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Investigation of Bio-ecological Factors influencing Infestation by the Passionvine Mealybug, Planococcus minor (Maskell)...

Permanent Link: http://ufdc.ufl.edu/UFE0042743/00001

Material Information

Title: Investigation of Bio-ecological Factors influencing Infestation by the Passionvine Mealybug, Planococcus minor (Maskell) (Hemiptera Pseudococcidae) in Trinidad for Application towards its Management.
Physical Description: 1 online resource (148 p.)
Language: english
Creator: FRANCIS,ANTONIO W
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: BIOLOGICAL -- COCCIDOXENOIDES -- CONTROL -- DACTYLOPII -- ENEMIES -- FITNESS -- HISTORY -- HOST -- LEPTOMASTIX -- LIFE -- MEALYBUG -- MINOR -- NATURAL -- PARAMETERS -- PASSIONVINE -- PERMINUTUS -- PHEROMONES -- PLANOCOCCUS -- SELECTION -- SEX
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: INVESTIGATION OF BIO-ECOLOGICAL FACTORS INFLUENCING INFESTATION BY THE PASSIONVINE MEALYBUG, PLANOCOCCUS MINOR (MASKELL) (HEMIPTERA: PSEUDOCOCCIDAE) IN TRINIDAD FOR APPLICATION TOWARDS ITS MANAGEMENT Planococcus minor (Maskell) is presently established throughout the Neotropics, where it is non-native and not considered as a pest of economic importance. However, it is regarded as a high risk pest to many areas of the continental U.S. and was recently discovered for the first time in Miami-Dade County, Florida. There is a lack of information on important aspects of the biology and ecology of P. minor. This dissertation focused on these bio-ecological factors on the island of Trinidad in order to gather relevant information in the event that the mealybug becomes a major pest concern. The life history study of P. minor indicated that it could successfully develop, survive, and reproduce across a relatively wide range of temperatures. Information gathered will provide the insight needed to better understand the life history of the mealybug in relation to temperature and enhance predictive distribution models, as well as optimize mass rearing procedures. Surveys conducted on the island determined that populations of P. minor were very low and sparse and found mainly on cacao. Additional surveys revealed that a well established natural enemy complex was exerting effective control on the mealybug populations. Based on the results of the field assessment of the primary parasitoids, L. dactylopii and C. perminutus exploited different host sizes/stages and persisted thoughout the sampling period when pest populations were low. These traits suggest that they have the potential to complement each other as biological control agents. The preferred host sizes/stages of L. dactylopii and C. perminutus varied, with the former attacking larger hosts (third instar nymphs to adult females), while the latter targeted smaller hosts (first instar to third instar nymphs). These preferences suggest minimal competition for suitable hosts and that a good level of complementarity exists between the two species. L. dactylopii had higher parasitism and a higher emergence rate than C. perminutus when the same mealybug size/stage was offered concurrently. When key fitness parameters were assessed, C. perminutus successfully completed its development in smaller mealybug sizes/stages, compared to L. dactylopii, which successfully developed in the larger hosts, with a female-biased sex ratio. Developmental time for L. dactylopii decreased as host size increased in parasitized mealybugs. Conversely, as host size increased, developmental time also increased for C. perminutus. While it had higher lifetime fertility, C. perminutus was relatively short-lived compared to L. dactylopii. The occurrence of P. minor in south Florida is the first record of the mealybug in the continental U.S. Information gathered from these studies will have an immediate impact, by allowing state as well as federal agencies to provide a wider range of control options if the need arises.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by ANTONIO W FRANCIS.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Kairo, Moses T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042743:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042743/00001

Material Information

Title: Investigation of Bio-ecological Factors influencing Infestation by the Passionvine Mealybug, Planococcus minor (Maskell) (Hemiptera Pseudococcidae) in Trinidad for Application towards its Management.
Physical Description: 1 online resource (148 p.)
Language: english
Creator: FRANCIS,ANTONIO W
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: BIOLOGICAL -- COCCIDOXENOIDES -- CONTROL -- DACTYLOPII -- ENEMIES -- FITNESS -- HISTORY -- HOST -- LEPTOMASTIX -- LIFE -- MEALYBUG -- MINOR -- NATURAL -- PARAMETERS -- PASSIONVINE -- PERMINUTUS -- PHEROMONES -- PLANOCOCCUS -- SELECTION -- SEX
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: INVESTIGATION OF BIO-ECOLOGICAL FACTORS INFLUENCING INFESTATION BY THE PASSIONVINE MEALYBUG, PLANOCOCCUS MINOR (MASKELL) (HEMIPTERA: PSEUDOCOCCIDAE) IN TRINIDAD FOR APPLICATION TOWARDS ITS MANAGEMENT Planococcus minor (Maskell) is presently established throughout the Neotropics, where it is non-native and not considered as a pest of economic importance. However, it is regarded as a high risk pest to many areas of the continental U.S. and was recently discovered for the first time in Miami-Dade County, Florida. There is a lack of information on important aspects of the biology and ecology of P. minor. This dissertation focused on these bio-ecological factors on the island of Trinidad in order to gather relevant information in the event that the mealybug becomes a major pest concern. The life history study of P. minor indicated that it could successfully develop, survive, and reproduce across a relatively wide range of temperatures. Information gathered will provide the insight needed to better understand the life history of the mealybug in relation to temperature and enhance predictive distribution models, as well as optimize mass rearing procedures. Surveys conducted on the island determined that populations of P. minor were very low and sparse and found mainly on cacao. Additional surveys revealed that a well established natural enemy complex was exerting effective control on the mealybug populations. Based on the results of the field assessment of the primary parasitoids, L. dactylopii and C. perminutus exploited different host sizes/stages and persisted thoughout the sampling period when pest populations were low. These traits suggest that they have the potential to complement each other as biological control agents. The preferred host sizes/stages of L. dactylopii and C. perminutus varied, with the former attacking larger hosts (third instar nymphs to adult females), while the latter targeted smaller hosts (first instar to third instar nymphs). These preferences suggest minimal competition for suitable hosts and that a good level of complementarity exists between the two species. L. dactylopii had higher parasitism and a higher emergence rate than C. perminutus when the same mealybug size/stage was offered concurrently. When key fitness parameters were assessed, C. perminutus successfully completed its development in smaller mealybug sizes/stages, compared to L. dactylopii, which successfully developed in the larger hosts, with a female-biased sex ratio. Developmental time for L. dactylopii decreased as host size increased in parasitized mealybugs. Conversely, as host size increased, developmental time also increased for C. perminutus. While it had higher lifetime fertility, C. perminutus was relatively short-lived compared to L. dactylopii. The occurrence of P. minor in south Florida is the first record of the mealybug in the continental U.S. Information gathered from these studies will have an immediate impact, by allowing state as well as federal agencies to provide a wider range of control options if the need arises.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by ANTONIO W FRANCIS.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Kairo, Moses T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042743:00001


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1 INVESTIGATION OF BIOECOLOGICAL FACTORS INFLUENCING INFESTATION BY THE PASSIONVINE MEALYBUG, PLANOCOCCUS MINOR (MASKELL) (HEMIPTERA: PSEUDOCOCCIDAE) IN TRINIDAD FOR APPLICATION TOWARDS ITS MANAGEMENT By ANTONIO WILLIAM FRANCIS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Antonio William Francis

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3 To my wife, Sandrene, my mother, Veronica, and my sister, Icilma, without whom this degree would not have been possible

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4 ACKNOWLEDGMENTS I would like to thank the following institutions and people for their support. I thank the USDA APHIS Center for Plant Health, Science and Technology (C PHST ) and the Center for Biological Control, College of Engineering Sciences, Technology and Agriculture ( CBCCESTAFAMU) for the funding that allowed me to conduct this research. I thank my committee members Dr. Moses Kairo, Dr. Raymond Hix, Dr. Amy Roda, Dr. Oscar Liburd, Dr. Lance Osborne, and Dr. Tim Momol, for their input and feedback on my research. I thank the staff at CAB International Regional Office for Latin America and at the Caribbean and at the Central Experiment Station, Trinidad for supplying facilities, equipment and transportation for my research. I thank Dr. Stuart Reitz and Mr. Gilbert Queeley for their assistance with the statistical analyses I thank my family and friends for their moral support. Finally, I would like to especially thank Dr. Moses Kairo for his efforts as my advisor and committee chair.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION .................................................................................................... 13 2 LITERATURE REVIEW .......................................................................................... 16 Origin, Distribution, and Taxonomy ......................................................................... 16 Description .............................................................................................................. 17 Biology and Ecology ............................................................................................... 18 Developmental Biology ..................................................................................... 18 Importance of Temperatur e/Development Data ............................................... 19 Reproductive Biology ....................................................................................... 20 Attendant Ants .................................................................................................. 20 Injury Symptoms ..................................................................................................... 21 Economic Importance and Host Range .................................................................. 22 Establishment in the Neotropics and Risk Potential to the U.S. .............................. 23 Control Options ....................................................................................................... 25 Monitoring and Detection .................................................................................. 25 Chemical Control .............................................................................................. 26 Biological Control ............................................................................................. 26 Predators ................................................................................................... 27 Parasitoids ................................................................................................. 28 Research Goal and Objectives ............................................................................... 32 3 LIFE HISTORY OF PLANOCOCCUS MINOR (MASKELL) (HEMIPTERA: PSEUDOCOCCIDAE) AT CONSTANT TEMPERATURES .................................... 35 Materials and Methods ............................................................................................ 37 Maintenance of Host Material and Colony of P. minor ..................................... 37 Development, Survival, and Sex Ratio ............................................................. 37 Thermal Requirements ..................................................................................... 39 Reproduction .................................................................................................... 40 Adult Longevity ................................................................................................. 41 Data Analysis ................................................................................................... 41 Life Table Parameters ...................................................................................... 42 Results .................................................................................................................... 42

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6 Development, Survival, and Sex Ratio ............................................................. 42 Thermal Requirements ..................................................................................... 43 Reproduction and Adult Longevity .................................................................... 44 Life Table Parameters ...................................................................................... 44 Discussion .............................................................................................................. 45 4 SURVEY FOR PLANOCOCCUS MINOR (MASKELL) (HEMIPTERA: PSEUDOCOCCIDAE) AND ITS NATURAL ENEMIES IN TRINIDAD .................... 53 Materials and Methods ............................................................................................ 55 Maintenance of Host Material and Colony of P. minor ..................................... 55 Occurrence and Pest Status of P. minor .......................................................... 56 Natural Enemies of P. minor ............................................................................. 58 Predators ................................................................................................... 60 Parasitoids ................................................................................................. 60 Mealybug Identification and Natural Enemy Identification ................................ 61 Sex Pheromonetrapping for Planococcus spp. ............................................... 61 Data Analys is ................................................................................................... 62 Results .................................................................................................................... 63 Occurrence and Pest Status of P. minor .......................................................... 63 Natur al enemies of P. minor ............................................................................. 63 Predators ................................................................................................... 63 Parasitoids ................................................................................................. 64 Sex Pherom one trapping for Planococcus spp. ............................................... 65 Discussion .............................................................................................................. 66 5 FIELD ASSESSMENT OF TWO PRIMARY PARASITO IDS OF PLANOCOCCUS MINOR (MASKELL) (HEMIPTERA: PSEUDOCO CCIDAE) ....... 84 Materials and Methods ............................................................................................ 85 Preparation of Host Plant Material .................................................................... 85 Mealybug Rearing ............................................................................................ 86 Preparation of P. minor Colonies on Potatoes for Field Deployment ................ 86 Field Sit e .......................................................................................................... 87 Field Protocol ................................................................................................... 87 Processing Infested Potatoes ........................................................................... 89 Data Anal ysis ................................................................................................... 89 Results .................................................................................................................... 90 Relative Abundance and Seasonal Occurrence of Parasitoids ........................ 90 Role of Attendant Ants ..................................................................................... 90 Discussion .............................................................................................................. 91 6 HOST STAGE SELECTION BY TWO ENCYRTID ENDO PARASITOIDS, LEPTOMASTIX DACT YLOPII HOWARD AND COCCIDOXENOIDES PERMINUTUS GIRAULT, ATTACKING PLANOCOCCUS MINOR (MASKELL) (HEMIPTERA: PSEUDOCO CCIDAE) ..................................................................... 98

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7 Materials and Methods ............................................................................................ 99 Maintenance of Host Material and Mealybug Colony ....................................... 99 Rearing of Parasitoids .................................................................................... 100 Host Stage Selection ...................................................................................... 101 Host Size Class Preference ............................................................................ 102 Interspecific Competition ................................................................................ 103 Data Analysis ................................................................................................. 104 Results .................................................................................................................. 104 Host Size Class Preference ............................................................................ 104 Interspeci fic Competition ................................................................................ 106 Discussion ............................................................................................................ 106 7 DEVELOPMENTAL TIME, LONGEVITY AND LIFETI ME FERTILITY OF LEPTOMASTIX DACTYLOPII HOWARD AND C OCCIDOXENOIDES PERMINUTUS GIRAULT, PARASITOIDS OF PLANOCOCCUS MINOR (MASKELL) ........................................................................................................... 111 Materials and Methods .......................................................................................... 112 Maintenance of Host Material and Insect Colonies ........................................ 112 Host Stage Selection ...................................................................................... 113 Developmental Time ...................................................................................... 114 Adult Longevity and Lifetime Fertility .............................................................. 115 Data Analysis ................................................................................................. 116 Results .................................................................................................................. 117 Developmental Time ...................................................................................... 117 Adult Longevity ............................................................................................... 118 Lifetime Fertility .............................................................................................. 118 Discussion ............................................................................................................ 119 8 CONCLUSION ...................................................................................................... 128 LIST OF REFERENCES ............................................................................................. 133 BIOGRAPHICAL SKETCH .......................................................................................... 148

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8 LIST OF TABLES Table page 3 1 Mean number of days ( SEM) for each developmental stadium of Planococcus minor reared on sprouted potatoes at five constant temperatures. ..................................................................................................... 48 3 2 Estimates ( SEM) of the fitted parameters of the linear thermal summation model and the nonlinear Logan 6 model for Pl anococcus minor reared on sprouted potatoes. .............................................................................................. 49 3 3 Mean ( SEM) survival rate (in %) for each developmental stadium of Planococcus minor reared on sprouted potatoes at five constant temperat ures. ..................................................................................................... 50 3 4 Mean ( SEM) proportion of females, preoviposition and oviposition periods (in days), fecundity, and adult longevity (in days) of Planococcus minor reared on sprouted potatoes at three constant temperatures. ............................ 51 3 5 Life table parameters of Planococcus minor reared on sprouted potatoes at three constant temperatures. .............................................................................. 52 4 1 List of plants inspected for Planococcus minor at field sites, June 30 July, 2006. .................................................................................................................. 72 4 2 Natural enemies recovered from field sites, June 28 October 19, 2006. ......... 73

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9 LIST OF FIGURES Figure page 2 1 Life stages of Planococcus minor .. ..................................................................... 34 4 1 Cacao pod infested with Planococcus minor and tended by ants. ...................... 74 4 2 Collection of mealybugs for identification from infested cacao pods. .................. 75 4 3 Cacao habitat types. .. ......................................................................................... 76 4 4 Sentinel trap with infested potato used to recover natural enemies of Planococcus minor ............................................................................................ 77 4 5 Primary parasitoids of Planococcus minor .. ........................................................ 78 4 6 Infestation levels of P. minor at cacao field sites categorized into habitat types (13) in Trinidad, June 30 to July 27, 2006.. .............................................. 79 4 7 Number of D. coccidarum recovered from cacao field sites categorized into habitat types (13) in Trinidad, June 28 to August 8, 2007. ............................... 80 4 8 Number of coccinellids recovered from cacao field sites categorized into habitat types (13) in Trinidad, June 28 to August 8, 2007. ............................... 81 4 9 Mealybug parasitism by L. dact ylopii from cacao field sites at different sampling dates in Trinidad, June 28 to October 19, 2007. ................................ 82 4 10 Number of P. minor males caught on baited traps from cacao field sites categorized into habitat types (13) in Trinidad, July 10 to August 8, 2007. ...... 83 5 1 Field deployment of wire cages with P. minor infested potatoes.. ...................... 94 5 2 Relative abundance and seasonal occurrence of primary parasitoids at Fishing Pond cacao field site, Trinidad, April 23, 2008 to March 18, 2009.. ....... 95 5 3 Mean ( SEM) percen t parasitism pooled across stages from ant excluded and ant tended treatments with P. minor infested potatoes throughout the sampling period at Fishing Pond cacao field site, Trinidad, April 23, 2008 to March 18, 2009. .................................................................................................. 96 5 4 Relative abundance of primary parasitoids from ant excluded and ant tended treatments with P. minor infested potatoes at Fishing Pond cacao field site, Trinidad, April 23, 2008 to March 18, 2009.. ....................................................... 97 6 1 Proportion of P. minor parasitized in nochoice preference tests.. ................... 109 6 2 Proportion of P. minor parasitized in choice preference tests... ........................ 110

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10 7 1 Proportion of L. dactylopii that emerged from mealybug host size classes.. ... 123 7 2 Sex ratio of L. dactylopii expressed as proportion of females... ........................ 124 7 3 Proportion of C. perminutus that emerged from mealybug host size classes.. 125 7 4 Devel opmental time (in days) of L. dactylopii from different host size classes. .......................................................................................................... 126 7 5 Developmental time (in days) of C. perminutus from different host size classes.. ........................................................................................................... 127

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11 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INVESTIGATION OF BIOECOLOGICAL FACTORS INFLUENCING INFEST ATION BY THE PASSIONVINE MEALYBUG, PLANOCOCCUS MINOR (MASKELL) (HEMIPTERA: PSEUDOCOCCIDAE) IN TRINIDAD FOR APPLICATION TOWARDS ITS MANAGEMENT By Antonio William Francis May 2011 Chair: Moses T. Kairo Major: Entomology and Nematology Planococcus minor (Maskell) is presently established throughout the Neotropics, where it is nonnative and not considered as a pest of economic importance. However, it is regarded as a high risk pest to many areas of the continental U S and was recently discovered for the fi rst time in MiamiDade County Florida. There is a lack of information on important aspects of the biology and ecology of P. minor This dissertation focused on these bio ecological factors on the island of Trinidad in order to gather relevant information in the event that the mealybug becomes a major pest concern. The l ife history study of P minor indicated that it could successfully develop, survive, and reproduce across a relatively wide range of temperature s. Information gathered will provide the insig ht needed to better understand the life history of the mealybug in relation to temperature and enhance predictive distribution models as well as optimize mass rearing procedures. Surveys conducted on the island determined that populations of P. minor w ere very low and sparse and found mainly on cacao. Additional s urveys revealed that a well

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12 established natural enemy complex was exerting effective control on the mealybug populations Based on the results of the field assessment of the primary parasitoid s, L. dactylopii and C. perminutus exploited different host sizes/ stage s and persisted thoughout t he sampling period when pest populations were low These traits suggest that they have the potential to complement each other as biological control agents The preferred host sizes/ stages of L. dactylopii and C. perminutus varied, with the former attacking larger hosts (third instar nymphs to adult females) while the latter targeted smaller hosts ( first instar to third instar nymphs ) These preferences sugg est minimal competition for suitable hosts and that a good level of complementarity exists between the two species. L. dactylopii had higher parasitism and a higher emergence rate than C. perminutus when the same mealybug size/ stage was offered concurrentl y When key fitness parameters were assessed, C. perminutus successfully completed its development in smaller mealybug sizes/ stages, compared to L. dactylopii which successfully developed in the larger hosts, with a femalebiased sex ratio. Developmental time for L. dactylopii decreased as host size increased in parasitized mealybugs. Conversely as host size increased, developmental time also increased for C. perminutus While it had higher lifetime fertility, C perminutus was relatively short lived com pared to L. dactylopii Th e occurrence of P. minor in south Florida is the first record of th e mealybug in the continental U S. I nformation gathered from these studies will have an immediate impact, by allow ing state as well as federal agencies to provide a wider range of control options if the need arises

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13 CHAPTER 1 INTRODUCTION Many species of mealybugs (Hemiptera: Pseudococcidae) have become serious invasive pests when introduced into new areas beyond their native (or natural) distribution (Miller et al. 2002). Based on such facts as well as interception data, several species of mealybugs were identified as posing a high risk of introduction to the United states including, the passionvine mealybug, Planococcus minor (Maskell) (Miller et al. 2002; USDA APHIS 2005). The passion vine mealybug is native to the Oriental region (Cox 1989), but it is now widely distributed in other areas including the Neotropics (Williams & Granara de Willink 1992 ; Ben Dov 1994). Worldwide, the reported host plant range includes more than 250 species (C ommonwealth Agricultural Bureau 2003), some of which include important agricultural crops (Venette & Davis 2004). Therefore, this insect has the potential to become a serious pest if introduced to new areas. Florida is particul arly concerned by the threats posed from invasive pests, not least being the fact that the states economy is heavily based on agriculture. Also, b ecause of its geographic location, the state is an important entry route for pests especially those originati ng in the Neotropics (Frank & McCoy 1992). This threat is further amplified by the large volume of traded commodities that enter the U S through Floridas ports and an amenable climate which makes it relatively easier for invaders to become established. These concerns were justified when P. minor was confirmed for the first time in the continental U.S. on Mussaenda sp. in Miami Dade County December, 2010 ( A. Roda, USDA ARS Center for Plant Health, Science and Technology, personal communication). In order to be ready to deal with this incursion of P. minor it was of strategic importance to develop technologies for management of the

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14 pest before it arrived on the U.S. mainland. As a group, invasive mealybugs have been amenable targets for classical biologic al control ( Willink & Moore 1988; Kairo et al. 2000; Neuenschwander 2001; Meyerdirk et al. 2004). This is also true for P. minor ; consequently, classical biological control is potentially one of the important approaches for dealing with the pest. Because of the concerns that P. minor might become a serious problem if it continues to expand its range, there is an urgent need to acquire information on aspects of its biology, ecology and management in order to develop mitigation measures. The strategic development of such measures is seen as a key step in implementing effective prevention efforts or rapid detection/eradication or management if it spreads to new geographic areas. Unfortunately, work on the biology and ecology of P. minor is limited to just a fe w recent studies (Maity et al. 1998; Martinez & Suris 1998; Sahoo et al. 1999 ; Biswas & Ghosh 2000). Furthermore, the pest status, host plant range and control by natural enemies remain unclear in the localities where this mealybug is present in the Neotropics such as the island of Trinidad where this research was undertaken. Against this background, the goal of this dissertation was to generate relevant knowledge to facilitate development of mitigation measures against P. minor The findings will allow p lant protection authorities to be better prepared for any major outbreaks and minimize delays in the implementation of control measures if the pest becomes a problem in the U S. It is also anticipated that the research carried out will serve as a model for investigating future pest threats from this particular region. To achieve these goal s, the life history of P. minor was investigated to determine the effect of temperature on its development, survival, reproduction, and to determine key life

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15 table paramet ers Additionally, surveys were carried out to assess the occurrence and pest status of the mealybug and to evaluate the potential for biological control by identifying any existing natural enemies and determining their abundance and potential impact A fi eld study was also undertaken to assess the impact of identified primary parasitoids on P. minor Finally, studies investigated host stage selection by the primary parasitoids of P. minor and several of their key fitness parameters.

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16 CHAPTER 2 LITERATURE REVIEW Origin, Distribution, and Taxonomy The passionvine mealybug, Planococcus minor (Maskell), is also known by the common names pacific mealybug and guava mealybug. P. minor is one of 35 species belonging to this genus which is native to the Old World (Cox 1989). The genus also includes many well known pests of economic plants such as Planococcus citri (Risso) and Planococcus ficus (Signoret) ( Williams & Watson 1988; Cox 1989). The passionvine mealybug was originally described by Cox (1981) as P. pacifi cus from material collected from the South Pacific region. Later, Cox (1989) placed the lectotype, Pseudococcus calceolariae var. minor Maskell, previously regarded as a synonym of P citri as P. minor and P. pacificus was synonymized with it. P lanococcus minor is widely distributed throughout the Oriental, AustroOriental, Australian, Polynesian, Nearctic, Afrotropical, Malagasian, and Neotropical regions ( Cox 1981; Cox & Freeston 1985; Williams 1985; Williams & Watson 1988; Cox 1989; Williams & Granara de Willink 1992; Ben Dov 1994; CAB 2003). The identification of many species in the genus Planococcus using morphological characters has been challenging (Cox & Wetton 1988). Two such examples are P. citri and P. minor which have been taxonomically confus ed and routinely misidentified due to similarity in appearance, host plant range, and geographic distribution ( Williams 1985; Cox 1989; Williams and Granara de Willink 1992 ; Ben Dov 1994). Several authors highlighted inaccuracies in past literature, where the species of Planococcus commonly occurring in the Austrooriental, Polynesian regions, and the Neotropics, was

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17 P. minor and not P. citri despite most published records listing the latter ( Williams 1982; Cox & Freeston 1985; Williams & Watson 1988). T hese early misidentifications emphasize the significance of the morphological scoring matrix developed by Cox (1983). This author first determined that P. minor could be distinguished from P. citri by differences in the number and size of key morphological characters on the body such as ventral tubular ducts multilocular disc pores and the ratio of length of hind tibia + tarsus to length of trochanter + femur. Because t hese characters vary appreciably under different environmental conditions she developed a matrix of six diagnostic characters and these characters w ere scored using a point system to identify adult females based on their total numbers, presence or absence, and width on the body. Specimens having a total score of 0 to 35 from both sides of the body were determined to be P. minor, while those having a total score of 35 to 120 were determined to be P. citri. This system is still relied upon by mealybug taxonomists to separate these two species. Molecular diagnostic techniques have been useful for distinguishing mealybugs in the genus Planococcus and a rapid and more dependable method to identify such cryptic species accurately has important ecological and diagnostic implications (Demontis et al. 2007). The utility and rapidity of this technique was further validated by Rung et al. (2008), who identified P. minor and P. citri specimens collected from diverse areas using two specific molecular markers the mitochondrial gene, cytochrome EF Description Eggs of P. minor are yellow and minute (Fig. 2 1 A ) and partially exposed in the ovisac underneath the posterior end of female. Immature nymphs undergo three and

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18 four successive molts prior to emergence of adult females and males, resp ectively (Sahoo & Ghosh 2001). Both sexes have a pinkish appearance during the first two instars and are indistinguishable. The male third instar is termed pupa and is protected in a silken cocoon (Fig. 21 B ) while the fourth instar from which the adul t emerges is termed prepupa and also develops in this coccoon. These stages develop in white cocoon like structures (Sahoo & Ghosh 2001). Adult males (Fig. 2 1 C ) are ~1 mm long with three distinct body divisions, three pairs of legs, and one pair of win gs (Gill 2004). Mouthparts are absent (Sahoo & Ghosh 2001); hence, they only live for a few days. Females are much larger than males, oval shaped, and wingless (Fig. 2 1 D ) They are distinctly segmented, with a dorsomedial bare area, three pairs of legs, and a pair of 8segmented antennae. When newly molted, they are pale yellow, but later turn brownish orange, and their skin becomes covered with a white, glandular secretion. Mouthparts are located ventrally between the first pair of coxae (Williams & Wats on 1988). The body margins of adult females produce 18 pairs of short lateral filaments with the two hindmost filaments being longer than the others. Biology and Ecology Despite the threat posed by this pest, there is still a lack of detailed life history data on P. minor Furthermore, because of the taxonomic difficulty of separating this species from P. citri there is uncertainty regarding the identity of some of the mealybugs used in earlier studies since the two species often occur together. Developmen tal Biology The few studies undertaken on the life history of P. minor were conducted at either a single temperature (Martinez & Suris 1998) or fluctuating temperature regimes on different readily available host plants ( Maity et al. 1998; Biswas & Ghosh 20 00) Eggs

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19 took as little as 25 days to hatch at 26oImportance of Temperature/Development Data C and 69% RH (Martinez & Suris 1998). The development time for males was longer than for females (Maity et al. 1998; Martinez and Suris 1998), and the time to complete a single generation ranged from 315 0 days (Maity et al. 1998; Martinez and Suris 1998; Biswas & Ghosh 2000). These differences in methodologies therefore complicate efforts in estimating key life history parameters of P. minor and the employment of these data in predicting its potential spr ead and distribution. An insects rate of development is affected by the temperature it is exposed to (Campbell et al. 1974), and its development occurs within a definite range of temperature (Wagner et al. 1984). The amount of heat required over time for an insect to complete some aspect of development is a thermal constant (Campbell et al. 1974). The developmental thresholds are the temperatures below or above which no development occurs and the upper and lower thresholds along with the thermal constant are useful indicators of potential distribution and abundance of an insect (Huffaker et al. 1999). Mathematical models that describe developmental rates as a function of temperature are therefore important in predicting the seasonal occurrence of insects (Wagner et al. 1984). Two such models are the thermal summation model (Campbell et al. 1974) and Logan 6 model (Logan et al. 1976), which are widely used to explain the relationship between developmental time and t emperature. Data such as thermal constants and temperature thresholds derived from these mathematical models can be used by simulation models such as CLIMEX and NAPPFAST to help predict potential spread and distribution of P. minor in the continental U S.

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20 Reproductive Biology Most mealybug species reproduce sexually (Gullan & Kosztarab 1997), but studies to determine if reproduction is achieved asexually through parthenogenesis have never been undertaken for P. minor However, both sexes occur in populations where males can mate multiple times and have been reported to be less numerous than females (Maity et al. 1998; Martinez & Suris 1998; Sahoo et al. 1999; Sahoo & Ghosh 2001). The preoviposition and oviposition periods of gravid females ranged from 611 and 8 14 days (Maity et al. 1998), and 68 and 89 days (Biswas & Ghosh 2000). Findings from the few studies conducted showed that f ecundity varied depending on the host plant. Biswas & Ghosh (2000) reported 66159 eggs on Ixora signaporensis soybean ( Glyc ine max ) and Acalypha wilkesiana. However, Maity et al. (1998) reported as many as 266426 eggs on taro ( Colocasia esculenta), sprouted potato ( Solanum tuberosum ), and pumpkin ( Cucurbita moschata). In warm climates, these mealybugs stay active and reproduce throughout the year (BenDov 1994). Sahoo et al. (1999) reported as many as 10 generations occurring per year in India. Attendant Ants The presence or absence of attendant ants can affect the relative abundance of different Planococcus spp. (Bigger 198 1). This mutualistic association benefits the ants, which are provided with a carbohydrate food source from the honeydew producing mealybug colonies, which in return are protected by the ants from natural enemies ( Way 1963; Traniello 1989). In some instanc es, several ant species are found tending mealybugs, such as Anoplolepis steingroeveri (Forel), Crematogaster peringueyi Emery and Linepithema humile (Mayr) (Hymenoptera: Formicidae) that form a mutualistic relationship with vine mealybug, Planococcus ficu s (Signoret) where they promote the

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21 latters infestations to unacceptable levels (Mgocheki & Addison 2009). Additionally, mealybug populations intimately associated with ants are usually larger than nontended ones of the same species (Buckley & Gullan 199 1). Some species aid in mealybug dispersal (Flanders 1951; Way 1963), which may improve their survival (Daane et al. 2007). Injury Symptoms Mealybugs have piercing sucking mouthparts which they insert into the plant vascular tissue, and they can remain in place through several molts (Arnett 1993), sucking up plant sap (Daane 2003). Feeding activity of P. minor causes reduced yield, lower plant or fruit quality, stunted growth, discoloration, and leaf loss (Venette & Davis 2004). In some cases, some species often reach high densities (BenDov 1994), killing perennial plants (Hodges & Hodges 2004; Walton et al. 2004). Plant death may also be caused by viral diseases because P. minor is known to be a vector of important viruses ( Williams 1985 ; Cox 1989). One s uch example known to occur in Trinidad is Cacao (Trinidad) virus and its various isolates (Kirkpatrick 1950 ). Similar to other pseudococcids, P. minor excretes copious amounts of honeydew onto the plant. Up to 90% of the ingested plant sap may be excreted in this way by mealybugs (Mittler & Douglas 2003). Sooty mold grows on the honeydew and builds up on the leaves, shoots, fruits, and other plant parts (Mani 19 89; Zada et al. 2004). This mold can cover so much of the plant that it interferes with the plants normal photosynthetic activity (Williams & Granara de Willink 1992). Honeydew and sooty mold cause cosmetic defects to plants and/or their fruits, which bec ome soiled even from In such cases, these mealybugs may be economic pests even at low densities (Franco et al. 2009).

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22 small mealybug populations, and they directly affect the sale of such produce (Millar et al. 2002; Zada et al. 2004). Economic Importance and Host Range Venette & Davis (2004) have compiled a list of more than 250 wild and cultivated host plants in nearly 80 families that are reportedly attacked by P. minor Franco et al. (2009) noted that most of the economically important mealybug species are associated with long lists of hosts, yet under low pressure of natural enemies they spread i n new areas and are observed on relatively large numbers of host plants. This polyphagous pest has also become established in some temperate areas in greenhouses where it can be a serious horticultural pest (Williams & Watson 1988). Additionally, plant hos t susceptibility to P. minor can vary widely (Venette & Davis 2004), and infestation levels can fluctuate spatially, even on plants in close proximity (Miller & Kosztarab 1979). With this potentially wide host plant range, it is reasonable to anticipate that P. minor will find and utilize additional new hosts as it spreads throughout the geographic limits of its distribution in new habitats ( USDA, APHIS 2002). Therefore, any local survey needs to also take into account those local susceptible plant species which may prove to be hosts ( USDA, APHIS 2002). Although some species of Planococcus have a wide host plant range, a few such as P. minor show distinct preferences, commonly occurring on cocoa ( Theobroma cacao) throughout its geographic range (Cox 1989). Since multiple species from the genus Planococcus may occur on the same host plant, it becomes difficult to estimate the economic impact of P. minor alone (Venette & Davis 2004). Although widely distributed, this mealybug is not reported as an economic pest in many countries. Additionally, some earlier host records in certain regions might be erroneous because of misidentification as P. citri (Cox 1989; Williams & Granara de

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23 Willink 1992 ; Santa Cecilia et al. 2002). For example, Szent Ivany (1956) reported it as P. citri from Papua New Guinea where it seriously damaged coffee ( Coffea spp.). Szent Ivany & Stevens (1966) again reported it as P. citri where it comprised more than 90% of a mixed population with another pseudococcid and two different soft scales on coffee, and caused 7075% reduction in crop yield. In India, this mealybug was reported as part of a Planococcus spp. complex or singly attacking custard apple ( Annona reticulata) (Shukla & Tandon 1984); grape ( Vitis spp.) (Batra et al. 1987; Tandon & V erghese 1987); ber ( Ziziphus sp.), guava ( Psidium guajava ), and mangoes ( Mangifera indica) (Tandon & Verghese 1987); and coffee (Reddy et al. 1997). In Taiwan, it was listed as a major pest of important crops, including banana ( Musa spp.), Citrus spp., man go, celery ( Apium spp.), melon ( Benincasa sp.), pumpkin ( Cucurbita spp.), soybean ( Glycine max ), betel nut ( Areca catechu), star fruit ( Averrhoa carambola), guava, and passionvine ( Passiflora spp.) (Ho et al. 2007). Establishment in the Neotropics and Ris k Potential to the U S It is likely that P. minor was introduced to the Neotropics from the Old World through trade many years ago (Cox 1989). This region encompasses all countries/territories in the Western Hemisphere south of the U S and also includes Bermuda. This mealybug was found in Argentina on Coffea sp.; Brazil on Araujia sericofera, Cassia imperialis Coffea spp., Dahlia sp., avocado ( Persea americana), and cocoa; Mexico on Aralia sp., Erythrina sp., and cassava ( Manihot esculenta); Costa Rica o n Coffea sp; Cuba on chayote ( Sechium edule) and a Tradescantia sp.; and Bermuda on cats tail ( Acalypha hispida), Brunfelsia sp., croton ( Codiaeum variegatum ), Cyperus sp., and Philodendron fonzii (Williams & Granara de Willink 1992). Martinez & Suris (20 00) reported that P. minor caused significant damage to the coffee crop in

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24 Cuba. In Trinidad, P. minor had been recorded from Epimeridi indicum Pachystachys coecinea, Pistia stratiotes and cocoa (Williams & Granara de Willink 1992) Due to their cryptic habits and small size, mealybugs are difficult to detect on plants during quarantine inspections, especially if their population density on plants is low (Gullan & Martin 2003). The most frequently intercepted genera included Nephelium Anona, Sechium Sy zgium Psidium Garcinia and Musa (Venette & Davis 2004). P. minor has been reported from Hawaii ( Wong 2009 ) and the US Virgin Islands (Williams & Granara de Willink 1992) and these areas are potential entry pathways into the continental U S. Planococcus minor was reported for the first time in the continental U.S. occurring on Mussaenda sp. in MiamiDade County Florida in December, 2010 ( A. Roda, USDA ARS CPHST, personal communication ). A risk assessment by Venette & Davis (2004) estimated that more tha n half of the mainland has favorable climate for its establishment, putting numerous crops of economic significance to domestic agriculture at risk. California and Florida are especially vulnerable because of their large agricultural sectors. It is possibl e that P. minor can enter California from Mexico or from the Pacific, while it might spread from the Caribbean to Florida ( USDA, APHIS 2002). These two states are the largest domestic producers of ornamentals and vegetables, along with crops such as citrus grapes, and avocados (USDA NASS 2007). These crops are listed hosts of P. minor along with many ornamentals and vegetables grown in tropical and subtropical areas of these states and in greenhouses throughout the U S (Venette & Davis 2004). The estim ated economic risk to domestic agriculture from a recent invasive mealybug ( M. hirsutus ) was $750 million per year in the absence of

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25 effective control measures (Moffitt 1999). While no comprehensive economic data exist for P. minor it is reasonable to expect similar figures for this invasive pest, making it a serious economic threat. Control Options A number of tactics have been employed to control mealybug pests, including cultural practices that involve cutting and burning of infested plant parts, chem ical control, biological control, and the use of sex pheromones ( Barrett 2000; Tenbrink & Hara 2007). For practical and economic reasons, insecticide use and biological control have been the most widely implemented for mealybug management. Monitoring and Detection Within the genus Planococcus sex pheromones have been identified and synthesized for P. citri (Bierl Leonhardt et al. 1981) and P. ficus (Hinkens et al. 2001) and successfully used in monitoring programs (Millar et al. 2002; Ortu et al. 2006). I n addition to monitoring purposes, these pheromones have other potential uses, such as for mating disruption and attract and kill technologies ( Walton et al. 2006) and complement other tactics used in IPM programs. Recently, the sex pheromone of P. minor was identified and synthesized (Ho et al. 2007; Millar 2008). Small doses of the pheromone were found to be very attractive to P. minor males in lab bioassays (Ho et al. 2007). Millar (2008) suggested that sex pheromonebaited traps would therefore provide a sensitive and effective method of detecting small populations of this pest. Although positive finds on a trap do not pinpoint the exact location of an infestation, they aid in defining the area where detailed field surveys need to be undertaken (Daane 2003). Such a monitoring system should have

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26 an immediate impact as a detection tool in threatened or highrisk areas for introduction of P. minor Chemical Control Despite its frequent use, chemical control is often ineffective (Krishnamoorthy & Mani 1989) because mealybugs are located primarily in protected sites on plants such as cracks and crevices, and under bark where insecticide penetration may be difficult ( Grasswitz & Burts 1995; Geiger & Daane 2001). They also have a protective wax covering and form dense colonies (Arnett 1993) and their eggs are laid in a protective ovisac (Mani 1989). Insecticides can also negatively impact natural enemy populations (Walton et al. 2004); therefore, applications should be timed carefully to minimize disruption of these beneficial insects (Walton & Pringle 1999). S ystemic insecticides that reach all parts of the plant are currently the most effective (Daane et al. 2004), and control of mealybugs has improved with the introduction of many new systemic products. Some ex amples include neonicotinoids such as acetamiprid, clothianidin, dinotefuran, imidacloprid, thiamethoxam, along with several insect growth regulators (IGR) that are used to control scale insects and mealybugs (Buss & Turner 2006). Imidacloprid and an IGR ( buprofezin) have provided promising results for suppression of P. ficus in Californian vineyards and are alternatives to inseason use of organophosphates (Daane et al. 2006). Biological Control Mealybugs are amenable candidates for biological control (Mani 1988), and this option is deemed the best form of long term control because it reduces the considerable costs associated with chemical control (Sagarra & Peterkin 1999). Also, unlike chemical control, biological control is relatively safe for human heal th and the environment (Flint

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27 & Dreistadt 1998). C lassical biological control would most likely be a major part of the overall control strategy against this mealybug pest. This approach involves the importation and establishment of nonnative natural enemy populations for suppression of non native or native organisms (Van Driesche & Bellows 1996). The steps involved in classical biological control are outlined by Van Driesche & Bellows (1996). There have been many such programs against invasive mealybugs in recent times, including the mango mealybug, Rastrococcus invadens Williams in West Africa (Willink and Moore 1988; BokononGanta et al. 2002), the pink hibiscus mealybug, Maconellicoccus hirsutus (Green) in the Caribbean and California (Kairo et al. 2000; Roltsch et al. 2006), the cassava mealybug, Phenacoccus manihoti Matile Ferrero in Africa (Neuenschwander 2001), and the papaya mealybug, Paracoccus marginatus Williams and Granara de Willink (Meyerdirk et al. 2004; Muniappan et al. 2006). The principal m ealybug natural enemies are predators and parasitoids, and the key species associated with Planococcus spp. are discussed below. Predators As many as 47 predators in diverse insect orders and families are known to feed on mealybugs. These include Coleoptera (coccinellids), Diptera (cecidomyiids), Neuroptera (chrysopids and hemerobiids), Lepidoptera (lycanids), and Hemiptera (Moore 1988; Mani 1989 ). Of these, coccinellid ladybird beetles are very important species. One of the most important is Cryptolaemus montrouzieri Mulsant, a generalist feeder, which has been utilized extensively against many mealybugs and scale insects. Successful control of P. citri was obtained through periodic releases of C. montrouzieri in citrus in California (Smith & Armitage 1931), and it has been reported to be effective in several crops infested with Planococcus spp. in India (Reddy & Seetharama 1997;

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28 Mani & Krishnamoorthy 2007). Much earlier, another member of the genus, Cryptolaemus affinis Crotch was also reported to be effec tive against P. minor in Papua New Guinea (Szent Ivany & Stevens 1966). The biology of another coccinellid, Brumoides suturalis (Fabricius) has also been investigated in some detail as a potential control agent for a number of mealybug pests including P. m inor (Chandrababu et al. 1997; Chandrababu et al. 1999). Parasitoids Parasitoids utilized for biological control belong to the orders Hymenoptera and, to a lesser extent, Diptera (Van Driesche & Bellows 1996). Important hymenopteran parasitoids of Planoco ccus spp. belong to the family Encyrtidae and include the solitary endoparasitoids Leptomastix dactylopii Howard, Leptomastidea abnormis (Girault), Anagyrus pseudococci (Girault), and Coccidoxenoides perminutus Girault (= Pauridia peregrina Timberlake, = Co ccidoxenoides peregrinus (Timberlake)) ( Berlinger 1977; Bartlett 1978; Noyes & Hayat 1994). Other reported genera that have been reared from Planococcus spp. include Aenasuis Gyranusoidea and Pseudaphycus ( USDA, APHIS 2002), and Pativana (Boucek & Bhuiya 1990). However, two of the most widely used of these encyrtid wasps in biological control programs against P. citri in particular have been L. dactylopii and C. perminutus (Noyes & Hayat 1994), but there is sparse information available on the use of these two biocontrol agents against P. minor L eptomastix dactylopii is thought to be of Afrotropical origin and has been used extensively in many countries to control P. citri (Noyes & Hayat 1994). In the US, this primary parasitoid was used successfully in c ontrol programs in California ( Clausen 1956; Bartlett and Lloyd 1958), Florida (Watson & Thompson 1940), and Texas (Meyerdirk et al. 1978). More recently, Krishnamoorthy & Singh (1987) reported that L.

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29 dactylopii was introduced from the West Indies and rel eased in citrus in India where complete control of P. citri was achieved in several months. It was also released to control Planococcus spp. in coffee and achieved levels of field parasitism as high as 85% (Reddy et al. 1992; Reddy et al. 1997). In Austral ia, L. dactylopii was the principal control agent used successfully against P. citri on citrus and custard apple (Smith et al. 1988). C occidoxenoides perminutus originated from Asia and has been used against P. citri since 1951 (Noyes & Hayat 19 9 4) in par ts of Africa, North and South America, Asia, and Europe ( Greathead 1971; 1976; Bartlett 1978). There has been some success with this primary parasitoid, such as in Texas ( Meyerdirk et al. 1978; Dean et al. 1983), Bermuda and Italy (Bennett & Hughes 1960; G reathead 1976; Cock 1985), but releases were usually made together with other species (Ceballo & Walter 200 4 ). More recently, C. perminutus was reported in India by Krishnamoorthy & Mani (1989), where it caused up to 30% parasitism of P. citri in citrus or chards. Several years later, it was the dominant species recovered and was most likely responsible for P. citri decline in citrus (Mani 1994). However, it achieved low levels of field parasitism in Australia (Davies et al. 2004), mainly due to the prevaili ng climate in the area (Ceballo & Walter 2005). Both parasitoids have also been evaluated in combination with other species. Meyerdirk et al. (1978) reported that a parasitoid complex consisting of L. dactylopii C. perminutus and Anagyrus sp. offered sea sonal control of P. citri on citrus in Texas. They reported that 21% of P. citri was parasitized by L. dactylopii in mid August, 49% was parasitized by C. perminutus in late August, which had replaced L. dactylopii as the dominant species, while only 4% was parasitized by by the only recovered species,

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30 Anagyrus sp. in mid September In greenhouse citrus, Summy et al. (1986) demonstrated that inoculative releases of L. dactylopii C. perminutus L. abnormis A pseudococci and Chrysoplatycerus splendens How ard rapidly suppressed populations of P. citri P. citri cohorts exposed to searching parasitoids for 8 weeks had 90% decline in populations, while cohort populations protected from parasitoids increased by 828%. Although these two parasitoids were introduced throughout the Neotropics for use against Planococcus spp. (Noyes & Hayat 1994), there are no records of intentional introduction of either species in Trinidad. L. dactylopii was recorded from mealybugs identified at that time as P. citri (Kirkpatrick 1953). L. dactylopii was also shipped from Trinidad to other areas for biological control of P. citri (Cock 1985). Rosen & DeBach (1977) reported that certain wasps in the superfamily Chalcidoidea, which includes the encyrtids, can discriminate cryptic sp ecies of Planococcus Because there is no available biological information for these parasitoids with P. minor it is important to investigate to what degree they will attack this species. Although the degree of host specificity of these primary parasitoids is not known with any certainty, they are able to develop in a number of different hosts (Noyes & Hayat 1994). For instance, L. dactylopii has been recorded from more than 20 mealybug hosts across many genera, but development was most successful in P. ci tri (Noyes & Hayat 1994). C. perminutus on the other hand, has been recorded from less than 10 hosts (Noyes & Hayat 1994), and its most successful development appears to be restricted to P. citri and to P. ficus (Golberg 1982; Walton & Pringle 2005). The biology of L. dactylopii was described by various authors (Lloyd 19 58 ; Tingle & Copland 1988; de Jong & van Alphen 1989; Tingle & Copland 1989). A female laid up

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31 to 10 eggs per day in P. citri (Kirkpatrick 1953), with oviposition taking place in 3rd in star and adult female mealybugs ( de Jong & van Alphen 1989). Fecundity was greatest at 30oC (Tingle & Copland 1989), and development was completed in as little as two weeks at 27oC ( de Jong & van Alphen 1989). More female parasitoids were reared from large hosts of P. citri while predominantly males were reared from smaller hosts ( de Jong & van Alphen 1989). Similarly, several authors have described the biology of C. perminutus (Flanders 1953; Golberg 1982; Ceballo & Walter 2004 ). Ceballo & Walter (2004) reported that the mode of reproduction was almost entirely thelytokous where females are produced from unfertilized eggs However, Flanders (1965) had earlier produced males by exposing a female population to temperatures as high as 35oC throughout the em bryonic and larval stages of the parasitoids development. Females oviposited into the first three instars of P. citri ; however, their productivity relied mainly on second instar hosts and they had an average fecundity of 239 eggs (Ceballo & Walter 2004). Relative humidity had a strong effect on the parasitoids survival, with females surviving best at 21.5oC and 92% RH (Golberg 1982). Krishnamoorthy & Mani (1989) reported that C. perminutus took 23 27 days to complete its development at 28oAlthough there are multiple parasitoids that are effective biocontrol agents against Planococcus spp., these parasitoids may vary with regard to suitability of mealybug species as hosts Furthermore, some parasitoids may be ineffective for some species due to a lack of preference to oviposit in a particular host species (Van Driesche et al. 1987) or an inability to escape encapsulation by the host ( Van Driesche et al. 1986; Blumberg et al. 1995). Encapsulation is a defense mechanism of mealybugs against C.

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32 their internal parasitoids (Blumberg 1997), and its frequency varies depending on the host species, its age, and conditions under which the host and parasitoid are reared (Blumberg 1988; 1991; 1997). Little information is available on the degree of suitability of L. dac tylopii and its various hosts, except for P. citri in which its eggs are seldom encapsulated (Blumberg & Van Driesche 2001). Conversely, Ceballo & Walter (2004) reported that eggs deposited into adult hosts of P. citri by C. perminutus were most likely en capsulated and destroyed. These findings on host immune response are especially significant given the many similarities between P. citri and P. minor Research Goal and Objectives In summary, P. minor is now present in the continental U S where it still poses a threat to new areas and uninfested areas in the Caribbean basin. Therefore, there is an urgent need to generate critical information to support efforts to prevent the spread of this pest, or to manage it should it become established in new localiti es. Two factors that favored the selection of Trinidad for this research were: knowledge that the pest was present in the country and the proximity to the U.S. and logistical ease of conducting the work there. Specific research needs include: clarification of its biology and ecology, specifically the life history, pest status and natural enemies (parasitoids). The first set of objectives was to determine the thresholds for development, thermal constant s, and survivorship of different stages from egg to adult; sex ratio, reproductive periods, fecundity ; adult longevity; as well as key life table parameters, over a range of different temperatures for P. minor The second set of objectives was t o assess the occurrence and pest status of the mealybug as a model for understanding its status across the region by a) determining the host plant range, infestation levels and damage, and distribution across the island,

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33 and b) investigating if populations of P. citri and P. minor occur together ; and to evaluate the potential for biological control by identifying existing natural enemies, if any, and determining their relative abundance and potential for use in biological control. The third set of objectives was to determine the relative abundance and seasonal occurrence of L. dactylopii and C. perminutus by comparing levels of stage specific parasitism of mealybugs ; and to determine the influence of resident ant populations on parasitoid activity. The fourth set of objectives was to determine if L. dactylopii and C. pe rminutus exhibit host size class preferences when selecting P. minor for oviposition; and to determine the degree of interspecific competition between the two species when offered the same host size class for oviposition based on the proportion of emerged adult parasitoids The final set of objectives was to determine the the developmental time, adult longevity and lifetime fertility of L. dactylopii and C. perminutus when provided with P. minor as the mealybug host.

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34 Figure 21 L ife stages o f P lanococcus minor A) Yellow e ggs in ovisac B) Coccoon made by 3rd instar ( prepupual ) males. C) Adult male. D) Adult female s. Photos Courtesy of Antonio Francis. A B C D

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35 CHAPTER 3 LIFE HISTORY OF PLANOCOCCUS MINOR (MASKELL) (HEMIPTERA: PSEUDOCOCCIDAE) AT CONS TANT TEMPERATURES The passionvine mealybug, Planococcus minor (Maskell) (Hemiptera: Pseudococcidae) is a polyphagous pest with more than 250 plants cited as hosts (Venette & Davis 2004). It was probably introduced to the Neotropics through trade (Cox 1989) and is found in at least 21 countries where it is commonly found on cocoa, Theobroma cacao L. and many other plants (Williams & Granara de Willink 1992). P lanococcus minor is morphologically very difficult to separate from P lanococcus citri (Risso) and the se cr yptic species share similarities such as host plants and geographical distribution (Cox 1989; Williams & Granara de Willink 1992). Therefore, estimating economic damage caused by P. minor alone is difficult (Venette & Davis 2004). Th is mealybug is considered a significant threat to noninfested countries in the Caribbean basin and the continental U.S. where it was recently identified for the first time in south Florida ( A. Roda, USDA ARS CPHST, personal communication), and where important agricultural crops might be affected in the absence of effective control measures. D espite the potential threat that P. minor poses, there is limited life history information, since no studies have been undertaken since its advent in the N eotropics. Limited Inform ation on its developmental biology was provided by Martinez & Suris (1998) whose study was conducted at a single temperature, by Maity et al. (1998) and Biswas & Ghosh (2000) at fluctuating temperatures and by Sahoo et al. (1999) under field conditions wit h seasonal changes. The host p lants used varied from a single (Martinez & Suris 1998) to multiple species (Maity et al. 1998; Biswas & Ghosh 2000). It also appears that studies to understand the reproductive biology and specifically

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36 whether or not partheno genesis occurs have never been undertaken for P. minor However, Sahoo & Ghosh (2001) reported that both males and females occurred together in populations. Data on development, survival, and reproducti on of P. minor at different temperatures are a necess ary first step to understand the effect of climate on these factors. This information can be incorporated into climate models to aid in determining potential spread and distribution, as well as risk assessment of P. minor Using the inductive biome approac h, Venette & Davis (2004) estimated that more than 50% of the continental U S would have a suitable climate for establishment of P. minor I f such a scenario were to occur, it would put California, Florida, Texas and a few other states at serious economic risk because of their production of tropical and subtropical fruits, vegetables, and ornamental plants. Given the polyphagous nature of P. minor and the potential economic losses that can result if it spreads to other U.S. states, there is an urgent need to elucidate key aspects of its biology in order to improve predictions of geographical distribution and limits in diverse climatic conditions such as are encountered in the continental U S. The goal of this study was to provide a better understanding of the life history of P. minor The specific objectives of the study were: 1) to determine the developmental thresholds, thermal constant s, and survivorship of different stages from egg to adult; 2) to determine sex ratio, reproductive periods, fecundity, an d adult longevity; and 3) to determine key life table parameters, over a range of different temperatures.

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37 Materials and Methods Maintenance of Host Material and Colony of P. minor Prior to sprouting, potatoes ( Solanum tuberosum L.) were soaked in a 1% bl each solution for 510 min. They were then washed and rinsed with clean water and left to dry. The potatoes were placed on plastic trays in a room at 25 2oA colony of P. minor was established on the sprouted potatoes under similar conditions as above, but in a s eparate dark room. Each week, 2025 sprouted potatoes were individually infested with 4 6 adult female P. minor with ovisacs transferred from the colony using a small camel hair brush. The infested potatoes were kept on plastic trays for mealybug development. This procedure was done on a weekly basis to maintain a continuous supply of different mealybug stages. Methods used were modified from those described by Meyerdirk et al. (1998). C 60 10% RH and complete darkness at CAB International Caribbean and Latin America Regional Off ice, Curepe, Trinidad Sprouting took 24 weeks, and potatoes were ready for use in the experiments when the blanched sprouts were 2.02.5 cm long. Development Survival and Sex Ratio Environmental growth chambers (TC1 model Chagrin Falls, OH and Precision model Thermo Scientific, Dubuque, IA ) at CAB International were maintained at five temperatures (15, 20, 25, 29, and 35oC) to determine the effects on the developmental and survival rates of P. minor during June 200 8 Chambers were also maintained at 60 10% RH and a photoperiod of 0:24 (L:D). Fluctuations across the range of temperatures never exceeded 1.0oC, and relative humidity was maintained within the range of 10% by placing plastic trays with water at the bottom of the chambers

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38 T emperature and relative humidity inside the chambers were verified with HOBO data loggers (Onset Computer Co., Bourne, MA) at 30min. intervals. All sprouts on individual potato es selected for use in these experiments were remov ed except for two to facilitate observations during regular scoring of mealybug development Twenty large gravid adult females were collected from the mealybug colony and held on sprouted potatoes until oviposition at each treatment temperature. Five eggs were carefully collected from each female with a small camel hair brush within 24 hours of oviposition at each treatment temperature. They were transferred onto the sprouts of individual potatoes and placed in polyethylene containers (12 cm dia. x 8 cm hei ght) with muslin cloth covered lids. The five eggs on each potato represented a cohort and 20 cohorts were prepared for each treatment temperature. The cohorts were examined every 24 hours under a dissecting microscope for egg eclosion and molting of immat ure stages Successful development from one instar to the next was indicated by the presence of cast skins or exuviae (inclusive of 3rd and 4thSurvival rate for each stadium was assessed as a percentage of individuals that succe ssfully developed to the next stadium. The sex of individual mealybugs could not be determined at egg and 1 instar males in puparia). st instar stages. The gender of each individual was determined by careful observation during the latter part of the second instar when the males change d their color from yellow to pink. At this point, the developmental times of males and females were established separately. Therefore, the cumulative survival rate from egg to adult (females and males) w as determined by dividing the total number of adult s by the number of eggs used to establish each cohort. The s ex ratio was

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39 determined by dividing the number of adult females successfully molted by the total number of surviving adults in each cohort. Treatment temperatures were replicated twice, once in each environmental growth chamber model Thermal Requirements The lower developmental threshold ( Tmin = a / b ) and the thermal constant (K = 1/ b ) of P. minor were estimated using the thermal summation model, which describes the relationship between the developmental rate of insects and the ambient temperature in a linear regression equation expressed as 1/D = b T + a (Wagner et al. 1984; Trudgill et al. 2005) where 1/D is the developmental rate, T is the ambient temperature (oThe upper developmental threshold ( T C), and a and b are the estimate d linear regression parameters (PROC REG, SAS Institute 2009). The lower developmental threshold is the minimum temperature at which no measurable development occurs while the thermal constant is the number of degreedays above the threshold summed over the developmental period. max) and the optimal temperature for development ( Topt) were estimated with the nonlinear Logan 6 model (Logan et al. 1976). The upper developmental threshold is the maximum temperature at whi ch the rate of development becomes zero. The optim al temperature is the temperature at which the maximum rate of development occurs Developmental rates of each developmental stage were fitted to the model 1/ D = [ exp( ) exp( max Tmax T / T ) ] an d the parameters ( Tmax, and T ) were estimated using the nonlinear regression procedure (PROC NLIN SAS Institute 2009). The parameter is the developmental rate at the base temperature, is the biochemical reaction rate as T increases to Topt, and T is the difference between Topt and Tmax when thermal breakdown becomes the overriding influence. The optimum temperature for

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40 development was calculated as Topt = Tmax [ 1 + (ln(o)/1 o) ] where T / Tmax and bo = TmaxReproduction The effect of temperature on reproductive periods and fecundity was evaluated at three treatment temperatures (20, 25, and 29oThe possibility of asexual reproduction was evaluated at a single temperature (25 C). Twenty females were collected from the cohorts produced at each treatment temperature immediately after adult molt Each femal e was paired with three newly emerged adult males on individual sprouted potatoes prepared as previously outlined in polyethylene containers at each treatment temperature. The females were examined every 24 hours for reproduction. The preoviposition perio d (duration between adult female molt and first day of egg production) and oviposition period (duration between start and end of egg production) were determined. The ovisacs produced by individual females were removed every 24 hours and placed in 70% alc ohol to dissolve them. The eggs were then counted under a dissecting microscope (10x). There were 20 cohorts per treatment temperature. Treatment temperatures were replicated twice, as was previously described. oC) in a nonmating experiment. Twenty virgin females were collected immediately from the cohorts after adult molt at that treatment temperature. Cocoons indicated the presence of immature males in a cohort and these were inspected to ensure that adult males had not emerged. E ach female was isolated without males on a sprouted potato prepared as previously outlined in a polyethylene container. The adult females were kept at the same temperature until death. Twenty cohort s (replicates) were prepared for this experiment.

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41 Adult Longevity The effect of temperature on adult longevity was evaluated at three treatment temperatures (20, 25, and 29oData Analysis C) The adult mealybugs from the mating experiment were kept at the assigned temper atures until death, and adult longevity was recorded as the duration between adult molt and death. One way analysis of variance (ANOVA) was used to determine the effect of temperature on the stagespecific and cumulative developmental times survival rate and sex ratio of P. minor ( PROC GLM, SAS Institute 200 2 ) The treatment temperature was the independent variable (source of variation), while the stagespecific and cumulative developmental times, survival rate, and sex ratio were the depen dent variables. The survival rate and sex ratio were arcsin transformed, and t ests for normality and homogeneity of variances of the dependent variables (PROC UNIVARIATE, SAS Institute 200 2 ) were performed prior to the analysis Tukeys honestly significant difference (HSD) test was used to separate the means when the statistical model indicated significant treatment effects on the dependent variables Likewise, ANOVA was used to determine the effect of temperature on pre oviposition and oviposition periods fecundity, and adult longevity ( PROC GLM, SAS Institute 200 2 ) The treatment temperature was the independent variable, while pre oviposition period, oviposition period, fecundity, adult female longevity, and adult male longevity were the dependent variables. T ests for normality and homogeneity of variances of the dependent variables (PROC UNIVARIATE, SAS Institute 200 2 ) were performed prior to the analysis Tukeys honestly significant difference (HSD) test was

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42 used to separate the means when the statisti cal model indicated significant treatment effects on the dependent variables Life Table Parameters The effect of the following temperatures (20, 25, and 2 9oC) on the population growth and age structure of P. minor was assessed based on six life table par ameters. Data on survivorship and reproduction were used to construct a life table of lx (age specific survival rate) and mxgross reproductive rate, GRR = (age specific fecundity) Age specific fecundity at a given temperature was obtained by multiplying the mean daily fecundity with t he proportion of females that was calculated from the developmental experiment at that temperature. The following life table parameters of female P. minor at each temperature were: xnet reproductive rate, R ; o = xmxmean generation time, T ); G = xmx)/ xmxintrinsic rate of increase, r ) ; m = (lnRo)/TG ; mdoubling time, DT = ln2/r ); and mResults Development Survival and Sex Ratio At 20oC eggs of P. minor eclosed in less than 13 days, which was longer than those held at 25 and 29oC (< 7 days) (Table 3 1). No eggs eclosed at 15 and 35oC. Developmental times decreased as the temperature increased between 20 and 29oC for both nymphal male and female mealybugs. The cumulative developmental time of female mealybugs was reduced from 48.8 0.3 at 20oC to 26.9 0.5 days at 29oC

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43 (Table 3 1). The cumulative developmental time of male mealybugs was shortest at 29oC (27.5 days), compared to 51.5 days at 20oThe high est (35 C (Table 3 1). oC) and low est (15oC) temperatures adversely affected the survival of P. minor (Table 3 3 ). Eggs turned brown and appeared dessicated after incubating for 45 and 15 days at 15 and 35oC, respectively. More than 90% of egg s hatched at 20 and 25oC, compared to only 84% at 29oC (Table 3 3 ). A t 20 and 25oC, 97100% of the third instar immature females and 9798% of the fourthinstar immature males successfully emerged as adults. At 29oC, 100 % of fourth instar male s and 89% of third instar females successfully emerged as adults. Overall, 68 and 71% of the eggs incubated at 20 and 25oC respectively, developed through to adults, and this was higher than 58% at 29oThe sex ratio of P. minor was significantly affected by temperature ( F = 3.38 ; df = 2,117; P < 0.0 5 ). Females made up 64.6 2.5, 73.4 4.3, and 60.1 8.5% of the total adult populations at 20, 25, and 29 C (Table 3 3 ). oThermal Requirements C, respectively (Table 3 4) The linear thermal summation equations fit (R2 > 0. 64) the developmental rates of all life stages of P. minor between 20 and 29oC (Table 3 2). The estimated lower developmental thresholds (Tmin) were 13.5 7.5 and 9.2oC for egg, female nymphal and male nymphal developments, respectively. The estimated Tm in for the cumulative developments of females and males were 8.0 and 9.0oThe nonlinear Logan 6 model also provided sufficient fit to the developmental rates of all life stages of P. minor (Table 3 2). The upper developmental threshold (T C, respectively. The estimated thermal constant (K) ranged from 90.9 DD for eggs to as high as 500 DD for the remaining stages to complete their development. max) and

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44 the optimal developmental temperature (Topt) of eggs were estimated at 35.4 and 27.3oC, respectively. The Tm ax for the nymphal and cumulative developments of males and females were all estimated at 3 8oC. The Topt for the nymphal and total development of males (both 2 8.8oC) were similar to those of the females (28.8 and 29. 3oReproduction and Adult Longevity C, respectively). Planococcus minor reproduced sexually. Virgin females did not reproduce after 2 months of isolation without males Temperature significantly impacted adult longevity, reproductive periods and fecundity of mated P. minor females The preovipos ition period was the longest at 20oC (14.8 0.1 days) and the shortest at 29oC (8.4 0.3 days) (Table 34) Similarly, the oviposition period was the longest at 20oC (< 14 days), while those at 25 and 29oC reproduced for 69 days ( Table 34 ). Female meal ybugs held at 25 and 29oC produced a significantly lower number of eggs (205.6 7.0 and 187.9 22.5 eggs per female, respectively) than those at 20oFemale longevity was the longest at 20 C (269.8 17.8 eggs per female) ( Table 34 ). oC where mean survival was 33.8 1.5 days after adult eclosion ( F = 334.56 ; df = 2, 117 ; P < 0.0 5 ). The longevities were similar at 25 (22.2 1.8 days) and 29oC (19.5 0.5 days) (Table 34) Adult male P. minor were short lived and their longevity decreased as the temperatur e increased ( F = 216.89; df = 2, 117 ; P < 0.0 5 ). They lived for 4.3 0.4 days at 20oC, followed by 1.7 0.7 days at 25 oC, and 1.3 0.3 days at 29oLife Table Parameters C (Table 34) The gross and net reproductive rates (GRR and Ro) increased with temp erature until the highest values were reached at 25oC (Table 3 5). These parameters were

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45 reduced to their lowest values at 29oC (Table 3 5). The generation time (TG) was increased from 39.5 days at 25oC to 63.5 days at 20oC. The intrinsic rate of increase (rm) was lower at 20oC (0.077) than at 25 (0.147) and 29oC (0.139) The finite rate of m, with the highest estimated value of 1.158 at 25 and 1.149 at 29oC. A population of P. minor required only approximately 5 day s to double its number at 25 and 29oC, but the doubling time (DT) was increased to approximately 9 days at 20oDiscussion C. Increased temperature accelerated the development of P. minor For example, t he duration of development for females was approximatel y 49 days at 20oC and 27 days at 29oC. The duration of development reported here was significantly different from other studies. For instance, w hen reared on different hosts at fluctuating temperatures between 19.7 to 28.9oThe T C, P. minor females completed dev elopment in as much as 22 days to as little as 16 days (Biswas & Ghosh 2000). However, these authors did not include the duration to egg eclosion, which meant that the cumulative development time for females was actually longer than reported. While the dif ferences in female development time when reared on different plant species indicated an influence of these hosts (Maity et al. 1998; Biswas & Ghosh 2000) it was more likely that the accelerated development reported by the latter when compared to this study was primarily due to the fluctuating temperature regime they employed. min estimated in this study suggested that P. minor was capable of surviving at ambient temperatures between 7.5 and 13 .5oC and it needed as many as 500 DD to complete its dev elopment These estimates were similar to several pseudococcid species T he Tmin and thermal constant of nymphal development were 11.7oC and 338

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46 DD for Pseudococcus citriculus Green, 7.7oC and 401 DD for P. citri and 8oVirgin female P. minor in the absence of males did not reproduce in this study, while mated females produced 188 to 270 eggs when reared on potato sprouts from 20 to 29 C and 519 DD for Planococcus kraunhi ae (Kuwana) (Arai 1996) Although the reported geographical range of P. minor suggests that it should be restricted to tropical and subtropical regions, these results indicate that it might be p ossib le for this mealybug to expand into moderate temperate regions than is generally expected for a tropical species. Ultimately, the utility of developmental data for P. minor will be that they provide a more realistic prediction of its potential distribution range, and therefore help to refine the output of predic tive models such as NAPPFAST, which are employed to estimate distribution ranges of many invasive species. oC. Martinez & Suris (1998) reported a fecundity of 219 eggs within 15 days when reared on potato sprouts at 26oC. Maity et al. (1998) reported a fecundity of 266 466 eggs when females were reared on several hosts from 14.5 to 31oC Othe r reports of P. minor fecundity varied greatly from 66 to 139 eggs between 19.7 and 28.9oL ife table parameters suggested that P. minor had a tremendous potential to increase its population s within a short period of time. The temperature range from 20 to 29 C on different plant s (Biswas & Ghosh 2000). As was demonstrated by these authors, the difference in fecundity of th e mealybug was likely due to the utilization of dif ferent host species. These findings are important from a pest management perspective because of the polyphagous nature of P. minor which includes many economic plants listed as potential hosts, oC appear ed favorable for its reproduction. However, at 25oC, females w ere able to

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47 achieve their highest net reproductive rate (greater than 325 female progeny per female) and a generation time of slightly less than 40 days. This reproductive rate together with a sex ratio that was significantly femalebiased at 25oAn understanding of the life history of P. minor has important implications in its management, not just in terms of deployment of resources where it is expected to establish but also understanding its patterns of invasion, distribution and spread. Its mass rearing for the production of natural enemies should be conducted in the range of 25 C implie d that mass production of P. minor for natural enemy production would be optimal within this temperature range. o C to maximize factors such as optimal developmental and reproductive rate s, and sex ratio. A comparison of the life history parameters o f P. minor and its natural enemies should also be helpful in selecting the most appropriate control agents.

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48 Table 3 1. Mean number of days ( SEM) for each developmental stadium of Planococcus minor reared on sprouted potatoes at five constant tempera tures. Developmental stadia Egg First Second Third Fourth Egg to adult T. ( o C) Female Male Female Male Male Female Male 15 20 12.8 0.1a 11.4 0.3a 12.4 0.1a 11.3 0.2a 11.1 0.1a 5.6 0.1a 10.2 0.2a 48.8 0.3a 51.5 0.1a 25 6.9 0.5b 7.7 0.1b 7.7 0.2b 7.6 0.2b 7.9 0.2b 4.6 0.6b 6.8 0.4b 30.8 0.2b 32.8 0.5b 29 5.7 0.5c 6.6 0.1c 7.2 0.1c 6 .4 0.4c 6.9 0.3c 2.6 0.2c 4.7 0.3c 26.9 0.5c 27.5 0.2c 35 ANOVA statistics n 552 547 279 254 279 242 236 270 23 4 F 1647.91 440.37 708.06 157.41 175.33 154.34 460.89 2319.17 1807.35 df 2,549 2,544 2,276 2,251 2,276 2,239 2,233 2,267 2,231 P < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 Means within a column followed by the same

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49 Table 3 2. Estimates ( SEM) of the fitted parameters of the linear thermal summation model and the nonlinear Logan 6 model for Planococcus minor reared on sprouted potatoes. Developmental stadia Statistics parameters Egg Total Total Thermal summation model F 900.53 1910.90 1484.44 2163.21 5289.3 P < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 R 0.6421 2 0.8770 0.8643 0.8898 0.9018 a SE 0.149 0.009 0.015 0.001 0.0183 0.001 0.016 0.001 0.018 0.001 b SE 0.011 0.004 0.002 0.00005 0.002 0.00006 0.002 0.00004 0.002 0.00004 Logan 6 model F 1182.03 348.44 280.96 343. 93 278.51 P < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 SS 2.0873 R 0.1598 0.1363 0.0968 0.0843 SS 10.9517 CT 0.4337 0.3365 0.2604 0.2070 Pseudo R 0.8094 2 0.6315 0.5949 0.6283 0.5928 5.71 1.78 0.09 0.03 0.08 0.04 0.07 0.04 0.06 0.04 0.12 0.03 0.11 0.04 0.10 0.05 0.10 0.04 0.10 0.03 T max 35.40 0.09 SE 38.00 0.12 38.00 0.02 38.50 0.06 38.00 0.01 8.13 0.14 9.25 0.32 9.24 0.3 4 9.16 0.31 9.18 0.34 SS R = residual sums of squares; SS CT = corrected total sums of squares; pseudo R 2 = 1 SS R /SS CT

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50 Table 3 3. Mean ( SEM) survival rate (in %) for each developmental stadium of Planococcus minor reared on sprouted potatoes at five constant temperatures. Developmental stadia Egg First Second Third Fourth Egg to adult T. ( o C) Female Male Male 15 0c 0c 20 91 1ab 88 6a 98 1a 100a 98 2a 97 2a 68 1b 25 93 5a 87 1a 87 5b 97 3ab 96 2a 98 2a 71 2a 29 84 5b 83 5a 80 1b 89 1b 97 1a 100a 58 1a 35 0c 0c ANOVA statistics n 120 120 120 120 120 120 120 F 3.43 0.78 8.00 3.49 0.37 0.62 2.82 df 2, 117 2, 117 2, 117 2, 117 2, 117 2, 117 2, 117 P 0.0357 0.4618 0.0006 0.0337 0.6916 0.5386 0.0639 Means within a column followed by the same letters are not

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51 Table 3 4. Mean ( SEM) proportion of females, pre oviposition and oviposition periods (in days) fecundity, and adult longevity (in days) of Planococcus minor reared on sprouted potatoes at thr ee constant temperatures. Proportion Pre oviposition Oviposition Adult longevity T. ( o of females C) period period Fecundity Female Male 20 64.6 2.5ab 14.8 0.1a 13.9 0.4a 269.8 17.8a 33.8 1.5a 4.3 0.4a 25 73.4 4.3a 10.2 0.4b 9.2 0.9b 205.6 7.0b 22.2 1.8b 1.7 0.7b 29 60.1 8.5b 8.4 0.3c 6.9 0.3c 187.9 22.5b 19.5 0.5c 1.3 0.3c ANOVA statistics n 120 120 120 120 120 120 F 3.38 218.34 190.05 55.90 334.56 216.89 df 2, 117 2, 117 2, 117 2, 117 2, 117 2, 117 P 0.0373 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

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52 Table 3 5. Life table parameters of Planococcus minor reared on sprouted potatoes at three constant temperatures. Temp. ( o C) Life table parameters 20 25 29 Gross reproductive rate, GRR ( 194.8 445.7 332.4 Net reproductive rate, R o ( 135.6 325.4 190.1 Generation time, T G (d) 63.5 39.5 37.7 Intrinsic rate of increase, r m ( 0.077 0.147 0.139 1.080 1.158 1.149 Doubling time, DT (d) 8.9 4.7 4.9

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53 CHAPTER 4 SURVEY FOR PLANOCOCCUS MINOR (MASKELL) (HEMIPTERA: PSEUDOC OCCIDAE) AND ITS NATURAL ENEM IES IN TRINIDAD Florida has long been a major entry point for invasive species into the U S with the largest proportion of recent insect arrivals originating from the Neotropics (Frank & McCoy 1992). The Caribbean in particul ar, is a major pathway for the entry of exotic insects into Florida (Miller et al. 1999; Skarlinsky 2003; Hoy et al. 2006). This can ultimately lead to the introduction of many unwanted pests such as Planococcus minor (Maskell). Work on this pest, including an assessment and the development of biological control options was considered a high priority and it was recently discovered in south Florida ( A. Roda, USDA ARS CPHST, personal communication). The invasion, spread and management of a similar pest, Maco nellicoccus hirsutus (Green) in the Caribbean Basin and U S during the last two decades highlighted the benefits of preemptive research on key pests while still offshore. B y working with Caribbean partners, USDA APHIS was able to develop and test the nec essary technology that ensured releases of encyrtid wasps and predatory coccinellid beetles could be made within several weeks of M. hirsutus appearing in the continental U S (Roltsch et al. 2006). A similar initiative reported on here was undertaken for P. minor on the island of Trinidad because of its confirmed populations of this mealybug and its relative proximity to the U S. P lanocccus minor has a broad native range, stretching from the South Pacific Islands to South Asia, and encompassing the Philippines, Indonesia, Malaysia, New Guinea, Australia, Thailand, and India (Cox 1981; Williams & Watson 1988 ; Cox 1989). The reported host plant list exceeds 250 plant species, including many crops such as banana, citrus, cocoa, coffee, corn, grape, mango, pota to (CAB 2003; Venette & Davis

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54 2004). In countries such as India (Shukla & Tandon 1984; Batra et al. 1987; Tandon & Verghese 1987; Reddy et al. 1999) and Taiwan (Ho et al. 2007) this mealybug is considered a serious pest of both agricultural and horticultural crops. In addition to its polyphagous nature, P. minor has many other characteristics of highly invasive species including: ease of spread on traded commodities such as fruit (Venette & Davis 2004), a relatively short life cycle (Martinez & Suris 1998) and high fecundity (Maity et al. 1998). Therefore, in the absence of effective control measures this mealybug is capable of spreading rapidly in newly infested areas and causing significant damage. It is not known when P. minor first invaded the Neotropi cs, but it was most likely introduced through trade activities (Cox 1989). It is now present in at least 21 countries/territories including Argentina in the south, Cuba in the north, and Mexico and Honduras in the west (Williams & Granara de Willink 1992). An important first step in developing strateg ies for the management of this and other pests is to understand their occurrence and pest status. Intriguingly, there have been no reports of crop losses caused by damage from P. minor in the Neotropics, includ ing Trinidad, where anecdotal evidence prior to this study suggested that this mealybug wa s not considered a major pest. Given its broad host range and key pest status, the question then arises as to why population levels of P minor have remained low throughout the region. One potential explanation is that populations of the pest are being limited by native or fortuitously introduced natural enemies. An important ecological consideration is the influence of plant composition at sites where P. minor is found. Altieri & Nicholls (2004) noted that the degree of plant diversity in agroecosystems influences the abundance and diversity

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55 of pests and their natural enemies The outcome of these survey activities should shed light on this hypothesis Conducting fiel d studies on P. minor and P. citri (Risso) has been a serious challenge because of their similar host range s and geographic distributions (Williams & Granara de Willink 1992) as well as the difficulty in distinguishing the two species (Cox 1983). However, the recent availability of synthetic sex pheromones for P. citri (Zada et al. 2004) and P. minor (Millar 2008) will allow for field separation and surveys of males of the two species for the first time. Challenges still exist especially on how to conduct surveys for natural enemies of the two species where it is imperative to know that the recovered natural enemies, especially parasitoids, are directly associated with a specific species. The goal of this study was to generate pertinent information that co uld facilitate the development of mitigation measures against P. minor The specific objectives were 1) to assess the occurrence and pest status of P. minor in Trinidad, including its distribution across the island, its host plant range, and its levels of infestation and damage on different host plants; and 2) to determine what, if any, natural enemies were attacking it P. minor in Trinidad, including their identity, relative abundance and potential for use in biological control. Materials and Methods Maintenance of Host Material and Colony of P. minor Prior to sprouting, potatoes ( Solanum tuberosum L.) were soaked in a 1% bleach solution for 510 min. They were then washed and rinsed with clean water and left to dry. The potatoes were placed on plastic trays in a room at 25 2oC 60 10% RH and complete darkness at the Central Experiment Station, Centeno, Ministry of Agriculture,

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56 Land and Marine Resources (MALMR), Trinidad. Sprouting took 24 weeks, and potatoes were ready for use in the experiments when the blanched sprouts were 2.02.5 cm long. A colony of P. minor was established on the sprouted potatoes under similar conditions as above, but in a separate dark room. A colony of P. minor was initiated in early May, 2006 on sprouted potatoes ( Solanum t uberosum L.) at the e xperiment station. This mealybug was inadvertently collected on field material infested with M. hirsutus and destined for use in a colony of the latter by personnel at the experiment station. Suspect P. minor females were isolated and offspring sent for identification to Douglass Miller, USDA ARS Systematic Entomology Laboratory (SEL) Beltsville, MD, to guarantee that a pure colony was established. The colony was maintained under constant environment conditions, 25 2oOccurrence and Pest Status of P. minor C, 65 5% RH and 24 h our darkness. It was transferred to the laboratory facilities at CAB International Caribbean and Latin America Regional Office, Curepe, during late May, 2007 and maintained under similar conditions. Based on protocols described by Meyerdirk et al. (1998), each week, 2030 sprouted potatoes were individually infested with 5 10 adult female mealybugs having well formed ovisacs. These potatoes were kept on plastic trays and placed on metal shelves. Weekly infestations of new potatoes ensured a continuous supply of different mealybug life stages. Consultations with staff at the experimental station indicated that P. minor was difficult to find but could be regularly found on cacao, Theobroma cacao L. Initial surveys verified this observation and the mealybug was collected from cacao trees during preliminary searches at the nearby LaReunion plant propagation station during late May 2006. Therefore, it was decided to use cacao as the primary indicator host

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57 plant for th is survey. A total of 33 cacao field sites were surveyed from June 30 to July 27, 2006. At ea ch site, ten cacao trees were randomly selected for visual inspection and scoring. Plant parts (pods, flowers, and leaves) on the main trunk of each tree from grou nd level to 1.5 m were examined for mealybugs (tentatively identified as P. minor ). All stages (2nd and 3rdA qualitative composite infestation score per tree ranging from 0 to 5 was devised based on the number of mealybugs counted on the pods on each tree. No mealybug s w ere scored as 0, <10 mealybugs were scored as 1, 10 to 100 were scored as 2, > 100 200 were scored as 3, 200500 were scored as 4, and >500 were s cored as 5. This score range was based on the range of infestation levels seen for other mealybugs such as M. hirsutus which was observed at very high levels on some cacao pods. When no pods were found on a tree, this plant part w as recorded as not available (NA ). In order to positively confirm the identity of the mealybug s surveyed as P. minor live specimens were collected from the plant into labeled brown paper bags using a camel hair brush (Fig. 4 2) or in some instances, infested plant parts were col lected and also placed into labeled bags for processing in the laboratory instar nymphs, and adult females with and without ovisacs) except first instar nymphs (crawlers) were counted with the aid of a hand lens (10x) and a tally counter. Very low and sparse numbers of mealybugs were found on flowers and leaves as compared to pods (Fig. 4 1) ; therefore, the latter was chosen as the principal sampling unit on the tree. Fruits, flowers, and leaves of different plant species also were visually inspected and scored for P. minor at cacao field sites using the procedure outlined above. Most of these plants were listed hosts of P. minor and were grown as part of mixed cropping

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58 systems within the same fields as cacao at 12 sites visited during the survey period. In addition, plant parts on grassy and herbaceous weeds were inspected and scored for P. mino r All weeds were identified using Fournet & Hammerton (1991). The cacao field sites included subsistence and commercial farms, and germplasm research stations. Insecticides were not applied to any of these field sites or known to have been used within the immediate area during this time period. In order to assess the influence of the plant diversity at these sites on the relative abundance of P minor the sites were categorized into three habitat types. Type 1 sites (commercial fields) were monocultures o f cacao trees receiving regular crop maintenance and weed management (Fig. 4 3 A) and were >2 hectares in size. Type 2 sites (abandoned fields) also had cacao trees, but with no crop maintenance or weed management (Fig. 4 3 B) and had from 0.25 to 2 hectar es of cacao planted. Type 3 sites (mixed crop systems) were cacao fields planted with vegetable and/or root crops with regular crop maintenance and weed management (Fig. 4 3 A) These sites were <0.25 hectares Most of the field sites, which included all habitat types, were planted 08 m from native tropical forests and had older cacao trees that were in the productive phase of the life cycle. Trees were greater than 5 m in height in most instances, and their canopies were supported by single trunks across most habitat types. Natural Enemies of P. minor From Jun e 26 to Oct ober 19, 2007, nine field sites were surveyed for predators and parasitoids of P. minor Eight sites surveyed for P. minor during the previous year were selected based on their central loc ations in the island and relative proximity to each other. The ninth site was added after natural enemies were recovered during a preliminary survey prior to the start of the main survey. Four sites were categorized as

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59 habitat type 1, four as type 2, and t he remaining site was categorized as type 3. In order to overcome the challenges of finding patchy, low populations of the mealybug at the field sites and ensure collections of natural enemies were positively associated with P. minor laboratory infested p otatoes were used in sentinel traps. The sentinel trap consisted of a rectangular shaped wire cage measuring 12 x 8 x 8 cm, with a mesh size of 1 cm2 (Fig. 4 4 ) in which a single infested potato was placed containing 200300 mealybugs of all life stages (egg to adult). These cages were securely hung from horizontal branches at 1.752.0 m above ground with a 12 cm long metal wire on randomly chosen trees (13 cages/site) in the center of each site and spaced 1015 m apart. The number of cages depended on the approximate size of each site. S ites less than 0.25 ha were allocated a single cage, 2 cages were placed at sites greater than 0.25 ha but less than 1 ha, while sites greater than 1 ha were allocated 3 cages. Tangle Trap insect trap coating (Tanglefoot C o., Grand Rapids, MI) was applied to the branch in a 5 cm wide band around the entire circumference on both sides of the cage attachment to exclude attendant ants from entering the cages because many species of ants negatively affect natural enemies (Buckl ey 1987; Buckley & Gullan 1991). The bands were separated by a distance of 12 cm and the coating was reapplied when it was no longer sticky to touch or if ants were found on the infested potato from the previous deployment. Every 10 14 days during the nearly four month survey period, newly infested potatoes were transported from the laboratory to the field in separate polyethylene containers in Styrofoam boxes and used to replace existing sentinel potatoes in the traps. Exposed potatoes were similarly transported back to the laboratory for evaluation.

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60 Predators Exposed sentinel potatoes were observed under a dissecting microscope and any adult predators found were immediately collected with a handheld aspirator. These predators were kept separately based on sentinel trap, field site, and sampling date in small plastic vials filled with 70% alcohol. Immature larvae and/or nymphs preying on remaining mealybugs were left undisturbed to develop to the adult stage under laboratory conditions of 25 1oParasitoids C, 60 70% RH, and a photoperiod 14:10 (L:D). The potatoes were kept in their original field containers. In some instances, infested potatoes from the lab colony were added to these containers to ensure that the immature predators were provided with an adequate suppl y of mealybugs to complete development. When all adults had emerged, they were collected, sorted by insect order, family and tentative species, and counted. Up to 100 mealybugs in two size classes (2nd instars, and 3rd instars to adult females ) were randomly removed from each infested potato and individually placed in gelatin capsules. The capsules were held in brown paper bags labeled according to size class and relevant collection data and kept at the same laboratory conditions as the predators. Capsules were inspected up to 4 weeks for emerged primary parasitoids. Percent parasitism was calculated by dividing the number of emerged parasitoids by the total number of encapsulated mealybugs from each sentinel trap. Mummies from parasitized mealy bugs found on the infested potatoes were placed individually into gelatin capsules to assess hyperparasitism. Percent hyperparasitism was calculated by dividing the number of emerged hyperparasitoids by the total number of mummies collected from each senti nel trap.

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61 Mealybug Identification and Natural Enemy Identification Representative specimens from the mealybug colony and field sites were preserved in 70% and 95% alcohol for subsequent identification. Specimens in 70% alcohol were identified by D Miller USDA ARS SEL while those in 95% alcohol were identified using molecular techniques by Alessandra Rung and subsequently outlined in Rung et al. (2008). Voucher specimens of the different species were retained by SEL. Other specimens were identified by Gr eg Hodges at the F lorida Department of Agriculture and Consumer Services Division of Plant Industry (F DACS DPI ) Gainesville, FL, and likewise, voucher specimens were kept at FDACS DPI Representative specimens of natural enemies were preserved in 70% al cohol and submitted for identification to relevant experts at USDA ARS S EL. Vouchers of each species were retained by SEL. Sex Pheromone trapping for Planococcus spp. In order to test for the co existence of P. minor and P. citri in Trinidad, pheromoneb aited traps were placed on cacao trees to attract male mealybugs at nine field sites where sentinel traps were deployed to recover natural enemies. The same habitat type designations were applicable to this study. Delta traps baited with sex pheromone lures for each species (23 of each type/field site) were hung in randomly chosen trees approx. 1.5 m in height and 1015 m apart from Jul y 10 to Aug ust 8 2007. A single trap with a blank septum (control) was also hung at each site. The P. minor sex pheromone was recently identified by Ho et al. (2007) and synthesized by Millar (2008). It was made available to us by the latter, while P. citri commercial lures were purchased from Aptiv Inc., Portland, OR. At 2 week intervals, traps were collected and transporte d to the

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62 laboratory where they were individually covered with clear polythene plastic and the number of males counted using a dis secting microscope ( Data Analysis The effect of habitat type on infestation levels of P. minor was analyzed using the chi square test of independence. This test compared the frequencies of the first nominal variable (habitat type) for the second nom inal variable (infestation score) using a 3x4 table (PROC FREQ, SAS Institute 200 2 ). Separate analyses of the natural enemy data from sentinel traps were performed based on the type of predators and parasitoids recovered. The numbers of cecidomyiid and c occinellids collected per sentinel trap were analyzed separately to determine the effect of habitat type and sampling date on th ese variables. The data did not meet parametric assumptions and were therefore squareroot transformed prior to performing ANOVA with habitat type and sampling date as the sources of variation (PROC GLM, SAS Institute 200 2 ). The dependent variable s in the statistical models w ere the adult cecidomyiid and the number s of adult coccinellids caught per sentinel trap. The models indicat ed significant treatment effects. Differences among means were separated by Tukeys studentized range (HSD) test. The parasitism by L. dactylopii C. perminutus and the hyperparasitoids were also analyzed separately to determine the effect of habitat type and sampling date on these variables. Parasitism d ata for all three were arcsine squareroot transformed prior to performing ANOVA with habitat type and sampling date as the sources of variation (PROC GLM, SAS Institute 2002 ). The dependent variable in the statistical models was the proportion of mealybugs parasitized per sentinel trap. The models indicated significant treatment effects, except for the hyperparasitoids. Differences among means for L. dactylopii parasitism were separated

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63 by Tukeys test For parasitism by C. perminutus a t test was used to compare the means from the two habitat types where this parasitoid was collected (TTEST, SAS Institute 200 2 ) The male mealybug trapping data did not meet the assumptions of ANOVA and were squareroot transformed prior to performing a oneway ANOVA with habitat type as the source of variation (PROC GLM, SAS Institute 2002 ). The dependent variable in the statistical model was males caught per baited trap and the model indicated significant treatment effects. Differences among means were separated by Tukeys test. Results Occurrence and Pest Status of P. minor Mealybug specimens collected from 20 of 33 cacao field sites on the island were confirmed as P. minor These field sites were found in 7 of the 8 cou nties (St. George, St. David, Caroni, St. Andrew, St. Patrick, Victoria, and Nariva) on the island. P. minor was only found on cacao and no specimens were recovered from any of the other plants inspected. A complete list of all plants inspected is given in Table 4 1 Habitat type had a significant effect on infestation levels ( 2Natural enemies of P. minor = 27.61; df = 6; P < 0.05). However, infestation levels scored 13 were very low (<0.5) for the three habitat types (Fig. 4 6 ) Twelve natural enemy species comprisi ed of predators and parasitoids in four orders and six families were recovered from cacao field sites (Table 4 2 ). Predators Diadiplosis coccidarum Cockerell (Diptera: Cecidomyiidae) was recovered from sentinel traps at all cacao field sit es (Table 3 2) and from all habitat types (Fig. 4 7 ). The

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64 sampling date did not affect the number of adults recovered; however, habitat type had a significant effect on their recovery ( F = 7.82; df = 2, 65; P < 0.05). Based on Tukeys HSD separation of means significantly greater numbers of adults were recovered from habitat type 2 sites than from either habitat type 1 or habitat type 3 sites (Fig. 4 7 ). Three common coccinellid species were recovered including: Diomus robert Gordon from two cacao f ield sites, Tenuisvalvae bisquinquepustulata Fabricius from four sites, and Diomus sp. from six sites (Table 4 2 ). The sampling date did not affect the number of adults recovered; however, there were significant differences among the three habitat types ( F = 14.62; df = 2, 65; P < 0.05). More adults ( from habitat type 2 sites than from either habitat type 1 or habitat type 3 sites (Fig. 4 8 ). The least common predator species recovered were Calliodis sp. (Hemiptera: Anthocoridae), Cryptognatha nodiceps Marshall (Coleoptera: Coccinellidae) and Ocyptamus stenogaster species group (Diptera: Syrphidae) (Table 3 2). Calliodis sp. is an undescribed species recovered as nymphs and adults from Maracas (2 adults/4 nymphs), Gran Couva ((1 adults/4 nymphs), and Santa Cruz ((3 adults/3 nymphs). Cryptognatha nodiceps was recovered from Gran Couva (8 adults), and O. stenogaster (9 adults) was recovered from LaReunion. Parasitoids The primary parasitoid, Leptomastix dactylopii Howard (Hymenoptera: Encyrtidae) (Fig. 4 5 A) was recovered from 3rd instar nymphs and adult females at five cacao field sites (Table 4 2). Mummified mealybugs with L. dactylopii were light brown in color. Habitat type did not affect the levels of parasitism; however, sampl ing date had a significant effect on their levels ( F = 2.81; df = 7, 104; P < 0.05). Based on Tukeys HSD

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65 separation of means Aug ust 8 22 recorded parasitism levels that were significantly higher than the other dates (Fig. 4 9 ). Another primary parasitoi d, Coccidoxenoides perminutus (Girault) (= Pauridia peregrina Timberlake, = Coccidoxenoides peregrinus (Timberlake)) (Hymenoptera: Encyrtidae) (Fig. 4 5 B) was recovered from 2ndThree species of hyperparasitoids were reared from mummified 3rd instar nymphs and adult female P. minor and associated only with L. dactylopii These were identified as Gahaniella tertia Kerrich and Coccidoctonus trinidadensis Crawford (Hymenoptera: Encyrtidae) and Signiphora n. sp. #11 (Woolley) mexicanus group (Hymenoptera: Signiphoridae), each recovered from one cacao field site (Table 4 2). Analysis of percent hyperparasitism at each field site showed that there were no significant interactions, and habitat type and sampling date did not affect hyperparasitism levels. No hyperparasitoids were found associated with C. perminutus instar nymphs at two cacao field sites (Table 4 2). The mummified mealybugs wit h C. perminutus were light green and smaller than those of L. dactylopii Parasitism levels were significantly different ( t = 3.48; df = 22; P < 0.05) with 25.6 8.5% recorded from the habitat type 2 site compared to 15.7 9.4% from the habitat type 1 si te. Sex Pheromone trapping for Planococcus spp. No male mealybugs were caught in P. citri sex pheromonebaited traps at any of the cacao field sites, but males were caught in P. minor sex pheromonebaited traps at all field sites for the entire trapping period. Analysis of P. minor males caught in baited traps showed that there were significant differences in the numbers caught among the three habitat types ( F = 10.39; df = 2; P < 0.05). Significantly more males (

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66 caught at habitat type 1 sites than at either habitat type 2 or habitat type 3 sites (Fig. 4 1 0 ). Discussion P lanococcus minor was widely distributed in Trinidad as evidenced by its recovery from cacao field sites in most of the counties on the island. However, this mealybug was found only on cacao despite its reported polyphagous nature. This plant is a reliable indicator host for Planococcus spp. in many areas (Strickland 1951; Donald 1955; Williams 1982 ; Cox & Freeston 1985), and it can be considered a primary host for P. minor Therefore, it is very likely that the host plant list provided by Venette & Davis (2004) includes many secondary hosts that may not be preferred by this mealybug. The very narrow host plant range of P. minor in Trinidad is indicative that the pest may have been introduced a long time ago. Franco et al. (2009) explained this phenomenon, where under low natural enemy pressure, mealybugs are found on relatively large numbers of host plants when they spread into new areas, but that host list declines once natural enemies become established. A recent example is M. hirsutus in the Caribbean, where Kairo et al. (2000) noted that although it a ffected numerous host plants when it spread throughout the islands, only a few were primary hosts and the vast majority were secondary hosts not capable of supporting high populations of the mealybug Once natural enemies were introduced and became establi shed, less than 20 plant species supported significant pest populations (Kairo et al. 2000). Another example that conforms to the long host plant list, but actually has a narrow range of damaged crops, is P. citri which has been found on plants from 70 fa milies, but actually attacks a smaller range of subtropical and tropical hosts (Franco et al. 2009).

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67 Although Bigger (1973) and Campbell (1983) reported that some mealybugs in the genus Planococcus favored the bark of canopy branches or green shoots of cacao trees, this study showed that pods were more likely to be infested and at heavier densities than other plant parts. These findings were similar to Kirkpatrick (1953), who reported that mealybugs identified at the time as P. citri mainly attacked the unripe pods, but also infested other plant parts to a lesser degree. Although cacao field sites categorized as habitat type 1 had higher mealybug infestation levels, the levels were generally low for the three habitat types. Various predators and parasitoi ds were consistently recovered at cacao field sites during the study period, and therefore validated this sentinel trap protocol as an effective recovery/monitoring tool for natural enemies. There was some loss of mealybugs from some potatoes due to factor s such as their natural degradation, movement of mealybugs away from the potatoes, and heavy rains. Other mealybug investigators have used similar survey methods in diverse settings with varying degrees of success (Walton & Pringle 2004 ; Ceballo & Walter 2 005). However, based on the results from this study, it is clear that this method can be used elsewhere in the region where the natural enemy complex of P. minor is not properly documented, and/or to collect different species for evaluation and introductio n into newly infested areas if the need arises. Additionally, it can be extended to noneconomic insect pests in their present locality to document the diversity of their natural enem ies Given that populations of P. minor were generally pretty low, it w as interesting that a diverse assemblage of predators and parasitoids were recovered and this suggested that these species may have a significant impact on field populations of P. minor It

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68 further supports the argument that P. minor is likely to have been introduced into Trinidad many years ago, because natural enemies tend to regulate pest populations that have been in an area for some time, which is not the typical situation with a recent invasive species (Franco et al. 2009). Nearly all of the predators collected from this study have been previously documented feeding on a variety of mealybug hosts in Trinidad (Kirkpatrick 1953; Bennett & Simmonds 1964). Of these, D. coccidarum and several coccinellids were the most widespread, and they were also recover ed in relatively high numbers. Their recovery in significantly greater numbers from habitat type 2 field sites than from the other habitat types support ed the findings of Altieri & Nicholls (2004) where more diverse crop settings tend to encourage greater and more diverse numbers of natural enemy populations. Although their impact on infested potatoes was difficult to quantify, the larvae of both groups of insects were observed in the laboratory feeding voraciously on various mealybug stages. L eptomastix dactylopii was the more dominant of the two primary parasitoids recovered at cacao field sites. Both species were recovered throughout the entire duration of the study despite the relatively low parasitism rates. Their persistence is important from a biol ogical control standpoint because it suggests that they were intimately associated with, and perhaps providing sustainable control of mealybug populations. These parasitoids have been reared from numerous mealybug hosts elsewhere (Noyes & Hayat 1994) L ept omastix dactylopii is thought to be of Afrotropical origin, while C. perminutus originated from Asia (Noyes & Hayat 1994). There are no records of intentional introduction of either parasitoid to the island, but L. dactylopii was

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69 shipped from Trinidad to other areas for biological control of P. citri (Cock 1985) Hence, these two parasitoids were probably introduced fortuitously with P. minor and have likewise been present on the island for quite some time. Fortuitous biological control, which is best descr ibed as the unintentional reduction and maintenance of a pest population by a natural enemy where both the natural enemy and pest are nonindigenous ( Nechols 2002) is not uncommon. Examples include the control of Nipaecoccus viridis (Newstead) (Hemiptera: Pseudococcidae) in the Pacific islands principally by Anagyrus indicus Shafee, Alam, and Agarwal (Hymenoptera: Encyrtidae) (Nechols & Seibert 1985) and the control of Phenacoccus solenopsis (Tinsley) (Hemiptera: Pseudococcidae) by another encyrtid wasp, A enasius bambawalei Hayat in India (Gautam et al. 2009). Despite the negligible hyperparasitism rates recorded during this study, the impact of these secondary parasitoids could be fairly significant as reported by Kirkpatrick (1953), where several species were recovered from greater than 90% of mealybug specimens. Despite reports of P. citri in Trinidad dating back to Kirkpatrick (1953), and the uncertainty regarding this species and P. minor only the latter was confirmed from specimens submitted for ident ification from 200609. Other mealybugs were also collected from cacao and identified, but they were not relevant to this study. Not only was P. minor identified using the morphological scoring matrix developed by Cox (1983), which addressed the taxonomic complexities of identifying these two species, but some specimens were identified using molecular diagnostics (Rung et al. 2008). This technique provided a high degree of certainty as to the identity of this species, and has proven to be a fast and reliabl e method to identify these cryptic mealybugs

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70 (Demontis et al. 2007). The fact that no male mealybugs were captured in P. citri sex pheromonebaited traps provided stronger evidence that this species most likely is not present on the island. The findings fr om this field study were supported by an extensive search of CAB Internationals pseudococcid records in Trinidad that revealed all references of P. citri were prior to the identification of P. minor as a separate species ( Cox 1981). Thereafter, all refere nces were only of the latter I n terms of applicability of P. minor sex pheromonebaited traps, Ho et al. (2007) demonstrated that small doses of the pheromone were very attractive to males of this species Later, Millar (2008) suggested that baited traps would be a sensitive tool for finding small populations of the pest. Trapped P. minor males were therefore a reliable indicator of this mealybugs presence at cacao field sites on the island. T hese baited traps also demonstrated that habitat type 1 field s ites supported higher mealybug populations than the other habitat types. The low populations of P. minor on cacao and its absence from other listed host plants suggests that it is not a pest of economic importance in Trinidad. This current pest status is probably attributable to the existing natural enemy complex. The survey method with sentinel material has immediate applications in the Neotropics and elsewhere for documenting more natural enemies of P. minor It is very likely that biological control wil l form part of major mitigation measures to suppress this important pest in new areas. The identified primary parasitoids, in particular, have been used successfully against Planococcus spp. with some degree of success (Meyerdirk et al. 1978; Smith 1991; M ani 1994). These parasitoids are also available from commercial insectaries at short notice for use in outbreak areas The sex pheromone trap will

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71 complement biological control activities by permit ting timely detection and monitoring of this pest in these outbreak areas. Given the high likelihood that P. minor will continue to pose a threat in new and sensitive areas data gathered from this study should have an immediate positive impact on management strategies

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72 Table 4 1. List of plants inspected for P lanococcus minor at field sites, June 30 July, 2006. Common name Scientific name Family cacao Theobroma cacao Sterculiaceae pigeon pea Cajanus cajan Fabaceae okra Abelmoschus esculentus Malvaceae eggplant Solanum melongena Solanaceae coffee Coff ea sp. Rubiaceae cassava Manihot esculenta Euphorbiaceae citrus Citrus spp. Rutaceae banana Musa sp. Musaceae yam Dioscorea sp. Dioscoreaceae pomerac Syzygium malaccense Myrtaceae dasheen Colocasia sp. Araceae sweet potato Ipomoea batatas Convolvula ceae sugar cane Saccharum sp. Poaceae papaya Carica papaya Caricaceae bhaji Amaranthus dubius Amaranthaceae morning glory Ipomoea tiliacea Convolvulaceae guinea grass Panicum maximum Poaceae fowlfoot grass Eleusine indica Poaceae

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73 Table 4 2. Natura l enemies recovered from field sites, June 28 October 19, 2006. Species Field site Hymenoptera: Encyrtidae Leptomastix dactylopii Howard Maracas, LaReunion, Santa Cruz, Lopinot, Fishing Pond Coccidoxenoides perminutus Girault Fishing Pond, Biche Gahaniella tertia Kerrich Maracas Coccidoctonus trinidadensis Crawford Lopinot Hymenoptera: Signiphoridae Signiphora n. sp. #11 (Woolley) LaReunion Diptera: Cecidomyiidae Diadiplosis coccidarum Cockerell LaReunion, S. Cruz, Biche, Maracas, Fishing P ond, Navet, Gran Couva, Plum Mitan, Lopinot Diptera: Syrphidae Ocyptamus stenogaster LaReunion Coleoptera: Coccinellidae Tenuisvalvae bisquinquepustulata Fabricius S. Cruz, Lopinot, Biche, Navet Diomus sp. LaReunion, S. Cruz, Biche, Maracas, Fishing Pond, Navet Diomus robert Gordon Gran Couva, Plum Mitan Cryptognatha nodiceps Marshall Gran Couva Hemiptera: Anthocoridae Calliodis sp. Maracas, S. Cruz, Gran Couva

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74 Fig ure 4 1. Cacao pod infested with P lanococcus minor and tended by ants Pho to Courtesy of Antonio Francis.

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75 Fig ure 4 2 Collection of mealybugs for identification from infested cacao pods Photo Courtesy of Antonio Francis.

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76 Fig ure 4 3. Cacao h abitat types. A) Type s 1 and 3 B) Type 2. Type 1 sites (commercial fields) were monocultures of cacao trees receiving regular crop maintenance and weed management T ype 2 sites (abandoned fields) also had cacao trees, but with no crop maintenance or weed management T ype 3 sites (mixed crop systems) were cacao fields planted with vegetable and/or root crops with regular crop maintenance and weed management. Photos Courtesy of Antonio Francis. A B

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77 Fig ure 4 4. Sentinel trap with infested potato used to recover natural enemies of P lanococcus minor Photo Courtesy of Antonio Francis.

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78 Fig ure 4 5. Primary parasitoids of P lanococcus minor A) L eptomastix dactylopii female. B) C occidoxenoides perminutus female. Photos Courtesy of Antonio Francis. A B

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79 Fig ure 4 6 Infestation levels of P. minor at cacao field sites categorized into habitat types (13) in Trinidad, June 30 to Jul y 27, 2006. Type 1 sites (commercial fields) were monocultures of cacao trees receiving regular crop maintenance and weed management T ype 2 sites (abandoned fields) also had cacao tr ees, but with no crop maintenance or weed management T ype 3 sites (mixed crop systems) were cacao fields planted with vegetable and/or root crops with regular crop maintenance and weed management.

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80 Fig ure 4 7 Number of D. co ccidarum recovered from cacao field sites categorized into habitat types (13) in Trinidad, June 28 to August 8 2007. Type 1 sites (commercial fields) were monocultures of cacao trees receiving regular crop maintenance and weed management T ype 2 sites (a bandoned fields) also had cacao trees, but with no crop maintenance or weed management T ype 3 sites (mixed crop systems) were cacao fields planted with vegetable and/or root crops with regular crop maintenance and weed management. Values are mean SEM an d means with the same letter are not significantly different from each other (Tukeys test, P 0.05).

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81 Fig ure 4 8 Number of coccinellids recovered from cacao field sites categorized into habitat types (13) in Trinidad, June 28 to August 8 2007. Type 1 sites (commercial fields) were monocultures of cacao trees receiving regular crop maintenance and weed management T ype 2 sites (abandoned fields) also had cacao trees, but with no crop maintenance or weed management T ype 3 sites (mixed crop systems) were cacao fields planted with vegetable and/or root crops with regular crop maintenance and weed management. Values are mean SEM and means with the same letter are not significantly different from each other (Tukeys test, P 0.05).

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82 Fig ure 4 9 Mealybug parasitism by L. dactylopii from cacao field sites at different sampling dates in Trinidad, Jun e 28 to Oct ober 19, 2007. Values are mean SEM and means with the same letter are not significantl y different from each other (Tukeys test, P 0.05).

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83 Fig ure 4 1 0 Number of P. minor males caught on baited traps from cacao field sites categorized into habitat types (13) in Trinidad, Jul y 10 to Aug ust 8 2007. Type 1 sit es (commercial fields) were monocultures of cacao trees receiving regular crop maintenance and weed management T ype 2 sites (abandoned fields) also had cacao trees, but with no crop maintenance or weed management T ype 3 sites (mixed crop systems) were ca cao fields planted with vegetable and/or root crops with regular crop maintenance and weed management. Values are mean SEM and means with the same letter are not significantly different from each other (Tukeys test, P 0.05).

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84 CHAPTER 5 FIELD ASSESSMENT OF TWO PRIMARY PARASITO IDS OF PLANOCOCCUS MINOR (MASKELL) (HEMIPTERA: PSEUDOCOCCIDAE) Two primary parasitoids, Leptomastix dactylopii Howard and Coccidoxenoides perminutus Girault (= Pauridia peregrina Timberlake, = Co ccidoxenoides peregrinus (Timberlake)), (Hymenoptera: Encyrtidae) were recovered from the passionvine mealybug, P lanococcus minor (Maskell), (Hemiptera: Pseudococcidae) at cacao field sites in Trinidad (Francis, Chapter 4 ). These two parasitoids appear to be critical natural enemies and together with several predator species were responsible for regulating populations of P. minor at low levels Based on field data and other records, it was speculated that their fortuitous introduction on the island was no t a recent event ( Francis, Chapter 4 ). The occurrence of these specialist natural enemies attacking P. minor i n Trinidad is interesting and it would be useful to assess their roles in controlling th e pest. A s noted by Neuenschwander et al. (1986), it is important to assess the impact of natural enemies on target pest populations under natural conditions In the context of P. minor a pre introductory assessment of these promising parasitoids could provide relevant data needed to determine if t hey are key r egulating natural enemies and therefore might be introduced into newly infested areas L eptomastix dactylopii is believed to be of Afrotropical origin while C perminutus originated from Asia (Noyes & Hayat 1994) L dactylopii is a solitary arrhenotokous endoparasitoid that primarily oviposits in 3rd instar and adult female hosts of P lanococcus citri (Risso), (Hemiptera: Pseudococcidae) (de Jong & van Alphen 1989). Alternatively, C perminutus is also a solitary endoparasitoid, but it appears to be almost entirely thelytokous with highest oviposit ional success occurring in 2nd instar hosts of P. citri ( Ceballo & Walter 2004). These encyrtid wasps have been used singly or together

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85 as biological control agents against P. citri with varying degrees of succes s and in different localities such as California ( Bartlett & Lloyd 1958), Texas ( Meyerdirk et al. 1978; Dean et al. 1983; Summy et al. 1986 ), India ( Krishnamoorthy & Mani 1989 ; Mani 1994), and Australia (Smith 1991; Davies et al. 2004; Ceballo & Walter 200 5). Due to the potential wide host range of P. minor (Venette & Davis 2004), this mealybug might pose a serious threat to numerous crops in the continental U S However, experimental data that elucidate the specific roles and impact of natural enemies inc luding the two parasitoid species on P. minor are lacking. T h e study reported on here was set up with the goal of assessing the impact of L dactylopii and C perminutus on P. minor at a commercial cacao field site in Trinidad Because P. minor is very rar e in Trinidad and difficult to distinguish from other mealybug species, simply collecting mealybugs from the field was not possible. Infested P. minor potatoes were used to overcome these issues as well as the technique provided an opportunity to assess st age specific and species interactions on parasitism rates. Specifically, this study addressed t he following objectives: 1) to determine the relative abundance and seasonal occurrence of th e two primary parasitoids by determining and comparing levels of sta ge specific parasitism of mealybugs in field deployed cages ; and 2) to determine the influence of resident ant s on parasitoid activity. Materials and Methods Preparation of H ost Plant M aterial Prior to sprouting, potatoes ( Solanum tuberosum L.) were soak ed in a 1% bleach solution for 510 min. They were then washed and rinsed with clean water and left to dry. The potatoes were placed on plastic trays in a room at 25 2oC 60 10% RH and complete darkness at CAB International Caribbean and Latin America Regional Office,

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86 Curepe, Trinidad Sprouting took 24 weeks, and potatoes were ready for use in the experiments when the blanched sprouts were 2.02.5 cm long. Mealybug R earing Adult female P minor were collected from potatoes coinfested with Maconelli coccus hirsutus (Green) at the Central Experimental Station, Centeno, Trinidad, were collected and used to initiate pure colonies. The identity of several specimens from the initial colony was confirmed by Douglas Miller at USDA ARS Systematic Entomology Laboratory, Beltsville, MD. Each week, 3648 sprouted potatoes were individually infested with five adult female mealybugs having well formed ovisacs using a small camel hair brush These infested potatoes were placed on plastic trays (12 15 potatoes per tr ay) supported on metal shelves. They were maintained in a separate dark room at 25 2oPreparation of P. minor Colonies on P otatoes for F ield D eployment C and 65 5% RH at CAB International Caribbean and Latin America Regional Office, Curepe. Weekly infestations ensured a continuous supply of different mealybug nymphal instars. This protocol was modified based on methods described by Meyerdirk et al. (1998). Twenty sprouted potatoes of uniform size were each infested with 34 adult female P. minor taken from the colony Large gravid females >2.5 mm in length were chosen and placed on sprouts 0.51.0 cm long The females were allowed to oviposit on the sprouts for 72 hours before they were carefully removed, leaving the ovisacs on the potatoes The mean nu mber of eggs laid in this 72 hour period was 115. The infested potatoes were kept in an incubator at 25 1C, 60 5% RH and a photoperiod of 0:24 (L : D). The procedure was repeated at 7 and 14 days to ensure that predominantly three stages (adult females, 3rd instar females, and 2nd instar nymphs) were available after at

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87 least four weeks from the initial infestation While male stages were present, preliminary observations showed that they were never parasitized beyond the 2ndField S ite instar nymphal stage and were therefore excluded during lab processing later. The mealybugs were observed daily until more than 75% had molted to the required stages. Mature cacao trees of the Trinidad Select Hybrid (TSH) variety were grown at Paul Manickchands E state, F ishing Pond located in the north east of Trinidad Th is estate had 14 hectares of commercial cacao and received regular crop maintenance and weed management. Trees were estimated at 15 years old and were uniformly spaced approx. 2 m apart within and between rows, respectively. Low population levels of P. minor were found on trees in previous years and this field site was the only one where L. dactylopii and C. perminutus were recovered together (Francis, Chapter 4 ). Field P rotocol To assess f ield parasit ism of P. minor over a 5day period, infested potatoes were plac ed in wire cages as described previously to survey for natural enemies (Francis, Chapter 4 ) The P. minor infested potatoes were transported to Fishing Pond in circular polystyrene containers stored in cardboard boxes. Twenty site s spaced 3550 m apart were selected for placement of cages. Fourteen locations were situated centrally within the cacao plantation 25 50 m from the margins, while the remaining six were situated along the margins of the field site. Three treatments were used. Treatment 1 had mealybugs excluded ants and assessed stage specific parasitism rates T reatment 2 had mealybugs attendant ants and their effect on parasitism rates w as assessed Treatment 3 had mealybugs but excluded both parasitoids and ants. Within each treatment, 3 separate mealybug stages

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88 were tested2nd instars, 3rdOn day 5, the cages were removed, infested potatoes were placed in separate labeled containers, stored in styrofoam boxes, and transported to the lab oratory This 5 day period limited pred ator feeding, hyper parasitoid activity, excessive mealybug loss from natural movement away from the potato and from periodic heavy rain showers during the rainy season. The study was repeated every 4 weeks from Apr il 2008 to instars, and adult females On day 1 at midmorning, three cages, each with one of the three mealybug stageinfested potatoes, w ere hung on the main trunk s of cacao trees (3 trees/ location in a triangular grid) at 8 completely randomized locations for both treatment 1 ( mealybug + ant excluded) and treatment 2 ( mealybug + ant tended ) Cages were random ly assigned with respect to tree placement at each location. To exclude ants, TangleTrap aerosol insect trap coating ( Tanglefoot Co., Grand Rapids, MI ) was sprayed on the main trunk of each tree in 5 cm bands above and below the cage to prevent ants from accessing the infested potatoes The sticky material was replenished at monthly intervals to ensure the barrier remained effective. N o sticky coating was applied to the tree trunk s in treatment 2 and ants had free access to the infested potatoes. The remaining f our locations were used for treatmen t 3 ( mealybug + parasitoid and ant excluded), and were also completely randomized by location. The mealybug stage infested potatoes were also randomized with respect to tree placement at each location. To exclude both ants and parasitoids, bands of sticky coating w ere similarly applied as previously described to exclude ants, in addition to covering the entire cage with a muslin screen cloth to prevent parasitoid entry. All cages for all three treatments were kept in place by tying a 12 cm long metal wire t o a wire loop fitted around the trunk at approx. 1.25 m in height.

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89 March 20 09 and spanned the ent ire duration of the islands rainy season (June Dec ember ). No sampling was done during May 2008 because heavy rains flooded the cacao fields. Weather data for the period were provided by the meteorological station on the island. Processing I nfested P otatoe s At least 100 randomly selected mealybugs from each stage were collected from individual potatoes and placed into separate gelatin capsules and into labeled paper bags for parasitoid emergence Parasitoids were collected over a 3 4 week period from the c apsules that were held in a room where temperature was maintained at 25 1oData Analysis C and 65 5% RH Percent parasitism of each mealybug stage was calculated by dividing the number of emerged parasitoids by the number of encapsulated mealybugs per infested potato Attendant a nts on infested potato es were preserved in 70% alcohol for identification to the genus level Analysis of variance (ANOVA) was used to determine the effect of mealybug stage, exclusion of ants and sampling date on parasitism le vels (PROC GLM, SAS Institute 200 2 ). Mealybug stage, ant exclusion and sampling date were analyzed as the treatment effects (sources of variation), while percent parasitism was the dependent variable. The interaction between sampling date and mealybug st age, sampling date and ant exclusion, mealybug stage and ant exclusion, and the interaction between all three variables were tested in the statistical model. Percent parasitism was arcsin transformed for statistical analysis

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90 Results Relative Abundance an d Seasonal Occurrence of Parasitoids Stage specific parasitism rates and the effect of ants on these parasitism rates from treatment 3 remained unchanged (0) and were therefore excluded from analysis. Stage specific parasitism rates from treatment 1 as wel l as the effects of ants on parasitism rates from treatment 2 were then analyzed. No interaction was found between sampling date, mealybug stage, and ant exclusion. However, the interaction between sampling date and mealybug stage ( F = 1. 86; df = 16, 96 ; P < 0. 05 ) and sampling date and ant exclusion ( F = 134.57; df = 1, 96; P < 0.05) were found to be significant Sampling date significant ly a ffect ed parasitism ( F = 2.46; df = 10, 96 ; P < 0.0 5 ) as did mealybug stage ( F = 21.43; df = 2, 96 ; P < 0.0 5) and ant exclusion ( F = 134.57; df = 1, 96 ; P < 0.0 5 ) L eptomastix dactylopii and C. perminutus were the only species of parasitoids collected from the potatoes. All parasitoids collected from 3rd instar nymphs and adult females were L. dactylopii while all parasitoids collected from 2nd instar nymphs were C. perminutus The levels of parasitism were generally low throughout the sampling period and parasitism never exceeded 20% for either species (Fig. 5 2 ). Parasitism of adult females peaked at 19% in September and again in March at 16.5%, and these rates w ere higher than those of 3rd instars, except for three sampling dates (Fig. 5 2 ). Parasitism of 2ndRole of At tendant Ants instars never exceeded 6% (Fig. 5 2 ), remaining relatively uniform throughout the sampling period. Exclusion of ants significantly affected parasitism ( F = 134.57; df = 1, 96; P < 0.0 5 ) and the interaction between sampling date and ant exclusion was significant ( F =

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91 3.51; df = 8, 96; P < 0.0 5 ). Mean percent parasitism from the ant excluded treatment fluctuated throughout the sampling period, but was generally higher than that of the ant tended treatment (Fig. 5 3 ). Mean percent parasitism from the ant tended treatment remained fairly constant and did not surpass 7% ( F ig. 5 3 ). The interact ion between mealybug stage and ant exclusion had a significant effect on parasitism ( F = 14.31; df = 2, 96 ; P < 0.0 5 ). Mean percent parasitism from the ant excluded treatment w ere similar (14 16%) for adult females and 3rdDiscussion instars attacked by L. dactylopii while parasitism from the ant tended treatment was less than 5% for both stages (Fig. 5 4 ). Mean percent parasitism by C. perminutus from the ant excluded and ant tended treatments were similar for second instars (>3%) (Fig. 5 4 ). The predominant ant species collected from the ant tended treatment were Anoplolepis Brachymyrmex, Camponotus, and Acanthomyops in the subfamily Formicinae and Strumigenys Cephalotes and Atta in the subfamily Myrmicinae (Hymenoptera: Formicidae) ). The cacao field s ite at Paul Manickchands estate, Fishing Pond was similar to other field sites surveyed (Francis, Chapter 4 ), in that there were low, patchily distributed populations of P. minor occurring primarily on cacao pods Given the general difficulty in locating these populations of P. minor a decision was made to use wire cages with mealybug infested potatoes as an attractant to the primary parasitoids Since each parasitoid exploits a particular range of host sizes/stages, the different aged mealybugs permitted the separate collection of each species. More importantly, these different aged mealybugs permitted an evaluation of each species. As reported for P. citri L. dactylopii attacked the larger mealybug stages on the infested potatoes, while C. perminutus parasitiz ed the smaller mealybug stages ( de Jong & van Alphen 1989 ;

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92 Golberg 1982; Ceballo & Walter 2004) T heir release together should ensure that different stages of P. minor are potential targets for parasitization over a broader period of time and there for e increase overall control Despite the short field deployment of infested potatoes, L. dactylopii and C. perminutus were consistently recovered throughout the sampling period. While the relative abundance of L. dactylopii was higher than C. perminutus the overall parasitism levels were low Two possible explanations for this were that existing pest populations were low and the exposure period of the potatoes to the parasitoids was too brief. The latter explanation was reported by Ceballo & Walter (2005) where lower percent parasitism by a parasitoid complex on P. citri infested lemons was recorded at 5 days when compared with those left out for 10 days. The presence of ants undoubtedly affected the foraging behavior of the parasitoids evident by t he differences in parasitism levels between the ant excluded and ant tended treatment for L. dactylopii Conversely, their impact on C. perminutus appeared to be negligible. A nts not only interfere with parasitism of mealybugs, but they can also reduce par asitoid abundance by causing direct mortality and low er reproductive success (Mgocheki & Addison 2009) These authors demonstrated that C. perminutus was more tolerant towards ants than Anagyrus sp., as indicated by its higher parasitism rates of P. ficus in the presence of ants. Daane et al. (2007) also demonstrated that Pseudaphycus flavidulus (Brethes) was better able to forage on ant tended grapevines than Leptomastix epona (Walker) based on parasitoid recovery rates.

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93 The mutualistic relationships betw een m ealybugs and ants are well documented. Mealybugs benefit because ants improve sanitation in their colonies and protect them from natural enemies ( Gonzalez Hernandez et al. 1999). An example of this protective behavior by ants was observed in Pheidole megacephala (F abricius ) which caused a significant reduction in the mortality of Dysmicoccus brevipes (Cockerell) from Anagyrus ananatis Gahan (Hymenoptera: Encyrtidae) and the coccinellid, Nephus bilucernarius Mulsant by disrupting the searching behavior of these natural enemies (Gonzalez Hernandez et al. 1999). Leptomastix dactylopii and C. perminutus appear to be important contributors in maintaining existing populations of P. minor at very low levels in this cacao field site B oth species persisted throughout the sampling period attacking different stages of the mealybug Therefore, they appear to have host size/ stage preferences and this finding suggests some level of complementarity. Th ese results should provide valuable information to guide appro priate biological control strategies in areas where P. minor is newly introduced. These encyrtid wasps are commercially available and can be release d into these areas if the need arises.

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94 Figure 5 1 Field deployment of wire cages with P. minor inf ested potatoes A) Mealybugs with ant exclusion treatment B) Mealybugs with ant and parasitoid exclu sion treatment. A B

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95 Fig ure 5 2 Relative abundance and seasonal occurrence of primary parasitoids at Fishing Pond cacao field s ite, Trinidad, Ap r il 23 2008 to March 18 2009. Parasitism was expressed as mean ( SEM). Adult female and third instar nymph represent ed parasitism by L. dactylopii Second instar nymph represent ed parasitism by C. perminutus

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96 Fig ure 5 3 M ean ( SEM) percent parasitism pooled across stages from ant excluded and ant tended treatments with P. minor infested potatoes throughout the sampling period at Fishing Pond cacao field site, Trinidad, Apr il 23, 2008 to March 18, 2009.

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97 Fig ure 5 4 Relative abundance of primary parasitoids from ant excluded and ant tended treatments with P. minor infested potatoes at Fishing Pond cacao field site, Trinidad, Apr il 23, 2008 to Mar ch 18 2009 Parasitism was expressed as mean ( SEM). Adult female and third instar nymph represent ed parasitism by L. dactylopii Second instar nymph represent ed parasitism by C. perminutus

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98 CHAPTER 6 HOST STAGE SELECTION BY TWO ENCYRTID ENDO PARASITOIDS, LEPTOMASTIX DACTYLOPII HOWARD AND COCCIDOXENOIDES PERM INUTUS GIRAULT, ATTACKING PLANOCOCCUS MINOR (MASKELL) (HEMIPTERA: PSEUDOCOCCIDAE) Leptomastix dactylopii Howard and Coccidoxenoides perminutus Girault (= Pauridia peregrina Timberlake, = Coccidoxenoides peregrinus (Timberlake)) (Hymenoptera: Encyrtidae) appear to play a key role in suppressing populations of Planococcus minor (Maskell) (Hemiptera: Pseudococcidae) in Trinidad ( Francis, Chapter 5 ). This mealybug was recently discovered for the first time in the continental U S in south Florida ( A. Roda, USDA ARS CPHST, personal communication), and there are many areas with suitable climate for its establishment putting numerous listed agricultural host crops at economic risk (Venette & Davis 2004). As with other Planococcus spp that are difficult to control with conventional methods such as insecticides (Grasswitz & Burts 1995; Geiger & Daane 2001), biological control should therefore be viewed as a key approach to manage this invasive pest. The biology and host selection behav ior of L. dactylopii using P lanococcus citri (Risso) (Hemiptera: Pseudococcidae) as a host has been investigated (Kirkpatrick 1953; Lloyd 1958; Tingle & Copland 1988; Tingle & Copland 1989; de Jong & van Alphen 1989). Likewise, t here is also a fair amount of information on the biology and host selection behavior of C. perminutus with P. citri as a host (Golberg 1982; Krishnamoorthy & Mani 1989; Ceballo & Walter 2004) and with Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae) (Joyce et al. 2001; Walt on & Pringle 2005) However, no biological data exist for these parasitoids attacking P. minor either singly or together. The importance of interspecific competition for parasitoids as a factor in population dynamics has long been a difficult issue to res olve ( Connell 1983 ; MacNaly 1983 ).

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99 C ompetitive interactions among parasitoids have generally involved laboratory studies i n which hosts are offered to female parasitoids ( BokononGanta et al. 1996 ). Elucidating if there are competitive interactions between L. dactylopii and C. perminutus will improve our knowledge of these species and thereby the success of biological control against P. minor The goal of this study was to understand how these parasitoids utilize P. minor as a host and to use this information to evaluate them as potential candidates and t o improve mass rearing techniques The specific objectives were: 1) to determine the host size class preferences of each parasitoid when attacking P. minor based on the proportion of mealybugs parasitized and the number of eggs oviposited; and 2 ) to determ ine the degree of potential interspecific competition between L. dactylopii and C. perminutus based on the proportion of emerged adult parasitoids of each species when offered the same host size class for oviposition. Materials and Methods Maintenance of H ost Material and Mealybug Colony Prior to sprouting, potatoes ( Solanum tuberosum L.) were soaked in a 1% bleach solution for 510 min. They were then washed and rinsed with clean water and left to dry. The potatoes were placed on plastic trays for sprouti ng in a room at 25 2oMealybugs were reared on the sprouted potatoes placed on plastic trays (1215 potatoes per tray) that were sup ported on metal Dexion shelves These infested potatoes were kept in a separate dark room at 25 3 C 60 10% RH and complete darkness at CAB International Caribbean and Latin America Regional Office, Curepe, Trinidad. oC, 65 5% RH, and 0:24 photoperiod (L:D) Weekly infestations of 2436 potatoes with 4 6 adult female

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100 mealybugs having well formed ovisacs ensured a conti nuous supply of different mealybug developmental size stages This protocol was adapted from Meyerdirk et al. (1998) Rearing of P arasitoids Cultures of the two parasitoid species were established using field collected mummified mealybugs recovered from P. minor infested sentinel potatoes traps placed at Fishing Pond, Trinidad (Francis, Chapter 5 ) Mealybug mummies from each species were separated by color ; L. dactylopii had light brown mummies, while those for C. perminutus were light green and kept ind ividually in gelatin capsules. The initial collection of parasitoids consisted of 10 pairs of male and female L. dactylopii and 6 female C. perminutus The latter species is thought to be thelytokous ( Ceballo & Walter 2004). Upon emergence, the parasitoids were removed from the capsules and placed in 30 cm3To initiate the experimental cultures, 40 L. dactylopii (1:1 F/M) were removed using a handheld aspirator. They were released into a single rearing cage containing 6 infested potatoes with mixed stages of P. minor Similarly, 20 female C. perminutus w ere released into a cage containing 6 infested potatoes supporting mixed stages After 12 days of exposure to L. dactylopii and 21 days to C. perminutus parasitized mealybugs were carefully collected with a fine paintbrush and were placed on sheets of pap er towel in separate plastic cages Within 24 hours of emergence, 100 pairs of L. dactylopii parasitoids were transferred into individual gelatin capsules with a minute drop of diluted honey placed at one extreme end of the capsule. They were held for 24 white plastic rearing cages (Bug Dorm 1, Bioquip, Rancho Dominguez, CA) containing 24 infested potatoes The parasitoids were supplied with supplemental food in the form of diluted honey on cotton wick s.

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101 h ours before the experiment to ensure successful mating. Within 24 hours of emergence, 100 C. perminutus females were similarly transferred into gelatin capsules. Because this species has a short life span (Ceballo & Walter 2004), females were tested within 4 hours after transferring to the capsules Both parasitoid species had no experience of oviposition so that they were nave at the start of the experiments. All cages and capsules were maintained under constant environment conditions at 25 1oHost Stage Selection C, 60 5% RH, under a photoperiod of 11:13 (L:D). The light source was provided by day light fluorescent lamps T here was some overlap in size within the different developmental instars of P. minor due to variation in size at molting. Size was also used as the criterion to categorize mealybug host stages as detailed for P. citri by de Jong & van Alphen (1989). Therefore, i ndividual mealybug were divided into the following instar size classes: 1st instar nymphs or size class 1 (0.25 0.50 mm), 2nd instar nymphs or size class 2 (0.50 0.80 mm), 3rd instar immature females or size class 3 (0.80 1.50 mm), adult females without ovisacs or size class 4 (>1.50 mm), and females with 2day old ovisacs or size class 5 (>3.0 mm including ovisac). Thirdand f ourthinstar immature males and adult males were not used in the experiments because preliminary tests showed that neither parasitoid attacked these stages. A method was devised to collect m ealybugs from infested potatoes using a fine camel hair brush and individually measure them by placing each on a measurement scale (0.004.00 mm) outlined with lead pencil on a sheet of white rectangular print paper. Two hundred mealybugs from each class were transferred into polyethylene container s (12 cm (dia.) x 8 cm (height) with a mesh covered lid) lab e led for each size class (1 5) and held for <1 hour prior to

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102 the start of the experiment. All experiments were conducted at 25 1oHost S ize C lass Preference C and 60 5% RH in a separate controlled environment room under day light fluorescent l amps with a photoperiod of 11:13 (L:D). Two types of experiments were completely randomized to study the host size class preferences of L. dactylopii and C. perminutus In nochoice tests the parasitoids were provided with P. m inor of a particular host size class, and in choice tests the parasitoids were allowed to choose the most preferred hosts from a population of P. minor of all 5 size classes. For all experiments, a 30 mm x 30 mm leaf disc of cacao was placed, abaxial side up on to a moistened filter paper held in a 50 mm x 15 mm Petri dish In the nochoice tests, ten mealybugs from one of the five host size classes were transferred from the container onto each leaf disc using a fine camel hair brush. In the choice tests, t wo mealybugs from each of the five host size classes were transferred onto each leaf disc Mealybugs in size classes 14 were allowed to settle on the leaf discs for at least 12 hours prior t o the start of the experiment. Because adult females (size class 5 ) had begun to produce waxy filaments for the ovisacs they were transferred onto leaf discs and isolated in their respective arenas 48 hours before the start of the experiment. This ensured that these females were allowed to deposit eggs in the ovisacs. O ne female parasitoid from each species was introduced into a separate arena. Twenty replicates were set up for the nochoice tests, and forty replicates were set up for the choice tests for each parasitoid species. A diluted honey solution was streaked ont o the inside of each cover as a food supplement. The parasitoids were removed from the arenas after 60 min. Mealybugs exposed to L. dactylopii were removed at 24 h ours when eggs were vis i ble Eggs of C. perminutus less than 24 h ours

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103 old were difficult to o bserve under magnification. Therefore, mealybugs exposed to this parasitoid were removed from the arenas at 48 h ours when the eggs were apparent Each mealybug was placed in a drop of 70% ethanol on a microscope slide and dissected along the dorsum with a micropin. The mealybug was gently pressed with a cover slip to expel the body contents and any parasitoid eggs into the ethanol. A compound microscope ( 100x ) was used to view and count the number of eggs. Eggs of L. dactylopii were larger than those of C. perminutus but both were oblong with short stalks. Size class preference was determined by the parasitism rate (the proportion of hosts parasitized) and by the number of eggs oviposited per host size class. Interspecific C ompetition Third instar immature females from host size class 3 (0.80 1.50 mm) were collected as previously outlined and held for < 1 hour in a polyethylene container before the start of the experiment. A potato with a single sprout 23 cm was placed in a similar container and 10 individuals were transferred onto each sprout. Mealybugs were allowed to settle on the potato sprouts for 12 hours before introduction of the parasitoids and a diluted honey solution was streaked into each container O ne female parasitoid of each species was intro duced into the same container and left to forage for 24 h ours T he experiment was replicated t hirty times After remov ing the parasitoids, the mealybugs were held on the potatoes under the enivironmental conditions described above. The potatoes were examin ed with a dissecting microscope ( 5 10x ) daily starting at day 12 and continuing through day 21 and any mummies p re sent were transferred into individual gelatin capsules and held until adult emergence. The adult parasitoid emergence rate (the proportion of adult parasitoids that emerged) for each species was

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104 used to determine which species had a higher success rate of parasitism when offered concurrently to mealybugs in host size class 3 Data Analysis The effect of host size class on the parasitism rates was analyzed using a chi square test of independence, which compared distributions of these rates across host size classes for the parasitoid species separately ( PROC FREQ SAS Institute 2002 ). The effect of host size class on the number of eggs per paras itized host was analyzed separately for each species by the Kruskal Wallis test (PROC NPAR1WAY, SAS Institute 200 2 ). Analys es 0.05. Tests for normality and homogeneity of variances ( PROC UNIVARIATE SAS Institute 200 2 ) were performed prior to using the non parametric analysis The difference in parasitism rates on h ost size class 3 for L. dactylopii and C. perminutus was analyzed using the chi square test for equal proportions, which compared distributions of the parasitism rates by the two species ( PROC FREQ SAS Institute 200 2 ). Results Host S ize C lass P reference In the no choice tests, the proportion of mealybugs successfully parasitized by L. dactylopii differed significantly among the host size classes ( 2 = 46.30; df = 2 ; P < 0.0 5 ). S ize class 4 was the most parasitized ( 0. 68), followed by size class 5 ( 0. 50) and size class 3 ( 0. 34) (Fig. 6 1 A ). L. dactylopii females were observed ovipositing via the pumping action of their abdomens, or at least attempting to oviposit in size classes 1 and 2, but no eggs were recovered from these size classes. In the no choice tests, the proportion of mealybugs successfully parasitized by C. perminutus differed significantly

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105 among the host size classes ( 2 When offe red a choice between all host size classes, the proportion of mealybugs successfully parasitized by L. dactylopii also differed significantly among the size classes ( = 13.25; df = 2 ; P < 0.0 5 ). S ize class 2 was the most parasitized ( 0. 52), while size classes 1 and 3 were parasitized at similar rates ( 0. 33) (Fig. 6 1 B ). C. perminutus females were observed ovipositing into size classes 4 and 5, but no eggs were recovered. 2 = 8.37 ; df = 2 ; P < 0.0 5 ). L. dactylopii females parasitized size class 4 ( 0. 6875) to a greater extent than either size class 5 ( 0. 4750) or size class 3 ( 0. 5125) ( Fig. 6 2 A ). Likewise, no eggs were recovered in the choice tests, but females were observ ed ovipositing or at least attempt ing to oviposit in size classes 1 and 2. In the choice tests, the proportion of mealybugs successfully parasitized by C. perminutus was also significantly different among the host size classes ( 2 The number s of eggs oviposited across host size classes by L. dactylopii w ere similar for no choice and choice tests; therefore, the data were pooled for analys i s. Host size class had a significant effect ( = 81.28 ; df = 2 ; P < 0.0 5 ). C. perminutus females parasitized a greater proportion of mealybugs in host size class 2 ( 0. 6625) than either size class 1 ( 0. 5125) or size class 3 ( 0. 3750) (Fig. 6 2 B ). Similar to the nochoice experiments, no eggs of C. perminutus were recovered from size classes 4 and 5. 2 The number s of eggs oviposited across host size classes by C. perminutus were also similar for no choice and choice tests A fter pooling the data, t he number of oviposited eggs was significantly different among the size classes ( = 6.53 df = 2, P < 0.0 5 ), with more eggs being recovered from host size class 4 (>1) than from the other two size classes (<1). 2 = 13.75; df = 2; P

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106 < 0.0 5 ), w ith 1.2 eggs/mealybug from size classes 2 and 3 and 1.02 eggs/mealybug from size class 1 Interspecific C ompetition Wh en mealybugs from host size class 3 were exposed to the two parasitoids, significantly more L. dactylopii emerged than C. perminutus ( 2Discussion = 28.17; df = 1; P < 0.0 5 ). A higher proportion of recovered parasitoids were L. dactylopii (0.70) as compared to C. p erminutus (0.29) Less than half of the exposed mealybugs (0.44) were either not parasitized or the immature parasitoids did not survive. Host selection behavior by L. dactylopii and the resulting egg distribution in P. minor demonstrated that this species preferred larger /older host stages Smaller instars (size classes 1 and 2) in nochoice and choice tests were never parasitized, and could be considered invulnerable to attack by L. dactylopii Female parasitoids were observed using their hind legs to remove these smaller mealybug hosts stuck in their ovipositors A preference for larger/older host stages has been reported for other encyrtids. For example, Anagyrus mangicola Noyes parasitized all developmental host stages of Rastrococcus i nvadens Williams but preferentially attacked the larger/older stages instead of the smaller/younger ones (BokononGanta et al. 1995). In this study, host selection behavior by C. perminutus and its resulting egg distribution in P. minor demonstrated that this parasitoids productivity relied primarily on host size class es 1 3 but with higher parasitism rates for size class 2. Availability of sufficient smaller/younger host stages will therefore maximize performance of C. perminutus Although de Jong & va n Alphen (1989) reported that the incidence of superparasitized P. citri hosts by L. dactylopii was negligible, females in this study were

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107 unable to recognize superparasitized hosts and oviposited slightly more eggs into their preferred host size/stage. Li kewise, C. perminutus females oviposited more eggs into their preferred host size/stage, but this behavior was previously documented across three preferred stages of P. citri by Ceballo & Walter (2004) Solitary mealybug parasitoids parasitizing larger /old er hosts generally oviposit multiple eggs into the same host (Sagarra & Vincent 1999; Heng Moss et al. 2001). However, these cases involved oviposition into the preferred sizes/stages and not necessarily into the larger/older hosts. This behavior suggested that the preferred host sizes/stages were more suitable in terms of host quality. Ultimately, these decisions affect f itness of the parasitoid progeny. T his study did not address mealybug immune responses, but there was some evidence of encapsulated C. perminutus eggs in the form of granular structures during dissection of the larger hosts from the no choice and choice tests However, there were no signs of encapsulation of L. dactylopii eggs by the larger P. minor sizes/stages that were attacked. The eg gs of this parasitoid were also not encapsulated by the related species, P. citri (de Jong & van Alphen 1989; Blumberg & Van Driesche 2001) However, P. citri P. ficus Planococcus vovae (Nasonov), and one unrelated species, Pseudococcus cryptus Hempel, e ncapsulated the eggs of Anagyrus pseudococci (Girault) (Blumberg et al. 1995). This immune response appears to be common in older mealybugs as noted by Sagarra et al. ( 2000) who reported that encapsulation was higher for Anagyrus kamali Moursi eggs ovipos ited in third instars and adult females of Maconellicoccus hirsutus (Green)

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108 Both L. dactylopii and C. perminutus emerged from size class 3 mealybug s when P. minor was offered concurrently to both species This finding implied that although the two parasi toids exploited preferred host sizes/ stages, there was some overlap for third instar female nymphs. A comparison of the host selection behavior of L. dactylopii and Leptomastidea abnormis (Girault) (Encyrtidae) another solitary endoparasitoid, showed that host sizes of P. citri parasitized by the two species differed considerably when evaluated separately ( de Jong & van Alphen 1989; Cadee & van Alphen 1997) Since L dactylopii d id not oviposit in hosts belonging to size class 2 (de Jong & van Alphen 1989) Cadee & van Alphen ( 1997) suggested that L abnormis could benefit numerically from that portion of the mealybug population that goes unparasitized by the former because it results in control of the number of mealybugs that attain further grow th. Further investigation is therefore needed to assess the implications of this apparent overlap of host size/stage of P. minor between L. dactylopii and C. perminutus under laboratory conditions. They appeared to target different sizes/stages when they were invest igated under field conditions (Francis, Chapter 5) If these two species are released together L. dactylopii will attack larger mealybug stages, while C. perminutus will target the smaller ones suggesting a good level of complementarity between the two. Similar patterns of host size preference have been reported for these parasitoids using P. citri as the host (Bartlett & Lloyd 1958; Lloyd 1958; de Jong & van Alphen 1989; Ceballo & Walter 2004). In the event of new outbreaks, biological control practi ti o ners can use this knowledge in the decisionmaking process to release the more suitable parasitoid at that time based on composition of the pest population in the field.

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109 Fig ure 6 1. Proportion o f P. minor parasitized in nochoice preference tests. A) Mealybug host size classes parasitized by L. dactylopii B) Mealybug host size classes parasitized by C. perminutus C hi square test of independence was used to compare distribution of parasitism rat es across host size classes. Individual mealybug s were divided into the following size classes: 1st instar nymphs or size class 1 (0.25 0.50 mm), 2nd instar nymphs or size class 2 (0.50 0.80 mm), 3rd instar immature females or size class 3 (0.80 1.50 mm), adult females without ovisacs or size class 4 (>1.50 mm), and females with 2day old ovisacs or size class 5 (>3.0 mm including ovisac). A B

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110 Fig ure 6 2 Proportion of P. minor parasitized in choice preference tests. A) Mealybug host size classes parasitized by L. dactylopii B) Mealybug host size classes parasitized by C. perminutus Chisquare test of independence was used to compare distribution of parasitism rates across host size classes. Individ ual mealybugs were divided into the following size classes: 1st instar nymphs or size class 1 (0.25 0.50 mm), 2nd instar nymphs or size class 2 (0.50 0.80 mm), 3rd instar immature females or size class 3 (0.80 1.50 mm), adult females without ovisacs or siz e class 4 (>1.50 mm), and females with 2day old ovisacs or size class 5 (>3.0 mm including ovisac). A B

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111 CHAPTER 7 DEVELOPMENTAL TIME, LONGEVITY AND LIFETI ME FERTILITY OF LEPTOMASTIX DACTYLOPII HOWARD AND COCCIDOXENOIDES PERM INUTUS GIRAULT, PARASITOIDS OF PL ANOCOCCUS MINOR (MASKELL) Leptomastix dactylopii Howard and Coccidoxenoides perminutus Girault along with several key predators were found attacking the passionvine mealybug, P lanococcus minor (Maskell) in Trinidad ( Francis, Chapter 4 ). Successful control of Planococcus citri (Risso) using these encyrtid wasps separately or together, along with other natural enemies has been reported in several countries (Bartlett & Lloyd 1958; Bennett 1959; Meyerdirk et al. 1978; Dean et al. 1983; Summy et al. 1986; Smith et al. 1988; Mani 1994). While these species have been well studied as parasitoids of P. citri and to a lesser extent, P ficus (Signoret) (Golberg 1982; Tingle & Copland 1988 ; Walton & Pringle 2005), there are no comparable biological studies that detai l their suitability as parasitoids of P. minor Developmental time, adult longevity and lifetime fertility are important fitness parameters when evaluating a biological control agent. The goal of this study was to investigate the suitability of these pop ulations of L. dactylopii and C. perminutus as biological control agents against P. minor The specific objectives were 1 ) to determine the optimal physiological host size classes for the development of each parasitoid species based on the proportion of em erged adult parasitoids, their developmental time, and the sex ratio for L. dactylopii only ; 2 ) to determine the adult longevity of each species ; and 3 ) to determine their lifetime fertility when provided with P. minor as the mealybug host The data from t his study will be useful in predicting the performance of these encyrtid parasitoids as biological control agents for P. minor and critical in the design and implementation of mass rearing and release programs

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112 Materials and Methods Maintenance of Host Material and Insect Colonies Prior to sprouting, potatoes ( Solanum tuberosum L.) were soaked in a 1% bleach solution for 510 min. They were then washed and rinsed with clean water and left to dry. The potatoes were placed on plastic trays for sprouting in a room at 25 2oMealybugs were reared on sprouted potatoes in an isolated laboratory room. Infested potatoes were maintained in the d ark at 25 3 C 60 10% RH and complete darkness at CAB International Caribbean and Latin America Regional Office, Curepe, Trinidad. oC and 65 5% RH. Each week, 2436 potatoes were individually infested with 46 adult female mealybugs having well formed ovisacs. Weekly infestations ensured a continuous supply of different mealybug developmental stages. The populations consisted mainly of 1st and 2nd instar nymphs 12 weeks after infestation. After three weeks, the populations consisted mainly of 3rdCultures of the two parasitoid species were established using mummified mealybugs collected from sentinel trap potatoes placed at Fishing Pond, Trinidad (Francis, Chapter 5 ) M ummies from each species were kept indi vidually in separate gelatin capsules. Mealybugs parasitized by L. dactylopii had light brown mummies, while those for C. perminutus were light green in color. The initial batch of parasitoids consisted of at least 10 pairs (F/M) of adult L. dactylopii and 6 adult female C. perminutus T he latter species is thought to be exclusively thelytokous (Davies et al. 2004). Upon emergence, adults were used to set up maintenance cultures, which supplied parasitoids for all experiments The parasitoid cultures were k ept separate in instar nymphs, while females with ovisacs appeared at 45 weeks after infestation. This procedure was modified based on metho ds described by Meyerdirk et al. (1998).

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113 30 cm3 white plastic rearing cages (Bug Dorm 1, Bioquip, Rancho Dominguez, CA) containing 24 potatoes infested with 1st to 3rd instars for C. perminutus and 3rd instars to adult females for L. dactylopii parasitization. These parasitoids were also supplied diluted honey on cotton wicks. Room conditions were kept at 25 1oHost Stage Selection C, 60 5% RH, under a photoperiod of 11:13 (L:D). The light source was provided by day light fluorescent lamps. T here was some overlap in size within the different developmental instars due to variation in size at molting. Therefore, s ize was used as the criterion to categorize mealybug host stages as detailed for P. citri by de Jong & van Alphen (1989). During Feb ruary 2009, individuals of the following instars were collected for the developmental time experiments: 1st instar nymphs or size class 1 (0.25 0.50 mm), 2nd instar nymphs or size class 2 (0.50 0.80 mm), 3rd instar immature females or size class 3 (0.80 1.50 mm), adult females without ovisac s or size class 4 (>1.50 mm), and females with 2day old ovisacs or size class 5 (>3.0 mm including ovisac). Thirdand fourthinstar immature males and adult males were not used in the experiments because preliminary tests showed that neither parasitoid attacked these stages. M ealybugs were collected from the colony potatoes using a fine camel hair brush They were individually measured by placing each individual on a measurement scale (0.004.00 mm) outlined with lead pencil on a sheet of white rectangular print paper. Once measured, 200 mealybugs from each class were transferred into labeled polyethylene containers (size classes 15) for brief storage period prior to the start of the experiments. Mealybugs were used within 1 h our after collection to minim ize any ill effects of not feeding. All experiments were

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114 conducted at 25 1oDevelopmental Time C and 60 5% RH in a separate controlled environment room under day light fluorescent lamps. Based on results of the host size class preference experiments (F rancis, Chapter 6) and preliminary experiments on developmental time in size classes 15 for each parasitoid species size classes 3 5 were selected for studies on developmental time in L. dactylopii while size classes 1 3 were selected for C. perminutus Neither parasitoid developed and successfully emerged from the omitted size classes. A potato with a single sprout 23 cm long was placed in a polyethylene container (12 cm (dia.) x 8 cm (height) with a meshcovered lid) and 10 mealybugs from each host si ze class were transferred onto the sprout using a fine paintbrush. Mealybugs were allowed to settle on the sprouts for 12 h ours before the addition of the parasitoids except for adult females with ovisacs (size class 5) Adult females that had begun to secrete the waxy filaments for the ovisacs were transferred onto the potato sprouts 48 hours before the start of the experiment. This ensured that these females were allowed to deposit eggs in the ovisacs. A diluted honey solution was streaked into each cont ainer O ne female parasitoid of each species was introduced and allowed to forage for 24 h ours Twenty replicates were set up for each size class/parasitoid species. After removal of the parasitoids, the mealybugs were kept under the same controlled envi ronment conditions. They were examined using a dissecting microscope at 12 days after exposure to L. dactylopii and at 21 days after exposure to C. perminutus All mummified individuals were isolated in individual gelatin capsules and held until adult emer gence. T he adult parasitoid emergence rate (the proportion of emerged adult parasitoids) from each host size class, the sex ratio (the proportion of

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115 females for L. dactylopii only) from each host size class, and the developmental time (in days) of each species were determined. Adult L ongevity and L ifetime F ertility Within 24 hours of emergence, 50 pairs of L. dactylopii (M/F) were collected from the parasitoid colony and transferred into individual gelatin capsules with a minute drop of diluted honey plac ed at one extreme end of the capsule as food They were held for 24 hours before the experiment to ensure successful mating. Similarly, within 24 hours of emergence, 50 C. perminutus females transferred and encapsulated, but they were held for only 2 4 hours because of the short life span of this species (Ceballo & Walter 2004). Mealybugs were not provided to either parasitoid species for oviposition during the holding period. Two treatments were used to assess the effect of supplemental diet on adult long evity and lifetime fertility in these species. Parasitoids were provided with honey in the first treatment and none in the second treatment. I nfested potatoes consisting mainly of 1st to 3rd instar nymphs for C. perminutus and infested potatoes with mainly 3rd instar nymphs to adult females for L.dactylopii were placed in separate labeled container s. A honey solution was streaked into each container on day 1 and replenished as necessary This procedure was repeated, but no honey was provided in the second set of containers. The pair of L. dactylopii (F/M) and the female C. perminutus were introduced into their separate containers and allowed to forage. There were 25 cohort s/replicates for each treatment. The containers were examined every 24 hours for dead parasitoids Live parasitoids were transferred to fresh containers with infested potatoes after 72 hours until the death of each female parasitoid. Thereafter, c ontainers were examined daily for the emergence of parasitoid progeny. Progeny was collected

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116 and counted for each species The sex of L. dactylopii was determined by antennal structure with f emales hav ing clavate antennae and males hav ing filiform antennae. Environmental c onditions were maintained under similar conditions as previously outlined. Dat a Analysis The effect of host size class on the adult parasitoid emergence rate and the sex ratio ( L. dactylopii only) were analyzed separately using the chi square test of independence, which compared distributions of these dependent variables across host size classes for each parasitoid species (PROC FREQ, SAS Institute 200 2 ). The effect s of host size class on developmental time and sex of L. dactylopii was analyzed by two way ANOVA (PROC GLM, SAS Institute 200 2 ). The effect of host size class on the dev elopmental time for C. perminutus was analyzed by oneway ANOVA (PROC GLM, SAS Institute 200 2 ) with host size class as the source of variation. Prior to doing the ANOVA, tests for normality and homogeneity of variances were performed (PROC UNIVARIATE, SAS Institute 200 2 ). Analyses of data from th e se experiment s were Tukeys test. A t test was used to determine the effect of supplemental diet on adult longevity and lifetime fertil ity (progeny production) of L. dactylopii Institute 200 2 ). Similarly, a t test was used to determine the effect of supplemental diet on adult longevity and lifetime fertility of C. perminutus Institute 2002 ). Adult diet was the independent variable (source of variation), while adult longevities and lifetime fertility were the dependent variables. Tests for normality and

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117 homogeneity of variances of each dependent variable (PROC UNIVARIATE, SAS Institute 2002 ) were performed prior to analysis. Results Developmental Time The proportion of L. dactylopii that successfully emerged from mummies differed significantly among the host size classes ( 2 = 54.25; df = 2; P < 0.05 ). P arasitoid emergence was highest from size class 4 ( 0.85), while size classes 5 and 3 had emergence rates of 0. 755 and 0. 525, respectively (Fig. 7 1 ). Host size class also had a significant effect on the proportion of female progeny ( 2The proportion of C. perm inutus that successfully emerged from mummies also differed significantly among the host size classes ( = 153.39; df = 4; P < 0.0 5 ). A low proportion (<0.09) of emerged L. dactylopii were females from size class 3, while a greater proportion (>0.74) of those that emerged from size classes 4 and 5 were females (Fig. 7 2 ). 2The developmental time of L. dactylopii differed significantly among the host size classes and by parasitoid sex ( F = 80.20; df = 5, 425; P < 0.0 5 ). Males developed slower than females from size class 3. Males took 19.45 0.17 days while females took 18.11 0.25 days (Fig. 7 4 ). Developmental time for females was slower than males when oviposition occurred in mealybugs from size classes 4 and 5. Females took 17.28 0.11 and 17.93 0.10 days from size classes 4 and 5, whil e males took 16.84 0.15 and 17.27 0.14 days from size classes 4 and 5, respectively (Fig. 7 4 ). = 81.29; df = 2; P < 0.0 5 ). P arasitoid emergence was highest from size class 2 ( 0. 755), while size classes 1 and 3 had emergence rates of 0. 375 and 0. 355, respectively (Fig. 7 3 ).

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118 The developmental time of C. perminutus differed significantly among the host size classes ( F = 219.12; df = 2, 296; P < 0.0 5 ). Individuals emerging from s ize classes 1 to 3 took 25.17 0.12 days, 27.15 0.08 days, and 28.80 0.14 days, respectively (Fig. 7 5). Adult Longevity Provision of supplemental diet did not affect longevity of L. dactylopii females ( t = 1.84; df = 48; P > 0.0 5 ). Their mean longevi ty when fed honey was 31.40 1.20 days, while females not fed honey lived 28.40 1.12 days. Likewise, supplemental diet did not affect longevity of L. dactylopii males ( t = 1.49; df = 48; P > 0. 05 ). The mean adult longevity of honey fed males was 19.24 1.08 days, while males not fed honey lived 17.32 0.69 days. However, longevity of C. perminutus females was significantly affected by supplemental diet ( t = 9.40; df = 48; P < 0.0 5 ). Honey fed females lived 7.44 0.41 days, while females not fed honey lived only 3.20 0.19 days Lifetime Fertility Progeny production of L. dactylopii females was not significantly affected by supplemental diet. Honey fed females averaged 142.04 5.56 eggs during their oviposition period, which was similar to those fem ales not fed honey (138.56 5.74 eggs). Progeny production of C. perminutus females was significantly affected by supplemental diet ( t = 7.76; df = 48; P < 0.0 5 ). Honey fed females averaged 242.04 5.52 eggs during their oviposition period, while females not fed honey averaged 149.8 10.52 eggs.

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119 Discussion F itness parameter s such as developmental time, longevity and lifetime fertility of parasitoids are important in the assessment of biological control agent s. T he preferred host sizes/stages that were parasitized at higher rates supported higher survivorship for both L. dactylopii and C. perminutus Most of the eggs oviposited into mealybugs in size classes 4 and 5 by L. dactylopii resulted in complete development by the parasitoid, while only about a half of those oviposited into younger hosts (size class 3) fully developed. Likewise, most of the eggs oviposited by C. perminutus into mealybugs in the preferred size class 2 emerged, while those oviposited into mealybugs in size classes 1 and 3 had an eme rgence rate of less than 40%. However, there were signs of encapsulated eggs in the larger size classes. These findings concur with Ceballo & Walter (2004), who reported signs of encapsulated C. perminutus eggs in the later host sizes/stages of P. citri Femalebiased sex ratios in older /larger hosts are frequently reported in hymenopteran parasitoids (King 1993). In parasitic Hymenoptera, female eggs are preferentially laid in larger hosts compared to male eggs, which are laid in smaller hosts (King 1987) Charnov et al. (1981) reported that more females may be produced from larger hosts because of the enhanced nutritional requirement and reproductive benefits for the offspring. As expected, the proportion of L. dactylopii females increased as host size in creased. The femalebiased sex ratios across host size classes were also similar to those reported by de Jong & van Alphen (1989). This is advantageous when mass producing L. dactylopii because the preferred mealybug sizes/stages will have sex ratios skewe d in favor of females.

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120 In terms of inherent advantages for thelytokous species such as C. perminutus producing progeny that are entirely female means that no mating is required to produce the next generation of ovipositing females. Although thelytokous, Flanders (1953) reported that males may be produced by exposing females to prolonged temperature extremes of 35oThe biological trend of decreased developmental time in larger hosts is fairly common in parasitoids (Liu & Stansly 1996; Karamaouna & Copland 2000; Neveu et al. 2000). Developmental times of both sexes of L. dactylopii were faster f or the largest host size classes (4 and 5) compared to size class 3. In a nother example, A nagyrus kamali Moursi had fast er developmental times when it parasitized thirdinstar nymphs and adult female M hirsutus than when it parasitized first and secondinstar nymphs (Sagarra & Vincent 1999). Likewise, Gyranusoidea tebygi Noyes had similar developmental times for secondand thirdinstar nymphs of R astrococcus i nvadens Williams but longer developmental time for first instar nymphs (Boavida et al. 1995). In contrast to these examples, developmental time of C. perminutus in mealybugs belonging to host size class 1 was faster than those mealybugs in size classes 2 and 3. Ceballo & Walter (2004) also reported that the developmental time for the same parasitoid was faster in the smaller stages of P. citri than in the larger ones. This suggests that parasitoids emerging from fir st instar nymphs possibly possess some added fitness value such as immune response mechanisms despite their relative small size and would be a useful next step of investigation. C throughout the embryonic and larval stages. N utritional status is a critical factor in determining the longevity and lifet ime fertility of parasitoids (Tingle & Copland 1989). Since s ynovigenic parasitoids emerge with none

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121 or only part of their total mature egg complement and develop eggs throughout their adult life, most species tend to maximize their reproductive potential by obtaining supplemental nutrition (Bartlett 1964; Jervis & Kidd 1986) Interestingly, L. dactylopii females provided with honey neither produced a greater number of progeny nor lived longer than those not fed honey. A possible explanation is that L. dact ylopii host feeds (de Jong & van Alphen 1989) and was therefore able to utilize mealybug hosts in this manner. Also, both sexes were observed feeding on the honeydew secreted by mealybugs on the sprouted potatoes. Therefore, females deprived of supplemental diet, relied on other available resources to maximize their longevity and egg production. Conversely, C. perminutus females, which are not known to host feed (Ceballo & Walter 2004), clearly benefitted when provided with supplemental diet with increased longevity and egg production when compared to those without the honey meal. As reported by Ceballo & Walter (2004) and corroborated by this study, this wasp tends to have a high reproductive potential, but is relatively short lived Females of L. dactylopi i outlived males. Greater female longevity has been reported in other parasitoid species. In longevity studies of A kamali females lived longer than males (Sagarra et al. 2000). I nformation on the basic biology of these parasitoids could be applied to im proving mass rearing program s involving P. minor In order to achieve highquality female progeny, the mass rearing protocol should be designed so that a high percentage of the mealybug population consists of the appropriate host size/stage to guarantee fe male progeny of optimal developmental time, high emergence rate, and a female biased sex ratio With regards to L. dactylopii the use of larger mealybug hosts should be the most productive way to produce mainly female offspring. Conversely, populations co nsisting

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122 primarily of secondinstars will benefit the production of C. perminutus Based on the longevity and fertility experiments supplemental diet appears to be more critical to the performance of C. perminutus Knowledge gained from this study also has the potential to improve additional aspects such as storage, shipping, and release of these natural enemies

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123 Figure 7 1 Proportion of L. dactylopii that emerged from mealybug host size classes Chisquare test of independence was used to compare distribution of emergence rates across host size classes. Mealybugs from the following size classes were used : 3rd instar immature females or size class 3 (0.80 1.50 mm), adult females without ovisacs or size class 4 (>1.50 mm), and females with 2 day old ovisacs or size class 5 (>3.0 mm including ovisac).

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124 Figure 7 2 Sex ratio of L. dactylopii expressed as proportion of females Chisquare test of independence was used to compare distribution of femal es across host size classes. Mealybugs from the following size classes were used: 3rd instar immature females or size class 3 (0.80 1.50 mm), adult females without ovisacs or size class 4 (>1.50 mm), and females with 2day old ovisacs or size class 5 (>3.0 mm including ovisac).

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125 Figure 7 3 Proportion of C. perminutus that emerged from mealybug host size classes Chisquare test of independence was used to compare distribution of emergence rates across host size classes. M ealy bugs from the following size classes were used: 1st instar nymphs or size class 1 (0.25 0.50 mm), 2nd instar nymphs or size class 2 (0.50 0.80 mm), and 3rd instar immature females or size class 3 (0.80 1.50 mm).

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126 Figure 7 4 Developmental time (in days) of L. dactylopii from different host size classes. Values are mean SEM M ealybug s from the following size classes were used: 3rd instar immature females or size class 3 (0.80 1.50 mm), adult females without ovisacs or size cl ass 4 (>1.50 mm), and females with 2day old ovisacs or size class 5 (>3.0 mm including ovisac).

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127 Figure 7 5 Developmental time (in days) of C. perminutus from different host size classes. Values are mean SEM M ealybug s fr om the following size classes were used: 1st instar nymphs or size class 1 (0.25 0.50 mm), 2nd instar nymphs or size class 2 (0.50 0.80 mm), and 3rd instar immature females or size class 3 (0.80 1.50 mm

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128 CHAPTER 8 CONCLUSION The passionvine mealybug, Plan ococcus minor (Maskell), is a polyphagous pest that can damage a large number of tropical and subtropical fruits, vegetables, and ornamental plants. A native of south Asia, P minor is presently established in several countries in the Caribbean, South and Central America However, it has not been recorded causing serious economic damage in any of these localities Given its invasive characteristics, the potential to cause serious economic and ecological damage, and its high rate of interception at U S port sof entry, this mealybug is regarded as a high risk pest. D espite the high pest priority, there was a lack of information on the biology and ecology of P minor and its natural enemies. This dissertation focused on generating relevant information that would be critical to address these shortcomings and ultimately lead to the development of mitigation measures to deal with this pests unwanted entry into the continental U S and its spread to uninfested Caribbean Basin countries. The first set of objectives sought to determine key developmental and reproductive parameters of P. minor over a range of different temperatures. The second set of objectives focused on assess ing the occurrence and pest status of P. minor in Trinidad and evaluat ing the potential for biological control via identif ication of existing natural enemies The third set of objectives sought to determine the relative abundance and seasonal incidence of two identified primary parasitoids and to determine the influence of resident ants on their activity The fourth set of objectives sought to determine the host size class preferences of each primary parasitoid, and the degree of interspecific competition between the species. The final set of objectives sought to d etermine the

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129 optimal physiologic al host size classes for development of each primary parasitoid, the adult longevity of each species, as well as their lifetime fertility The l ife history study of P minor indicated that it could successfully develop, survive, and reproduce across a rel atively wide range of temperature. It complet ed its life cycle at temperatures ranging from 20 to 29C. Egg to adult emergence occurred in approximately 30 days or less at optimal temperatures, indicating it has the potential for rapid development on ideal host plants and under suitable conditions. However, the low minimum temperature threshold and high thermal constant s suggested that this mealybug possibly can tolerate or even thrive in higher latitudes. Information gathered from th is stud y will provide t he insight needed to better understand the life history of P minor in relation to temperature. It will also go a step further by utilizing developmental and reproductive data to incorporat e into predictive distribution models such as NAPPFAST and also to optimize mass rearing procedures for biological control purposes Surveys conducted on the island determined that populations of P. minor were very low and sparse and found mainly on cacao, which appear ed to be a preferred host plant. Additional s urveys at cacao field sites throughout the island later revealed that a well established natural enemy complex consisting of generalist predators and two key primary encyrtid parasitoids was exerting effective control on these populations This discovery was made possible by the use of sentinel traps consisting of wire cages containing infested potatoes with the target mealybug to attract the natural enemies. The rationale for this method was to compensate for the low, sparsely distributed populations of P. minor that also resembled other morphologically similar mealybug

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130 species The success of this method means that similar exercises can be undertaken in other infested areas to identify new candidate natural enemies or to document the existing natural enemy complex It is possible that P. minor was accidentally introduced a very long time ago to Trinidad and misidentified from the onset as P. citri given the taxonomic complexities that exist between the two species. Using sex pheromonebaited traps only males of P. minor were recovered and this finding further supports th e argument that only P. minor is present on the island. The baited traps also provide a highly sensitive detection tool for this mealybug. Given that P. minor was not a recent introduction, it is also likely that some of its natural enemies, namely the two identified primary parasitoids, L eptomastix dactylopii and C occidoxenoides perminutus were introduced fortuitously and together with other resident and introduced natural enemies prevented P. minor from becoming a pest of economic importance Given their host specificity, these encyrtid parasitoids appear to be promising candidate natural enemies However, the role of several coccinellids and the ubiquitous D iadiplosis coccidarum should not be discounted, as they contribute to the suppression of P. minor Based on the results of the field assessment of the primary parasitoids, L. dactylopii was the main contributor to the mortality of P. minor These species exploited different host sizes/ stage s and persisted thoughout t he sampling period at very low pest populations These traits suggest that they have the potential to complement each other as biological control agents In the cacao agroecosystem, ants when present, affected the foraging ability of the parasitoids Depending on the setting, c ountermeasures to ant interference should therefore be implemented when biological control is being

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131 contemplated as a component of the control strategy for P. minor Although there was little evidence to indi cate whether seasonal differences affected the performance of these parasitoids this factor should be taken into account to minimize its effects on a natural enemy release program The p referred host sizes/ stages of L. dactylopii and C. perminutus var ied, with the former attacking larger hosts, while the latter targeted smaller hosts. These preferences suggest ed minimal competition for suitable hosts and that a good level of complementarity exist ed between the two species However, L. dactylopii had hi gh er parasitism and a higher emergence rate than C. perminutus when the same mealybug size/ stage was offered concurrently Despite this apparent overlap between the two species given a choice and depending on the availability of hosts, neither species app ear to preferentially target this particular host size /stage In mass rearing systems, this type of knowledge on preferred host size/stage is critical to maximize parasitoid production and achieve high levels of efficiency. When key fitness parameters were assessed separately, C. perminutus successfully completed its development in smaller mealybug sizes/ stages, compared to L. dactylopii which successfully developed in the larger hosts, with a female biased sex ratio Developmental time decreased as host s ize increased in L. dactylopii Conversely as host size increased, developmental time increased in C. perminutus These findings have important implications for mass rearing programs where optimal developmental time is a key requisite, and a high proporti on of females is desirable given that they are responsible for direct mortality of the target pest. The thelytokous nature of C. perminutus appears to be beneficial under insectary conditions where this parasitoid

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132 can amass very large numbers over a few generations. While it had higher lifetime fertility, C perminutus was relatively short lived compared to L. dactylopii However, t he provision of supplemental food prolonged longevity and productivity of this species Planococcus minor was confirmed from south Florida in December 2010. This is the first record of th e mealybug in the continental U S. Whether it will develop into a major pest in south Florida and elsewhere is still unknown. However, given the invasive characteristics of P. minor Florida and the other southeastern states will need to implement contingency plans for its possible spread into new areas Fortunately, information gathered from these studies was quite timely and will allow state and federal agencies to provide a wider range of cont rol options for P. minor if the need arises

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148 BIOGRAPHICAL SKETCH Antonio William Francis was born in Basseterre, St. Kitts, West Indies, and graduated from Basseterre Senior High School in 1990. Thereafter, he worked for two years as a registry clerk in the Registrars Office of the Eastern Caribbean Supreme Court, Basseterre, St. Kitts. Antonio journeyed to Barinas State, Venezuela in 1992, where he completed the Diploma in Farm Management in 1 995. From 19952000, Antonio worked as a crops extension officer, and later as an entomology assistant on projects such the biological control of the pink hisbicus mealybug and the citrus blackfly in St. Kitts. The urge to pursue higher education ultimately led Antonio to Cameron University, Lawton, OK from 2000 to 2003 where he obtained a Bachelor of Science degree in Agricultural Sciences with a concentration in Environmental Science. After completing his Master of Science degree in Agricultural Science s with a concentration in Entomology at Florida A&M University, Tallahassee, FL in 2005, he officially entered the joint Ph.D. program in Entomology at the University of Florida/Florida A&M University in 2006. Antonio was involved in research of invasive mealybugs during the pursuit of his Ph D in Entomology. He received his Ph.D. from the University of Florida in the spring of 2011.