, 40, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, Mar 138 13.2 42.2 2.0 0 315 53, 54 2, 3, 4, 6, 7, 8, 9, 10,11, 12, 13, 15, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 48, 49, 50, 52, 53, Apr 81 20.0 29.0 8.0 0 165 54, 55 1, 3, 4, 5, 7, 8, 9, 10, 12, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 32, 36, 37, 39, 44, May 38 14.7 19.6 8.0 1 87 45, 46, 48, 49, 54 1, 2, 3, 4, 5, 6, 9, 10, 11, 8, 10,14, 20,21,22 24, 28, 30, 31, 32, 33, 36, 38, 39, 41, Jun 56 23.5 36.9 10.5 3 170 42, 43, 48, 49, 53, 55 1, 2, 9, 13 , 15 , 23, 28, 31, 35, 36, 39, 43, 44, 49, 51, Jul 28 37.3 43.8 23.5 4 198 52, 53, 55 1, 5,10, 14,15, 19, 20, 32, 36, 38, 39, 41, 43, 45, 49, Aug 22 57.5 93.9 19.0 2 368 52, 55 1, 3, 7, 13, 14, 16, 17, 21, 22, 23, 27, 28, 29, 30, 36, 37, 38, 39, 40, 42, 46, 50, Sep 41 17.3 54.8 0.0 0 254 52, 54

68 Month n Mean SD Median Min Max Host-plant species no. 1, 2, 6, 7,10, 11, 13, 14, 15,16, 17, 21, 22, 23, 24, 28, 29, 30, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, Oct 66 19.0 51.0 0.0 0 289 46, 49, 52, 54 7, 11, 13, 17, 22, 23, 28, Nov 15 12.8 25.0 4.0 0 79 29, 30, 36, 40 11, 17, 18, 21, 22, 23, 28, Dec 12 17 21.2 9.5 0 65 29, 30, 35, 39, 45 n = number of spots (1host plant species at 1 locality) recorded for infestation of CMB For host-plant species names, see the s. no.s in Table 14 above End of Table 19.

In Table 19 (above), the magnitude of the standard deviation values reflects the host-acceptance flexibility of CMB, and n give the frequency of its occurrence on the available host plants. The host number refers to the S. no. of that host in Table 14 above. The table shows how the continuous availability of alternate host plants ensured successful carry over of CMB in the agro-ecological conditions of Pakistan. It also revealed the way that the pest managed to carry over in different months of the year on different host-plant species. The results might be statistically biased, as the observations were made from purposefully selected localities rather than those chosen at random. Even so, it indicates trends in the ecological conditions most favoured, in terms of both the availability of suitable hosts and the prevailing meteorological conditions.

4.2.2 Ecology 4.2.2.1 Study of CMB developmental stages in different seasons A general survey of the CMB population on the early sown cotton (May), peak activity season (July) and at the end of the cotton season (October), was described in 3.2.7 above. It yielded data that are summarized in Table 20 below. ANOVA (details in Appendix Table 7.0) followed by comparison of means analysis (details in Appendix Table 7.4) showed that the different environmental conditions in May, July and October induced significant variation (p = 0.000, 0.000 and 0.000 respectively) in the numbers of reproductive CMB, pre-reproductive feeding stages of CMB, the number of first instars and the total damage-inducing CMB population (see Table 20 below).

69 Table 20. Average number of different stages of the pest damaging cotton in three different months, on the top three inches of a cotton twig

Month (1) Females (2) 2nd , 3rd instar & (3) First-instar (4) Total in 2007 with crawler non-reproductive crawlers damaging sacs females population May 51.7 ± 11.7 a 166.3 ± 16.1 a 1107.3 ± 92.5 cd 1325.3 ± 105.1de July 49.7 ± 8.5 a 116.3 ± 20.7 a 1579.0 ± 67.9 ef 1745.0 ± 79.3 f October 79.0 ± 9.8 a 110.3 ± 13.7 a 865.0 ± 69.6 bc 1054.3 ± 81.5 cd In each category, values sharing the same letters were statistically similar.

Fig. 14 (below) illustrates how the total damage-inducing CMB populations were significantly different between different months (p = 0.000).

Total population of CMB

1800

1700

1600

1500

1400

1300

1200

1100

1000

population of CMB (number)in situ counts situ (number)in of CMB population 900 May Jul Oct Month

July = mid-cotton season; May = early cotton season; October = end of cotton season

Figure 14. Averaged total damage-inducing CMB population on a three-inch cotton twig in May, July and October 2007 at Faisalabad, Pakistan

4.2.2.2 Population Dynamics of CMB 4.2.2.3 Intensive survey of CMB on shoe flower The year-round survey of CMB on shoe flower (Hibiscus rosa-sinensis) were described in 3.2.7 above. The population curve in Fig. 15 (below) is based on month-wise pooled data, and shows the variation in the CMB population throughout the year.

70

Date of observation Av ht inch = average height in inches; Br. 1 in D = branches of 1 inch diameter; Drd. Twigs = dried twigs; Wh caps = white caps; Pop/twig = population per twig; Beneficials = number of beneficial organisms

Figure 15. Graph to show Population Dynamics of CMB and various Features of the Host Plant, Shoe Flower (Hibiscus rosa-sinensis)

As indicated in Table 11 above, the behaviour of CMB on shoe flower in field conditions was similar to that on cotton. In spring CMB was detected in mid-March and the population increased rapidly (Fig. 15 above). The main population growth and spread of CMB occurred in June to July, but there was little change in the number of dried twigs and white caps (twig tips totally covered by mealybugs, forming a cap-like structure). Infested twigs developed ever greater numbers of CMB and eventually became desiccated and died. Although the total number of twigs on the host-plant increased, no such increase was seen in the number of white caps or dried twigs. It was also observed that in the cotton fields, the pest occurred in patches. The mealybugs remained in those patches to the extent that the entire patch of infested plants died. The week-wise data from the shoe flower study (Appendix Table 11.0) were analyzed to examine the population dynamics of CMB. The analysis revealed that the population dynamics of CMB on shoe flower fit a regression model (p = 0.000, Appendix Table 11.1). The four-in-one graph of residual plots in Fig. 16 below examine various features of the data, which suggested that the assumptions necessary for a good regression model were met by the data. The necessary assumptions for a good regression model and interpretation of the figures generated from the data are explained below Fig. 16.

71

Residual Plots for Pop/twig Normal Probability Plot Versus Fits 99 20 90 10

50 0 Percent Residual -10 10 -20 1 -20 -10 0 10 20 0 25 50 75 100 Residual Fitted Value

Histogram Versus Order

12 20 10 9 0 6 Residual

Frequency -10 3 -20 0 -20 -10 0 10 20 51 10 15 20 25 30 35 40 45 Residual Observation Order

Figure 16. Graph to show properties of the data of residuals of population dynamics of CMB on shoe flower (Hibiscus rosa-sinensis) at UAF in 2007, against a regression model fitted line

1. Normal probability plot of residuals (top left graph, Fig. 16): The points in the plot should generally form a straight line if the residuals (the difference from the fitted line or 0 line) are normally distributed. If points of the plot depart from the straight line, normality assumption (necessary for a good regression line/model) may be invalid. The graph showed that the data followed a normal distribution, hence it qualifies for the assumption necessary for a good regression model. 2. Residuals versus fitted values (top right graph, Fig. 16): This plot show a random pattern of residuals from both sides of the 0 or fitted line, there should not be any recognizable patterns in the residual plot. For instance, if the spread of residual values tend to increase as the fitted values increase, then this may violate the constant variance assumption. The graph represents the residual versus fit of this data; there is a somewhat of a spread in the residual values with the increase in the fitted values, but it is homogeneous on both sides (not skewed or clustered to any one side). 3. Histogram of the residuals (bottom left graph, Fig. 16): This plot should show a random pattern of residuals on both sides of line 0. If a point lies far from the majority of points, it may be an outlier. There should not be any recognizable

72 patterns in the residual plot. For instance, if the spread of residual values tend to increase as the fitted values increase, then this may violate the constant variance assumption. The graph shows that the residuals generated from the shoe flower data follow a random (bell shaped) distribution, which satisfies the assumption and is a good regression model. 4. Residuals versus order of the data (bottom right graph, Fig. 16): This is a plot of all residuals in the order that the data were collected, which can be used to find non- random error, especially of time-related effects. This plot helps to check the assumption that the residuals are uncorrelated with each other, as required for generating a regression model. The graph shows that the data figures from shoe flower were random and non-patterned, as required for a good regression model. In view of the above analysis, which suggests that the CMB population dynamics data was a good fit for a regression model, a subset of regression models were generated for selection of an appropriate model (Table 21 below). The model in the bottom row of Table 21 had the highest R 2 value (0.945), that is it governed the 94.5% variability in the response (population of the pest per twig), and minimum p value (0.000), so it was selected as the most appropriate regression model for the shoe flower data. The graphs in Fig. 16 (above) show the different properties of residuals corresponding to the regression line fitted by this model, using the regression equation in the bottom row of Table 21 below. (More details of these analyses are given in Appendix Tables 11.0 to 11.4).

Table 21. Linear Multiple Regression Models between the CMB Population on Shoe Flower and Ecological Factors, along with Coefficient of Determination Values

Role of Regression equations R2 100 R 2 individual factor (%) Y = -8.0+9.09 *X1 0.782 78.2 78.2

Y = -8.0+9.09 *X1+8.05 * X2 0.900 90.0 11.8X1

Y = -8.0+9.09 * X1+8.05 *X2-1.01 X3 0.936 93.6 3.6X2

Y = -8.0+9.09 *X1+8.05 *X2-1.01 X3+0.696X4 0.942 94.2 0.8X3

Y = -8.0+9.09 *X1+8.05 *X2-1.01 0.943 94.3 0.1X4 X3+0.696X4+0.43 *X5

*Y = -8.0+9.09 * X1+8.05 * X2-1.01 0.945 94.5 0.2X5 X3+0.696X4+0.43 *X5+0.055X6 Where X1 = White cap-like clusters (obviously visible) on terminals of the plants X2 = Number of beneficial fauna found (beetles, ant lions and spiders)

73 X3 = Average maximum temperature of the study period (one week) X4 = Average minimum temperature of the study period (one week) X5 = Average relative humidity % in the study period (one week) X6 = Average rainfall (millimeters) in the study period (one week) * = Significant at P < 0.05.

Analysis of the data (Appendix Table 11.1) revealed that three parameters of the data had a significant effect as a predictor of this regression model: 1. Wh. caps: formation of white caps on the terminals of the host plant, which was denoted in the regression equation as X1 (p = 0.000) 2. Beneficials: the population of beneficial insects, which was denoted in the regression equation as X2 (p = 0.000) 3. RH%: percent relative humidity, which was denoted in the regression equation as X5 (p = 0.045). Graphs of the residuals versus two important predictors, X2 (beneficial insects) and X5 (relative humidity), are shown in Figs 17 and 18 below, respectively. The figures show that the data were randomly distributed, not having a patterned formation nor clustered or skewed to any one corner.

Residuals Versus Benificials (response is Pop/twig)

20

10

0 Residual

-10

-20

0 1 2 3 4 5 6 7 Benificials

Figure 17. Graph of the residual of the CMB population per twig (variable) versus population of beneficials (predictor) for CMB on shoe flower, against a regression model fitted line

74 Residuals Versus RH % (response is Pop/twig)

20

10

0 Residual

-10

-20

20 30 40 50 60 70 80 RH %

Figure 18. Graph of the residual of the CMB population per twig (variable) versus percent relative humidity (predictor) for CMB on shoe flower, against a regression model fitted line

4.2.2.4 Intensive Survey of CMB on Cotton Intensive surveys on cotton in the research area at UAF (as described in 3.2.7 above) provided data for the following population curve. Fig. 19 (below) which shows that the peak of the CMB population size on cotton was from the end of July to the third week of September, after which the population declined gradually.

7000

6000

5000

4000

3000

2000 Population of CMB 1000

0

7 7 7 7 7 7 7 7 7 .0 0 .0 0 .0 0 0 0 0 8.07 9.07 .07 .07 08 0 0 .09. .10. 4. 10.07.0717 24.07. 31 07.08. 1 21.08. 28. 04.09. 11. 18 25.09.0702 Weeks of observation

Figure 19. Population dynamics of CMB on cotton (Gossypium hirsutum )

75

It took time for mealybug numbers to build up from the small starting population. The maximum weekly percentage growth rate was 218.3% in the third week of observations (in late July), but the graph could not show this because the total population at that time was so small (63 compared to later population totals of over 1,500) that the scale of the graph obscured the steepness of the line at that point (for details see Appendix Table 12.0). There was a gradual increase in population early in the season, when the calculated number of adults was only 25 per plant (Fig. 20 below). Up to the third week of observations there was only a nominal increase in population while the first progeny produced by those adults developed; once the progeny matured and reproduced, there was a geometric increase in the mealybug population that continued until the third week of September, when the population went into negative growth. From the end of September the population growth rate decreased from the peak and declined to -8.8 % in the first week of October 2007 (more detail is given in Appendix Table 12.0).

Weekly Growth% = weekly CMB population growth %; CommulativeG.% = cumulative CMB population growth %; Plant Growth = growth of the host plant; Population = total CMB population

Figure 20. Population dynamics of CMB as compared with cumulative population growth, and growth of the host-plant (cotton)

Statistical analysis of the data from the experiment described in 3.3.1, which measured the resistance of the ten cotton cultivars to CMB attack, also yielded information on CMB population dynamics on cotton in the field (Fig. 21 below).

76

450

400

350

300

250

200 PGR of CMB population of CMB PGR 150

100

1 2 3 4 5 6 7 8 9 10 11 12 13 Weeks(Jul. 10,2007 to Oct. 2,2007)

PGR=Percent Growth Rate (percent increase in CMB Population) after an initial inoculation of 100% on each variety, calculated as: PGR={population of (i +1) week- population of week (i )/population of week (i)}* 100 Figure 21. Weekly growth rate of CMB on ten cotton cultivars in 2007 There was a sharp initial increase in the CMB population growth rate (Fig. 21 above), peaking 7-15 days after initial infestation. A second, smaller peak occurred four weeks later, reflecting production of the second generation. The slowing of the population growth rate while the second generation matured was reflected in Fig. 22 (below).

Normal Probability Plot (response is Growth rate) 99.9

99

95 90 80 70 60 50 40

Percent 30 20 10 5

1

0.1 -200 -150 -100 -50 0 50 100 150 Residual

77 Figure 22. Weekly population growth rate of CMB through the cotton season, averaged from ten cotton cultivars in 2007

The analysis revealed that the different cotton cultivars had a non significant role in the PGR (percent population growth) of CMB after it was released in equal numbers on all the varieties (p = 1.000, Appendix Table 13.0). At the same time, there was a significant difference in the PGR of CMB in different weeks of observation (p = 0.000, Appendix Table 13.1). It also found that there was a strong correlation between the CMB population and meteorological factors, which had different dimensions, some negative and some positive; the detail is summarized in Table 22 below. Table 22. Interactions between various biotic and abiotic factors and CMB population growth Factors Population ofWeekly Temp. Temp. RH of CMB growth% max. ºC min. ºC % Weekly -0.711** growth % 0.006 Plant 1.00 -0.711** growth * ** 0.006 Temp. -0.667 * 0.439 ns max. 0.013 0.133 Temp. -0.637* 0.463ns 0.893** min.. 0.019 0.411 0.000 RH -0.513ns 0.463ns 0.606ns 0.818** % 0.073 0.111 0.228 0.001 Rainfall -0.388 ns 0.470 ns 0.213 ns 0.010 ns 0.161 ns mm 0.190 0.105 0.484 0.974 0.599 Cell contents: Pearson correlation * = significant, ** = highly significant : P value ns = non-significant (correlation)

The analysis also showed that the response (the population of CMB) followed a line fitted by a regression equation (p = 0.000; 100 R 2 value = 41.6, Appendix Table 13.2), but due to a low 100 R 2 value (which is the measure of reliability) this regression model is not dependable. Fig. 23 (below) shows various features of the data which render it less reliable (the detailed analysis is in Appendix Table 13.1.) The possible reasons and suggestions for improvement of this model are discussed in 6.6.10 below.

78 Residual Plots for Pop Normal Probability Plot Versus Fits 99 3000

90 1500

50 0 Percent Residual 10 -1500

1 -3000 -5000 -2500 0 2500 5000 0 2000 4000 6000 8000 Residual Fitted Value

Histogram Versus Order 3000 4

3 1500

2 0 Residual Frequency 1 -1500

0 -3000 -2000 -1000 0 1000 2000 3000 10987654321 11 12 13 Residual Observation Order

Figure 23. Different features of the weekly CMB population data through the cotton season, averaged from ten cotton cultivars In Fig. 23 above, the normal probability plot (top left) is reasonably good but there is a great dispersal in the plot of residual versus fit (top right). The frequency of residuals in the histogram (bottom left) does not follow a normal (bell-shaped) distribution. There is a pattern in the observation order plot (bottom right); initially the observed values produce negative residuals but as the values of observation increase the residuals produced become positive. This was because the CMB population growth rate was fastest in the second week after initial infestation of the plants (Fig. 23 above). From the third week onwards, the CMB population growth rate decreased (although the population continued to increase), probably because of strong intra-specific competition. Fluctuations in the CMB population growth rate throughout the trial (Fig. 23 above) were the result of the interaction of various biological and ecological factors (Table 23 below).

79 Table 23. Comparison of means of the weekly population growth rate of CMB through the cotton season

S. Date of weekly Average Av. Average Av. population no. observation temp. °C RH% rainfall in mm growth rate in 2007 1. 10.07.07 32.1 55.6 76.8 100.00 ef 2. 17.07.07 20.1 53.3 49.6 267.73 b 3. 24.07.07 32.0 58.6 15.8 351.99 a 4. 31.07.07 32.5 53.4 7.5 198.24 c 5. 07.08/07 32.3 53.9 0.0 182.45 cd 6. 14.08.07 33.2 53.1 0.7 183.07 cd 7. 21.08.07 32.7 50 .0 0.5 184.22 cd 8. 28.08.07 32.6 48.9 4.8 148.91 de 9. 04.09.07 32.3 54.4 15.7 138.98 de 10. 11.09.07 30.2 54.6 7.8 130.95 ef 11. 18.09.07 30.4 42.1 0.0 122.57 ef 12. 25.09.07 29.6 53.7 2.2 102.29 ef 13. 02.10.07 28.4 40.0 0.0 88.47 f Means sharing same letters have non significant difference at alpha = 0.05.

Whether the decline in the CMB population growth rate was principally due to overpopulation or environmental factors is not clear and needs further research. If it shows that the early-season population growth rate peak is due to favorable environmental conditions, then it can be assumed that last week of July (or thereabouts) provides the environmental conditions in which CMB thrives best; otherwise, the peak might be attributable to a lack of intra-specific competition early in the season, as population growth rate may well be a density dependent phenomenon. 4.2.2.3 Distribution of CMB in the Cotton Field In the field, CMB infestation occurred in patches, showing no regular, sequential or spatial trend in the incidence of either infestation or damage. Fig. 24 (below) depicts averaged values of various biological features from ten selected, CMB-infested cotton plants (all of the same cultivar, showed on the same date, subject to same agronomic practices and the same micro climate), which were observed over three consecutive weeks. Fig. 24 illustrates the situation in the field, which showed a patchwork of different intensities of damage inflicted by CMB. The infestation occurred in patches so the damage level varied significantly in different parts of the field. The data from different plants was haphazard and without any sequence, not showing any statistically significant differences. The damage observed was directly proportional to the duration of CMB infestation and the mealybug population density.

80

Population/inch 5/8 Population/inch 8/8 Branches Leaves Height cm Immature Bolls Squares Duration of damage

100 90 80 70 60 50 40 30 Number observed 20 10 0 P1 P 2 P 3 P 4 P 5 P 6 P 7 Plants

Figure 24. Graph to show various features of cotton plants infested by CMB

4.2.2.4 Distribution of CMB in Pakistan The surveys for CMB (P. solenopsis) were carried out throughout 2005-2008. Different districts were visited to locate CMB host plants and their role in the carry over of the pest. The distribution of CMB in the cotton-growing areas of the Punjab, documented by extensive surveys by the Directorate General of Pest Warning and Quality Control of Pesticides, Punjab; and in the Sindh by the Govt. of Sindh Agriculture Dept., were used as guidelines for CMB surveys in this study. The spots indicated by these departments were taken as records of the occurrence of the pest. The above surveys were concerned with economic crops only, whereas in this study all available host plants were recorded and the site localities were noted to record the distribution of the pest. A summary of the CMB distribution survey data for 2005- 2008 is illustrated in Fig. 25 below.

81 400

300

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100 max. pop. of CMB on 20g biomass of host plant 0

r r r r t i r l r s i l z r i l r i t r i t i n a a n i d d d g a h a n a r e h n n a r n n h a d h r a r a i n t a n d a a a b a K a a g r a d g u k a D a a a n l l c s u w r a d o a r a a h i a t a t a a r w a a h r o a h t a u n w h h b d y t h i n a r a a l p k h b b b a a a a e a z h y h r d K i t l l o b s h e e k i s e h p i i g d i w a a s h i a B n a a K l a r a a h K r K n h u a y a M u u G r r a h F P u K n l p h o S S h h e Z l w h a e b J a a K h L a d r M M r a e b s O Q r j a a a a n g m l a T e k V a a B G is d a K h K L L o M u f a N e a o r o a a w S S a r m l T h D a y k K P f s w N Y R a s a A a w a F a c i r z a a a e h R h o a h B H J u N N h i m u d b a M s h o B M o a . M a n T N R T T Districts visited

Lower and upper lines from both ends of the box (where applicable) show the first and fourth quartiles. The host plant no. denotes the s. no. of the host in Table 14 above. * indicates an outlying observation.

Figure 25. Graph to show the CMB infestation levels at survey sites in pakistan, based on summarized survey data for 2005- 2008

Although Fig. 25 did not provide a complete picture of the CMB infestation in Pakistan, it did indicate the trend of the dispersal of the pest in different districts. This data embodies of 506 sites in 44 districts. The detailed district-wise intensity of the surveillance data is summarized in Table 24 below.

82 Table 24. Maximum CMB population intensities recorded at different locations in Pakistan on various host plants during field surveys during 2005- 2008 (continued on the next page)

Population S. No. of intensity no. District hosts Mean SD Median 1. Badin 8 12.6 10.8 7.0 2. Bahawal nagar 14 36.8 81.7 7.5 3. Bahawlpur 14 41.6 51.2 23.5 4. Bhakkar 10 9.4 21.0 0.0 5. D G Khan 10 113.0 119.2 59.0 6. Dir 4 0.0 0.0 0.0 7. Faisalabad 34 26.4 36.6 9.0 8. Hyderabad 5 20.8 25.0 8.0 9. Jackababad 4 16.7 7.8 17.0 10. Jhang 6 5.8 4.0 4.5 11. Kallat 7 0.0 0.0 0.0 12. Karachi 1 45.0 0.0 45.0 13. Kasur 7 31.3 55.2 8.0 14. Khanewal 12 21.7 29.7 7.5 15. Kharan 5 0.0 0.0 0.0 16. Khuzdar 5 0.0 0.0 0.0 17. Lahore 9 30.7 27.2 19.0 18. Layyah 12 16.9 21.2 9.5 19. Lodhran 52 1.8 1.8 2.0 20. Mardan 4 0.0 0.0 0.0 21. Mir Pur Khas 4 2.8 1.5 2.0 22. Mitiari 3 14.0 17.3 5.0 23. Multan 59 17.5 42.1 5.0 24. Muzaffar Garh 58 14.8 24.1 4.0 25. Narowal 3 22.7 28.9 7.0 26. Naseer abad 3 43.0 3.2 3.0 27. Nawab shah 3 110.0 122.6 76.0 28. Noshehra 7 0.0 0.0 0.0 29. Noshero Feroz 6 36.0 59.0 15.0 30. Okara 12 13.7 15.1 7.5 31. Pishin 7 0.0 0.0 0.0 32. Quetta 5 0.0 0.0 0.0 33. Rahim Yar Khan 22 34.8 59.8 8.0 34. Rajan Pur 17 45.2 95.7 10.0 35. Rawal pindi 4 16.3 9.7 18.0 36. Sahiwal 9 25.9 23.4 17.0 37. Sanghar 9 31.7 33.7 12.0 38. Sargodha 2 18.0 15.6 18.0 39. Sibbi 8 0.0 0.0 0.0 40. Swat 9 0.0 0.0 0.0 41. T. Muhammad Khan 2 19.0 18.4 19.0

83 42. Tando Allah Yar 6 40.2 64.8 12.5 S. No. of no. District hosts Mean SD Median 43. Thatta 6 5.0 2.0 5.0 44. Toba Tek Singh 1 24.0 - 24.0 45. Vehari 9 13.0 13.0 8.0 46. Ziarat 7 0.0 0.0 0.0 End of Table 24.

The distribution information from survey data, personal communications and collections received for identification were combined to indicate the distribution of the pest in Pakistan, as listed below and illustrated in Fig. 26 below. Districts in Punjab : Narowal, Lahore, Kasur, Okara, Pakpattan, Sahiwal, Vehari, Khaneval, Lodhran, Bahawal Nagar, Rahimyar Khan, Bahawalpur, Rajanpur, Dera Ghazi Khan, Muzaffargarh, Multan, Layyah, Bhakkar, Sargodha and Rawalpindi showed CMB distribution. Districts in Sindh : Karachi, Thatta, Badin, Hyderabad, Tando Allahyar, Tando Muhammad Khan, Matiari, Mirpur Khas, Sanghar, Nawabshah, Naushehro Feroze, Sukker, Ghotki and Jacobabad showed CMB infestation. . North West Frontier Province (NWFP) : Dera Ismail Khan district showed CMB infestation (Mamoon ur Rashid (Ph.D. scholar, Gomal University, Dera Ismail Khan, Pakistan), 2008, personal communication). Baluchistan : Naseerabad district (unpublished data, A.S. Buriro (Director, Entomology Dept. Research Wing, Govt of Sindh, Tando Jam), 2007, personal communication).

Adapted from World Atlas (2008).

84 Figure 26. Distribution of CMB-infested areas on a map of Pakistan 4.2.2.5 Overwintering and carry over of CMB in Pakistan Information on overwintering and carry-over of cotton mealybug in Pakistan was provided by data from the intensive and extensive surveys described in 3.2.7 above. The agro-ecological zones of Pakistan are shown in Fig. 27 below.

. I- Indus Delta V - Barani Lands IX - Dry Western Plateau II - Southern Irrigated Plain VI - Wet Mountains X - Sulaiman Piedmont III - Sandy Desert (a and b) VII - Northern Dry Mountains IV - Northern Irrigated Plain VIII - Western Dry (a and b) Mountains Source: PARC (2007)

Figure 27. Agro-ecological Zones of Pakistan

85

Cotton mealybug was observed in various cropping systems adapted to the different agro-ecological zones, and overwintering successfully in the zones listed in Table 25 below.

Table 25. Occurrence and average population of CMB in various agro-ecological zones of Pakistan

Zone Agro-ecological General cropping Occurrence Means of no. zone pattern & population overwintering I Indus delta Vegetables Observed - Tomato, lady finger, low cucurbits, weeds, ornamentals II Southern Cotton-wheat Observed - Weeds, sunflower irrigated plains /sunflower high vegetables III Sandy Desert Not applicable Observed - Weeds (a & b) (shrubs) traces IV Northern irrigated Cotton-wheat Observed - Weeds, vegetables, plains (a & b) high sunflower, potato V Barani Lands Wheat-mixed Observed - Weeds,Ornamentals low VI Wet mountains Mixed Not observed Not applicable VII Northern dry Mixed Not visited Not reported mountains VIII Western dry Wheat-vegetables Traces Ornamentals Mountains observed IX Dry western Shrubs Not observed Not applicable Plateau X Suleman Shrubs Not observed Not applicable Piedmount

Most of the economic damage CMB caused in Pakistan occurred in the cotton- growing zones on irrigated land, where there is maximum use of pesticides and hence maximum loss of biodiversity. 4.2.2.6 Parasitoids and Predators of CMB in Pakistan During the intensive and extensive surveys mentioned above, the fauna associated with CMB (parasites and predators) was observed. The insects, mites, spiders, fungi etc were recorded, together with brief observation notes. The beneficial fauna observed attacking CMB in the field is listed in Table 26 below. Representatives of the unidentified species have been deposited in the IPM Lab., Dept. of Agri. Entomology, for future reference and identification by subsequent researchers.

86 Table 26. List of beneficial fauna associated with CMB in the field in Pakistan

S. Common name Scientific name Higher taxa Distribution no. 1. Transverse LBB Coccinella Coleoptera: Cotton zones of transversalis Fabricius Coccinellidae Punjab & Sindh 2. Stripped beetle Brumus suturalis Coccinellidae Plains of Punjab (Fabricius) & Sindh 3. Zigzag beetle Menochilus Coccinellidae Cotton zones of sexmaculatus (Fabricius) Punjab & Sindh 4. Eleven-spotted Coccinella Coccinellidae Throughout Beetle undecimpunctata L. Pakistan 5. Convergent Hippodamia Coccinellidae Throughout LBB convergens Pakistan Guérin-Méneville 6. Seven-spotted Coccinella Coccinellidae Throughout Beetle septempunctata (L.) Pakistan 7. Mealybug Scymnus spp. Coccinellidae Throughout beetles Pakistan 8. Spotted Hippodamia variegata Coccinellinae Northern Punjab amber LBB 9. Red Chilochorus Coccinellidae : Lower Sindh Chilochorus sp. circumdata Schbn. Chilochorinae 10. Steel-blue LBB Chilochorinae Lower Sindh (Boisduval) 11. Ant lion Chrysoperla spp. Neuroptera: Plains of Punjab Chrysopidae & Sindh 12. Encyrtid wasp Unidentified Hymenoptera: Plains of Punjab Encyrtidae & Sindh 13. Gall midges Unidentified Diptera: Plains of Punjab Cecidomyiidae & Sindh 14. Big-eyed bugs Geocoris spp. Hemiptera: Plains of Punjab Lygaeidae & Sindh 15. Minute pirate Orius spp. Hemiptera: Plains of Punjab bugs Anthocoridae & Sindh 16. Spiders (numerous species) Order Araneae Throughout Pakistan 17. Mites (numerous species) Order Acari Throughout Pakistan

General predators like spiders, coccinellid beetles and ant lions were found feeding voraciously on CMB. Mealybug crawlers were preferred over other soft-bodied insect prey. Unfortunately most of this beneficial fauna was ruthlessly wiped out by routine pesticide-spraying of cotton. This was why CMB was most prevalent in cotton- growing areas. Even in the non-core cotton-growing districts of the Punjab like

87 Faisalabad, Toba Tek Sing, Jhang, Okara etc. the pest occurred mainly in pockets where cotton was a regular component of the general cropping pattern of most of the farmers.

4.2.3 Ecobiology Discussion 4.2.3.1 Life Cycle Study The laboratory study in this work fed the mealybugs on cotton leaves detached from the plant. The variability recorded was, therefore, natural variation between individuals from a population, all kept in the same environmental conditions on same food source. However, this meant that the leaf water content, osmotic pressure, opening of the stomata, natural reaction of the living plant to a foreign injury, predators and parasitoids present, inter-specific competitors, and temperature and humidity differed in the laboratory compared to those in the field. Two studies of the life cycle and life span of P. solenopsis have been published. One was a study on cotton, most probably at Cotton Research Station Multan, but that is not clearly mentioned (ICAC Recorder, 2008). Although in this publication, the mealybug is referred to as P. solani, actually it is about the mealybug pest on cotton in Pakistan [now identified as P. solenopsis (Hodgson et al. , 2008)]. The second publication documented the life cycle of P. solenopsis on shoe flower (Hibiscus rosa-sinensis) in Nigeria (Akintola and Ande, 2008). The features of the life history in those studies are compared to the findings in this work in Table 27 below.

Table 27. Comparison of Studies on Features of the Life Cycle of P. solenopsis

S. no. Feature compared Abbas ICAC Recorder, Akintola and 2008 Ande, 2008 1. Female total life span 25.1 ± 13.7 days 30-48 days 37 days 2. Duration of immature 16.0 ± 2.5 days 13-17 days Not available Stages of male 3. Duration of first 6.8 ± 1.8 days NA 6 days instar 4. Duration of second 6.4 ± 0.7 days NA 8 days instar 5. Duration of third 7.3 ± 2.5 days NA 10 days instar 6. Duration of winged 2.2 ± 1.8 days 3-5 days Not available adult male 7. No. crawlers per sac 77.9 ± 22.2 98-329 Not available 8. Mating period 5 minutes 10-45 minutes Not available

88 Table 27 shows that the findings of the three studies were largely similar, except for a difference in the maximum number of crawlers per sac. In this study, in repeated observations in the field and laboratory, the maximum number of crawlers per female was 113 in the laboratory and 129 on cotton in the field (average of 10 females in each location). This information was shared with the author’s community through popular articles (Abbas et al., 2007a) and no consistent research work has refuted it. 4.2.3.2 Number of Instars in CMB The results of the laboratory studies in this work, in finding that CMB has four instars in the female, were similar to those of other researchers (ICAC recorder, 2008; Hodgson et al., 2008; Akintola and Ande, 2008). The number of instars in both sexes determined in this study was also in agreement with the general biology of mealybugs (Osborne et al., 1994; Oetting, 2004) and with other published research on P. solenopsis (ICAC Recorder, 2008; Akintola and Ande, 2008). Mealybugs are hemimetabolous insects and do not undergo complete metamorphosis. However, the male undergoes a great morphological change to the winged adult, so it passes through two inactive, non-feeding stages known as the “prepupa” and “pupa”. 4.2.3.3 Sexual Dimorphism in CMB Cotton mealybug was observed to be sexually dimorphic, having a wingless, larviform adult female and a very small, short-lived, winged male. This dimorphism has been documented in a number of publications (Abbas et al., 2006; Abbas et al., 2007a and b; Hodgson et al., 2008; Abbas et al., 2009) and has been confirmed by all the researchers working on this species (Buriro, 2006; Khaskheli, 2007; ICAC recorder, 2008; NCIPM, 2008; Akintola and Ande, 2009). 4.2.3.4 Time and Duration of Mating in CMB In the field, mating usually occurred in the early morning on hot summer days, but in months with mild temperatures (25-30 ºC), mating was observed to occur at any time of the day. In laboratory studies, the author observed that the male was attracted to the female, perhaps by pheromones she produces, but this was not proved and the biochemical nature of any pheromone is unknown. The mating period of 10-45 minutes mentioned in ICAC Recorder (2008), observed in field conditions, differs considerably from that found in controlled laboratory conditions in the present study (Table 27 above, s. no. 8). The limited number of observations in this study (3) did not support the findings in ICAC Recorder (2008) and

89 this value needs further research for confirmation. Since there was no methodology involved, the difference was based on observations. In the present study, it was seen that the male takes a long time to position himself before insertion of the aedegus into the vulva. The time taken for mating reported by ICAC Recorder (2008) may have included this positioning time in the recorded mating time; or the difference might be due to different meteorological conditions in the two studies. 4.2.3.5 Mode of Reproduction of CMB ICAC Recorder (2008) reported that the pest mealybug on cotton in Pakistan (which they called P. solani) reproduced asexually as well as sexually. No explanation was given as to how they arrived at this conclusion, but it might have been based on the finding that males were seldom observed in the field in summer. However, in field work in the present study, the author observed that males were present in the cool, humid conditions very early in the morning in summer but disappeared as the temperatures rose and the humidity fell during working hours. The adult male lacked mouthparts and could not feed; he was seen to live only 2 hours to 3 days in the field because he was very vulnerable to high summer temperatures, low humidity, rainfall and pesticides. In contrast, in September when temperatures were mild (below 30˚C), large numbers of males were seen flying in clouds near the fields. Based on both field and laboratory studies, the present work found that CMB reproduced sexually all year around, in agreement with reports on other mealybug species by Osborne et al. (1994) and Oetting (2004). The author observed that mating usually occurred only once in the female’s lifetime. However, in the field additional matings were sometimes seen, which could result in the female producing up to 20% of additional crawlers compared with a singly mated female. This observation needs more experimentation for confirmation. After mating, the female’s body was observed increasing in size 3 - 10 times, depending on the number of embryos developing within. 4.2.3.6 CMB Crawler Emergence and Movement Khaskheli (2006a) and Khushk and Mal (2006a) reported that an adult female cotton mealybug produced 500-600 eggs, but this was the result of misidentification of CMB as Maconellicoccus hirsutus, and erroneous use of information about M. hirsutus taken from Internet websites. This was pre-research information that has been disproved by a number of researchers since. In the present work, consistent field observations found that when the adult female becomes detached from its ‘crawler sac’, the crawlers were released and started crawling hither and thither in search of a suitable feeding site. These

90 observations agreed with a number of researchers (Khaskheli, 2007; Buriro et al., 2006; Yousuf et al., 2007; ICAC Recorder, 2008; Parvez, 2008a; Akintola and Ande, 2008). The findings were published by our research team (Abbas et al., 2007a and b, 2008; Arif et al., 2007a, b and c) and were undisputed in Pakistan. However, in the opinion of some Indian researchers (A.K. Dhawan, S. Saini, 2008, personal communications) the crawler of P. solenopsis takes 6-8 hours to hatch from an egg, which is not the case in Pakistan. This difference may be attributed to meteorological conditions such as differing temperature ranges in the cotton zones of India and Pakistan. 4.2.3.7 Deposition of Wax on CMB Crawlers Wax is a biochemical substance which is produced by all developmental stages of mealybugs. It is secreted from glands inside the body, through various types of pore. The waxy coating protects the insect from desiccation and penetration of pathogens and toxic chemicals through the integument (Osborne, 2004, Oeting et al., 2004; Watson and Kubiriba, 2005) . It has been mentioned to occur in CMB by a number of researchers (Arif et al., 2006; NCIPM, 2008; Tanwar et al., 2007). These records are consistent and unanimous but there may be some differences in the number of days taken for the freshly emerged crawlers to develop the wax coating. Our studies carried out in a controlled environment (25 ± 2 ºC and 65 ± 5 % R.H.) found that the crawlers took 2.7 ± 0.7 days to turn white with wax, whereas field observations in the cotton zone (at about 40 ± 8 ºC) found that new crawlers became white with wax within 24 hours of emergence from the crawler sac. The different times recorded by different studies are probably attributable to different environmental conditions between the studies. 4.2.3.8 Mortality in CMB The female CMBs reared in isolation for the sex ratio study showed relatively low mortality (36.7% in Table 9 above), probably because the only significant cause of injury was abrasion caused by the camel-hair brush during specimen transfers to new leaves. In contrast, the mortality level recorded in the life cycle study, in which crawlers were reared in crowded cages, was much higher (63.3%). The increased level of mortality was probably dues to factors like competition for feeding sites, honeydew fouling and consequent fungal infection, and abrasions caused by the camel-hair brush during transfer of the crawlers to new leaves, probably increased the mortality. The death of mealybugs before they could reproduce can be termed as premature mortality. These individuals still contributed significantly to host-plant damage and economic injury, but they did not contribute to increasing the pest population.

91 4.2.3.9 The Average Duration of one CMB Instar Akintola and Ande (2008) worked on P. solenopsis in Nigeria, and reported on the average period of the first three instars; their data agreed with the values found in this study (see Table 6, s. nos 6, 7 and 8 above). 4.2.3.10 Total Lifespan of CMB The study on P. solenopsis in Nigeria by Akintola and Ande (2008) did not indicate the average duration of the adult female stage or include the period of immaturity in the average life span of the female. In the present study, the life expectancy of the adult female after production of the first crawler batch, excluding immature mortality, averaged 16.4 ± 4.2 days (see Table 6 above, s. no.1) and her total lifespan from hatching to death averaged 25.1 ± 13.7 days (Table 6, s. no. 5). The lowness of the latter value was due to the high level of mortality in the early instars. Field observations confirmed these figures, as an adult female was more resistant to environmental stresses like high temperature, rainfall, starvation, physical disturbances and low doses of pesticides than the crawlers were. The total life span was highly dependent upon the immature mortality ratio of the population and the prevailing environmental conditions; even so, the finding of a female life span of 25.1 ± 13.7 days in this study fell within the range reported by other researchers (ICAC Recorder, 2008; Akintola and Ande, 2009), that is 30-48 days and 37 days respectively. The maximum life span of a female mealybug in controlled conditions was noted as 52 days, but this was an outlier, an exceptional case in the population. 4.2.3.11 The Sex Ratio of CMB There is no published sex ratio study on CMB available for comparison, but in general the progeny of sexually reproducing species have equal chance of sex determination, that is the numbers of males and females occur in a 1 : 1 ratio. In this study the sex ratio of CMB was found to be 1 male : 1.29 females. Most of the mortality in the instars that could be sexed occurred in males, thereby reducing their numbers and hence the sex ratio. However, this is a generalization, and more research is needed to confirm the causes of high male mortality. The minute morphological differences between the sexes in the second instar were described by Hodgson et al. (2008), making a repetition of this study to include the second-instar mortality possible; that would provide a more accurate result.

92 4.2.3.12 The Effect of Host-Plant Species on CMB Fecundity In the study of host-plant effects on CMB fecundity, similar trends were observed between the number of developing eggs inside a dissected female and the total number of crawlers produced per female on each of the ten plant species tested (Figs. 8 and 9 above). Field-collected material also showed this correlation. However, the number of offspring produced also depended upon environmental conditions as well as host plant quality (Table 14 above). In unfavorable conditions, both the number of offspring produced and their survival was greatly reduced. No comparable published study is available, but Akintala and Ande (2008) recorded the preference of P. solenopsis for shoe flower (Hibiscus rosa-sinensis) in Nigeria, as did Moghaddam (2006) in Iran and Wang et al. (2009) in China. Similarly, ICAC Recorder (2008) said that the favorite hosts of CMB were shoe flower (H. rosa- sinensis), aksun (Withania somnifera), lantana (Lantana camara), itsit (Trianthema patulacastrum), hazardani (Euphorbia granulate) and lady’s finger (H. esculentus) but gave no order of preference. These papers support the findings in the present study. The variation in the number of CMB crawlers in a single batch, observed in different months on different hosts in this study, is resembles the findings of Nava- Camberos et al. (2001) in their study of Bemisia argentifolii Bellows and Perring [a junior synonym of B. tabaci (Gennadius), Hemiptera: Sternorrhyncha: Aleyrodidae]. They found that temperature and host-plant species substantially affected development, fecundity and survival. The present study found that cotton was the most nutritious and favored host of CMB in all months in Pakistan; this agrees with other published reports from Pakistan (Buriro et al, 2006; Khaskheli, 2007; Saeed et al., 2007; ICAC Recorder, 2008; Johnson et al., 2008; Parvez, 2008b; and Arif et al., 2009), India (Dhawan et al., 2008; Nagrare et al., 2009) and China, where P. solenopsis has been reported to be a serious (NCIPM, 2008) or potentially serious pest of cotton (Wang et al., 2009). 4.2.3.13 Alternate Host Plants When the present study was started in 2005, there were no alternate hosts of CMB known because the identity of the pest was uncertain. The alternate host plants found were shared with other stake holders immediately (Abbas et al., 2007a; Arif et al., 2006; Arif et al, 2007a, b and c) even though the list was still incomplete, as the information was urgently needed. As a result of the present study, the total of 55 host-plants in 18 families was reported (Hodgson et al., 2008: 34, Annexure 2). Most of the economically important alternative hosts (like sunflower, brinjal, lady’s finger, cucurbits, chillies, and

93 weeds like mako, itsit etc.) were also published by the agricultural scientists in the Dept. of Agriculture to raise the awareness of farmers (Buriro et al., 2006; Parvez, 2008a). The host-plants of CMB listed by ICAC Recorder (2008) included 22 plant species, 18 of which agree with the present study. Muhammad (2007) indicated that there were 300 host plants of the mealybug but this number has been quoted for Maconellicoccus hirsutus . Some host plants of CMB were also mentioned by Muhammad (2007) but most the plants listed were incompletely named, unlike the list of plants determined in the present study. The diversity of host plants observed during the CMB surveys (listed in Table 14 above) reflected the preferences of the pest in natural conditions. These findings are comparable with the host preferences of CMB on the basis of fecundity (Table 11 above). Cotton (G. hirsutum) and shoe flower (H. rosa-sinensis) were the top two preferred host- plant species, when measured either in the laboratory by the number of developing eggs within a dissected adult female, or in the field by the CMB infestation intensity and percentage CMB infestation. The results of these two studies support each other. The effect of tobacco (Nicotiana tabacum) on egg- and crawler production was not observed, so it cannot be compared with cotton in this way. The CMB survey results also support a generalization that the host plant-species found to be most heavily infested were most conducive to the proliferation of CMB. The percentage CMB infestation and CMB infestation intensity will both be important parameters for decision-making in pest management. Attacks by CMB were reported from several states in India by Nagrare et al. (2009), but they did not list alternate hosts of the pest. ‘ScaleNet’ (Ben-Dov et al., 2009) listed 31 host-plants of P. solenopsis (in 14 plant families) from the world literature, including 9 species already reported as CMB hosts, confirming the findings of the present study. Of these, H. rosa-sinensis had already been documented as a preferred host by several researchers (Williams and Granara de Willink, 1992; Ben-Dov, 1994; Akintola and Ande, 2008; and Wang et al., 2009). Tomato (Lycopersicon esculentum) was a favored host in Brazil (Culik and Gullan, 2005). Infestation of cucurbits has also been reported (Williams and Granara de Willink, 1992; Ben-Dov, 1994). The most comprehensive study of alternate hosts of CMB, conducted at Central Cotton Research Institute (CCRI), Multan, Pakistan, was published recently by Arif et al. (2009). It documented 154 host-plant species including 20 economically important field crops, 64 weeds, 45 ornamental plants and 25 shrubs and trees, belonging to a total of 53 plant families. When analyzed critically, this list is approximately similar to the list of 55

94 confirmed host plants determined in the present study and included all the plants listed in Table 14 above. Arif et al. (2009) divided the hosts into four categories: 1. Incidental i: Only a few individuals of the mealybug found ii: No breeding individuals observed 2. Low i: All stages of CMB found in low numbers ii: No adverse symptoms observed on the plant 3. Medium i: All stages of CMB found in large numbers ii: Wilting and yellowing of plant leaves observed iii: Infested plants normally survived 4. High i: All stages of CMB found in very large numbers ii: Almost all plant parts appeared white with mealybugs iii: Excessive leaf and fruit shedding observed iv: Most of the infested plants died. Among the reported CMB host plants reported in Arif et al. (2009), 72 species fell in category 1 (incidental), 58 in category 2 (low), 15 in category 3 (medium) and 9 in category 4 (high). In contrast, the criteria used to designate a CMB host plant in the present study was that “a host plant is a plant where CMB feeds and breeds”, that is, a plant species on which all the stages of the pest were found including the breeding female in at least five different localities (see 3.2.6 above). In this way, the present study only reported as hosts members of categories 3 and 4 described above (medium and high) and a few from category 2 (low). All the plants reported in category 4 (high) by Arif et al. (2009) [that is, Xanthium strumarium (Asteraceae); Trianthema portulacastrum (Aiozoaceae); Abutilon indicum , A. muticum , Gossypium hirsutum , Hibiscus mutabilis , H. rosa-sinensis (Malvaceae); S. melongena and Withania somnifera (Solanaceae)] were included in the list in Table 14 except A. muticum, another species of the genus Abutilon which was already represented in Table 14. Similarly out of 15 host-plant species reported in category 3 (medium) by Arif et al. (2009), 66% were included in Table 14 and a further 13.4% were additional species in genera already represented in Table 14. In category 4 in Arif et al. (2009), many of the plant species are different from hosts listed in the present study. However, 60% of the host-plants reported in the present study were listed as CMB hosts by Arif et al. (2009). There are some differences in nomenclature between the studies, mainly in generic combinations and some family names, which may reflect different opinions of the botanists or literature sources of different ages.

95 In category 1 (incidental) in Arif et al. (2009), only few of the plants reported (for example, Salvadora oleoides Decsn.) were included in the list of CMB hosts in the present study, because the other species did not fulfill the definition of a CMB host applied in this study. As indicated by Arif et al. (2009), these were incidental hosts. It has been observed in the field that CMB can be carried by visiting birds and rodents to nearby trees like jangli kikir (Acacia leucophloea Wild .), phulai (A. modesta), siris [Albizzia lebbek Benth.], (Mimosaceae); mango [Mangifera indica L.] (Anacardiaceae); simbol [Salmalia malabarica (DC) Schott & Endl.] (Bombacaceae); shisham (Dalbargia sisso Roxb.) (Fabaceae); date palm (Phoenix dactylifera L.) (Palmae) etc., where it can survive for a few days. Although they play a role as a temporary host for the mealybug, these plants do not fall in the criteria of true ‘host plants’ as defined in this study. The observations made by Arif et al. (2009) were correct, but as explained in the section on field observations in this study in 6.5 (below), CMB had a remarkable ability to survive starvation; a mature adult female was observed to survive up to 12 days of starvation in October (at a mean temperature of 27.8 ºC and 50.6% Relative Humidity). A confusing observation was that when a mature adult female was near to death in winter it produced its crawler sac, which was sheltered under its dead body through the unfavorable conditions while the development of the crawlers was prolonged by the low temperatures. When favorable conditions returned, the crawlers emerged from beneath the body of the dead female in search of a favorable feeding site. Cotton mealybug infestations of other plant species were reported by other entomologists during the present study, where the author was not able to verify the records himself for various reasons including shortage of time or resources. The acceptability of these host-plant species have not been confirmed in the laboratory. The species have been listed in Table 28 (below) so that future investigators can confirm the status of these reported hosts and their role in CMB management.

96 Table 28. List of reported Host Plants of CMB, not verified Personally or in the Laboratory

S. Vernacular Latin name Family Reported No. observations No. name by & intensity 1. Akas Bail Cuscuta spp. Convolvulaceae Shahid 1 : Cr.> 20 2. Jangli Kikir Acacia spp. Fabaceae Ishaque 1 : Cl. Cr. > 25 3. Neem Azadirachta Meliaceae Buriro 1 : (Cl. >100 ) indica A. Juss. 4. Malvastrum Malvastrum Malvaceae Monga ? : coromandelianum May-December (L.) Garcke 5. Rose Rosa indica L. Rosaceae Shahid 1 : Is. Cr.> 5 Note: Is. = isolated; Cr. = Crawler; Cl. = cluster; > = more than; < = less than; Is. ad. = isolated adult

4.2.3.14 Study of Developmental Stages in Different Seasons The presence of different stages of the pest at any particular time was very variable, according to host plant identity and quality, the presence or absence of natural enemies, what plant protection measures were used and the meteorological conditions. There is no comparable published study available for comparison, but field observations on early sown cotton, and the population dynamics studies in this work, agreed that CMB had its highest rate of multiplication in the initial stages of infestation of the cotton crop, after which its productivity gradually declined, probably because of intra-specific competition. Similarly since the most of the mortality occurred in the first 1-8 days (75% in the laboratory study, Figs. 5 and 6 above), the greatest numbers of CMB were in the early instars. In October, towards the end of the cotton season, there were a large number of mature females reproducing, which would have increased the chance of some of their progeny surviving the winter. 4.2.3.15 Population Dynamics of CMB: Intensive Surveys In the present study it was noted that, as the CMB population on shoe flower increased, no such increase was seen in the number of white caps or dried twigs even though the total number of twigs on the host-plant increased during this interval. This was because the wingless CMB failed to disperse on its own. It was also observed that in the cotton fields, the pest occurred in patches. If dispersal was not assisted by some external agent, the mealybugs remained in those patches to the extent that the entire patch of infested plants died. The dispersal ability of CMB crawlers on their own was very poor; they needed to be assisted by various agents like birds, rodents, insects and other

97 animals including humans, water and air currents. The availability of a suitable host determined whether the crawler survived and started a new colony. The population dynamics of CMB on shoe flower (Hibiscus rosa-sinensis) were similar to those of other insect pests active in summer (Mohyuddin and Mahmood, 1993; Muhammad, 2007; Pedigo, 2003; Parvez, 2008b). In Appendices Table 1 below, it was apparent that the appearance of CMB on shoe flower coincided with the initiation of active growth of the plant (in the fifth week of observation), but the appearance of beneficial insects was not recorded until the eighth week of observation; then both populations increased gradually until there was a check on population growth in observation weeks 21 to 24. After this point, the population of beneficial insects continued to grow while that of CMB declined. It was observed that shoe flower plants around the entire campus were heavily attacked by CMB, to the extent that a number of tender twigs showed white caps and eventually dried out. However, in mid-July the damaged plants sprouted again and the new shoots flourished; within two months the shoe flower looked as if it had never been attacked by a serious pest (see weeks 34-37 in Appendices Table 1). The same pattern of population growth and lack of homogeneous dispersal as the study on shoe flower, was observed in the results of the experiment described in 3.3.1 (which measured the resistance of the ten cotton cultivars to CMB attack). The CMB population growth rate decreased from the third week onwards (although the population continued to increase), probably because of strong intra-specific competition since the flightless mealybugs multiplied rapidly and could not disperse to find new food sources and living space. Overpopulation occurred in localized patches, where shortage of food resulted and the heavily attacked tissues became weak and ultimately dried out and died. Although the dramatic recovery from CMB damage described above occurred on shoe flower, unfortunately it did not happen in the case of cotton (Gossypium hirsutum) for the following reasons. 1. Seasonal cotton (comprising on 80 percent of total cotton production, particularly in the Punjab - see Table 3 above) is sown in May and June. Each seedling becomes a vigorous plant after the first irrigation (in 28-35 days), and after thinning eliminates the surplus plants (after 40-45 days) the surviving plants achieve economic status requiring care to maintain the recommended population (usually 18,000-21,000 plants per acre, depending upon the variety). Based on the shoe flower model (Appendices Table 1), after the establishment of CMB on cotton, there is a delay of three weeks

98 before the natural enemies to become established, and further 3-4 weeks before the CMB population begins to decline. Such a long period of growth loss in a highly economic plant is unacceptable (unpublished field observations of the author). 2. The cotton plant is extremely sensitive to injuries inflicted by sucking pests like the jassid Amrasca devatans (Hemiptera: Auchenorrhyncha: Jassidae), whitefly Bemisia tabaci (Hemiptera: Sternorrhyncha: Aleyrodidae) and cotton mealy bug (P. solenopsis), as well as to herbicides. It cannot even withstand the fumes of herbicides from a nearby field being sprayed, and responds to this exposure by deformation of the leaves (unpublished field observations of the author). 3. Cotton is a seasonal, herbaceous plant with a shallow, not very extensive root system resulting in relatively passive uptake of nutrients compared to shoe flower, which is a perennial plant with a deep and extensive root system and better uptake of nutrients. Through its superior ability to take up nutrients, shoe flower probably has a faster recovery rate compared to cotton (unpublished, an opinion of author). In view of the circumstances and field realities mentioned above, the control of CMB on cotton cannot be left to the sole responsibility of natural enemies. However, there is a great need to exploit and manipulate this natural beneficial fauna as part of the IPM program on cotton, by working out a program that is more or less compatible with the natural beneficial fauna. In the pest management results in this study, Table 33 in 6.2 below provides a glimpse of what can be achieved with use of the most appropriate pesticide. 4.2.3.16 Distribution of CMB in Pakistan The data recorded during the CMB surveys in various districts of Pakistan lacked a necessary condition required for statistical analysis, that is, randomization. The mandate of the surveys was documentation of the distribution of the pest in order to study its means of carry over on alternate host plants, and the dispersal and distribution of CMB in different districts. The districts and localities were purposely selected for survey, mostly based on information received. This meant that not all the districts had an equal chance of observation and the data collected was not randomized, making it unsuitable for statistical analysis. In addition, the survey visits were made in different months in different districts. Consequently the picture portrayed in Fig. 13 and Table 18 may not accurately reflect a true picture of the intensity of the pest infestation. Nevertheless, it indicates the trend of CMB infestation intensity and the distribution of the pest in various districts of Pakistan.

99 The distribution of CMB was surveyed in different districts of the Pakistan and was confirmed by reports from a number of sources. The distribution of the pest in Pakistan documented by Hodgson et al. (2008) agreed with a number of reports (CCRI, 2006; Khaskheli, 2006, 2007; Yousuf et al., 2007; ICAC Recorder, 2008; Naqvi and Nausheen, 2008). The CMB-infested districts reported in Punjab agree with those in the official reports from various sources (Muhammad, 2007; Saeed et al., 2007; Parvez, 2008b; Raza, 2008; Arif et al., 2009). The infestation in Sind was also reported by other sources, which confirm the reported localities (Buriro et al., 2006; Khushk and Mal, 2006 a, b; GOS, 2008). The record from Baluchistan was based on unpublished data (A.S. Buriro, 2007, personal communication), and that from NWFP was also based on an unpublished report and CMB samples from that locality [Mamoon ur Rashid (Ph.D. scholar at Gomal University, Dera Ismail Khan, Pakistan), 2008, personal communication]. 4.2.3.17 Overwintering and Carry Over of CMB in Pakistan As explained in 5.3.15 above, the CMB surveys in various districts of Pakistan were not randomized. The mandate was documentation of the distribution of the pest in order to study its means of carry over on alternate host plants, and the dispersal and distribution of CMB in Pakistan, not the comparison of various months. Survey visits were made in different months in different districts, so not all the months were documented equally. Consequently the picture portrayed in Table 24 may not accurately reflect the true intensity of the CMB infestation in any particular locality. Nevertheless, the survey data does indicate the means of carry over on different host-plant species throughout Pakistan. The mealybug pest of cotton colonizes a large number of host-plant species, where it can easily overwinter for infestation of cotton in the following year. The polyphagy of CMB is highly advantageous behavior that helps to make it a successful pest. In terms of IPM, polyphagous insects tend to be dangerous pests because there is no bottleneck in their life cycle. In monophagous pests there may be a bottleneck in the biology that provides an opportunity for control; for example, pink bollworm [Pectinophora gossypiella (Lepidoptera: Gelechiidae)], a common pest of cotton in the 1980s, was successfully managed by interrupting its carry over (based on unpublished official reports by Pest Warning and Quality Control of Pesticides (PWQCP), Govt. of Punjab; Dr. I. Pervez, 2009, personal communication).

100 4.2.3.18 Recording the Natural Enemies of CMB The most serious CMB infestations occur where there is a shortage or complete absence of natural enemies (Alene et al., 2004). When this study was carried out, there was considerable doubt as to whether CMB had any natural enemies in Pakistan. However, during field surveys for CMB between 2005 and 2008 a number of natural enemies were recorded feeding on the mealybugs (listed in Table 26 above). This valuable discovery was shared with the farming community and researchers through popular articles in daily newspapers (Abbas et al., 2007a; Arif and Abbas, 2007). The natural enemies recorded in this study agree with the findings of other researchers working on cotton in Pakistan [Dr. I. Parvez (Director General, Pest Warning and Quality Control of Pesticides, Punjab, Lahore), Dr. R. Ahmad (Director, CABI Bioscience, Islamabad), A.S. Buriro, 2008, personal communications]. The predatory beetles Brumus suturalis, Coccinella transversalis, C. undecimpunctata, Cheilomenes sexmaculatus, Hippodamia convergens were confirmed as CMB predators in the field. Linked with this work, additional studies were carried out at the IPM laboratory, UAF, to determine the role and pest control potential of the predatory beetles Brumus suturalis, Coccinella septempunctata, Cheilomenes sexmaculatus and Hippodamia convergens on CMB. The study found that B. saturalis has the greatest biotic potential against CMB in the laboratory, whereas C. sexmaculatus had the most biotic potential for the control of CMB in the cotton fields (Imran, 2008). These results confirm the findings in the present study. A lady beetle (Coleoptera: Coccinellidae) from Australia, known as “Australian lady beetle” or “mealybug destroyer” (Cryptolaemus montrouzieri Mulsant), has been reported feeding on CMB in India (Nagrare et al., 2009) but it has not been found in the field in Pakistan. This insect is being reared in different public sector laboratories in Pakistan (personal observations of the author; Dr. R. Ahmad and Dr. I. Parvez, 2008, personal communications) for future neoclassical biological control of CMB. The list of beneficial fauna associated with CMB found in this study (Table 26 above) can be compared with records from India in Tanwar et al. (2007), who listed beneficial fauna active against mealybug infestations in general (not specifically against P. solenopsis); however, the mealybugs they recorded did include P. solenopsis as a major pest. This paper mentioned that in some cases the beneficial fauna kept the pest mealybug populations below economic injury levels. Coccinellid beetles such as Cheilomenes sexmaculata , Rodolia fumida , Scymnus coccivora and Nephus regularis

101 were reported as important predators of mealybug nymphs (Tanwar et al., 2007). Of these, in the present study Scymnus sp. was found to be a very efficient predator of CMB. The predator Cheilomenes sexmaculata was recorded by Tanwar et al. (2007) under the old generic combination, Menochilus sexmaculata. Tanwar et al. (2007) recommended biological control of mealybug pests through the release of additional natural enemies. Among the control agents, introductions of Cryptolaemus montrouzieri (Australian ladybird or mealybug destroyer), Anagyrus pseudococci , Leptomastix dactylopii, Hypoaspis sp. (a small mite that feeds on mealybug crawlers), and the pathogens Verticillium lecanii and Beauveria bassiana have been reported to be effective in managing mealybug infestations in India. These natural enemies may be useful and effective for use in the IPM of CMB. The present study did not record any of these biological agents as present in the field in Pakistan, particularly not in the cotton-growing areas. It was observed that CMB was most abundant in the core of the cotton-growing districts, where the general cropping pattern is entirely cotton-wheat-cotton, in contrast to lower levels of CMB infestation in other areas with a more diverse cropping system [with a larger number of crops grown in different rotations, for example sugarcane, maize, vegetables and orchards (see Table 25 above)]. This difference in CMB-infestation level corresponded with the population levels of the natural beneficial fauna, which is massively depleted in cotton-growing areas because of 450% pesticide coverage of the cotton fields (the average for the last ten years, based on unpublished data of PWQCP, Govt. of Punjab). In the areas with diversified cropping patterns the cotton receives almost the same quantity of pesticides but the other crops are not sprayed, so they serve as a reservoir of beneficial fauna. For example, in one district of Faisalabad CMB was found everywhere on its alternate host plants in very small numbers and only 30% of the farmers were aware of its presence. In contrast, in the Mamon Kanjan area (the cotton- growing area in Faisalabad), CMB-infestation levels were much higher and 70% farmers were aware of the pest (unpublished data of the author, based on interviews with the farmers). This supports the recommendation that Pakistan should evolve an IPM strategy with a component that conserves the existing natural beneficial fauna in reservoirs amongst the cotton fields.

102 RESULTS AND DISCUSSION: 4.3 PEST MANAGEMENT

Part of the role of this initial study of CMB was to explore possible methods for short-term management of this pest. Long-term management of the mealybug, which will be necessary for sustainable and environmentally friendly agriculture, was beyond the scope of this project of limited duration; its development will fall to subsequent researchers. The following aspects of the management of CMB were investigated in this study: host-plant resistance; the impact of narrow-spectrum insecticides and Insect Growth Regulators (IGRs); the optimum volume for sprayable material; and the role of additives in sprayable material.

4.3.1 Relative resistance of various cotton cultivars to CMB infestation In the experiment described in 3.3.1, the resistance of the ten cotton cultivars to CMB attack was measured as the percentage growth rate of the CMB population (that is, the increase in population per week) on each cultivar. The percentage growth rate figures obtained are given in Appendix 1. The analysis of variance of this data are presented in Table 29 below.

Table 29. Analysis Of Variance of CMB population on each of ten cotton cultivars

SOV DF SS MS F ratio P value Variety 9 1785 198 0.030 1.00 ns Error 120 779200 6493 Total 129 780985 ns = not significant

The comparison of means analysis in Table 30 (below) showed that there was no significant difference between the populations of CMB on the different cultivars. This indicated that CMB had nearly equal preference to all the cultivars, and all of them were susceptible to attack. None of the cultivars tested could be declared as resistant to the pest.

103 Table 30. Comparison of means of CMB population on each of ten cotton cultivars, as an indicator of relative resistance against CMB

Cotton cultivar Mean CMB population BT 121 167.92a FH 901 170.82a FH 1000 171.97a BH 160 170.25a FDH 170 170.14a FDH 228 172.35a MNH 786 174.42a CIM 541 169.05a CIM 554 162.42a CIM 496 162.89a P = 0.30 (not significant), df = 9. Means sharing the same letter have no significant difference; for details of the analysis, see Appendix Table 13.0.

Fig. 28 (below) shows that early in the experiment, the rate of CMB population growth was fastest on the FH cultivars (FH 901 and FH 1000), with growth rates of 400% and 364% respectively, followed by BH 160 with a growth rate of 334.7%. This trend soon disappeared, however, as in the following week CIM 506 showed the fastest growth rate (424.5%) followed by MNH 786 and CIM 541 with growth rates of 389.2% and 358.1% respectively.

PGR= Percent Growth Rate

Figure 28. Graph to show the population dynamics of CMB on ten cotton cultivars

104 This experiment yielded a lot of data on the population dynamics of CMB feeding on cotton, which were analyzed and discussed in section 4.2.2.

4.3.2 Narrow-spectrum pesticides and IGR impacts on CMB and beneficials Pesticides available on the market that are regarded as relatively safe for the environment were applied to CMB-infested fields, and the mortality of pest and its natural enemies were determined as described in 3.3.2 above. The data were subjected to analysis of variance and comparison of means analyses. The analysis of variance (Table 31 below) revealed that the pre-spray populations of CMB and the beneficial fauna showed no significant difference between treatments, probably because plants having reasonable populations on them were pre-selected and tagged before the pesticide application. Since the population consisted of pre-screened, selected plants, it could not be inferred that either CMB or the beneficial fauna had homogeneous population levels in nature.

Table 31. Analysis of Variance for the percentage mortality of CMB, recorded 24, 72 and 168 hours after application of various pesticides

Data SOV S S DF M S F Sig. CMB: pre -spray Treatments 2085.367 9 231.707 0.160ns 0.996 Error 29024.000 20 1451.200 CMB: 24 HAS Treatments 14327.467 9 1591.941 3.953* 0.005 Error 8054.000 20 402.700 CMB: 72 HAS Treatments 28098.300 9 3122.033 7.121* 0.000 Error 8768.000 20 438.400 CMB: 168 HAS Treatments 39760.833 9 4417.870 8.275* 0.000 Error 10677.333 20 533.867 Beneficial s: pre-spray Treatments 13.467 9 1.496 1.663ns 0.164 Error 18.000 20 0.900 Beneficial s: 24 HAS Treatments 34.800 9 3.867 16.571* 0.000 Error 4.667 20 0.233 Beneficial s: 72 HAS Treatments 35.200 9 3.911 39.111* 0.000 Error 2.000 20 0.100 Beneficial s: 168 HAS Treatments 65.500 9 7.278 24.259* 0.000 Error 6.000 20 0.300 HAS = hours after spray * = significant at α = 0.05 (5% confidence interval) ns = not significant

The data collected 24, 72 and 168 hours after application of the insecticides showed a significant difference in the CMB populations between the control and the

105 insecticide treatments (Table 32 and Fig. 29 below). The most toxic and long-lasting pesticides for the control of CMB were the organophosphates (Methidathion and Profenophos), one of the pyrethroids (Fenpropathrin) and one of the neonicotenoids (Acetamiprid). The pyrethroid bifenthrin and the carbamate methomyl both gave moderate control. The carbamate carbosulphan and the neonicotenoid imidacloprid killed some CMB, while the IGR buprofezin gave a low level of control of the mealybugs.

Table 32. Comparison of means for the percentage mortality of CMB, recorded 24, 72 and 168 hours after application of various pesticides

S. Insecticides Dose/100 liters % Population reduction after no. Common name Brand name of water 24 hours 72 hours 168 hours 1. Buprofezin @ Buprofezin 500 g 47.53a 55.87a -32.10ab 2. Fenpropathrin # Fenpropathrin 300 ml 52.09a 72.92a -49.62ab 3. Bifenthrin # Talstar 250 ml 56.98a 73.20a -53.86b 4. Acetamiprid N Rani 125 g 56.88a 73.09a -52.55ab 5. Imidacloprid N Confidor 250 ml 71.83a 82.81a -68.87ab 6. Carbosulphan * Advantage 400 ml 74.94a 80.37a -67.54ab 7. Methomyl * Lannate 500 g 63.93a 88.88a -78.66a 8. Methidathion ° Supracide 150 ml 85.07a 93.73a -88.82ab 9. Profenophos ° Curacron 1000 ml 80.56a 92.56a -85.75a 10. Control - -16.88b -47.17b 88.06 c 11. Total Average - - 57.29 66.63 -48.97 Values sharing same letter have no significant difference at α = 0.05 @ = chitin synthesis inhibitor (an IGR) # = pyrethroid N = neonicotinoid * = carbamate ° = organophosphate

Figs. 29 and 30 (below) illustrate in two different ways, the significant difference between the changes in CMB population caused by the various insecticide treatments and that of the control.

106 Fenpropathrin Methomyl Bifenthrin Buprofezin Acetameprid Amidacloprid Carbosulfan Methidathion Profenophos 5.0 Control 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Populationdata of CMB 0.5 0.0 Prespray 24 hrs 72 hrs 7 days Positions of the field

Figure 29. Graph to show the effect of different pesticides on the population of CMB on cotton

Figure 30. Graph to show the population dynamics of CMB after various pesticide treatments

The populations of beneficial fauna on treated and untreated plants were significantly different (Tables 31 above and Fig. 31 below). The beneficial organisms were unharmed in the control 24 hours after the treatment application (Table 33). All the pesticide treatments killed beneficial organisms but they were least impacted by Buprofezin (Table 33). The order of the insecticides as regards the safety of the beneficial fauna, 168 hours after application, was as follows: the control (least impact), then in

107 order of increasing impact, Buprofezin, Imidacloprid, Methomyl, Fenpropathrin, Bifenthrin, Acetameprid and Profenophos, with Methidathion as the most damaging treatment to the beneficial organisms.

Table 33. Comparison of Means for the population of beneficial fauna present on cotton before and after application of various insecticides

S. Insecticides Pre-spray Population of the no. population beneficial fauna after Common name Dose/100 liters of beneficial s f 24 hours 72 hours 168 hours of water no./6 in. twig 1. Buprofezin @ 500 g 3.000a 3.000c 2.000b 3.666c 2. Fenpropathrin # 300 ml 1.666a 0.000a 0.000a 1.000ab 3. Bifenthrin # 250 ml 1.667a 0.333a 0.000a 0.667ab 4. Acetamiprid N 125 g 2.000a 0.000a 0.000a 0.333a 5. Imidacloprid N 250 ml 2.667a 1.333bc 0.333a 1.333ab 6. Carbosul ph an * 400 ml 1.333a 1.000a 0.333a 2.000b 7. Methomyl * 500 g 1.000a 0.333a 0.000a 1.333ab 8. Methidathion ° 150 ml 1.000a 0.000a 0.000a 0.000a 9. Profenophos ° 1000 ml 1.667a 0.000a 0.000a 0.000a 10. Control 2.667a 2.667bc 3.333c 4.6667c Total Average - 1.867 0.867 0.600 1.500 Values sharing the same letter have no significant difference at α = 0.05 @ = chitin synthesis inhibitor (an IGR) # = pyrethroid N = neonicotinoid * = carbamate ° = organophosphate

Figure 31. Graph to show the population dynamics of the beneficial fauna after various pesticide treatments

108

In terms of the impact on the beneficial fauna, the most toxic and long-lasting pesticides were the organophosphates (Methidathion and Profenophos), one of the pyrethroids (Fenpropathrin) and one of the neonicotenoids (Acetamiprid). The pyrethroid Bifenthrin and the carbamate Methomyl both had moderate impact. The carbamate Carbosulphan and the neonicotenoid Imidacloprid both killed slightly fewer beneficials, while the IGR Buprofezin had the lowest impact on the beneficial fauna. This experiment therefore confirmed that all the insecticides tested impacted the beneficial fauna as well as CMB, but some had more impact than others. An orthogonal contrast analysis of the pesticide treatments that did not kill all the beneficial fauna is shown in Table 34 below.

Table 34. Comparison of orthogonal contrasts of different groups of pesticides and their relative efficacy against CMB

Dependent variable: Population of CMB 72 hours after treatment -95.00% +95.00% Contrast Estimate Std. err. t p Cnf. Lmt Cnf. Lmt 1. 593.37000 15.08870 39.32540* 0.00000 561.89500 624.84500 2. 57.51100 12.31990 4.66814* 0.00015 31.81220 83.20990 3. -23.13100 10.05920 -2.29950* 0.03238 -44.11400 -2.14810 * shows significant difference in the groups at α = 0.05 Contrast 1: Control vs all insecticide treatments Contrast 2: Buprofezin vs Carbamates (Methomyl and Carbosulphan) Contrast 3: Carbamates (Methomyl and Carbosulphan) vs Pyrethroids (Fenpropathrin and Bifenthrin)

The analysis revealed that the control was significantly different from all the other treatments, having no impact on the CMB population (Table 34 above). Buprofezin impacted the mealybugs but not the beneficial organisms; it behaved significantly differently from the carbamates (Methomyl and Carbosulphan), which killed both the mealybugs and the beneficials. When the pyrethroids (Fenpropathrin and Bifenthrin) were compared with the carbamates (Methomyl and Carbosulphan), there was no significant difference in CMB percentage mortality up to 72 hours after the pesticide application. The outcome of this trial was shared with the stakeholders for onward transmission to the farmers, and was published in popular articles (Abbas et al. , 2006, 2007a, 2008; Arif et al ., 2006, 2007b, 2008).

109 In a comparison of orthogonal contrasts of different groups of the pesticides, the organophosphate chemicals (profenophos and methidathion) behaved significantly differently from most of the other groups, as shown in Table 35 below.

Table 35. Comparison of orthogonal contrasts of different groups of pesticides and their relative effects on the population of beneficial fauna, 168 hours after the pesticide application

Dependent variable: population of beneficial fauna 72 hours after treatment Population of beneficial fauna Contrast 168 hours after pesticide Description of contrast no. application Mean 1 Mean 2 P value 1. Pyrethroids (Fenpropathrin and Bifenthrin) vs neonicotenoid 0.833 0.833 1.000 ns (Imidacloprid and Acetamiprid) 2. IGR(Buprofezin) vs Carbamates 1.667 0.000 0.001* (Methomyl and Carbosulphan) 3. Neonicotenoid(Imidaclopridand Acetamiprid) vs organophosphates 0.833 0.000 0.022* (Profenophos and methidathion) 4. IGR (Buprofezin) vs all 8 other 3.667 0.833 0.000* insecticides 5. Pyrethroids (Fenpropathrin and Bifenthrin) vs Organophosphates(Profenophos and 0.833 0.000 0.001* methidathion) 6. Control (water treatment) vs all 9 4.667 1.148 0.000* pesticide treatments *significant difference between the means of contrasting groups ns = non-significant difference between means of contrasting groups

There was no significant difference between pyrethroids and neonicotinoids in their effect on the population of beneficial fauna (Table 35, contrast 1); but there was a significant difference in populations of beneficial fauna between the plots treated with pyrethroids and those treated with organophosphates (contrast 5). The IGR impacted the population of beneficial fauna significantly differently from the carbamates (contrast 2) and from all the other insecticides together (contrast 4). The neonicotenoids had a significantly different effect on the population of beneficial fauna as compared with organophosphates (contrast 3) and the control treatment was significantly different from all other treatments including the IGR (contrast 6).

110 4.3.3 Optimum Volume of Sprayable Material The experiment described in 3.3.3 above, tested the efficacy of different spray volumes for pesticide application to control CMB on cotton in the field. Five different volumes were tested with the pesticide Curacron (Profenophos) 40 EC. The data obtained were subjected to ANOVA (details are in Appendix Table 15.0). A comparison of means analysis of the CMB population data recorded 168 hours after pesticide application using different volumes of water, is shown in Table 36 below .

Table 36. Comparison of means for the CMB population present after application of various volumes of insecticide spray

S. no Volume of spray material used in the trial Average population of CMB 1. 80 liters of spray volume / 4360 sq ft (acre) 35.97 a 2. 100 liters of spray volume / 4360 sq ft (acre) 7.57 b 3. 120 liter s of spray volume / 4360 sq ft (acre) 7.57 b 4. 140 liters of spray volume / 4360 sq ft (acre) 22.93 ab 5. 160 liters of spray volume / 4360 sq ft (acre) 24.53 ab Values sharing the same letter are statistically similar. P = 0.002

The analysis suggests that 100 and 120 liters water used in spray material gave better results in terms of better CMB population reduction and maintaining the lowest population of the pest, compared to a lower (80 liters) and higher spray volumes (140, 160 liters), as shown in Fig. 32 below.

Prespray 24hrs 72hrs 7days

120.0

100.0

80.0

60.0

40.0

CMB Population Population CMB per plant 20.0

0.0 80 lit 100 lit 120 lit 140 lit 160 lit

spray volumes in litres

Figure 32. Average population reduction of CMB on cotton after spraying different volumes of water with pesticides

111

Maximum CMB control was attained 72 hours after spraying. The optimum spray volume was 100-120 liters, which gave better control and suppression of the CMB population than lower (80 liters) or higher (140, 160 liters) spray volumes. The higher spray volumes (140, 160 liters) were almost as effective as the optimum volumes (100, 120 liters); all four of these volumes differed from the lowest spray volume tested (80 liters), which failed to suppress the CMB population.

4.3.4 The Effects of additives in sprayable material The trial to assess the effects of additives to applications of Imidacloprid on cotton in the field, at 330 grams per acre, was described in 3.3.4 above. The analysis of variance is given in Table 37 (below).

Table 37. Analysis of variance for the effects of various additives in population reduction of CMB on cotton

Data SOV S S DF M S F Sig. 72 HAS Treatments 20.763 3 6.921 0.586ns 0.641 Error 94.567 8 11.821 168 HAS Treatments 32.233 3 10.744 0.394ns 0.761 Error 217.947 8 27.243 HAS = hours after spray α =0.05; ns = not significant

The comparison of means analysis (Table 38 below) showed that the difference between the means for all the treatments was not significant, so none of the additives tested increased CMB mortality. It was concluded that the use of additives in insecticide applications was of no benefit.

Table 38. Comparison of means of CMB population reduction on cotton by various additives to insecticide treatments

S. no. Additives used with the insecticide % CMB population reduction after Confidor at 330 gm/120 liters water 72 hours 168 hours 1. 100 grams detergent (Burq) 73.07a 66.17a 2. 100 ml mineral oil (Kerosene oil) 69.93a 62.07a 3. 100 ml Vegetable oil (Canola oil) 73.23a 63.43a 4. Control (insecticide only) 71.97a 65.53a 5. Overall average 72.05 64.30 Values sharing same letter have no significant difference at α = 0.05

112 4.3.5 Pest Management Discussion 4.3.5.1 Relative Resistance of various Cotton Cultivars to CMB Infestation The present study found that, of the ten cotton cultivars tested, none were resistant to attack by CMB. There is no publication comparable to this study but the findings above have been agreed with by many researchers (Dr. S. Saeed and Dr. I. Parvez, 2008, personal communications). Observations in the field during extensive surveys (described in 3.2.7 above) found that CMB behaved similarly on both BT and non-BT cotton, and on early and late, monopodial and dipodial, broad-leaved and short-leaved cotton cultivars. Both the transgenic (Bts) and local (non-Bts) cultivars grown in Pakistan performed similarly when infested by CMB (unpublished, consistent field observations made by the author, 2005-2008). Based on the cotton cultivars tested, and the field observations of CMB performance on other cultivars, it appears that one component of IPM (selection of a resistant crop variety) does not work against CMB. In another, linked study carried out at the IPM laboratory, UAF, Pakistan, a study was made of the infestation level of CMB on the same cultivars, correlating their physicomorphic characters. This showed that there were significant differences in these characters between cultivars. There was a negative correlation between hair density on the leaf lamina and mid rib and the infestation level by CMB; however, none of the varieties tested could be regarded as a truly CMB-resistant variety. The cultivar MNH- 786 was relatively more susceptible and FDH-228 was relatively more resistant to CMB infestation (Hussain, 2008). Work is also in progress on chemical analysis of various host-plants of CMB to determine the chemical basis for the host preferences of CMB (unpublished, research synopsis for PhD, defended by M.R. Shahid, IPM Laboratory, UAF). Such information might not be immediately helpful to a conventional cotton breeder, but it could help a genetic engineer select genes from the plants least preferred by CMB, for inclusion in the genome of new transgenic cultivars. It can result in introduction of new cotton cultivar relatively resistant to the pest. Further hunting in the varieties may prove fruitful, similarly excess of physicomorphic characters enhancing resistance in plants against other sucking pests like aphids and whiteflies may prove useful. 4.3.5.2 Narrow-Spectrum Pesticides and IGR Impacts on CMB and Beneficials Of the insecticides tested against CMB in the present study (see 6.2, Fig. 29 above), the most toxic and long-lasting against CMB were the organophosphates

113 (Methidathion and Profenophos), one of the pyrethroids (Fenpropathrin) and one of the neonicotenoids (Acetamiprid). However, of these four pesticides, the first three also destroyed the beneficial fauna completely and the last impacted it severely. The pyrethroid Bifenthrin and the carbamate Methomyl both gave moderate control of CMB but still impacted the beneficial fauna considerably. The carbamate Carbosulphan and the neonicotenoid Imidacloprid both impacted the beneficial fauna more severely than CMB. The IGR Buprofezin was safest for the environment and the beneficial fauna. Even though it had least impact on the CMB population, this might be regarded as a desirable outcome for use against such a rapidly multiplying pest, especially when the crop was an edible commodity such as vegetables or fruit. For eco-friendly IPM, three sorts of bio-pesticides were suggested by Muhawi (2004): 1. Microbial pesticides (eg., fungi, viruses, bacteria etc.) 2. Plant-incorporated proteins (PIPs) such as Bt toxins (a gene for pesticidal chemicals incorporated in the crop’s genetic material) 3. Biochemical pesticides that interfere with the pest’s life cycle or development (like insect sex pheromones and IGRs). Muhawi (2004) included IGRs amongst the environmentally safe pesticides that present least risk to the beneficial fauna. The present study supported his conclusion. The effect of various pesticides on CMB populations had already been tested by a number of researchers (see the Review of Literature, 2.4.2 above). The findings in the present study were compatible with those of Tanwar et al. (2007), who reported Profenphos to be the most effective against CMB in the laboratory bioassay and in the field 72 hours after application, followed by Methidathion (Supracide) and Bifenthrin (Talstar). Arif et al. (2008) found Methidathion was apparently slightly more effective than Profenophos; however, the performance of these pesticides was statistically similar, as found in the present study. Their CMB mortality rate for Methidathion and Profenophos was 94.7 % and 92.87% respectively 168 hours after application, whereas in the present study the rate of population reduction was 93.73% and 92.56% respectively after only 96 hours. The two studies differed in the results 168 hours after application, with Arif et al. (2008) recording a check or decrease in the CMB population through the full 168 hours, whereas in the present study the CMB population increased earlier than 168 hours after application. A possible reason for this difference was that in the present study,

114 experiments were conducted in the first week of September, whereas their trial was conducted earlier in the season when thecottonplants were smaller and CMB populations were lower. The two studies also differed in the level of pesticide coverage with spray material, with the present study using a lower coverage due to the dense canopy of the mature plants. This permitted some female CMB to escape the pesticide spray and continue breeding, resulting in a resumption in population increase earlier than 168 hours after the application. Saeed et al. (2007) concluded that Bifenthrin, Profenofos and Chlorpyrifos followed by Methomyl were the best insecticides for CMB control, based on laboratory and field experiments. The present study also showed that all these pesticides controlled CMB effectively at the recommended doses. Emmamectin Benzoate was not tested, so the present study could not be compared with Dhawan et al. (2008). 4.3.5.3 Optimum Volume of Sprayable Material The present study found that the lowest volume of spray material (80 liters of water) failed to suppress CMB because of inadequate coverage of the crop with insecticide. The optimum spray volume was found to be 100-120 liters, which gave the best control of CMB because it ensured that the crop was adequately covered with the pesticide. This finding agreed with earlier field observations, that the basic factor in the chemical control of CMB had been proper coverage with the spray material; plant parts that were not covered adequately with spray bore the highest number of living mealybugs. Field observations made during the extensive surveys (described in 3.2.7 above) also found that variation in the completeness of pesticide spray coverage in the field can have different effects on CMB populations. The results obtained in the present study were published in a popular article (Arif and Abbas, 2007). These findings have been agreed with by other researchers (Parvez, 2008a), and have taken as recommendations in the literature distributed by public sector departments for the awareness of the farmers (Parvez, 2008a). Similar information has also been conveyed in literature distributed to cotton farmers by private firms dealing in pesticides (Syngenta, 2008; Ali Akbar Group, 2008). The present study tested the hypothesis ‘more volume of sprayable material can work better on CMB infested fields’ and found evidence that upheld the hypothesis to some degree, by showing that too low a volume resulted in inadequate pesticide coverage and poor CMB control. However, it also showed that the hypothesis did not hold true for volumes over 120 liters per acre, probably because of excessive dilution of the pesticide.

115 4.3.5.4 The Effects of Additives in Sprayable Material The use of various additives in spray material was recommended by field workers and experts in the public and private sectors as hit-or-miss control methods against CMB (unpublished observations of the author). The addition of detergents was clearly recommended by public sector agriculture departments throughout 2006 (J. Khalid, District Officer Agriculture, personal communication with reference to the Director General, Agriculture (Extension and Adaptive Research) and Director Entomological Research Institute, AARI, Faisalabad) and the demand for detergent drove up its price (unpublished observation of the author). The present study found that none of the three additives tested (detergent, and mineral and vegetable oils) enhanced control of CMB. This was shared with the farming community and public sector department through popular articles in daily newspapers (Abbas et al, 2007a; Arif and Abbas, 2007). This finding, and the observations of other researchers, caused the recommendation of the addition of detergent to spray material to be abandoned, and trials on other additives also ceased because they were recognized to be useless and a waste of resources. During this study it was noticed that some additives (like mineral oil and detergent) adversely affected the general health of the cotton plants, increasing production costs and causing economic loss. Droplets of vegetable oil on the plant surfaces accumulated dust particles, blocking light from the leaves, so decreasing photosynthesis and hence yield. In some cases, the extra dust on the leaves also resulted in multiplication of mite pests. It was concluded that the use of additives in insecticide applications (often recommended by the field workers) was of no benefit and was sometimes deleterious. These results were shared with the farmers through popular articles in print media (Abbas et al., 2007a; Arif and Abbas, 2007). 4.3.6 Future Work Scientific knowledge develops through observation leading to the development of hypotheses, which are then tested by experimentation. In the present study, limitations in time and resources and other factors prevented experimental investigation of all the field observations made. The observations that were not investigated experimentally in this work are discussed below, to provide future researchers with a number of subjects for investigation. Experimentation on these topics will yield additional information on various aspects of the biology and ecology of CMB that may improve its control, and mitigate its spread and impact on cotton and other crops. 4.3.6.1 The Origin of CMB

116 The original cause for the appearance and population build-up of CMB on cotton in Pakistan is still unknown. Hodgson et al. (2008) said that P. solenopsis was possibly an introduced species to Pakistan, and Nagrare et al. (2009) said that P. solenopsis was an American native that had been introduced to India . The following information (provided by Dr G.W. Watson, 2009, personal communication) also suggests that CMB is probably not native to Pakistan but was accidentally introduced from the New World: • CMB had not been recorded from Pakistan before the outbreak on cotton • the nearest relatives of P. solenopsis are all native to the New World • CMB passed from being unknown to being a major pest in only a few years, which is typical of a new introduction in the absence of its natural enemies • no hymenopteran endoparasitoids were recorded attacking the pest in Pakistan during the research for this study (a native species of mealybug would be likely to have native endoparasitoids) • the hymenopteran endoparasitoid discovered in Pakistan and India in 2008 (Anaesius sp., Hymenoptera: Encyrtidae) is an undescribed species belonging to a genus native to the New World (Dr G.W. Watson, 2009, personal communication). If CMB was accidentally introduced to Pakistan, it would be valuable to know by which pathway it entered so that precautions could be taken to avoid similar damaging introductions in the future. The biological differences between CMB in Pakistan and P. solenopsis in the U.S.A. (discussed in 4.1.5 above) raise some uncertainty about whether CMB in Pakistan is an introduction or not. If it is a native species (the valid name would be P. gossypiphilus), then it is surprising that it had not been recorded before the outbreak on cotton in 2005. Molecular analysis should help to clarify whether the biological differences recorded between the two continents reflect a species difference, or a single species showing modified behaviour in response to different environmental conditions. If only one species is involved (P. solenopsis) , its extreme adaptability and wide host-plant range give it the potential to become a significant agricultural pest unless managed successfully. 4.3.6.2 Factors determining CMB Infestation Levels In the present study, the degree of CMB infestation in areas with a mixed cropping system was observed to be less severe than that in areas with a cropping pattern

117 of cotton-wheat-cotton (see 5.2.5 above). Also in this study, the maximum CMB population growth rate on cotton was recorded in the first three weeks of July, possibly because of environmental factors or the initial low population density of the pest (see 5.1.8 above). Investigation is needed to determine how different cropping patterns, vegetation types, natural enemies, and different growing conditions affect CMB infestation levels. Such information might lead to modification of cultural practices to minimize CMB population build-up without the use of pesticides. 4.3.6.3 Factors Facilitating Dispersal of CMB The spread of CMB on cotton has probably been facilitated by the transport of mealybugs from infested patches to the healthy plants elsewhere in the field, on agricultural machinery (tractors, cultivators, ridgers and seed drills) and workers’ clothes (consistent observations of the author in the field, unpublished). Similar problems with machine-facilitated dispersal of vine mealybug [Planococcus minor (Maskell) (Hemiptera: Pseudococcidae)] between vineyards in California have resulted in changes in harvesting practice and machine hygiene (Daane et al., 2008a). The role of machinery in the dispersal of CMB, and changes in practice in order to minimize it, need to be investigated. Irrigation apparently played an important role in promoting secondary infestation within the cotton field; the crawlers and adults of the CMB were easily washed off one plant and carried to another plant in the same field (consistent observations of the author in the field, unpublished). Investigation may lead to a recommendation to change the irrigation method in order to reduce this problem. The weeds growing along the irrigation water channel appeared to be substantially responsible for the transition of CMB across distances greater than one kilometer (consistent observations of the author in the field, unpublished). The weedy strip provided a continuous corridor of alternative host-plants for CMB between one area and another. Experimentation with field hygiene may show that clean practice will minimize the spread of the mealybug. Birds and invertebrates (particularly moths) have been seen to carry CMB crawlers long distances, but in these cases the initial primary infestation was very low (consistent observations of the author in the field, unpublished). Ants may also be instrumental in local dispersal of CMB (see 6.6.5.6 below). If ants prove to be an important factor in CMB dispersal, it may be possible to reduce this problem through the use of ant-bait stations in the fields, using the method described by Daane et al. (2008b).

118 4.3.6.4 Factors Facilitating Carry Over of CMB The commonest method of carry over of CMB was on infested weeds (consistent observations of the author in the field, unpublished). Vigilant farmers who kept their land clean of weeds were less subject to heavy infestation of the cotton crop and resultant loss of yield (consistent observations of the author in the field, unpublished). Weeds growing along the irrigation channel provide a reservoir of alternative hosts for CMB overwintering and carry over beside every field. Experimentation may show that clean practice will minimize CMB carry over of the mealybug between cotton seasons. On the other hand, mealybugs on these plants may maintain an overwintering reservoir of beneficial organisms, ready to migrate into the cotton crop and control the mealybugs early in the growing season. Experimentation is needed to determine whether the benefits of infested weeds near the fields outweigh the risks. During the present study, an adult female CMB was found to be able to survive without feeding for up to 12 days and still produce crawlers successfully. The mealybug was remarkable in its ability to survive prolonged starvation. Care is necessary therefore when identifying host-plants of CMB, as the pest can be encountered on plant species that are not capable of supporting a CMB population for more than one generation. The basic information on the carry over of CMB on alternate host-plant species from the CMB survey (see 4.2.2.4 and 4.2.2.5 above) will be helpful in pest management, where the CMB population size and presence of the alternate host plants will help predict the successful carry over of the pest. 4.3.6.5 Appropriate Pesticide Use “Mealybug” is a general term and there are a large number of species, even just within the genus Phenacoccus (more than 180 species have been described). Without authoritative identification of the insects, it cannot be assumed that any plant found to be infested by mealybugs is the victim of CMB attack. It was observed in the present study that in the initial stages of CMB infestation on cotton, treatment of infested patches was all that was needed. It was sufficient (and more economic) to treat only the infested patch and a surrounding radius of 1-3 meters of adjacent healthy plants, rather than the entire field. Future workers may wish to develop threshold values for CMB infestation levels to determine when pesticide application is necessary, to minimize pesticide use and promote beneficial fauna on the crop. In the chemical control of CMB on field cotton, the main causes of problems are: inadequate spray coverage; improper spraying technique; inadequate spraying machinery;

119 negligence, and delay in action taken. The development of education methods for growers and extension workers should reduce the incidence of such problems; field schools have been found to address these needs effectively (FAO, 2004). Crops that harbor a large number of beneficial fauna, in spite of being infested with CMB, do not require a chemical treatment because a natural equilibrium will become established after a short interval of multiplication of the beneficial fauna. For example, sunflowers (Helianthus annuus) are grown in the winter, when pesticide applications fall to 0-12% of the summer rate, so the beneficial fauna has the chance to survive. During this study, sunflower was sown on a large acreage in 2007-08 and a considerable area was reported to be infested with CMB; but within three weeks the mealybugs were under natural control by the resident beneficial fauna. In Pakistan, this natural source of control on cotton is wasted in the summer because of continuous blanket spraying of the crop with non-selective pesticides. Development of cultural methods to reduce pesticide use should allow natural control to develop in the cotton fields, so reducing the health and environmental hazards associated with heavy pesticide use, and expenditure on pesticides. With reference to the CMB survey findings and the discussion in section 4.2.2 and 4.2.3 above, the percentage CMB infestation and CMB infestation intensity are likely to be important parameters for decision-making in pest management. 4.3.6.6 Proposal for an Experiment in Sustainable Control of CMB on Cotton Cotton production in Pakistan is highly dependent upon the use of chemical poisons sprayed to control the cotton pest complex; fully PKR198.9 billion is spent on the import of these chemicals annually (FBS, 2008a). The indiscriminate use of pesticides is contributing significantly to the severity of the cotton mealybug pest problem. This observation is seconded by other entomologists (S. Suresh, Tamil Nadu Agricultural University, India; G.W. Watson, California Department of Food & Agriculture, USA, 2008, personal communications). After evaluation of the results above, and their practical implications, the following hypothesis is suggested for future researchers to test: “Sacrifice one merla and save one acre” (one merla is 1/160 th of an acre = 272 sq. ft). Since this is a hypothesis, it cannot be recommended to the general farming community until it is tested. It should be tested on public sector research farms and by large farm owners of cotton who can afford the possible losses involved in testing. This suggestion is based on the observed low dispersal ability of CMB in the field.

120 It is proposed that whenever there is a CMB infestation in a cotton field, the infested patch should be treated with pesticide to stop significant economic damage. However, the less heavily infested plants surrounding the treated patch, up to the extent of one merla , should be left untreated to provide a reservoir population of CMB; this will attract and sustain beneficial fauna in the field at the cost of one merla of cotton plants sacrificed to the pest. The reservoir of infested plants will serve as a hatchery for the existing natural beneficial fauna, with a CMB population to feed them. The resultant predators and parasitoids will multiply and spread to control the CMB in the rest of the field. This is only a hypothesis, recommended for future testing; no liability of any adverse results falls on the author. Success cannot be guaranteed, but it seems likely that this tactic would work in the conditions in cotton fields in Pakistan. 4.3.6.7 The Role of Ants The nature of the association between ants and CMB (whether it is mutualistic, antagonistic etc.) has yet to be explored. During this study, P. solenopsis was observed in association with more than 20 (unidentified) species of attendant ants (Hymenoptera: Formicidae). The ants were seen feeding on the sugary honeydew egested by the mealybugs. It is not known whether the good health of the mealybug colony was dependent on the presence of ants or not; this could be tested experimentally using sticky trapping material to exclude ants from some infested cotton plants. In rare cases, ants have been observed keeping CMB captive inside their nest during severe winter weather. At the end of February, the ants were been seen to carry crawlers, in their mouthparts, from the nest to the growing tip of a host plant, so helping the mealybugs to survive the winter and find good feeding sites in the spring. The role of ants in facilitation of the successful overwintering of CMB requires investigation. A single (unidentified) species of black ant, with three 3 or 4 white streaks on the dorsum of the abdomen, was sometimes seen apparently feeding on CMB crawlers and on the wax on the body of the adult mealybugs, exposing the cuticle. The true nature of this relationship requires investigation. 4.3.6.8 Other Insect Associations of CMB The dusky cotton bugs, Oxycarenus hyalinipennis (Costa) and O. laetus (Hemiptera: Heteroptera: Lygaeidae) have been observed in association with CMB but the nature of association is not understood. Experimentation might establish whether there is a direct association between these bugs and CMB, or if the connection between them is through their relationships with ants. Understanding the relationship may permit

121 development of methods of disrupting it, if there are economic implications. In Malaysia, some of the ants attracted into cocoa plantations by mealybugs producing honeydew have been found to reduce the damage caused by the bug Helopeltis theobromae Miller (Hemiptera: Heteroptera: Miridae) (Khoo and Chung, 1989). 4.3.6.9 A Possible Impact of a Change in Microclimate A number of factors need research, such as what factors might have triggered the outbreak of CMB. Possibly a change in microclimate might have disturbed the natural equilibrium of the existing fauna, so the meteorological data for the last 13 years was analyzed statistically. The data (Table 39 below) was divided into the following three distinct periods: i. 1996-2004 average of data for ten years preceding the attack of CMB on cotton ii. 2000-2004 average of data for five years preceding the attack of CMB on cotton iii. 2005-2008 average of data for four years from the onset of CMB attack on cotton.

Table 39. Average meteorological conditions in cotton fields at Faisalabad from 1996 to 2008, in three distinct periods

Average values 2005-2008 2000-2004 1996-2004 Month Temp. RH RF Temp. RH RF Temp. RH RF °C % mm °C % mm °C % mm Jan . 12.4 58.6 21.9 13.1 71.0 10.4 13.2 78.0 11.0 Feb . 16.0 56.1 28.1 13.3 72.5 6.3 14.0 78.0 18.4 Mar . 21.3 45.5 31.7 21.7 50.0 9.0 21.0 54.0 12.0 Apr . 27.7 31.8 6.7 28.6 34.7 11.6 27.4 41.1 27.1 May 32.2 27.7 33.5 31.3 31.3 11.0 31.4 37.2 12.6 Jun . 34.2 35.9 47.4 34.2 43.9 74.0 33.9 45.6 73.8 Jul . 32.9 54.3 88.8 33.2 63.0 92.2 33.1 62.4 94.8 Aug . 32.7 54.2 88.7 32.9 62.8 76.5 32.2 66.7 94.9 Sep . 30.5 50.9 50.5 30.6 60.8 27.7 30.6 64.0 20.1 Oct . 27.8 50.6 7.4 26.0 62.0 0.9 26.0 61.0 4.0 Nov . 21.6 47.3 3.2 20.8 63.0 4.1 20.7 65.0 3.7 Dec . 15.1 56.9 14.6 16.2 64.0 3.3 15.3 70.0 3.1 Temp. = average temperature; RH = average Relative Humidity; RF = average rainfall.

Statistical analysis of the climatic factors in Table 39 above revealed that there was a non-significant difference in average temperature ( °C) and rainfall (mm) between the three periods (p = 0.989 and 0.838, Appendix Tables 16.0 and 16.2 respectively).

122 However, the comparison of means analysis revealed that there was a significant difference in Relative Humidity between the three periods (p = 0.048, Appendix Table 16.1), as shown in Table 40 below.

Table 40. Comparison of means analysis of average relative humidity in cotton fields at Faisalabad from 1996 to 2008, for three distinct periods

P. no. Category of period of comparison of RH% Mean RH% ± SD 2005- 2008 four years from the onset of 1. 47.48 ± 10.34a CMB attack on cotton 2000-2004 five years preceding the attack of 2. 56.58 ± 13.51ab CMB on cotton 1996-2004 ten years preceding the attack of 3. 60.25 ± 13.35b CMB on cotton P = 0.048; pooled standard deviation = 12.48. RH% = Relative Humidity %. Values sharing the same letter are significantly different.

Although the above analysis is based on insufficient data, it suggests that detailed exploration of the effect of climatic factors, especially Relative Humidity, may be very important for future researchers. This agrees with the findings of the present study (see 5.1.4 above), that Relative Humidity (RH%) was significantly correlated with the population dynamics of CMB on shoe flower (p = 0.001) and on cotton (p = 0.003, for details refer to Appendix Tables 11.5 and 13.2 respectively). When the meteorological data was analysed in greater detail, this revealed that Relative Humidity showed significant variation (p = 0.000) between different months over the entire data set for 1996-2008. The comparison of means analysis of monthly mean values of Relative Humidity are given in Table 41 below. This is an indication for further researchers in ecology to explore whether this change in RH% played any role in the outbreak of CMB. If so, Relative Humidity might be useful as a predictor of potential CMB outbreaks in the future.

Table 41. Comparison of Means Analysis of Monthly Mean Relative Humidity for the period 1996-2008 Month Mean ± SD of RH% Month RH% mean ± SD January 69.2 ± 9.8 a July 59.9 ± 4.9 bc February 68.9 ± 11.4 a August 61.2 ± 6.4 bc March 49.8 ± 4.3 d September 58.6 ± 6.8 bc April 35.9 ± 4.8 ef October 57.9 ± 6.3 bc May 32.1 ± 4.8 f November 58.4 ± 9.7 bc June 41.8 ± 5.2 de December 63.6 ± 6.6 ab

123 P = 0.000; pooled standard deviation = 7.10. RH% = Relative Humidity %. Values sharing the same letter are significantly different. 4.3.6.10 Tools for CMB Management The host plant list and order of CMB host preference (Table 17 above) may serve as a base line for scientists interested in the use different host plants as trap crops in the organic culture of vegetables in their experiments. The data from the intensive survey on cotton (see 3.2.7 and 5.2.2.2 above) did not generate an effective regression line, in spite of an indication of significant correlations existing in the pest population and its corresponding climatic factors, because there is a statistical problem with population data from pests that undergo a geometric increase in their populations and have a non-random distribution (Southwood, 1976; Pedigo, 2003). Both Southwood (1976) and Pedigo (2003) suggested that, to overcome this problem, the population values should be transformed into log values to obtain a normal distribution in the data set. This technique did not work in the present study, as the log of the population regression line gave a non-significant result (p = 0.233, Appendix Table 13.3). This might have been due to failure to include some other important factors in the analysis, like the beneficial fauna which has a significant effect on the population, or the population of alternate host plants in the study area, etc. Since this study was not intended to evolve a regression model, future researchers with such an objective should ensure that all other relevant factors are included in order to produce an effective regression model, which is an important tool in pest management. It is anticipated that future researchers will wish to develop population indices or various indicators to estimate the CMB population or number of adults or honeydew production or damage symptoms etc., which are important tools in pest management. It is recommended that such work should be based on data from at least one year of monthly observations, to ensure that the resultant population indices or relative estimates are effective.

124 SUMMARY

The proper identity of a pest was one the most important mandates of these studies. The pest mealybug was a new record in Pakistan and from the morphological differences it was named as P. gossypiphilus , spec. nov. because initially the widespread view was that it was undescribed. Because the type series of the neotropical species Phenacoccus solenopsis Tinsley lacks multilocular disc-pores along the abdominal submargins (Williams & Granara de Willink, 1992), it was initially thought straightforward to separate the material from Pakistan from P. solenopsis . However, during this study (see Appendix II), it was pointed out (D.R. Miller (USDA) and D.J. Williams (BMNH), pers. com.) that many specimens from the Neotropics, which appeared to be otherwise similar to P. solenopsis , have some multilocular disc-pores submarginally on the abdomen. Such specimens - with the additional multilocular disc-pores on the submargins - are now known to be quite widespread in parts of North, South and Central America and apparently similar specimens are also now known from other parts of Asia and from West Africa. After a fairly exhaustive survey of morphological characters on the material from Pakistan, India and from several other sites in Asia, it became clear that this material from Asia was rather variable, the variation appearing to depend on the environmental conditions (similar to that shown for other mealybugs by Cox, 1983). Thus, specimens taken from the field tended to have many multilocular disc-pores along the submargins of the abdomen (a mean of about 15 per side (range 12-24)) and more oral collar tubular ducts medially on the thorax, whereas specimens studied during the non-cotton-growing season (Nov.-March) in Pakistan, taken from cultures in screen houses but initially collected off cotton in the field, had many fewer multilocular disc-pores (a mean of about 4 per side) and oral collar ducts. Indeed, whereas the field collected specimens appeared to have significantly more multilocular disc-pores on the abdominal submargins than was known on specimens of P. solenopsis from the Neotropics, cultured specimens appeared to be identical (C.J. Hodgson, pers. com.). These studies based on speciomens from all regions (Hodgson et al ., 2008, Appendix 2) have shown that the morphological variability of the species in Pakistan falls within that of Phenacoccus solenopsis , and it is recommended that, until the DNA studies currently being undertaken in the United States are completed, the name P. solenopsis Tinsley should be used.

125 It is a notorious pest of cotton Gossypium hirsutum L in Pakistan. It has also been recorded as a pest in most of the cotton growing and non cotton growing areas of India, however its level of infestation in non-cotton growing areas is low.( G W Watson pers. com. with reference to Dr. Suresh). Phenacoccus solenopsis is an aerial pest and passes all of its life cycle on aerial parts of the host plants, on tender shoots, leaves, flower buds and even on stems. It has been noted to reproduce sexually. Its mode of reproduction is ovoviviparous, ie. it retains the eggs in the body until they are ready to hatch. It produces a sac covered with cottony covering protruded from anal end of the pest. As soon as it is torn or the adult female moves forward the sac is detached and the offspring technically called crawlers, come out and spread for search of food. Number of crawlers is variable and depends upon source of food and environmental conditions; the average on cotton, Gossypium hirsutum L., in local conditions of Multan in July is 99.1 (range 56-129) whereas, on sunflower Helianthus annus in the month of April (off season hosts), the average is 51.3 (range 22-83). Its life cycle is variable with response to changing environmental conditions, availability of preferred host and its physical health. In lab conditions it has taken 4 days in 1 st instar, 4.4 -5.6 days in 2 nd instar, 4.1-7.3 days in 3 rd instar and 7.3 to 8.23 days in 4 th instar whereas, it has taken 21-23 days in total from hatching to ovisac production at 25± 2 C temperature and 65± 5 % Relative humidity. Anyhow, these conditions were subject to scheduled or unscheduled power failure due to load shedding of electricity. It is a dimorphic insect having a winged male and wingless female. The second instar nymphs can be identified for their sex with a very careful examination under microscope (Hodgson et al ., 2008) but after the second instar the male can be identified with naked eye as the female moults into 3 rd instar nymph, whereas, the male transforms into prepupa. Usual life span of the female is 29.7± 13.7 days. It is most active in earlier instars and most of the dispersal occurs through initial instars. A mature female tends to be sluggish and prone to anchor the host plant with its sharp opisthognathous labial shield inserted in host tissues. Most of the mortality also occurs in initial instars. In an experiment it was observed that 75% mortality occurred when the nymphs were of 1-8 days of age and 20 % mortality occurred when the nymphs were 9-16 days of age. Usually a female tends to be a reproducing female within 16-21 days. The cotton mealybug Phenacoccus solenopsis is ovoviviparous; it retains the eggs in its body until they are ready to hatch. The number of eggs developing in one female is variable depending upon the

126 type of the host plant. The number of developing eggs counted in mature females on different plants ranged from 46.7 to 92.5. Host plants in descending order of suitability are as follows, cotton, Gossypium hirsutum > Chinese rose, Hibiscus rosa-sinesis > Lady finger Hibiscus esculentus > Aksun Withana somnifera >Itsit Trianthema patulacastrum>Hazar dani Euphorbia granulata> Gule Dupehri Portulaca grandiflora>Lantana Lantana camera>Tandla Digera muricata> Chillies Capsicum annum. Similarly total number of crawlers determined in the field from one female in cotton Gossypium hirsutum was significantly more than the average number of crawlers found on sunflower Helianthus annus. The numbers of crawlers taken from different host plant species in different localities were significantly different from one another, whereas, taken from same host plant species on different localities has a non significant variation. Sex ratio is variable, as counted from three hosts in three different months the average male to female sex ratio came to be 1.0:1.3. but it is statistically non significant, as there is a great variation in percentage of both sexes in various months. Newly emerged crawlers are capable of moving and feeding freely. The newly emerged crawlers are tiny (0.5 mm) and relatively transparent, and therefore they can hardly be observed with an overview except a careful observation. In 1-2 days size is increased and wax is deposited on the body which increases its visibility. This mealybug has been recorded on 55 host plants in 18 families. There are still some reports of their alternate hosts which need to be verified further. It is wide spread in all the cotton growing areas of Pakistan. In addition to the cotton tract it has also been recorded in other districts. It has been observed in 20 districts of Punjab, 14 districts of Sindh, one district each from NWFP and Baluchistan. These districts have been confirmed by the author, and still there are some districts and localities which are prone to the occurrence of this pest. As already discussed morphologically it is inseparable from Phenacoccus solenopsis , as the later has a plenty of range in its morphological variability (Hodgson et al, 2008, full length paper is attached as annexure II), a DNA analysis can disclose the situation. It has also been reported from India, Thailand, USA, Brazil, Chile, New Guinea, New Caledonia, Nigeria and the New World. It has been reported devastating cotton in India also, from a number of cotton growing districts. This pest can find a large number of alternate host plants in agro ecological conditions of Pakistan. Its occurrence has been observed in 6 out of 10 agro ecological zones of Pakistan. In 2 zones its occurrence is in traces, in another 2 agro ecological zones its intensity of damage is low, in remaining two agro ecological zones it is a

127 serious pest of the economic crops. Its severe damage has been found in the cotton or vegetable growing areas where there is maximum use of pesticides. A number of beneficial insects and spiders have been observed feeding on the pest particularly the beetles, Scymnus spp; Menochilus spp; Brumus suturalis , Hippodamia spp; Coccinella spp.; green lace wings Chrysoperla spp. and a number of spiders have been noted in the field but these are wiped out by the indiscriminate spraying process adopted to protect the crops. The results of the relative efficacy of the insecticides revealed that their efficacy falls in the following sequence. Relative resistance of the present 10 cotton cultivars shows that they are nearly equal in their response towards infestation of cotton mealybug, none of them is resistant to this pest. The rate of growth in the population of the pest shows that maximum growth rate is in first three weeks July 2007, it might be due to environmental factors or low population density of the pest, but it is to be explored. The relative efficacy of the insecticides shows that the pesticides used fall in the following sequence of efficacy after 72 hours of the application; Methidathion> Profenophos > Methomyl > Imidacloprid > Carbosulfon > Bifenthrin > Acetameprid > Fenpropathrin >Buprofezin > Control. However, for safety to beneficial fauna the sequence was different ie., Control> buprofezin> Imidacloprid> Methomyl> Fenpropathrin > Bifenthrin> Carbosulfon> Acetameprid> Profenophos> Methidathion.

The research trial for optimum quantity of spray volume showed that 100 & 120 liters water used as spray material with recommended pesticides, in one acre (43560 sq ft), was the optimum volume, whereas, more than this was also good but less than this volume resulted in low control as there was no proper coverage of the spray material on the target pest and the pest escaped and resulted in build up of population again. Similarly, it was revealed that there is no additional effect of additives like detergent, vegetable oil and mineral oil in the spray material, which were recommended as hit and trial from various agencies and persons, but rather these affected the plant health so these should be avoided.

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139 APPENDIX 1. ADDITIONAL DATA AND DETAILS OF ANALYSES

Table 1.0. Summary of Life cycle of Cotton Mealy Bug (CMB) Variable N* Mean SE Mean St Dev Minimum Median Maximum Life aop 0 16.400 1.320 4.17 0 12.000 15.50 0 26.00 0 Crwl sacs 0 2.600 0.306 0.966 2.000 2.000 4.000 Crwl Mx 0 39.100 3.810 12.04 0 20.000 40.50 0 53.00 0 Crwl Mn 0 22.500 3.380 10.69 0 8.000 23.00 0 39.00 0 Crwl T 0 77.900 7.030 22.22 0 39.000 80.50 0 113.00 0 Wax d 1 2.667 0.236 0.707 2.000 3.000 4.000 S Ex 1 2 7.125 0.441 1.246 6.000 7.000 10.000 Pupation 6 16.000 1.220 2. 45 0 13.000 16.50 0 18.00 0 D O 1 -2 0 7.600 0.452 1.430 5.000 7.500 9.000 D O 2 -3 7 2.667 0.667 1.155 2.000 2.000 4.000 D O 3 -4 7 5.330 3.330 5.77 0 2.000 2.000 12.000 Frst Ins 2 6.750 0.620 1.753 4.000 7.000 10.000 Snd Ins 2 6.375 0.263 0.744 5.000 6.500 7.000 Trd Ins 4 7.330 1.020 2.50 0 4.000 7.00 0 11.00 0 D O prod 9 23.000 * * 23.000 23.000 23.000 Males 7 42.000 4.160 7.21 0 36.000 40.00 0 50.00 0 L sp Crw 0 25.100 4.330 13.71 0 12.000 18.00 0 52.00 0 D M fem 2 18.630 1.030 2.92 0 15.000 18.50 0 23.00 0

Table 1.0. Explanation of variables/Abbreviations Life aop= Life after ovisac production Crwl sacs=No of crawler sacs produced by one female in its life time Crawl Mx= Maximum crawlers batch produced by a female in its life time Crawl Mn= Minimum crawlers batch produced by a female in its life time Crawl T = Total number of crawlers produced in all batches Wax d= Number of days taken to produce white mealy powder on its body S Ex 1= Number of days taken in shedding exuvium of first instar Pupation= Number of days taken in pupation D O 1-2= Number of days taken from first instar to second instar D O 2-3 = Number of days taken from second instar to third instar D O 3-4 = Number of days taken from third instar to fourth instar First ins = Maximum number of days in first instar

140 Table 1.0 continued. Snd ins = Maximum number of days in second instar Trd ins = Maximum number of days in third instar D O prod = Total days taken upto crawlersac /ovisac production Males = percentage of males produced from the progeny L sp Crw = Total life span of a crawler D M fem= Days taken to become a mature female

Table 1.1: Data of in vitro mortality of CMB No of Cumulat ive Days Mortalities Mo rtality 1 38 38 2 77 115 3 104 219 4 60 279 5 83 362 6 36 398 7 89 487 8 67 554 9 35 589 10 45 634 11 11 645 12 10 655 13 13 668 14 11 679 15 12 691 16 11 702 17 8 710 18 0 710 19 12 722 20 5 727 21 3 730 22 3 733 23 1 734 24 1 735 25 3 738 26 2 740 27 0 740 28 1 741 29 0 741 30 0 741 31 1 742

141 Table 3.0. Descriptive Statistics on the basis of intensity population of CMB/ 20g fresh biomass of host plant from the data of CMB surveys, 2005-2008 Variable: CMB / 20g fresh biomass Month N Mean St Dev Minimum Median Maximum Apr. 61 16.15 20.91 0.00 9.00 79.00 Aug. 22 57.50 93.90 2.00 19.00 368.00 Dec. 12 16.92 21.21 0.00 9.50 65.00 Feb. 26 15.38 29.26 3.00 9.00 156.00 Jan. 2 40.50 54.40 2.00 40.50 79.00 Jul. 28 37.29 43.80 4.00 23.50 198.00 Jun. 26 27.27 42.44 3.00 12.50 170.00 Mar. 89 15.35 47.72 0.00 2.00 315.00 May 31 11.87 18.16 2.00 7.00 87.00 Misc. 49 29.16 40.53 1.00 14.00 198.00 Nov. 10 9.40 21.00 0.00 0.00 67.00 Oct. 65 19.22 51.35 0.00 0.00 289.00 Sept. 37 18.68 57.60 0.00 0.00 254.00

Table 3.1. Descriptive statistics: percentage CMB infestation Variable: % CMB infestation

Year N Mean St Dev Minimum Median Maximum 2005 3 3.93 2.50 1.08 5.00 5.71 2006 87 9.08 19.45 0.00 2.56 100.00 2007 387 13.62 23.49 0.00 4.00 100.00 2008 33 13.32 26.28 1.00 4.00 100.00

142 Table 3.2. Descriptive Statistics: CMB/ 20g CMB/ 20g fresh biomass of host plant Variable: CMB / 20g fresh biomass

Year N Mean St Dev Minimum Median Maximum 2005 3 91.30 140.90 8.00 12.00 254.00 2006 87 19.09 38.35 0.00 7.00 246.00 2007 384 19.56 45.43 0.00 5.00 368.00 2008 33 16.18 21.09 2.00 8. 00 79.00

Table 3.2. Descriptive Statistics: CMB/ 20g CMB/ 20g fresh biomass of host plant Variable: CMB / 20g fresh biomass

Month N N* Mean St Dev Minimum Median Maximum Jan. 2 0 40.50 54.40 2.00 40.50 79.00 Feb. 26 0 15.3 8 29.26 3.00 9.00 156.00 Mar. 89 45 15.35 47.72 0.00 2.00 315.00 Apr. 61 1 16.15 20.91 0.00 9.00 79.00 May 31 0 11.87 18.16 2.00 7.00 87.00 Jun. 26 1 27.27 42.44 3.00 12.50 170.00 Jul. 28 0 37.29 43.80 4.00 23.50 198.00 Aug. 22 0 57.50 93.90 2.00 19.00 368.00 Sept. 37 4 18.68 57.60 0.00 0.00 254.00 Oct. 65 1 19.22 51.35 0.00 0.00 289.00 Nov. 10 0 9.40 21.00 0.00 0.00 67.00 Dec 12 0 16.92 21.21 0.00 9.50 65.00 Misc. 49 0 29.16 40.53 1.00 14.00 198.00

143 Table 3.3. One-way ANOVA: CMB/ 20g fresh biomass versus Host No. Source DF SS MS F P Host no. 55 489841 8906 8.08 0.00 Error 451 496981 1102 Total 506 986822

S = 33.20 R-Sq = 49.64% R-Sq(adj) = 43.50%

Individual 95% CIs For Mean Based on Pooled St Dev Level N Mean StDev -----+------+------+------+---- 0 27 2.56 2.34 (--*-) 1 15 14.87 20.59 (--*--) 2 8 36.88 31.65 (---*----) 3 9 4.33 4.85 (---*---) 4 7 6.57 5.06 (----*----) 5 8 4.75 3.73 (----*----) 6 8 12.38 9.78 (---*----) 7 8 4.13 5.38 (----*---) 8 6 3.17 5.00 (-----*----) 9 8 13.63 9.98 (----*---) 10 7 5.71 4.57 (----*----) 11 20 1.80 1.74 (--*--) 12 12 3.67 6.85 (---*--) 13 8 7.62 6.05 (----*---) 14 6 2.67 2.34 (-----*----) 15 7 5.14 5.37 (----*----) 16 7 6.29 5.31 (----*----) 17 8 5.00 4.07 (----*----) 18 8 5.50 7.73 (----*----) 19 6 7.33 10.03 (----*-----) 20 8 26.38 29.32 (---*----) 21 7 34.71 28.48 (----*----) 22 17 3.00 2.60 (---*--) 23 6 8.50 5.72 (-----*----) 24 5 9.20 4.44 (-----*-----) 25 8 9.00 7.56 (----*---) 26 7 7.14 4.85 (----*----) 27 9 4.33 4.15 (---*---) 28 16 4.06 3.40 (--*--) 29 7 6.86 8.13 (----*----) 30 12 8.50 10.89 (---*--) 31 7 3.14 2.27 (----*----) 32 5 6.00 4.12 (-----*-----) 33 5 6.60 4.04 (-----*-----) 34 11 2.36 2.01 (--*---) 35 7 28.43 22.26 (----*----) 36 24 114.54 107.10 (--*--) 37 10 41.50 44.30 (---*---) 38 9 11.78 9.82 (---*----) 39 14 105.71 61.95 (-*---) 40 5 5.60 5.90 (-----*-----) 41 5 8.00 9.25 (-----*----) 42 10 16.20 18.05 (---*---) 43 10 5.00 4.92 (---*---) 44 5 5.20 4.76 (-----*-----) 45 6 17.50 14.68 (-----*----) 46 17 15.06 21.84 (--*--) 47 5 141.60 116.07 (-----*-----) 48 7 31.00 24.83 (----*----) 49 11 9.91 9.02 (---*---) 50 6 11.00 9.59 (----*-----) 51 5 73.60 116.19 (-----*-----) 52 9 11.89 11.66 (---*----) Continued on next page

144 Table 3.3. Continued 53 6 17.67 13.54 (-----*----) 54 7 23.43 21.34 (----*----) 55 6 9.17 7.47 (----*----) -----+------+------+------+---- 0 50 100 150

Pooled St Dev = 33.2

Table 4. ANOVA for the effect of host plant species on CMB fecundity

SOV DF SS MS F P Host 9 8863.7 984.9 22.58 0 Error 20 872.5 43.6 Total 29 9336.3

Table 5. Tucky comparison for the average numbers of developing embryos dissected from adult females feeding on the studied host plants at Faisalabad in 2007

Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ------+------+------+------+--- 1 3 92.40 6.90 (----*----) 2 3 81.70 5.80 (----*----) 3 3 90.40 7.10 (-----) 4 3 63.90 6.30 (----*----) 5 3 52.80 5.90 (----*---) 6 3 46.80 7.20 (----*----) 7 3 68.60 6.80 (----*----) 8 3 51.60 7.30 (----*---) 9 3 48.40 6.80 (----*----) 10 3 46.70 5.70 (----*----) ------+------+------+------+--- 48 64 80 96

Pooled St Dev = 6.60

145 Table 6.1. General Linear Model: pop/female versus Month, Host

Factor Type Levels Values Month random 3 1, 2, 3 Host random 3 1, 2, 3

Analysis of Variance for pop/female, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P Month 2 3658.81 3658.81 1829.40 46.98 0.002 Host 2 3788.03 3788.03 1894.01 46.64 0.002 Month*Host 4 155.75 155.75 38.94 0.96 0.454 Error 18 73 1.56 731.56 40.64 Total 26 8334.15

S = 6.37513 R-Sq = 91.22% R-Sq(adj) = 87.32%

Table 6.2. Tukey 95.0% Simultaneous Confidence Intervals Response Variable pop/female All Pairwise Comparisons among Levels of Host

Host = 1 subtracted from:

Host Lower Center Upper ------+------+------+------2 -31.00 -23.33 -15.66 (------*-----) 3 -34.27 -26.60 -18.93 (------*-----) ------+------+------+------24 -12 0

Table 6.3. Tukey 95.0% Simultaneous Confidence Intervals, Response Variable pop/female. All Pairwise Comparisons among Levels of Month

Month = 1 subtracted from:

Month Lower Center Upper ------+------+------+------2 -33.30 -25.63 -17.96 (---*---) 3 -9.67 -2.00 5.67 (---*---) ------+------+------+------20 0 20

Month = 2 subtracted from:

Month Lower Center Upper ------+------+------+------3 15.96 23.63 31.30 (---*---) ------+------+------+------20 0 20

146 Table 6.4. Descriptive Statistics: pop/female Variable: pop/female

Host Mean St Dev Minimum Median Maximum

1. 54.57 10.73 37.30 57.10 67.70

2. 31.23 15.09 8.40 37.60 47.50

3. 27.97 15.01 4.20 30.40 45.40

Table 6.5. Descriptive Statistics: pop/female Variable: pop/female

Month Mean St Dev Minimum Median Maximum 1. 47.13 12.63 30.40 43.00 67.70

2. 21. 50 16.44 4.20 14.60 47.90 3. 45.13 12.44 30.20 45.40 67.20

Table 6.6. Descriptive Statistics: pop/female

Variable Mean St Dev Minimum Median Maximum pop/female 37.92 17.90 4.20 38.90 67.70

147 Table 7.0. General Linear Model: POP versus Month, Group (Population of CMB population groups in different months)

Factor Type Levels Values Month fixed 3 1, 2, 3 Group fixed 4 1, 2, 3, 4

Analysis of Variance for POP, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Month 2 726656 726656 363328 101.00 0.000 Group 3 12798646 12798646 4266215 1185.93 0.000 Month*Group 6 79828 6 798286 133048 36.98 0.000 Error 24 86336 86336 3597 Total 35 14409924

S = 59.9779 R-Sq = 99.40% R-Sq(adj) = 99.13% Table 7.1. Tukey 95.0% Simultaneous Confidence Intervals Response Variable POP All Pairwise Comparisons among Levels of Month Month = 1 subtracted from:

Month Lower Center Upper +------+------+------+------2 148.7 209.9 270.97 (--*---) 3 -196.6 -135.5 -74.38 (--*--) +------+------+------+------400 -200 0 200

Table 7.2. Tukey 95.0% Simultaneous Confidence Intervals Response Variable POP All Pairwise Comparisons among Levels of Group Group = 1 subtracted from:

Group Lower Center Upper +------+------+------+------2 -7.14 70.83 148.8 (-*-) 3 1045.66 1123.63 1201.6 (-*-) 4 1236.76 1314.73 1392.7 (-*-) +------+------+------+------0 400 800 1200

148 Table 7.3. Tukey Simultaneous Tests Response Variable POP All Pairwise Comparisons among Levels of Group Group = 1 subtracted from:

Difference SE of Adjusted Group of means Difference T-value P-value 1. 70.83 28.27 2.505 0.0845 2. 1123.63 28.27 39.741 0.0000 3. 1314.73 28.27 46.500 0.0000

Table 7.4. Tukey 95.0% Simultaneous Confidence Intervals Response Variable POP All Pairwise Comparisons among Levels of Month*Group Month = 1 Group = 1 subtracted from:

Month Group Lower Center Upper -----+------+------+------+- 1 2 -62.0 114.60 291.2 (-*) 1 3 879.0 1055.60 1232.2 (-*) 1 4 1097.0 1273.60 1450.2 (-*) 2 1 -178.6 -2.00 174.6 (*) 2 2 -112.0 64.60 241.2 (-*) 2 3 1350.7 1527.30 1703.9 (-*) 2 4 1516.7 1693.30 1869.9 (*-) 3 1 -149.3 27.30 203.9 (*-) 3 2 -118.0 58.60 235.2 (*-) 3 3 636.7 813.30 989.9 (-*) 3 4 826.0 1002.60 1179.2 (*-) -----+------+------+------+- -1200 0 1200 2400

Table 7.5. Tukey Simultaneous Tests Response Variable POP All Pairwise Comparisons among Levels of Month*Group Month = 1 Group = 1 subtracted from:

Difference SE of Adjusted Month Group of means difference T-value P-value 1 2 114.60 48.97 2.3401 0.4777 1 3 1055.60 48.97 21.5553 0.0000 1 4 1273.60 48.97 26.0068 0.0000 2 1 -2.00 48.97 -0.0408 1.0000 2 2 64.60 48.97 1.3191 0.9681 2 3 1527.30 48.97 31.1874 0.0000 2 4 1693.30 48.97 34.5771 0.0000 3 1 27.30 48.97 0.5575 1.0000 3 2 58.60 48.97 1.1966 0.9842 3 3 813.30 48.97 16.6075 0.0000 3 4 1002.60 48.97 20.4730 0.0000

149

Table 7.6. Descriptive Statistics: POP Results for Month = 1 Variable POP

Group Mean St Dev Median 1 51.70 11.70 51.70 2 166.30 16.10 166.30 3 1107.30 92.50 1107.3 0 4 1325.30 105.1 0 1325.3 0

Results for Month = 2 Variable POP

Group Mean St Dev Median 1 49.70 8.50 49.70 2 116.3 0 20.7 0 116.3 0

3 1579.00 67.90 1579.00

4 1745.0 0 79.3 0 1745.0 0

Results for Month = 3 Variable POP Group Mean St Dev Median 1 79.0 0 9.80 79.00

2 110.30 13.70 110.30

3 865.00 69.60 865.00

4 1054.30 81.50 1054.30

Descriptive Statistics: POP

Variable POP Month Mean St Dev Median 1 663 588 599 2 873 828 824 3 527 460 460

Table 7.6 iscontinued on the next page.

150 Table 7.6. Continued.

Descriptive Statistics: POP

Variable POP Group Mean St Dev Median 1 60.13 16.65 58.20 2 131.0 0 30.5 0 124.0 0 3 1184.0 0 322 .00 1107 .00 4 1375.0 0 311 .00 1325 .00

Table 8.0. General Linear Model: Ratio versus sex, condition

Factor Type Levels Values Sex fixed 2 1, 2 Condition fixed 2 1, 2

Analysis of Variance for Ratio, using Adjusted SS for Tests Source DF Seq SS Adj S S Adj MS F P Sex 1 0.27848 0.27848 0.27848 17.59 0.003

Condition 1 0.06172 0.06172 0.06172 3.90 0.084 Sex* Condition 1 0.06172 0.06172 0.06172 3.90 0.084 Error 8 0.12663 0.12663 0.01583 Total 11 0.52855

Table 8.1. General Linear Model: Ratio versus Sex, Host (Sex ratio of CMB on different hosts)

Factor Type Levels Values Sex fixed 2 1, 2 Host fixed 3 1, 2, 3

Analysis of Variance for Ratio, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sex 1 0.27848 0.27848 0.27848 8.22 0.029 Host 2 0.02339 0.02339 0.01169 0.35 0.721 Sex*Host 2 0.02339 0.02339 0.01169 0.35 0.721 Error 6 0.20330 0.20330 0.03388 Total 11 0.52855

151 Table 9.0. One-way ANOVA: reprod versus mating (Reproduction of CMB female in isolation and in conditions where chances of mating provided in vitro)

Source DF SS MS F P Trt 1 3.000000 3.000000 * * Error 14 0.000000 0.000000 Total 15 3.000000

S = 0 R-Sq = 100.00% R-Sq(adj) = 100.00%

Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev +------+------+------+------1 12 0.00000 0.00000 * 2 4 1.00000 0.00000 * +------+------+------+------0.00 0.25 0.50 0.75

Pooled St Dev = 0.00000

Table 10.0: Data of weekly growth of CMB in Cotton in 2007 Date Weeks Pop Weekly G. % Comm. G % Plant G 10.07.07 1 25.0 0 100 5 17.07.07 2 63.5 154.1 254.1 12.7 24.07.07 3 202.2 218.3 218.3 40.4 31.07.07 4 405.1 100.3 318.6 81 07.08.07 5 841.6 107.8 426.4 168.3 14.08.07 6 1521.2 80.8 507.1 304.2 21.08.07 7 2493.1 63.9 571 498.6 28.08.07 8 3570.3 43.2 614.2 714.1 04.09.07 9 4767.4 33.5 647.8 953.5 11.09.07 10 6002.4 25.9 673.7 1200.5 18.09.07 11 6517.5 8.6 682.2 1303.5 25.09.07 12 6234 -4.3 677.9 1246.8 0210.07 13 5686.7 -8.8 669.1 1137.3

152 Table 11.0. Weekly data of population dynamics observed in CMB on Hibiscus rosa-sinensis

at UAF, Pakistan

Date W H" B1D Tw DTw WCp P/Tw Benf. Tem Mx Tem Mn RH % R.fall mm

07.01.07 1 31.5 3.3 1.5 0.3 0.0 0.0 0.0 20.4 ± 1.1 2.4 ± 1.6 72.3 ± 2.1 0.0 14.01.07 2 32.0 3.3 1.5 0.3 0.0 0.0 0.0 19.7 ± 1.0 2.3 ± 1.3 72.1 ± 1.4 0.0 21.01.07 3 32.0 3.3 1.5 0.3 0.0 0.0 0.0 19 ± 0 4.4 ± 1.3 67 ± 6.5 0.0 28.01.07 4 32.0 3.3 1.5 0.3 0.0 0.0 0.0 21.5 ± 1.9 6.3± 2.1 56.9 ± 6.6 0.0 04.02.07 5 34.0 3.3 2.8 0.0 0.0 0.3 0.0 27.4 ±2.6 16.3 ±8.8 45.5 ±26.3 0.0 11.02.07 6 34.5 3.3 3.3 0.0 0.0 0.8 0.0 21.4 ±2.4 11.7 ±1.7 68.1 ±2.9 5.2 ±10.1 18.02.07 7 27.3 2.3 2.0 0.0 0.0 1.0 0.0 17.5 ±1.4 9.6 ±1.5 74.7 ±4.1 1.6 ±3.6 25.02.07 8 29.0 2.8 2.9 0.0 0.0 1.0 0.1 20.8 ±1.7 8.9 ±1.2 59.4 ±1.3 0.3 ±0.8 04.03.07 9 29.0 2.8 3.0 0.0 0.0 1.3 0.1 21±2.4 12 ± 1.3 62.4±12.4 1.4 ±3.3 11.03.07 10 29.5 3.0 3.1 0.0 0.0 1.4 0.2 24.2± 3.2 12 ± 1.6 47.1± 12.5 .2 ± .5 18.03.07 11 32.5 3.0 3.5 0.0 0.0 1.9 0.2 21.3±3.0 11.6 ±2.3 50 ±8.3 3.4 ± 5.8 25.03.07 12 32.8 3.0 3.6 0.0 0.0 2.1 0.3 27 ±2.2 14.1 ± 1.9 43.6 ± 8.9 2.2 ±5.9 01.04.07 13 35.5 3.0 5.7 0.0 0.0 4.6 0.6 32.2 ±2.1 17.9 ±3.0 41 ±7.5 0.0 08.04.07 14 37.0 3.3 5.7 0.0 0.0 4.6 0.6 31.4 ±1.5 18 ±3.1 40 ±6.2 0.0 22.04.07 15 38.8 3.5 6.8 0.0 0.0 6.8 1.2 38.5 ±2.3 21.6 ±1.7 37.9 ±6.2 0.0 30.04.07 16 41.0 4.3 7.9 0.0 1.3 8.7 1.3 40.1 ±1.9 22.1 ±1.6 22.0 ±4.2 0.0 06.05.07 17 44.0 4.3 9.1 0.0 4.0 10.8 1.4 41.7±3.5 24.6±1.4 23.0±6.7 2.5±6.1 13.05.07 18 37.3 3.9 9.3 0.0 5.3 20.1 1.8 39.8±1.5 24.6±1.9 26.7±2.8 0.1±0.3 27.05.07 19 40.8 4.2 9.5 0.5 6.3 35.4 1.9 40.4±2.2 26.1±2.2 24.7±5.6 0.0±0.1 03.06.07 20 43.0 4.3 9.8 0.8 7.8 48.9 2.6 39.9±2.5 24.7±1.6 23.4±4.8 1.0±2.6 10.06.07 21 44.8 4.3 10.0 0.8 9.0 80.1 2.5 42.8±3.1 29.9±2.3 24.7±2.9 0.0 24.06.07 22 30.8 2.3 33.8 1.8 8.8 95.5 3.0 39.0±5.0 27.6±3.7 40.3±7.4 1.5±3.5 01.07.07 23 31.8 2.5 10.3 2.0 9.0 104.8 3.4 29.4±5.2 35.8±4.6 36.1±9.0 44.4±20.6 08.07.07 24 34.5 2.7 10.7 1.5 8.8 128.3 3.8 36.4±1.8 28.1±2.1 55.1±4.1 1.0±1.9 22.07.07 25 36.8 3.6 11.0 1.5 7.8 107.3 4.5 36.1±2.1 26.9±2.9 53.4±6.2 9.6±20.3 29.07.07 26 39.5 3.6 11.6 2.0 7.5 120.3 4.8 36.0±2.1 27.6±1.7 58.4±9.2 1.3±2.3 12.08.07 30 41.8 4.2 11.9 2.0 6.8 125.5 5.5 36.9±1.6 28.7±1.7 53.6±4.7 0.1±0.2 19.08.07 31 44.3 4.8 12.3 2.0 5.8 96.3 6.1 37.5±0.8 28.2±1.6 50.3±3.9 0.1±0.2 26.08.07 32 46.3 5.0 13.0 1.3 2.5 71.5 7.0 37.1±1.3 27.5±0.3 50.6±4.7 0.7±1.8 02.09.07 33 47.5 5.0 13.3 0.8 2.0 52.0 6.6 37.1±1.1 28.7±1.5 49.3±7.2 1.9±5.1 09.09.07 34 48.0 5.0 13.5 0.0 1.3 41.3 5.3 35.1±2.5 26.6±2.6 56.6±6.0 0.3±0.8 16.09.07 35 47.8 5.3 13.1 0.0 0.5 33.0 4.6 35.0±1.4 24.9±1.8 51.1±5.0 1.1±2.9 23.09.07 36 47.8 5.3 13.4 0.0 0.3 23.5 4.0 35.4±3.3 26.1±2.4 53.1±6.1 0.3±0.8 30.09.07 37 47.0 5.3 13.2 0.0 0.0 15.5 3.7 34.1±2.0 23.1±0.9 43.7±7.1 0.0 07.10.07 38 46.8 5.3 12.9 0.0 0.0 13.8 3.4 33.1±3.1 19.7±1.4 32.3±5.0 0.0 14.10.07 39 46.5 5.3 12.9 0.0 0.0 11.8 2.8 33.8±0.6 16.3±0.8 26.4±6.1 0.0 21.10.07 40 46.3 5.3 12.9 0.0 0.0 9.8 2.7 32.7±1.0 16.1±1.2 35.4±3.5 0.0 28.10.07 41 45.5 5.3 12.9 0.0 0.0 8.3 2.1 32.1±1.0 16.7±0.8 34.6±2.8 0.0 04.11.07 42 44.8 5.3 12.9 0.0 0.0 5.5 1.8 31.3±0.4 13.6±0.9 30.6±6.1 0.0 11.11.07 43 44.5 5.3 12.9 0.0 0.0 3.0 1.3 29.2±1.1 15.3±1.6 40.1±6.0 0.0 18.11.07 44 43.8 5.3 12.9 0.0 0.0 2.5 0.4 26.7±0.6 13.6±1.0 53.6±5.3 0.0 25.11.07 45 43.8 5.3 12.9 0.0 0.0 1.8 0.2 26.1±0.5 11.6±0.6 54.1±2.9 0.0 02.12.07 46 43.3 5.3 12.9 0.0 0.0 1.3 0.0 23.9±1.7 12.2±0.6 54.3±5.4 0.0 09.12.07 47 43.0 5.3 12.9 0.0 0.0 0.8 0.0 21.4±1.1 8.4±1.6 43.9±7.8 0.0 16.12.07 48 43.0 5.3 12.9 0.0 0.0 0.0 0.0 18.6±1.2 8.2±2.4 51.0±7.3 0.9±1.9 23.12.07 49 43.0 5.3 12.9 0.0 0.0 0.0 0.0 20.9±1.7 5.9±0.8 58.9±7.4 0.0 30.12.07 50 43.0 5.3 12.9 0.0 0.0 0.0 0.0 51.5±2.3 4.9±1.2 67.3±8.4 0.0

153 Table 11.1. Regression Analysis: Pop/twig versus Wh caps, Benificials, ...

The regression equation is Pop/twig = - 8.0 + 9.09 Wh caps + 8.05 Benificials - 1.01 Tem Mx + 0.696 Tem Mn + 0.430 RH % + 0.055 R.fall mm

Predictor Coeff. SE Coeff. T P VIF Constant -8.04 25.52 -0.31 0.754 Wh caps 9.0934 0.7437 12.23 0.000 2.578 Benef icials 8.050 1.490 5.40 0.000 4.320 Temp. -1.0149 0.8043 max. -1.26 0.214 16.911 Temp. 0.6958 0.6773 min. 1.03 0.311 14.658 RH % 0.4299 0.2076 2.07 0.045 3.924 R.fall mm 0.0553 0.2682 0.21 0.838 1.397 S = 10.1217 R-Sq = 94.5% R-Sq(adj) = 93.6%

Analysis of Variance Source DF SS MS F P

Regression 6 70027 11671 113.92 0.000

Residual 40 4098 102 Error

Total 46 74125

154 Table 11.3. Stepwise Regression: Pop/twig versus Benificials, Tem Mx, ... Stepwise Regression: Pop/twig versus Wh caps, Benificials, ...

Alpha-to-Enter: 0.15 Alpha-to-Remove: 0.15 Response is Pop/twig on 6 predictors, with N = 47

Step 1 2 3 Constant 5.616 -4.232 -34.592 Wh caps 11.02 0 8.41 0 9.150 T-value 12.69 0 12.09 0 16.530 P-value 0.000 0.000 0.000 Beneficials 7.760 7.940 T-value 7.200 9.540 P-value 0.000 0.000 RH % 0.600 T-value 5.550 P-value 0.000 S 19.000 13.000 10.000 R-Sq 78.160 89.980 94.160 R-Sq(adj) 77.680 89.520 93.750 Mallows Cp 115.000 31.500 3.300

155 Table 11.4. Best Subsets Regression: Pop/twig versus Wh caps, Benificials, ...

Response is Pop/twig Mallows S Wh Vars R-Sq cp Caps Bene- Temp. Temp. RH R.fall R-Sq (adj) ficials max. min. % mm 1 78.2 77.7 115.0 18.967 X 1 57.9 57.0 261.5 26.330 X 2 90.0 89.5 31.5 12.995 X X 2 81.8 81.0 90.6 17.505 X X 3 94.2 93.8 3.3 10.033 X X X 3 93.6 93.1 7.3 10.507 X X X 4 94.3 93.7 4.3 10.043 X X X X 4 94.2 93.7 4.6 10.079 X X X X 5 94.5 93.8 5.0 10.003 X X X X X 5 94.3 93.6 6.1 10.128 X X X X X 6 94.5 93.6 7.0 10.122 X X X X X X

Table 11.5. A farmer friendly regression model Regression Analysis: Pop/twig versus Tem Mx, Tem Mn, RH %, R.fall mm (an attempt to develop a farmer-friendly model that requires the meteorological conditions only) The regression equation is Pop/twig = - 142 + 1.37 Tem Mx + 3.45 Tem Mn + 1.37 RH % + 1.08 R.fall mm SE Predictor Coeff. Coeff. T P VIF Constant -141.91 47.20 -3.01 0.004 Temp. max. 1.372 1.676 0.82 0.418 15.824 Temp. min. 3.453 1.274 2.71 0.010 11.177 RH % 1.3670 0.3871 3.53 0.001 2.943 R.fall mm 1.0785 0.5424 1.99 0.053 1.232 S = 21.7994 R-Sq = 73.1% R-Sq(adj) = 70.5% PRESS = 24597.8 R-Sq(pred) = 66.82%

Analysis of Variance Source DF SS MS F P

Regression 4 54166 13542 28.50 0.000

Residual 42 19959 475 Error

Total 46 74125

Lack of fit test Possible curvature in variable Temp. Min. (P-Value = 0.013 ) Overall lack of fit test is significant at P = 0.013

156

Table 12.0. Population Dynamics of CMB on Cotton (Gossypium hirsutum)

Date Weeks Pop weeklyG% Comm.G % Plant G T. Mx T. Mn RH % RF mm 10.07.07 1 25 0 100 5 36.4 28.1 55.1 1 17.07.07 2 63.5 154.1 254.1 12.7 36.6 25.9 53.1 17.1 24.07.07 3 202.2 218.3 218.3 40.4 35.7 27.9 53.7 2.1 31.07.07 4 405.1 100.3 318.6 81 36 27.6 58.4 1.3 07.08.07 5 841.6 107.8 426.4 168.3 36.9 28.7 53.6 0.1 14.08.07 6 1521.2 80.8 507.1 304.2 37.5 28.2 50.3 0.1 21.08.07 7 2493.1 63.9 571 498.6 37.1 27.5 50.6 0.7 28.08.07 8 3570.3 43.2 614.2 714.1 37.1 28.7 49.3 1.9 04.09.07 9 4767.4 33.5 647.8 953.5 35.1 26.6 56.6 0.3 11.09.07 10 6002.4 25.9 673.7 1200.5 35 24.9 51.1 1.1 18.09.07 11 6517.5 8.6 682.2 1303.5 35.4 26.1 53.1 0.3 25.09.07 12 6234 -4.3 677.9 1246.8 34.1 23.1 43.7 0 02.10.07 13 5686.7 -8.8 669.1 1137.3 33.1 19.7 32.3 0

Table 13.0. One-way ANOVA: PGR versus Variety (Percent growth rate of various varieties of cotton and CMB population dynamics)

Source DF SS MS F P

Variety 9 1785 198 0.03 1.000

Error 120 779200 6493

Total 129 780985

S = 80.58 R-Sq = 0.23% R-Sq(adj) = 0.00% Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev -+------+------+------+------1 13 167.91 83.98 (------*------) 2 13 170.81 89.53 (------*------) 3 13 171.96 86.21 (------*------) 4 13 170.24 75.31 (------*------) 5 13 170.13 72.08 (------*------) 6 13 172.37 74.56 (------*------) 7 13 174.43 82.22 (------*------) 8 13 169.05 87.07 (------*------) 9 13 162.40 71.13 (------*------) 10 13 162.88 81.27 (------*------) -+------+------+------+------120 150 180 210 Pooled St Dev = 80.58

157 Table 13.1. One-way ANOVA: PGR versus Weeks

Source DF SS MS F P

Variety 12 653072 54423 49.78 0.000

Error 117 127913 1093

Total 12 9 780985 S = 33.06 R-Sq = 83.62% R-Sq(adj) = 81.94%

Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev --+------+------+------+------1 10 100.00 0.00 (--*-) 2 10 267.73 104.94 (-*--) 3 10 352.00 40.83 (--*--) 4 10 198.22 20.18 (--*-) 5 10 182.44 14.32 (--*-) 6 10 183.08 21.60 (--*-) 7 10 184.22 15.88 (--*--) 8 10 148.91 6.67 (--*-) 9 10 138.96 4.51 (-*--) 10 10 130.94 5.58 (-*--) 11 10 122.57 7.19 (-*--) 12 10 102.29 7.00 (--*-) 13 10 88.47 2.08 (--*--) --+------+------+------+------80 160 240 320 Pooled St Dev = 33.06

158 Table 13.2. Regression Analysis: PGR versus T. Mx, T. Mn, RH %, RF mm

The regression equation is PGR = 1209 - 58.7 T. Mx + 51.3 T. Mn - 6.20 RH % + 12.4 RF mm

SE Predictor Coeff. Coeff. T P

Constant 1209.3 352.0 3.44 0.001 Temp. max. -58.65 14.78 -3.97 0.000 Temp. min. 51.30 10.33 4.97 0.000 RH % -6.204 2.072 -2.99 0.003 R.fall mm 12.355 1.708 7.23 0.000 S = 60.4058 R-Sq = 41.6% R-Sq(adj) = 39.7%

Analysis of Variance Source DF SS MS F P

Regression 4 324878 81219 22.26 0.000

Residual 125 4561 07 3649 Error

Total 129 780985

Source DF Seq SS Temp. max. 1 105310 Temp. min. 1 16451 RH % 1 12272 R.fall mm 1 190845

159 Table 13.3. Regression Analysis: Log pop versus T. Mx, T. Mn, RH %, RF mm

The regression equation is Log pop = - 2.2 + 0.346 T. Mx - 0.330 T. Mn + 0.0222 RH % - 0.116 RF mm

Predictor Coeff. SE Coeff. T P

Constant -2.20 13.44 -0.16 0.874

Temp. max. 0.3455 0.5641 0.61 0.557

Temp. min. -0.3300 0.3941 -0.84 0.427

RH % 0.02223 0.07910 0.28 0.786

R.fall mm -0.11627 0.06521 -1.78 0.112

S = 0.729102 R-Sq = 46.6% R-Sq(adj) = 19.9%

Analysis of Variance Source DF SS MS F P

Regression 4 3.7104 0.9276 1.74 0.233

Residual 8 4.2527 0.5316 Error

Total 12 7.9631

Source DF Seq SS Temp. max. 1 1.3785 Temp. min. 1 0.0768 RH % 1 0.5652 R.fall mm 1 1.6900

Unusual Observations

Obs T. Mx Log pop Fit SE Fit Residual St Resid 1 36.4 0.699 2.210 0.258 -1.511 -2.22R

R denotes an observation with a large standardized residual.

160 Table 14.0. General Linear Model: B 7 D AS versus Pesticides (Population of beneficial fauna after 7 days of the application of Pesticides)

Factor Type Levels Values Pesticides fixed 10 Acetameprid, Amidacloprid, Bifenthrin, Buprofezin, Carbosulfan, Control, Fenpropathrin, Methidathion, Methomyl, Profenophos

Analysis of Variance for B 7 D AS, using Adjusted SS for Tests

Source DF Seq SS Adj SS F P

B 24 HAS 1 51.3091 0.1667 0.56 0.463

B 72 HAS 1 7.0161 0.6429 2.17 0.158

Pesticides 9 7.8415 0.8713 2.94 0.025 Error 18 5.3333 0.2963 Total 29 71.5000 S = 0.544331 R-Sq = 92.54% R-Sq(adj) = 87.98%

Table 14.1. One-way ANOVA: B 72 HAS versus Pesticides

Source DF SS MS F P

Pesticides 9 35.200 3.911 39.11 0.000

Error 20 2.000 0.100

Total 29 37.200

S = 0.3162 R-Sq = 94.62% R-Sq(adj) = 92.20%

Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ---+------+------+------+------Acetameprid 3 0.0000 0.0000 (--*--) Amidacloprid 3 0.3333 0.5774 (--*--) Bifenthrin 3 0.0000 0.0000 (--*--) Buprofezin 3 2.0000 0.0000 (---*--) Carbosulfan 3 0.3333 0.5774 (--*--) Control 3 3.3333 0.5774 (--*--) Fenpropathrin 3 0.0000 0.0000 (--*--) Methidathion 3 0.0000 0.0000 (--*--) Methomyl 3 0.0000 0.0000 (--*--) Profenophos 3 0.0000 0.0000 (--*--) ---+------+------+------+------0.0 1.2 2.4 3.6 Pooled St Dev = 0.3162

Table 14.1 is continued on the next page

161 Table 14.1 continued.

Tukey 95% Simultaneous Confidence Intervals All Pairwise Comparisons among Levels of Pesticides

Individual confidence level = 99.80% Pesticides = Acetameprid subtracted from:

Pesticides Lower Center Upper

Amidacloprid -0.5814 0.3333 1.2480 Bifenthrin -0.9147 0.0000 0.9147 Buprofezin 1.0853 2.0000 2.9147 Carbosulfan -0.5814 0.3333 1.2480 Control 2.4186 3.3333 4.2480 Fenpropathrin -0.9147 0.0000 0.9147 Methidathion -0.9147 0.0000 0.9147 Methomyl -0.9147 0.0000 0.9147 Profenophos -0.9147 0.0000 0.9147

Pesticides ------+------+------+------+-- Amidacloprid (--*---) Bifenthrin (---*---) Buprofezin (---*---) Carbosulfan (--*---) Control (--*---) Fenpropathrin (---*---) Methidathion (---*---) Methomyl (---*---) Profenophos (---*---) ------+------+------+------+-- -2.5 0.0 2.5 5.0

Table 15.0: Table of ANOVA of Various Volumes of Spray against CMB, 168 Hours after Application of Insecticide

Source DF SS MS F P

Treatments 1074.89 4 268.72 4.547* 0.002

Blocks 467.77 2 233.89 3.958* 0.021

Blocks*trt 327.03 8 40.88 0.692 0.698

Error 7977 135 59.10

162 Table 16.0. One-way ANOVA: tem versus period (Meteorological conditions of Faisalabad from 1996 to 2008 tested in three major groups)

Source DF SS MS F P

Period 2 1.300 0.700 0.010 0.989

Error 33 2012.900 61.000

Total 35 2014.200

S = 7.810 R-Sq = 0.07% R-Sq(adj) = 0.00%

Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ------+------+------+------+ 1 12 25.367 7.775 (------*------) 2 12 25.158 7.882 (------*------) 3 12 24.900 7.772 (------*------) ------+------+------+------+ 22.5 25.0 27.5 30.0 Pooled St Dev = 7.810

Tukey 95% Simultaneous Confidence Intervals All Pairwise Comparisons among Levels of period Individual confidence level = 98.04%

Period = 1 subtracted from:

Period Lower Center Upper ------+------+------+------+-- 2 -8.032 -0.208 7.615 (------*------) 3 -8.290 -0.467 7.357 (------*------) ------+------+------+------+-- -5.0 0.0 5.0 10.0

163 Table 16.1. One-way ANOVA: RH% versus period

Sou rce DF SS MS F P

Period 2 1037 518 3.33 0.048

Error 33 5142 156

Total 35 6179

S = 12.48 R-Sq = 16.78% R-Sq(adj) = 11.74%

Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ---+------+------+------+------1 12 47.48 10.34 (------*------) 2 12 56.58 13.51 (------*------) 3 12 60.25 13.35 (------*------) ---+------+------+------+------42.0 49.0 56.0 63.0 Pooled St Dev = 12.48

Period = 1 subtracted from:

Period Lower Center Upper ---+------+------+------+------2 -3.40 9.10 21.60 (------*------) 3 0.26 12.77 25.27 (------*------) ---+------+------+------+------12 0 12 24

164 Table 16.2. One-way ANOVA: RF mm versus period

Source DF SS MS F P

Period 2 380 190 0.18 0.838

Error 33 35222 1067

Total 35 35602

S = 32.67 R-Sq = 1.07% R-Sq(adj) = 0.00%

Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ---+------+------+------+------1 12 35.21 29.22 (------*------) 2 12 27.25 33.30 (------*------) 3 12 31.29 35.20 (------*------) ---+------+------+------+------12 24 36 48 Pooled St Dev = 32.67

Period = 1 subtracted from:

Period Lower Center Upper +------+------+------+------2 -40.68 -7.96 24.77 (------*------) 3 -36.64 -3.92 28.81 (------*------) +------+------+------+------40 -20 0 20

Period = 2 subtracted from:

Period Lower Center Upper +------+------+------+------3 -28.68 4.04 36.77 (------*------) +------+------+------+------40 -20 0 20

165 Table 16.3. One-way ANOVA: RH% versus month

Source DF SS MS F P

Period 11 4969.2 451.7 8.96 0.000

Error 24 1209.7 50.4

Total 35 6178.9

S = 7.100 R-Sq = 80.42% R-Sq(adj) = 71.45%

Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ----+------+------+------+----- 1 3 69.200 9.824 (-----*-----) 2 3 68.867 11.393 (-----*-----) 3 3 49.833 4.252 (----*-----) 4 3 35.867 4.759 (-----*-----) 5 3 32.067 4.796 (----*-----) 6 3 41.800 5.180 (-----*-----) 7 3 59.900 4.859 (-----*-----) 8 3 61.233 6.396 (-----*----) 9 3 58.567 6.830 (-----*-----) 10 3 57.867 6.313 (-----*----) 11 3 58.433 9.693 (-----*-----) 12 3 63.633 6.558 (----*-----) ----+------+------+------+----- 30 45 60 75

Pooled St Dev = 7.100

166