<%BANNER%>

Life History of Papaya Mealybug (Paracoccus marginatus) and the Effectiveness of Three Introduced Parasitoids (Acerophag...

Permanent Link: http://ufdc.ufl.edu/UFE0021652/00001

Material Information

Title: Life History of Papaya Mealybug (Paracoccus marginatus) and the Effectiveness of Three Introduced Parasitoids (Acerophagus papayae, Anagyrus loecki, and Pseudleptomastix mexicana)
Physical Description: 1 online resource (114 p.)
Language: english
Creator: Amarasekare, Kaushalya
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: acerophagus, anagyrus, biological, control, effectiveness, field, history, host, life, loecki, marginatus, mealybug, mexicana, papaya, papayae, paracoccus, plants, pseudleptomastix, temperature
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: Native to Mexico and Central America, papaya mealybug (Paracoccus marginatus) is an adventive pest insect that can damage a large number of tropical and subtropical fruits, vegetables, and ornamental plants in the US, the Caribbean, and the Pacific islands. It is an important pest in Florida, and potentially poses a threat to other states such as California, Hawaii, and Texas. Currently, three introduced parasitoids are used as biological control agents. Information on papaya mealybug and its parasitoids is scarce. In this dissertation, the life history of papaya mealybug in relation to temperature and host plants, and the biology and the effectiveness of its parasitoids were investigated. Temperature is one of the important abiotic factors that may decide the establishment and distribution of papaya mealybug into other areas in the US. Adult males and females required 303.0 and 294.1 degree-days, and 14.5 and 13.9?C, minimum temperature threshold, respectively. In addition, papaya mealybug was able to complete its life cycle on three ornamental plants, hibiscus, acalypha, plumeria, and the weed parthenium, which are commonly found plants in many US states. In the field, Acerophagus papayae provided better control than the other parasitoids. Pseudleptomastix mexicana was not observed, while Anagyrus loecki had lower parasitism. In the laboratory, all parasitoids were able to develop and emerged successfully in all stages of P. marginatus except for first-instar nymphs. Acerophagus papayae and P. mexicana preferred second-instar P. marginatus while A. loecki preferred third instars. Developmental times of A. papayae and A. loecki were similar but P. mexicana had a longer developmental time. Overall, A. papayae provided better control of the host, when alone or with the other two parasitoids. Pseudleptomastix mexicana was less competitive when mixed with A. papayae and A. loecki. Considering its low thermal requirement and high minimum temperature threshold, papaya mealybug has a smaller distribution range than anticipated. Southern parts of Texas and California, South Florida, and Hawaii are suitable areas for its development. Its final establishment and distribution may be influenced by other factors such as host plant range, and the rules and regulations governing plant movement from state to state.
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 Kaushalya Amarasekare.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Mannion, Catharine M.
Local: Co-adviser: Osborne, Lance S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021652:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021652/00001

Material Information

Title: Life History of Papaya Mealybug (Paracoccus marginatus) and the Effectiveness of Three Introduced Parasitoids (Acerophagus papayae, Anagyrus loecki, and Pseudleptomastix mexicana)
Physical Description: 1 online resource (114 p.)
Language: english
Creator: Amarasekare, Kaushalya
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: acerophagus, anagyrus, biological, control, effectiveness, field, history, host, life, loecki, marginatus, mealybug, mexicana, papaya, papayae, paracoccus, plants, pseudleptomastix, temperature
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: Native to Mexico and Central America, papaya mealybug (Paracoccus marginatus) is an adventive pest insect that can damage a large number of tropical and subtropical fruits, vegetables, and ornamental plants in the US, the Caribbean, and the Pacific islands. It is an important pest in Florida, and potentially poses a threat to other states such as California, Hawaii, and Texas. Currently, three introduced parasitoids are used as biological control agents. Information on papaya mealybug and its parasitoids is scarce. In this dissertation, the life history of papaya mealybug in relation to temperature and host plants, and the biology and the effectiveness of its parasitoids were investigated. Temperature is one of the important abiotic factors that may decide the establishment and distribution of papaya mealybug into other areas in the US. Adult males and females required 303.0 and 294.1 degree-days, and 14.5 and 13.9?C, minimum temperature threshold, respectively. In addition, papaya mealybug was able to complete its life cycle on three ornamental plants, hibiscus, acalypha, plumeria, and the weed parthenium, which are commonly found plants in many US states. In the field, Acerophagus papayae provided better control than the other parasitoids. Pseudleptomastix mexicana was not observed, while Anagyrus loecki had lower parasitism. In the laboratory, all parasitoids were able to develop and emerged successfully in all stages of P. marginatus except for first-instar nymphs. Acerophagus papayae and P. mexicana preferred second-instar P. marginatus while A. loecki preferred third instars. Developmental times of A. papayae and A. loecki were similar but P. mexicana had a longer developmental time. Overall, A. papayae provided better control of the host, when alone or with the other two parasitoids. Pseudleptomastix mexicana was less competitive when mixed with A. papayae and A. loecki. Considering its low thermal requirement and high minimum temperature threshold, papaya mealybug has a smaller distribution range than anticipated. Southern parts of Texas and California, South Florida, and Hawaii are suitable areas for its development. Its final establishment and distribution may be influenced by other factors such as host plant range, and the rules and regulations governing plant movement from state to state.
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 Kaushalya Amarasekare.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Mannion, Catharine M.
Local: Co-adviser: Osborne, Lance S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021652:00001


This item has the following downloads:


Full Text





LIFE HISTORY OF PAPAYA MEALYBUG (Paracoccus marginatus), AND THE
EFFECTIVENESS OF THREE INTRODUCED PARASITOIDS (Acerophagus papayae,
Anagyrus loecki, AND Pseudleptomastix mexicana)





















By

KAUSHALYA GUNEWARDANE AMARASEKARE


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

2007

































2007 Kaushalya Gunewardane Amarasekare

































Affectionately dedicated to my late parents









ACKNOWLEDGMENTS

The completion of this research would have been impossible but for the help of my

advisors, friends, colleagues and family. Whatever credit this research deserves should be shared

amongst all those whose help and support was invaluable throughout the course of my study.

A very special word of thanks goes to Dr. Catharine Mannion, my major advisor, for her

advice and guidance, throughout the study period. I thank my graduate advisory committee, Drs.

Lance Osborne (co-chair), Robert McSorley, Wagner Vendrame, and Nancy Epsky for their

advice, guidance, and encouragement throughout. A word of thanks must also be offered to the

staff at University of Florida, Entomology and Nematology Department, Gainesville, and the

Tropical Research and Education Center, Homestead, for their assistance and support at all

times. Special thanks are due to Joan Barrick and Kenneth Brown for helping me through the

bad times and sharing my joy through good times. Last but not least, I thank my family and all

my friends whose warmth of heart made all this possible: their loyalty is unforgotten and

unforgettable.









TABLE OF CONTENTS

page

ACKNOW LEDGM ENTS .......................... ........................... ... .... ............. 4

L IST O F T A B L E S ......... ...... ...................................................................................

ABSTRAC T ..........................................................................................

CHAPTER

1 INTRODUCTION ............... ................. ........... ................................. 11

M e a ly b u g s ................................................................ ................................................ 1 1
Genus Paracoccus ............................................................................ .............. ......... ........ 13
Paracoccus marginatus Williams and Granara de Willink..................... ...................13
H ost P lant Species ............................................................................ 14
T em perature ................................................................................................17
Chem ical Control of Papaya M ealybug...................................................................... ...... 18
B biological C control ........................ ................................................. ........... 19
Classical Biological Control of Papaya M ealybug............... .............................................. 19
P ara sito id s ............................. .. ........ ............. ........................................2 0
Acerophagus papayae Noyes and Schauff ............................. ................................... 21
Pseudleptomastix mexicana Noyes and Schauff ..................................................21
Anagyrus loecki N oyes and M enezes...................................... ........................ ......... 22
Developmental Time, Longevity, and Lifetime Fertility............................................ 22
Host Stage Susceptibility, Host Stage Suitability, and Sex Ratio .......................................24
Interspecific C om petition ............................................ .. .. ........... ......... 25
Research Objectives.............................. .. .... ..... ..................26

2 LIFE HISTORY OF Paracoccus marginatus WILLIAMS AND GRANARA DE
WILLING (HEMIPTERA: PSEUDOCOCCIDAE) ON FOUR HOST PLANT
SPECIES UNDER LABORATORY CONDITIONS .........................................................28

Introduction .......... ................................ ...............................................28
M materials and M methods ........................ .. ........................ .. .... ........ ........ 29
R results ................. .. .. ...... ................. .................................32
D discussion ................................... ...................................... ................ 34

3 EFFECT OF CONSTANT TEMPERATURE ON THE DEVELOPMENTAL
BIOLOGY OF Paracoccus marginatus WILLIAMS AND GRANARA DE WILLING
(HEM IPTERA : PSEUDOCOCCIDAE) ......... .. .............. .................. .....................40

Introduction.............................................................40
M materials and M methods ........................ .. ........................ .. .... ........ ....... .. 4 1
R e su lts ............................................ ................................................................ 4 5
D iscu ssion .......... ..........................................................47









4 HOST STAGE SUSCEPTIBILITY AND SEX RATIO, HOST STAGE SUITABILITY,
AND INTERSPECIFIC COMPETITION OF Acerophaguspapayae, Anagyrus loecki,
AND Pseudleptomastix mexicana: THREE INTRODUCED PARASITOIDS OF
Paracoccus marginatus WILLIAMS AND GRANARA DE WILLINK ..............................56

Introduction .......... ............................... ...............................................56
M materials and M methods ........................ .. ........................ .. .... ........ ........ 58
R e su lts ........................ ............................ ......................................... 6 3
D discussion .................................... ..................................... ................. 64

5 DEVELOPMENTAL TIME, LONGEVITY, AND LIFETIME FERTILITY OF
Acerophagus papayae, Anagyrus loecki, AND Pseudleptomastix mexicana; THREE
INTRODUCED PARASITOIDS OF Paracoccus marginatus WILLIAMS AND
G R A N A R A D E W IL L IN K ......... ................. ....................................................................72

Introduction .......... ................................ ...............................................72
M materials and M methods ........................ .. ........................ .. .... ........ ........ 73
R e su lts ........................ ............................ ......................................... 7 9
D discussion .................................... ..................................... ................. 80

6 FIELD ASSESSMENT OF THREE INTRODUCED PARASITOIDS OF Paracoccus
marginatus WILLIAMS AND GRANARA DE WILLINK (HEMIPTERA:
P SE U D O C O C C ID A E ) ...........................................................................................................87

Introduction....................................................................87
M materials and M methods ........................ .. ........................ .. .... ........ ........ 88
R e su lts ................ ...... ......... ............................................................................... 9 3
D discussion ................................................................... 95

7 SUMMARY AND CONCLUSIONS ..... .................... ............... 103

REFERENCE LIST .................................. .. .... .... ..................105

BIOGRAPHICAL SKETCH .............................. ................. 114









LIST OF TABLES


Table page

2-1 Mean number of days ( SEM) for each developmental stadium ofP. marginatus
reared on four host species......................................................................... .................. 38

2-2 Mean ( SEM) percent survival for each developmental stadium ofP. marginatus
reared on four host species ......... ................. ........................................ ............... 39

3-1 Mean number of days ( SEM) for each developmental stadium ofP. marginatus
reared at different constant temperatures.............................. ......................52

3-2 Mean ( SEM) percent survival for each developmental stadium ofP. marginatus
reared at different constant tem peratures ........................................ ........ ............... 53

3-3 Mean ( SEM) proportion of females, adult longevity, fecundity, pre-oviposition
and oviposition periods of P. marginatus reared at four constant temperatures ...............54

3-4 Summary of statistics and the estimates ( SE) of the fitted parameters of the linear
thermal summation model and the nonlinear Logan 6 model............... ... ............ 55

4-1 Mean percent parasitism ( SEM) of A. papayae, A. loecki, and P. mexicana reared
in different developmental stages of P. marginatus to evaluate host stage
susceptibility using no-choice tests........ ... ............... ............... 68

4-2 Mean proportion of females (sex ratio) ( SEM) of A. papayae, A. loecki, and P.
mexicana reared in different developmental stages of P. marginatus to evaluate host
stage susceptibility using no-choice tests. .............................................. ............... 69

4-3 Mean percent parasitism ( SEM) of A. papayae, A. loecki, and P. mexicana reared
in different stage combinations of P. marginatus to evaluate host stage suitability
using choice tests. .......................................... ............................ 70

4-4 Mean percent parasitism ( SEM) of combinations ofA. papayae, A. loecki, and P.
mexicana reared in second and third-instar P. marginatus to evaluate interspecific
com petitions of parasitoids. .................... ................................................... ..................... 71

5-1 Mean developmental time (egg to adult eclosion) in days ( SEM) for male and
female A. papayae, A. loecki, and P. mexicana reared in second instar, third-instar
female, and adult-female P. marginatus......................................... ........................ 84

5-2 Mean longevity in days ( SEM) for male (unmated and mated), and female
(unmated, mated-without oviposition, and mated-with oviposition) A. papayae, A.
loecki, and P m exicana. ......................... ...... ................................... .. .....85









5-3 Mean ( SEM) number of male and female progeny, cumulative progeny, sex ratio,
and reproductive period of mated and unmated A. papayae, A. loecki, and P.
m exicana ........................................................ .................................86

6-1 Mean ( SEM) number of mealybug destroyer (Cryptolaemus montrouzieri) adults
and larvae collected per cage from open sleeve cage and no cage treatments using
pooled data of 2005 and 2006 in three experimental locations ............ ................100

6-2 Mean ( SEM) number of ants and spiders collected from open sleeve cage and no
cage treatments using pooled data of 2005 and 2006 in three experimental locations....101

6-3 Individual and cumulative mean percent parasitism ( SEM) of P. marginatus by A.
papayae, A. loecki, and P. mexicana in open sleeve cage, and no cage treatments
using pooled data of 2005 and 2006 in three experimental locations...........................102









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

LIFE HISTORY OF PAPAYA MEALYBUG (Paracoccus marginatus), AND THE
EFFECTIVENESS OF THREE INTRODUCED PARASITOIDS (Acerophagus papayae,
Anagyrus loecki, and Pseudleptomastix mexicana)

By

Kaushalya Gunewardane Amarasekare

December 2007

Chair: Catharine Mannion
Cochair: Lance Osborne
Major: Entomology and Nematology

Native to Mexico and Central America, papaya mealybug (Paracoccus marginatus) is an

adventive pest insect that can damage a large number of tropical and subtropical fruits,

vegetables, and ornamental plants in the US, the Caribbean, and the Pacific islands. It is an

important pest in Florida, and potentially poses a threat to other states such as California, Hawaii,

and Texas. Currently, three introduced parasitoids are used as biological control agents.

Information on papaya mealybug and its parasitoids is scarce. In this dissertation, the life history

of papaya mealybug in relation to temperature and host plants, and the biology and the

effectiveness of its parasitoids were investigated.

Temperature is one of the important abiotic factors that may decide the establishment and

distribution of papaya mealybug into other areas in the US. Adult males and females required

303.0 and 294.1 degree-days, and 14.5 and 13.90C, minimum temperature threshold,

respectively. In addition, papaya mealybug was able to complete its life cycle on three

ornamental plants, hibiscus, acalypha, plumeria, and the weed parthenium, which are commonly

found plants in many US states.









In the field, Acerophagus papayae provided better control than the other parasitoids.

Pseudleptomastix mexicana was not observed, while Anagyrus loecki had lower parasitism. In

the laboratory, all parasitoids were able to develop and emerged successfully in all stages of P.

marginatus except for first-instar nymphs. Acerophagus papayae and P. mexicana preferred

second-instar P. marginatus while A. loecki preferred third instars. Developmental times of A.

papayae and A. loecki were similar but P. mexicana had a longer developmental time. Overall,

A. papayae provided better control of the host, when alone or with the other two parasitoids.

Pseudleptomastix mexicana was less competitive when mixed with A. papayae and A. loecki.

Considering its low thermal requirement and high minimum temperature threshold, papaya

mealybug has a smaller distribution range than anticipated. Southern parts of Texas and

California, South Florida, and Hawaii are suitable areas for its development. Its final

establishment and distribution may be influenced by other factors such as host plant range, and

the rules and regulations governing plant movement from state to state.









CHAPTER 1
INTRODUCTION

Mealybugs

Mealybugs are soft-bodied insects, which belong to the family Pseudococcidae in the order

Hemiptera (Borror et al. 1992). The name "mealybug" is derived from the mealy or waxy

secretions that cover the bodies of these insects (Borror et al. 1992). This layer of fine mealy

wax often extends laterally to form a series of short filaments. The mealy wax covering is

frequently white and the color may vary among some species (Williams and Granara de Willink

1992). The body of the adult female is normally elongate to oval, and membranous (Williams

and Granara de Willink 1992). Antennae normally have 6 to 9 segments. Legs are present; each

with a single tarsal segment and a single claw (Williams and Granara de Willink 1992).

In common with other hemipterans, female mealybugs have piercing and sucking

mouthparts and are generally active throughout their life (Ben-Dov 1994). In the tropics, their

life cycle may be reduced to less than one month. They often attain high numbers, killing the

host plant by depleting the sap and occasionally by injecting toxins, transmitting viruses, or by

excreting honeydew, which is a suitable medium for the growth of sooty mold (Ben-Dov 1994).

The mold often covers the plant to such an extent that normal photosynthesis is severely reduced

(Williams and Granara de Willink 1992). Although some mealybugs are host plant specific,

mealybugs such as Maconellicoccus hirsutus (Green), and Phenacoccus madeirensis Green are

polyphagus mealybugs that can damage a large number of economically important plants

(Sinacori 1995, Serrano and Lapointe 2002).

Reproduction of mealybugs under greenhouse conditions is year round, and in certain

species is by the production of living nymphs or young often without fertilization. Some

mealybug species reproduce parthenogenetically. The cassava mealybug, Phenacoccus manihoti









Matile-Ferrero reproduces by thelytokous parthenogenesis (Calatayud et al. 1998, Le Ru and

Mitsipa 2000). Many species form an ovisac in which to lay the eggs. In sexually reproducing

species, the adult males are normally minute without functional mouthparts. Male mealybugs

are often winged but occasionally apterous. In contrast, females are always wingless (Williams

and Granara de Willink 1992).

Many of the 2000 mealybug species already described are important insect pests of many

agricultural crops (Williams and Granara de Willink 1992). Infestations may occur within

vegetative shoots or apexes and can be extremely difficult to detect. This ability of mealybugs to

form dense colonies, particularly within the shoot and apex, often makes chemical control of this

pest quite difficult. With the introduction of many new systemic insecticides, control has

improved; however, with insects that are polyphagus, and have numerous hosts, it becomes a

challenge to manage them with just chemical control.

Many times mealybug populations in their countries of origin are not pest problems due to

their parasitoids and predators. The most serious outbreaks occur when mealybugs are

accidentally introduced to new countries without their natural enemies. The introduction of pests

on infested plant material has unfortunately become fairly common. Florida is one of the

important agricultural states in this country and it has weather and climatic patterns that are

conducive for the establishment of many insects. In South Florida, the more subtropical climatic

condition facilitates the growth of a variety of tropical and subtropical crops. This agricultural

pattern, subtropical climatic condition, increase of world trade, and geographic location of the

state, are the main reasons for the regular invasion of insect pests to Florida. Invasive insect

species such as the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae)

(Mead 2007) and the pink hibiscus mealybug, Maconellicoccus hirsutus (Green) (Hemiptera:









Pseudococcidae) (Hoy et al. 2006), which were accidentally introduced to Florida in 1998 and

2002, respectively are good examples of the pest invasions to Florida. Paracoccus marginatus

Williams and Granara de Willink is one of the mealybug species that has been accidentally

introduced into the Caribbean, the US and the Pacific islands, from Central America.

Genus Paracoccus

The genus Paracoccus was first described by Ezzat and McConnell in 1956 by using the

type species Pseudococcus burnerae Brain, by original designation (Ben-Dov 1994). Generic

characters of Paracoccus were later described by Williams and Granara de Willink (Ben-Dov

1994). Paracoccus has a varied distribution from the "Austro-Oriental", Ethiopian, Madagasian,

Nearctic, Neotropical, New Zealand, Pacific, Palaearctic, and Oriental regions (Ben-Dov 1994).

There are about 79 species recorded from the genus Paracoccus (Ben-Dov 1994). Most of the

Paracoccus species are not recognized as major economic pests except for two species. In South

Africa, Paracoccus burnerae (Brain) is considered as a serious pest of citrus (Ben-Dov 1994).

Paracoccus marginatus Williams and Granara de Willink (papaya mealybug) is a pest of papaya

and other economically important fruits, vegetables and ornamentals in the Caribbean, the US,

and several Pacific islands.

Paracoccus marginatus Williams and Granara de Willink

Specimens of papaya mealybug were first collected from Mexico in 1967, which were

believed to be native to Mexico and/or Central America (Miller et al. 1999). Papaya mealybug is

not a serious pest in Mexico, probably because of the availability of its natural enemies (Miller et

al. 1999). This species was first described by Williams and Granara de Willink in 1992 from the

specimens collected from neo-tropical regions in Belize, Costa Rica, Guatemala and Mexico

(Williams and Granara de Willink 1992). In 2002, Miller and Miller re-described this mealybug

species (Miller and Miller 2002).









In early 1990, papaya mealybug invaded the Caribbean region and became a pest of many

tropical and subtropical fruits, vegetables, and ornamental plants (Miller and Miller 2002). Since

1994, it has been recorded in 14 Caribbean countries. In 2002, a heavy infestation of papaya

mealybug was observed on papaya (Caricapapaya L. (Caricaceae)) in Guam (Meyerdirk et al.

2004). Subsequently, papaya mealybug infestations were reported from the Republic of Palau in

2003 and in Hawaii in 2004 (Muniappan et al. 2006, Heu et al. 2007).

The papaya mealybug is an adventive pest insect species that has been found in the US. It

was first recorded on hibiscus in Palm Beach County in Florida in 1998 (Miller et al. 1999) and

subsequently spread into several other counties in the state. It has been collected from more than

25 different plant genera in many counties in Florida since then (Walker et al. 2003).

Paracoccus marginatus is yellow in color and has a series of short waxy filaments around

the margins of the body, which are less than 1/4 the length of the body (Miller et al. 1999). The

female papaya mealybug passes through three immature stages (first, second, and third instar)

before emerging as an adult. The ovisac produced by the adult female is on the ventral side of

the body and is generally two or more times the body length (Miller et al. 1999). Generally, first

instars of mealybugs are called "crawlers". There is no distinguishable difference between male

and female crawlers, and male and female early second instars. In the latter part of the second

instar, the color of the male changes from yellow to pink. Later, it develops a cottony sack

around itself. Male third instars are termed as "prepupa". Unlike the female, the male has a

fourth instar termed as "pupa", from which the adult male emerges (Miller et al. 1999).

Host Plant Species

Food is a component of the environment and may influence an animal's chance to survive

and multiply by modifying its fecundity, longevity or speed of development (Andrewartha and

Birch 1954). The economically important host range of the papaya mealybug includes papaya,









hibiscus, acalypha, plumeria, avocado, citrus, cotton, tomato, eggplant, pepper, beans and peas,

sweet potato, mango, cherry and pomegranate (Miller and Miller 2002). In addition, weed

species such as Parthenium hysterophorus L. are also recorded as host plants of papaya

mealybug (Miller and Miller 2002). Infestations of papaya mealybug have been observed on

papaya, plumeria, hibiscus and jatropha in Hawaii with the favored hosts appearing to be papaya,

plumeria, and hibiscus (Heu et al. 2007). However, insects may settle, lay eggs, and severely

damage plant species that are unsuitable for development of immatures (Harris 1990). There is

no specific information about the life history of papaya mealybug on different host plant species.

Although, papaya is the dominant host plant species of papaya mealybug, it is important to find

out how it can develop on popular ornamental plants such hibiscus, acalypha, and plumeria as

well as on a commonly found invasive annual weeds such as parthenium.

Hibiscus, which is believed to be native to China, is a popular ornamental and landscape

shrub, and widely grown in the tropics and subtropics (Ingram and Rabinowitz 2004). Different

hibiscus species are grown in many areas of the US (USDA 2007a). Hibiscus has been grown in

Florida for many years (Ingram and Rabinowitz 2004), and its potential planting range in the US

includes some areas of Texas and California (Gilman 1999b). Hibiscus is widely grown in

Hawaii. Hibiscus is sold nationwide as potted flower plants, and maintained in greenhouses

around the country. Pink hibiscus mealybug, Maconellicoccus hirsutus (Green) (Hemiptera:

Pseudococcidae) is another important mealybug species that was introduced to Florida in 2002,

and has been identified as one of the most important insect pests of hibiscus (Goolsby et al.

2002, Hoy et al. 2006).

Acalypha L. is a large, fast growing evergreen shrub, which can provide a continuous

splash of color in the landscape with the bronze red to muted red and mottled combinations of









green, purple, yellow, orange, pink or white (Gilman 1999a). It is believed to be native to Fiji

and nearby Pacific islands. Acalypha L. is grown in many parts of the United States (USDA

2007a). Aphids, mites, scales, and mealybugs are recorded as pests of acalypha (Gilman 1999a).

The genus Plumeria L. originates from Central America and its different species are

popular ornamental plants that are widely distributed in the warmer regions of the world (Begum

et al. 1994). Plumeria belongs to the family Apocynaceae dogbanee) (Criley 1998) and the sap

of most of the plants belonging to this family is milky, and may contain toxic alkaloids or

glycosides. In Southwestern Puerto Rico, a caterpillar of the sphinx moth, Pseudosphinx tetrio

L. (Lepidoptera: Sphingidae), two mealybug species (P. marginatus and Puto sp.) and one

unidentified Margaroididae are the frequently encountered herbivores of Plumeria alba (Sloan et

al. 2007). The most common homopteran attacking P. alba in Puerto Rico is the papaya

mealybug (Sloan et al. 2007). These homopterans attack the leaves, inflorescences, flowers,

fruits and sometimes the stem ofP. alba (Sloan et al. 2007). They feed on the sap ofP. alba

leaves when the standing crop of leaves is the greatest, causing the leaves to be frequently

contorted, misshapen, and not fully expanded (Sloan, et al. 2007). Triterpenoids are chemicals

commonly found in plants that belong to the family Apocynaceae, and in plumeria, these

compounds can be feeding deterrents to most generalist insects. The aposematic coloration of P.

tetrio suggests that it is able to detoxify and sequester secondary compounds in P. alba, but these

compounds can make P. alba unpalatable to other generalist herbivores (Sloan et al. 2007).

Parthenium hysterophorus L. is an introduced, invasive weed species, which can be

found in more than 17 states in the Eastern, Southern, and South Central US (USDA 2007a).

Parthenium is considered a noxious annual weed because of its prolific seed production and fast

spreading ability, allelopathic effect on other plants, strong competitiveness with crops and









health hazard to humans as well as animals (Tefera 2002, Raghubanshi et al. 2005). Parthenium

contains sesquiterpene lactones and phenolic acids (Picman and Picman 1984, Mersie and Singh

1988). Terpinoids, from volatile monoterpenoids to involatile triterpenoids, are broadly

defensive against herbivory on plants (Harbone 2001). Parthenin is a terpinoid found in

parthenium weed, which is identified as a barrier to herbivore feeding (Harbone 2001). A leaf

feeding beetle, Zygogramma bicolorata Pallister (Coleoptera: Chrysomelidae) and a stem-galling

moth, Epiblema strenuana Walker (Lepidoptera: Tortricidae) are some of the natural enemies

used in the biological control of parthenium in Australia (Dhileepan 2001, Dhileepan et al.

2005).

Temperature

Temperature is one of the important environmental factors that can affect the movement,

establishment, and abundance of insects. Insect biology is influenced by various environmental

factors and temperature is one of the most important and critical of the abiotic factors (Huffaker

et al. 1999). The rate of insect development is affected by the temperature to which the insects

are exposed (Campbell et al. 1974). Insect development occurs within a definite temperature

range (Wagner et al. 1984). The temperature below which no measurable development occurs is

its threshold of development. The amount of heat required over time for an insect to complete

some aspect of development is considered a thermal constant (Campbell et al. 1974). The

thresholds and the thermal constant are useful indicators of potential distribution and abundance

of an insect (Huffaker et al. 1999). The importance of predicting the seasonal occurrence of

insects has led to the formulation of many mathematical models that describe developmental

rates as a function of temperature (Wagner et al. 1984). The thermal summation model

(Campbell et al. 1974) and Logan 6 model (Logan et al. 1976) are widely used models to explain

the relationship between developmental time and temperature of arthropods. Temperature had









pronounced effects on the development, survival, and reproduction of Madeira mealybug,

Phenacoccus madeirensis Green (Chong et al. 2003). The female P. madeirensis was able to

complete its development in temperatures ranging from 15 to 250C within 66 to 30 days

respectively (Chong et al. 2003). Between 15 to 250C, survival rates of P. madeirensis were not

affected by temperature but the temperature had a strong influence on fecundity, pre-oviposition

time, and the duration of reproduction (Chong et al. 2003). Between 20 and 250C, the cassava

mealybug, Phenacoccus manihoti Matile-Ferrero, and Phenacoccus herreni Cox and Williams,

complete development within 46 to 36 days (Lema and Herren 1985) and 91 to 41 days

respectively (Herrera et al. 1989). Comparison of whole-life developmental times of P. herreni

to those of P. manihoti suggests that P. herreni develops slower than P. manihoti at cooler

temperatures but faster than P. manihoti at warmer temperatures (Herrera et al. 1989). This is

supported by the more tropical distribution of P. herreni (Columbia, The Guyana, and northern

Brazil) compared to that of P. manihoti, which has subtropical distribution (Herrera et al. 1989).

Chemical Control of Papaya Mealybug

Organophosphate and carbamate insecticides such as dimethoate, malathion, carbaryl,

chlorpyrifos, diazinone, and acephate (Walker et al. 2003) were commonly used insecticides to

control mealybugs. Currently neonecotinoid insecticides such as acetamiprid, clothianidin,

dinotefuran, imidacloprid, thiamethoxam, and insect growth regulators (IGR) such as

pyriproxyfen are used to control scale insects and mealybugs (Buss and Turner 2006). However,

there is no specific insecticide currently registered for control of papaya mealybug (Walker et al.

2003). Mealybugs are generally difficult to control chemically due to their thick waxy secretion

covering the body, and their ability to hide in the damaged buds and leaves without being

exposed to the insecticide. The adult mealybugs were more difficult to control than the young

and repeated applications of chemicals targeting immatures were required in suppressing P.









madeirensis (Townsend et al. 2000). In addition, with polyphagous insects such as papaya

mealybug, it would be difficult to manage it with just insecticides and to achieve long-term

control with the wide variety of host plants. Development of insecticide resistance and non-

target effects of insecticides on natural enemies make chemical control a less feasible option for

the long-term control of papaya mealybug (Walker et al. 2003). Because of these reasons,

biological control was identified as a preferred method to control the papaya mealybug.

Biological Control

Biological control is the use of parasitoid, predator, pathogen, antagonist, or competitor

populations to suppress a pest population, making it less abundant and thus less damaging (Van

Driesche and Bellows 1996). It is widely accepted that there are three general approaches to

biological control: importation, augmentation, and conservation of natural enemies. Importation

biological control is often referred to as "classical biological control" reflecting the historical

predominance of this approach (Orr and Suh 1998). Classical biological control can be defined

as importation and establishment of non-native natural enemy populations for suppression of

non-native or native organisms (Orr and Suh 1998). Augmentation includes activities in which

natural enemy populations are increased through mass culture, periodic release, and colonization.

Conservation biological control can be defined as the study and modification of human

influences that allow natural enemies to realize their potential to suppress pests (Orr and Suh

1998). Currently, the "classical" approach is probably the most recognized and heralded form of

biological control among biological control practitioners.

Classical Biological Control of Papaya Mealybug

Many adventive insect species become pests because they are unaccompanied by natural

enemies from their native home (Orr and Suh 1998). In the classical biological control of an

adventive pest species, most often the natural enemies of the pest are searched for in its native









homeland by examining the pest population in its native environment (Van Driesche and

Bellows 1996). These natural enemies are then collected and shipped to the country where the

pest has invaded. After being subjected to appropriate quarantine and testing to ensure safety,

these natural enemies are released and established. This type of introduction of natural enemies

is self-maintaining and less expensive than chemical control over the long term (Van Driesche

and Bellows 1996).

The United States Department of Agriculture (USDA), Animal Plant Health Inspection

Service (APHIS) initiated a classical biological control program for papaya mealybug using

several natural enemies in 1999. The identified natural enemies of papaya mealybug are solitary

endoparasitic wasps that belong to the family Encyrtidae in the Order Hymenoptera. These

wasps were collected in Mexico as potential biological control agents. They were Acerophagus

papayae Noyes and Schauff, Anagyrus loecki Noyes and Menezes, Anagyrus californicus,

Pseudophycus sp. and Pseudleptomastix mexicana Noyes and Schauff (Meyerdirk et al. 2004).

Acerophaguspapayae, A. loecki and P. mexicana are three parasitoid species that are currently

used in the biological control of papaya mealybug. They are mass reared in Puerto Rico and

released in papaya mealybug infested areas in the Caribbean, the US, and the Pacific islands as

needed (Meyerdirk et al. 2004).

Parasitoids

The term "parasitoid" embraces an exceedingly large number of insect species (Gauld

1986). Parasitoids are arthropods that kill their hosts and are able to complete their development

on a single host (Vinson 1976). Parasitoids have been the most common type of natural enemy

introduced for biological control of insects. They have been employed in the management of

insect pests for centuries (Orr and Suh 1998). The last century, however, has seen a dramatic

increase in their use as well as an understanding of how they can be manipulated for effective,









safe use in insect pest management systems (Orr and Suh 1998). Most parasitoids that have been

used in biological control are in the orders Hymenoptera and, to a lesser degree, Diptera (Van

Driesche and Bellows 1996). Of these, certain groups stand out as having more species

employed in biological control projects than others. The most frequently used groups in the

Hymenoptera are Braconidae and Ichneumonidae in the Ichneumonoidea, and the Eulophidae,

Pteromalidae, Encyrtidae, and Aphelinidae in the Chalcidoidea. In the Diptera, Tachinidae is the

most frequently employed group (Greathead 1986). Although parasitoids have been recorded in

the orders Strepsiptera and Coleoptera, parasitism is not common in them (Van Driesche and

Bellows 1996).

Acerophagus papayae Noyes and Schauff

This species of parasitoid is named for the papaya plant (C. papaya L.) on which its host

feeds. It is the smallest species out of the three introduced parasitoids of papaya mealybug. The

female A. papayae is 0.58 to 0.77 mm long including its ovipositor, and males are generally 0.44

to 0.66 mm in length (Noyes and Schauff 2003). The male and female A. papayae are generally

pale orange in color. Other than the un-segmented clava, and genitalia, males are very similar to

their females (Noyes and Schauff 2003). Acerophaguspapayae was originally recorded from P.

marginatus in Mexico (Noyes and Schauff 2003).

Pseudleptomastix mexicana Noyes and Schauff

This is the second parasitoid out of the three introduced parasitoids of P. marginatus; P.

mexicana is named for its country of origin, Mexico (Meyerdirk 2003, Noyes and Schauff 2003).

Larger than A. papayae, the length of the male and female P. mexicana is 0.56 to 0.84 and 0.76

to 1.03 mm, respectively. The head and thorax of the female are black in color and the gaster is

dark brown with a coppery and purple or brassy sheen. Pseudleptomastix mexicana also was

originally recorded from P. marginatus in Mexico (Noyes and Schauff 2003). In 2000, P.









mexicana was introduced into Puerto Rico with other exotic natural enemies from Mexico to

control P. marginatus (Meyerdirk 2003). There are no other known introductions of exotic

Pseudleptomastix species into various countries for the control ofP. marginatus or any other

mealybug species (Meyerdirk 2003).

Anagyrus loecki Noyes and Menezes

The largest out of the three species, female A. loecki is 1.45 to 1.76 mm in length, and the

male is 0.94 to 1.08 mm long respectively (Noyes 2000). In the female, the head and thorax are

mostly orange in color and the gaster is light brown. The male is dark brown in color and varies

from the female in its size and color (Noyes 2000). This species was recorded from several

mealybug species. The holotype was reared from Dysmicoccus hurdi and some of the paratypic

material was laboratory reared on Phennacoccus madeirensis and P. marginatus (Noyes 2000).

Developmental Time, Longevity, and Lifetime Fertility

Developmental time, longevity, and lifetime fertility are important fitness parameters when

evaluating a parasitoid as a biological control agent (Hemerik et al. 1999). Developmental time

of a parasitoid is the duration of time from oviposition to adult emergence. The time between

adult emergence and death is termed as adult longevity. The lifetime fertility of an insect is the

total number of progeny produced during its lifetime.

In koinobiont parasitoids that consume the entire host before pupation, adult parasitoid size

and developmental time are often strongly correlated with host size at the time when it is

developmentally arrested through destructive feeding by the parasitoid larva (Hemerik et al.

1999). The development of Venturia canescens (Gravenhorst) (Hymenoptera: Ichneumonidae),

a solitary endoparasitoid ofPlodia interpunctella (Hubner) (Lepidoptera: Pyralidae) depends on

the ability of early stadia of its host to grow after parasitism and to reach their final stadium

(Hemerik et al. 1999). The early emerging females of Trichogramma evanescens Westwood









(Hymenoptera: Trichogrammatidae), a gregarious egg parasitoid ofEphestia kuehniella Zeller

(Lepidoptera: Pyralidae) were larger and produced more progeny and had higher fitness than late

emerging females (Doyon and Boivin 2005). The adult size and the developmental time of the

solitary endoparasitoid, Aphidius ervi Haliday were affected by the size of its host,

AcyI 1, it hniJh ,pisum (Harris) (Sequeira and Mackauer 1992).

The developmental time, longevity and the progeny production of parasitoids can be

affected by the developmental temperature of the host (Hansen 2000). Between 15 to 300C, the

developmental time of the female Trichogramma turkestanica on the host Ephestia kuehniella,

ranged from 32.9 to 7 days (Hansen 2000). The developmental time decreased with increasing

temperature for the gregarious encyrtid endoparasitoid Tachinaephagus zealandicus reared on

Chrysomyaputoria (Ferreira de Almeida et al. 2002). Amitusfuscipennis MacGown and

Nebeker, a potential biological control agent of Trialeurodes vaporariorum (Homoptera:

Aleyrodidae), had longer developmental time and adult longevity at lower temperatures

(Manzano et al. 2000). The lifetime fecundity and the reproductive life were significantly

affected by temperature for Anagyrus kamali Moursi, a parasitoid ofMaconellicoccus hirsutus

Green reared at 26 and 32C (Sagarra et al. 2000a). Early emerged Tachinaephagus zealandicus

lived longer than late emerged T. zealandicus (Ferreira de Almeida et al. 2002).

The host diet affected the developmental time, fecundity, sex ratio, and size ofApanteles

galleriae Wilkinson (Hymenoptera: Braconidae), a parasitoid ofAchroia grisella (F.) (Uckan

and Ergin 2002). The mating status of a parasitoid can affect its fitness parameters. The mated

solitary endoparasitoid female Anagyrus kamali Moursi had higher progeny production and had a

female biased sex ratio in comparison with unmated females, which had lower progeny

production and male only progeny (Sagarra et al. 2002). Unmated A. kamali lived longer than









the mated ones (Sagarra et al. 2002). Fecundity and survival ofAnagyrus kamali was also

affected by higher feeding and storage temperatures of 270C than 200C (Sagarra et al. 2000b).

Host Stage Susceptibility, Host Stage Suitability, and Sex Ratio

Although a specific stage or stages of a mealybug are preferred by a parasitoid for

oviposition, all or most of its stages can be susceptible to oviposition and subsequent parasitoid

development. Parasitoids that develop in early instar mealybugs have a tendency to produce

male progeny compared to those that develop in the late instars, in which they can produce more

female progeny (Charnov et al. 1981, Sagarra and Vincent 1999). In no choice tests, A. kamali a

parasitoid of the pink hibiscus mealybug, M. hirsutus Green, was able to parasitize all nymphal

stages and adult females, while choice tests indicated that A. kamali prefers third instar and pre-

oviposition adult females (Sagarra and Vincent 1999). Parasitoids emerged from hosts that were

parasitized as second-instar P. herreni were strongly male-biased for A. vexans while apparently

preferred later host stages yielded significantly more females than males (Bertschy et al. 2000).

Increased size of the host translates into both increased male and female fitness. For

females, this measure is the lifetime production of eggs while for the male it is longevity

(Charnov et al. 1981). The later the developmental stage of the host at oviposition, the faster the

parasitoids develop and emerge (Bertschy et al. 2000). Within a particular host stage, the male

had a shorter developmental time than the female for Aenasius vexans Kerrich, an encyrtid

parasitoid of cassava mealybug, Phenacoccus herreni Cox and Williams (Bertschy et al. 2000).

Depending on the instar they attack, the parasitoid progeny can be either male or female biased.

The solitary endoparasitoid of cassava mealybug (Phenacoccus herreni Cox and Williams),

Aenasius vexans Kerrich (Hymenoptera: Encyrtidae), shows male-biased sex ratio when it

attacks second-instar P. herreni, and female-biased sex ratio when it parasitizes third instars

(Bertschy et al. 2000).









The haplodiploid sex determination system of most parasitoid wasps provides females a

means of controlling the offspring sex ratio, because they can adjust the proportion of fertilized

eggs at oviposition (King 1987). Parasitoid wasps provision their young with food by

ovipositing in or on a host. Upon hatching the wasp larva feeds on the host, usually killing it

prior to the wasp's pupation. Because a few males can fertilize many females, female-biased

broods facilitate the use of parasitoids wasps as biological control agents (King 1987). The

factors that may influence the offspring sex ratio are parental characteristics, environmental

characteristics, host characteristics, and factors influencing local mate competition. The parental

characteristics are time delay between emergences and insemination, number of times a female

has mated, maternal and paternal age, maternal size, maternal diet, and genetics (King 1987).

Photoperiod, temperature, and relative humidity are the environmental characteristics that can

affect sex ratio. Host characteristics such as host size, age, sex, and species can affect the

progeny sex ratio of the parasitoids. Local mate consumption theory predicts that isolated

females should produce primarily daughters with only enough sons to inseminate those

daughters. Superparasitism, female density, number of offspring per host, and host density are

factors affecting local mate consumption theory (King 1987). Sex ratio of the progeny can also

be affected when a female hymenopteran lacks sperm and lays male eggs (Ridley 1988).

Interspecific Competition

According to Dent (1995), when two species compete with one another intensely enough

over limited resources, then with time, one or the other can become extinct. When there is a

dominant parasitoid, which can displace other parasitoid species, the releasing of several species

might not provide the expected efficiency of a biological control program. In solitary insect

parasitoids, generally only one offspring survives in a host (Vinson 1976). Females normally

deposit one egg per host and this reduces the host availability to conspecific and heterospecific









parasitoids. The successful oviposition of a female, therefore, would be increased if she were the

first to identify and oviposit only in hosts with no previously laid eggs (Lawrence 1981).

Although, coexistence of several parasitoid species in the system can be more productive than a

single parasitoid species, coexistence requires that some difference exist in niches among the

species. When several parasitoid species attack the same host species, and one parasitoid prefers

to attack early instars of the host and others prefer late instars or vice versa, there can be efficient

control of the host species (Bokonon-Ganta et al. 1996). The pest instar they attack is the most

important factor to decide the coexistence or competitive exclusion of biological control agents

when several agents are released together. The competition of parasitoids can be affected by the

temperature. Some parasitoids compete more for hosts at lower temperatures and some prefer to

attack hosts when temperatures are higher (Van Strien-van Liempt 1983). The parasitoids of

Drosophila melanogaster Meigen and Drosophila subobscura Collin, Asobara tabida Nees von

Esenbeck, and Leptopilina heterotoma (Thomson) compete differently at different temperatures.

Asobara tabida is a better competitor at lower temperatures and Leptopilina heterotoma

performed better at higher temperatures (Van Strien-van Liempt 1983).

Research Objectives

Research studies on papaya mealybug and its parasitoids are lacking. There is no

information on the life history of papaya mealybug, either in relation to its host plant species or

to temperature. Understanding the life history of an insect is important in insect predictions,

distribution, and its management. Determining thermal constants and temperature thresholds is

also useful in predicting insect emergence, distribution, and its management. In addition, there is

very little published research on papaya mealybug parasitoids. Information on the biology ofA.

papayae, A. loecki, and P. mexicana, and their interspecific competition, and the effectiveness in

the field is scarce. It is important to find out whether populations of these parasitoid species are









established in the field, and if there is a need for inoculative releases. The goal of this study was

to understand the life history of papaya mealybug and to identify the efficient parasitoids for

successful utilization of currently used biological control agents to obtain an effective and

sustainable biological control program for papaya mealybug infestation in the US. Therefore,

research was conducted to determine the life history of papaya mealybug, and then to evaluate

the effectiveness of three introduced parasitoids of papaya mealybug. There were five objectives

for this study.

The first objective was to define the life history of papaya mealybug using four host plant

species commonly found in Florida. The second objective was to understand the effect of

constant temperature on development, reproduction and survival of papaya mealybug, and then

to estimate its thermal constants and temperature thresholds for development. The third

objective was to evaluate the effectiveness of currently released parasitoids of papaya mealybug,

A. papayae, A. loecki and P. mexicana in the field. The fourth objective was to study the

developmental time, longevity and the lifetime fertility ofA. papayae, A. loecki and P. mexicana.

The fifth and final objective was to investigate the host stage susceptibility and suitability, sex

ratio, and interspecific competition ofA. papayae, A. loecki and P. mexicana.









CHAPTER 2
LIFE HISTORY OF Paracoccus marginatus WILLIAMS AND GRANARA DE WILLING
(HEMIPTERA: PSEUDOCOCCIDAE) ON FOUR HOST PLANT SPECIES UNDER
LABORATORY CONDITIONS

Introduction

Paracoccus marginatus Williams and Granara de Willink (Hemiptera: Pseudococcidae)

is a polyphagus insect and a pest of various tropical fruits, vegetables and ornamental plants

(Miller and Miller 2002). Its host range includes Caricapapaya L. (papaya), Citrus spp. L.

(citrus), Persea americana P. Mill. (avocado), Solanum melongena L. (eggplant), Hibiscus spp.

L. (hibiscus), Plumeria spp. L. (plumeria), and Acalypha spp. L. (acalypha) (Miller and Miller

2002). Paracoccus marginatus was first described by Williams and Granara de Willink (1992)

and re-described by Miller and Miller (2002). Paracoccus marginatus was originally reported

from the neotropical regions in Belize, Costa Rica, Guatemala, and Mexico (Williams and

Granara de Willink 1992). This species was introduced to the Caribbean in the early 1990's, and

spread among many of the Caribbean islands by 1994 (Walker et al. 2003). In 1998, P.

marginatus was first reported in the US in Florida, in Palm Beach County on hibiscus (Miller et

al. 1999). Thereafter, it was recorded in several other counties in Florida from more than 25

genera of plants (Walker et al. 2003). Heavy infestations of P. marginatus on C. papaya were

recorded in Guam in 2002 (Walker et al. 2003, Meyerdirk et al. 2004) and in the Republic of

Palau in 2003 (Walker et al. 2003, Muniappan et al. 2006). In 2004, P. marginatus was reported

in Hawaii on papaya, plumeria, hibiscus, and Jatropha sp. L. (Heu et al. 2007).

Since its introduction to the Caribbean, the US, and the Pacific islands, P. marginatus has

established in most of the Caribbean islands, Florida, Guam, the Republic of Palau, and Hawaii.

Paracoccus marginatus potentially poses a threat to numerous agricultural products in the US

especially in Florida, and states such as California and Hawaii, which produce similar crops. In









southern parts of Texas, where the country's third largest citrus production exists (CNAS 2007)

is also a susceptible area for P. marginatus. The potential planting range of hibiscus includes

Southern Texas (Gilman 1999b).

Life history ofP. marginatus has not been investigated. Understanding the life history of a

pest insect is important in predicting its development, emergence, distribution, and abundance.

Life history information also plays an important role in pest management, especially when

applying chemical and biological control methods. Since there is a high possibility of spreading

P. marginatus into other areas in the US, it is important to study its life history using host plant

species that are either widely grown in the susceptible areas, or potted plant species that are

commonly transported to these areas. In this study, three ornamental plants Hibiscus rosa-

sinensis L (hibiscus), Plumeria rubra L. (plumeria), Acalypha amentacea Roxb. ssp. wilkesiana

(Muell.-Arg.) cutivar Marginata (acalypha), and one weed species, Parthenium hysterophorus L.

(parthenium) were selected to study the life history ofP. marginatus. These four plant species

were previously recorded as host plants of P. marginatus (Miller and Miller 2002) and are

widely grown in many areas in the US.

Materials and Methods

Rearing Mealybugs. Paracoccus marginatus was initially collected from a papaya

(Caricapapaya L.) field in Homestead, FL. Red potatoes (Solanum tuberosum L.) (Ryan Potato

Company, East Grand Forks, MN) were allowed to sprout and then used in rearing a colony of P.

marginatus. Potatoes were soaked in 1% solution of bleach (Clorox The Clorox Company,

Oakland, CA; 6% sodium hypochlorite) for 15 minutes, and then rinsed with water, air-dried and

placed in bags made from black cotton cloth to encourage sprouting. Bags were kept inside a

dark room at 27 1IC and 65% + 2 R.H. Each week, 30 newly sprouted potatoes were infested

with ovisacs ofP. marginatus to maintain the colony. Each sprouted potato was infested with 3









to 5 ovisacs depending on the size of the potato and ovisacs. Infested potatoes were kept in 3.8-

L plastic containers at the rate of 10 per container (Rubbermaid Newell Rubbermaid Inc.

Atlanta, GA). Prior to placing the infested potatoes, screens (Amber Lumite Bio Quip,

Gardena, CA) were glued to cut sections of lids in these containers to facilitate air circulation.

The mealybug colony was held in an environmental growth chamber (Percivel I-36LL, Percival

Scientific Inc. Perry, NC) at 250 1IC, 65 2% R.H., and a photoperiod of 12:12 (L:D).

Eggs to be used in the studies were obtained from gravid females identified by a body

length (2-2.5 mm) which is approximately twice the size of newly emerged virgin females (1.1-

1.3 mm). To obtain eggs, gravid females from the colony (each from a different infested potato)

were placed individually on newly sprouted potatoes.

Development and Survival. All plant material was collected and prepared 24 hours

before the experiment. Hibiscus cuttings were obtained from 1-yr old container-grown hibiscus

and maintained in a shadehouse. Acalypha and plumeria cuttings were obtained from plants in

the landscape on TREC premises. Parthenium seedlings were collected from the field. A fully

expanded young leaf with a stem 4-cm long was used for each replicate of hibiscus and acalypha.

For parthenium, a whole plant approximately 8-cm in height with an intact root system was used

as each replicate. A tender leaf was selected from each parthenium plant and the remaining

leaves were removed. For plumeria, a 5-cm long terminal shoot with one tender leaf was

selected as each replicate.

Host tissue was placed in arenas (9-cm-diam Petri dish with a 0.6-cm-diam hole in the

bottom for hibiscus, acalypha, and parthenium; 18-cm-diam Petri dish for plumeria). The stem

of each leaf of hibiscus and acalypha was inserted through the hole and the lid was placed on the

Petri dish. For parthenium, the main stem of the plant was inserted through the hole in the Petri









dish until the leaf was completely placed inside the Petri dish. Each Petri dish was kept on a 162

ml translucent plastic souffle cup (Georgia Pacific Dixie, Atlanta, GA) filled with distilled water

into which the stem was submerged. For plumeria, each terminal shoot was hydrated using a ball

of cotton tied to the cut end of the shoot, and moistened daily with distilled water.

Eggs collected from a single female were placed on the leaves of all four hosts with 10

eggs per leaf using a paintbrush (No.000) (American Painter 4000, Loew-Cornell Inc.,

Englewood Cliffs, NJ). Eggs were collected within 24 h of oviposition. Dishes were checked

daily for egg hatch and shed exuviae. The number of days to egg hatch, and emergence and

survival of each instar, and number of emerging adult males and females were recorded. The

developmental time and the survival of eggs and first instars were not separated by gender. The

gender of each individual mealybug was determined during the latter part of the second instar

when males change their color from yellow to pink. At this point, the developmental times of

males and females were counted separately. For each plant species, 35 Petri dishes (replicates)

each with 10 eggs were used. This experiment was repeated twice at the end of the preceding

experiment. All experiments were carried out inside an environmental growth chamber as

above.

Reproduction. Newly emerged virgin females obtained from the developmental study of

each plant species were used to assess reproduction. Virgin females were placed individually in

Petri dishes with either a leaf or a terminal shoot of each plant species prepared as mentioned

above. Females were held alone to assess asexual reproduction or were provided with three

newly emerged males from the same plant species for sexual reproduction. Petri dishes were

kept in an environmental growth chamber as above. The date oviposition began, the number of

eggs laid, and adult mortality were recorded. For each of the two treatments (sexual and asexual)









35 females were used, and each female was considered a replicate. This experiment was

repeated twice using newly emerged males and females collected from developmental time

experiments.

Statistical Analysis. The experimental design was completely random for all experiments.

The 10 eggs or mealybugs in each Petri dish were considered as a single unit/replicate and the

mean of the response variable was calculated and used in subsequent analyses in all experiments.

Data of the initial and repeated experiments were pooled together after a two-way analysis of

variance (ANOVA) indicated no interaction among the experiments (F = 0.69, df = 6, 408, P =

<0.6539). One-way ANOVA was performed using a general linear model (GLM) for all

experiments (SAS Institute 1999). Means were compared at P = 0.05 significance level using

the Tukey's HSD test. Data for proportions of females (sex ratio) and survival were square-root

arcsine-transformed, when necessary prior to ANOVA (Zar 1984).

Voucher Specimens. Voucher specimens of P. marginatus were deposited in the

Entomology and Nematology Department insect collection, at Tropical Research and Education

Center, University of Florida.

Results

Preliminary studies demonstrated that it takes approximately one month for eggs of P.

marginatus to hatch and develop into adults. Use of tender leaves could avoid leaf senescence

during this time. Hibiscus cuttings can root within two to three weeks time in water. Even after

30 days, acalypha cuttings were not rooted. Use of rooting hormones could have accelerated the

process of rooting, however the impact of rooting hormones on the development of insects is not

known. Therefore, the fresh cuttings were used. Cuttings obtained from parthenium, a soft

herbaceous plant, were unable to survive 30 days in water. When parthenium plants with intact

root system were used, the leaves were able to withstand this period. Plumeria cuttings were









able to survive more than 30 days without leaf senescence with the provision of daily hydration

through a ball of cotton tied around the cut end of the plumeria terminal. During this time, new

leaves grew from the shoots indicating that these shoots were continuously growing and alive.

Use of hard water in the containers to which the stems of the cuttings were submerged, could

stain the bottom of the Perti dish and disturb the checking procedures for the mealybugs, which

were dislodged from the leaf into the Petri dish. Use of distilled water did not significantly affect

the development, reproduction, and survival of P. marginatus compared to hard water.

Therefore, distilled water was used instead of hard water.

Development. There were differences in the developmental times of P. marginatus

reared on four host species (Table 2-1). Males had longer developmental time than females.

Adult females emerged earlier from the eggs on acalypha and parthenium than from the eggs on

hibiscus and plumeria. Adult males had longer developmental time on acalypha and plumeria

than on parthenium and hibiscus (Table 2-1).

Survival. Eggs survived similarly on all four plants (Table 2-2). The lower survival of the

first and second instars on plumeria was reflected in the cumulative adult survival on plumeria.

Survival for the third-instar males and females, and the fourth-instar males were not affected by

the host species (Table 2-2).

Proportion of Females and Adult Longevity. Adults emerged on plumeria with a higher

proportion of females than on the other three host species (F = 8.15, df= 3, 416, P <0.0001).

The mean proportion of adult females ranged from 53-59 % (acalypha: 53.9 1.3, hibiscus: 53.7

+ 1.1, parthenium: 53.4 1.0, and plumeria: 58.9 1.7). No difference in adult longevity of

males (F = 0.69, df= 3, 416, P = 0.5562) and females (F = 0.52, df= 3, 416, P = 0.6659)









occurred among the hosts. Mean longevity of adult males and females was 2.3 + 0.1 and 21.2 +

0.1 d, respectively.

Reproduction. Virgin females did not lay any eggs on any of the four plant species.

Mated females reared on plumeria laid a lower number of eggs (186.3 1.8) than the number of

eggs laid by females reared on hibiscus (244.4 6.8), acalypha (235.2 3.5), and parthenium

(230.2 + 5.3) (F = 29.9, df= 3, 416, P= <0.0001). The mean pre-oviposition (6.3 0.1) and

oviposition periods (11.2 0.1) were not affected by the plant species (F = 0.23, df= 3, 416, P =

0.8739, F = 0.12, df = 3, 416, P = 0.9496).

Discussion

Determining the life history of an insect is important to understand its development,

distribution and abundance. In polyphagus insects, life history can vary with the plant species it

feeds on. There were differences in the life history parameters of P. marginatus reared on four

plant species, however, P. marginatus was able to develop, survive and reproduce on all four

plants. Different plant species provide different nutritional quality and chemical constituents,

which can affect the development, reproduction and survival of an insect. The differences

observed in the life history of P. marginatus may be due to nutritive factors, allelochemical

compounds, and physical differences in leaf structures, which may be involved in the variation in

plant suitability, although these factors were not investigated for P. marginatus in this study.

Use of different presentations may have confounded the results but preliminary studies found

that these were the best ways to maintain these hosts in a condition suitable for the tests.

Different host plant species have affected the life history parameters of other mealybug

species. Longer pre-reproductive period and a higher progeny production were observed for

Rastrococcus invadens Williams reared on different varieties ofMangifera indica L. (Bovida

and Neuenschwander 1995). Mortality of the of citrus mealybug Planococcus citri (Risso) was









higher on green than on red or yellow variegated Coleus blumei "Bellevue" (Bentham) plants,

and developed faster and had a higher fecundity when developed on red-variegated plants (Yang

and Sadof 1995). The developmental time of female Planococcus kraunhiae (Kuwana) was

shorter when reared on germinated Viciafaba L. seeds than on leaves of a Citrus sp. L. and on

Cucurbita maxima Duchesne, and it survived better when reared on germinated V faba seeds

than on citrus leaves (Narai and Murai 2002). The pink hibiscus mealybug, Maconellicoccus

hirsutus (Green), was able to develop equally well on Cucurbitapepo L. as on C. maxima

(Serrano and Lapointe 2002). There was no difference in survival, development, and fecundity

of cohorts of the mealybug, Phenacoccus parvus Morrison when reared on Lantana camera L.

Lycopersicon esculentum Miller, and Solanum melongena L (Marohasy 1997). However,

Gossypium hirsutum L., Ageratum houstonianum Miller, and Clerodendrum cunninghamii Benth

were identified as less suitable host plants for the development ofP. parvus compared to L.

camera (Marohasy 1997).

Although the eggs of P. marginatus on plumeria hatched in a similar manner to the eggs

on other three plant species, there was less survival of the first and second instars on plumeria.

Stickiness observed on plumeria leaves may have contributed to this low survival. This

stickiness may have resulted from the experimental conditions such as the hydration method

used in this experiment. In the Republic ofPalau, P. marginatus has caused serious damage to

plumeria (Muniappan et al. 2006), and it is found to be the most common homopteran found on

Plumeria alba L. in Puerto Rico (Sloan et al. 2007) indicating its ability to develop well on this

plant species. A loss of 17 to 18% of the first instars was also observed on hibiscus, acalypha,

and parthenium. A low survival rate of first-instar mealybugs was also observed when P.

kraunhiae were reared on V. faba seeds (Narai and Murai 2002). The loss of first instar P.









marginatus may be due to the movement of crawlers (first instars) away from the leaf tissues and

they falling off the plants. This movement was observed on all plant species, although it was

more evident on plumeria. Crawlers have a tendency to move toward light so the 12-h

photoperiod used in this experiment may have caused them to move toward light and dislodge

from the leaves or the shoots. Preliminary studies demonstrated that the crawlers of P.

marginatus, which were dislodged from the leaf, were not be able to survive, unless they moved

back or were placed back on the leaf. Ultimately, the low percent survival of eggs and first

instars was reflected in the low egg to adult survival ofP. marginatus.

Insects may settle, lay eggs, and severely damage plant species that are unsuitable for

development of immatures (Harris 1990). However, males and females that emerged from

hibiscus, acalypha, plumeria, and parthenium were able to mate and reproduce successfully.

Under experimental conditions, the mean number of eggs produced by an insect could be lower

than its actual capacity due to restricted conditions and the experimental arena used. With a

female developmental time of 24 to 25 d, even with the lowest fecundity observed from the

females reared on plumeria, the number of eggs obtained was large enough to build up a

substantial population in the field in a short time.

Although some mealybugs such as the cassava mealybug, Phenacoccus manihoti Matile-

Ferrero can reproduce by thelytokous parthenogenesis (Calatayud et al. 1998, Le Ru and Mitsipa

2000), no virgin females produced eggs in the current study. The sex ratio was slightly female

biased, thus there is no evidence for parthenogenetic reproduction in this species.

The ability of P. marginatus to develop on these plant species demonstrates the

possibility of movement, distribution, and establishment of P. marginatus into new areas in the

US. Hibiscus, acalypha, and plumeria are popular ornamental plants widely grown in Florida,









California, and Hawaii (Criley 1998, Gilman 1999a, USDA 2007a). Different hibiscus species

are grown in many US states (Gilman 1999b, USDA 2007a), and potted hibiscus plants are

transported to other parts of the US and Canada. Parthenium is a noxious annual weed

commonly found among the ornamental plants in the landscape of urban areas, agricultural

lands, and in disturbed soil in more than 17 states in the Eastern, Southern, and South central US

(USDA 2007a). There is a possibility that P. marginatus can spread from weeds such as

parthenium to economically important fruits, vegetables and ornamental plants. However, the

ultimate movement, distribution, and establishment of P. marginatus in the other areas in the US

could be decided by the other abiotic and biotic factors, such as temperature, availability of host

plants, and the rules and regulations governing the movement of plant material from one state to

the other.

Life history of P. marginatus is affected by host plant. However, it has the ability to

develop, survive, and reproduce on a variety of host plant species. The information gathered

from this study will be important in the management of P. marginatus, by providing a better

understanding of its life cycle, and its ability to survive on different host plant species. This

information is needed in the development of integrated pest management of this pest.










Table 2-1 Mean number of days ( SEM) for each developmental stadium ofP. marginatus reared on four host species (gender
could not be determined before the second instar).
Host Stadia Cumulative


First


Male


Second


Female


Third


Male


Female


Fourth
Male


M


Acalypha 8.6 + 0.1b 5.9 + 0.1c 6.5 + 0.1c 3.8 + 0.1c 2.8 + 0.1b 6.3 O0.1a 4.5 O0.1a 28.4
Hibiscus 8.4 0.1c 6.2 0.1b 6.8 0.1bc 5.0+ 0.1b 2.3 0.1c 5.9 0.1b 3.9 0.1b 27.6
Parthenium 8.8 0.1a 5.8 + 0.1c 5.6 0.1d 5.2 + 0.lab 3.4 + 0.1a 4.7 + 0.1d 4.1 0.1b 27.7
Plumeria 8.5 + 0.1b 6.6 + 0.2a 9.6 + 0.1a 5.3 0.1a 2.7 + 0.1b 5.1 + 0.1c 2.6 + 0.1c 30.0
F 25.44 63.78 358.88 122.56 32.37 109.51 128.68
df 3,416 3,416 3,413 3,416 3,415 3,416 3,415
P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <
n= 105
Means within a column followed by the same letters are not significantly different at a = 0.05 (Tukey's HSD test).


ale
+ 0.1b
+ 0.1c
+ 0.1c
+ 0.1a
239.96
3,415
0.0001


Female
24.5 + 0.lb
25.5 0. la
24.4 + 0.lb
25.5 O0.1a
74.78
3,416
<0.0001









Table 2-2 Mean ( SEM) percent survival for each developmental stadium of P. marginatus reared on four host species.
Host Egg First Second Third Fourth Egg to Adult
Male Female Male
Acalypha 82.8 0.7 83.2 + 0.9a 89.4 l.a 89.6 1.4 89.3 1.4 89.7 1.6 49.9 + 0.8a
Hibiscus 83.3 + 0.6 82.7 0.9a 89.4 + 1.0a 89.8 + 1.5 89.0 + 1.5 89.7 + 1.6 50.4 + 0.8a
Parthenium 83.5 + 0.6 82.7 0.9a 89.1 0.9a 89.5 + 1.4 89.7 + 1.4 89.6 + 1.5 50.1 + 0.7a
Plumeria 82.2 0.7 58.4 + 1.4b 64.9 + 1.7b 84.5 2.3 81.8 2.4 81.4 3.5 20.0 + 0.5b
F 0.89 73.44 58.67 0.42 1.55 0.48 379.44
df 3,416 3,416 3,416 3,416 3,416 3,416 3,416
P 0.4475 <0.0001 <0.0001 0.7398 0.1998 0.6955 <0.0001
n= 105
Means within a column followed by the same letters are not significantly different at a = 0.05 (Tukey's HSD test).









CHAPTER 3
EFFECT OF CONSTANT TEMPERATURE ON THE DEVELOPMENTAL BIOLOGY OF
Paracoccus marginatus WILLIAMS AND GRANARA DE WILLING (HEMIPTERA:
PSEUDOCOCCIDAE)

Introduction

Understanding the developmental biology of an insect can provide useful information for

pest management. Developmental biology, however, can be influenced by various

environmental factors. Temperature is one of the most important and critical of the abiotic

factors that can affect insect development. Insects require a certain amount of heat units to

develop from one stage of their life cycle to another, which can be measured in degree-days

(Gordan 1999). The ability of an insect to develop at different temperatures is an important

adaptation to survive varying climatic conditions, and is important in insect population

predictions and control strategies (Mizell et al. 1978). Temperature also influences the

population dynamics of insect pests and their natural enemies (Huffaker et al. 1999).

Temperature range and climatic condition of an area determine the ability of an adventive insect

species to invade that area. There is no information on the effect of temperature on the

development and survival of one such adventive pest species, Paracoccus marginatus Williams

and Granara de Willink (Hemiptera: Pseudococcidae), which has been recently introduced in to

the US. Paracoccus marginatus is a polyphagus insect that has been recognized as a significant

pest of a large number of tropical and subtropical fruits, vegetables, and ornamental plants

(Miller and Miller 2002).

First described by Williams and Granara de Willink in (1992) and re-described by Miller

and Miller (2002), P. marginatus is believed to be native to Mexico and Central America (Miller

et al. 1999). Its economically important hosts include papaya, hibiscus, avocado, citrus, cotton,

tomato, egg plant, beans and peas, sweet potato, mango, cherry, and pomegranate (Walker et al.









2003). It is an important pest in the Caribbean, the US (Miller and Miller 2002) and some

Pacific islands such as the Republic of Palau (Muniappan et al. 2006), Guam (Meyerdirk et al.

2004) and Hawaii (Heu et al. 2007). Since 1994, P. marginatus has been recorded in 14

Caribbean countries (Walker et al. 2003). In 1998, P. marginatus was first discovered in the US

in Palm Beach County, Florida, on hibiscus plants, and since then has been recorded on more

than 25 genera of hosts (Miller and Miller 2002). Paracoccus marginatus was subsequently

found in several other counties in Florida, and potentially poses a threat to numerous agricultural

products in Florida as well as to the other US states producing similar crops (Walker et al. 2003).

The ability of P. marginatus to spread into other states in the US may depend on its

ability to develop and survive at different temperatures. Determining the effect of temperature

on the life history of P. marginatus and estimating its thermal requirements will be useful in

predicting where this pest can potentially spread in the US. This study focuses on the effect of

constant temperature on the developmental biology and thermal requirements of P. marginatus.

Materials and Methods

Insect Rearing. Paracoccus marginatus was initially collected from a papaya (Carica

papaya L.) field in Homestead, FL. Sprouted red potatoes (Solanum tuberosum L.) (Ryan Potato

Company, East Grand Forks, MN) were used to rear a colony of P. marginatus at the University

of Florida, Tropical Research and Education Center (TREC), Homestead, FL. Prior to sprouting,

potatoes were soaked in a 1% solution of bleach (Clorox The Clorox Company, Oakland CA;

6% sodium hypochlorite) for 15 minutes, and then rinsed with clean water and dried. Potatoes

were placed in black cotton cloth bags, and kept inside a dark room at 270 1IC to encourage

sprouting. Each week, 30 sprouted potatoes were infested with P. marginatus ovisacs collected

from the previously infested potatoes selected from the colony to maintain the mealybug

population. The mealybug colony was held in an environmental growth chamber (Percivel I-









36LL, Percival Scientific Inc. Perry, NC) at 250 1IC, 65 2% R.H., and a photoperiod of

14:10 (L:D).

Development and Survival. Hibiscus (Hibiscus rosa-sinensis L.) leaves were used as the

host tissues. These leaves were taken from hibiscus plants that were obtained from a local

nursery and maintained outdoors. The experimental arena consisted of a 9-cm-diam Petri dish.

A 0.6-cm-diam hole was made in the bottom of the Petri dish using a heated cork borer. A

tender hibiscus leaf with a 4 cm long stem was placed in each Petri dish with the stem inserted

through the hole at the bottom of the Petri dish and the lid was replaced. Each Petri dish with a

hibiscus leaf was placed on a cup of water so that the stem below the petiole was immersed in

water. The development and survival ofP. marginatus was initially evaluated at five constant

temperatures of 15, 20, 25, 30, and 350C, all 1IC. Eggs maintained at 15 and 350C hatched but

these nymphs were unable to complete their first instar development. Three new temperatures of

18, 34, and 370C, all 1IC were later included in the experiment to find out more about the egg

and the first instar development. For each temperature, 35 gravid females were collected from

the colony to obtain eggs. To acclimatize them for each temperature, gravid females were kept

individually on hibiscus leaves prepared as above, and were transferred to environmental growth

chambers (TCI model, Environmental Growth Chambers, Chagrin Falls, OH) at the experimental

temperatures, 65 2% R.H., and a photoperiod of 14:10 (L:D), 48 hours before the experiment.

Eggs were collected within 24 hours of oviposition. Ten eggs collected from a single female

were placed on each hibiscus leaf arranged in a Petri dish prepared as above. There were 35

replicates for each temperature. Immediately after placing the eggs, the Petri dishes were

transferred to each environmental growth chamber with the specific temperature. Petri dishes

were checked daily for egg hatch and molting. When eggs started to hatch, chambers were









maintained in complete darkness for 72 hours to encourage the first-instar nymphs (crawlers) to

settle on the leaves. The gender of each individual was determined during the latter part of the

second instar when the males change their color from yellow to pink. At this point, the

developmental times of males and females were counted separately. The developmental time

and the survival of eggs and first instars were not separated by gender. The number of surviving

individuals at each stage was counted.

Reproduction. To determine the effect of constant temperatures on reproduction of P.

marginatus, newly emerged males and females were separated as soon as they emerged as adults

at 18, 20, 25, and 30+1C. Each adult female was placed on a hibiscus leaf, which was

arranged in a Petri dish prepared as above. Each female was provided with 2-3 newly emerged

adult males to ensure mating. Each female represented a replicate. There were 35 replicates for

each temperature. The number of eggs laid by each female was counted daily. The number of

days for the pre-oviposition period (number of days from adult emergence to oviposition) and the

oviposition period (time between beginning and end of oviposition) were also counted.

Adult Longevity. The number of days from adult emergence to death was evaluated at

four constant temperatures (18, 20, 25, and 30+1C) for both males and females. Each

individual was placed in a Petri dish prepared as mentioned above. For each temperature, 35

individuals (replicates) each of both males and females were evaluated.

Developmental Thresholds and Thermal Constant. A linear regression analysis (PROC

REG) (SAS Institute 1999) was carried out to calculate the thermal constant and lower

developmental threshold (Tmin) for P. marginatus, using rate of development (reciprocal of

development) from egg to adult against the constant temperatures used. The linear degree-day

model (thermal summation model) estimates the relationship between temperature and the rate of









development in a linear relationship (Campbell et al. 1974). This linear relationship is Y= a +

bT, where Yis the rate of development (1/days), T, ambient temperature (C), and the regression

parameters intercept (a) and slope (b). The thermal constant K (1/b) is the number of degree-

days above the threshold summed over the developmental period. Lower developmental

threshold Tmin (-a/b) is the minimum temperature at which the rate of development is zero or no

measurable development occurs.

To describe the developmental rate over a wider temperature range, a nonlinear model

(Logan 6 model) was used to calculate the upper developmental threshold (Tmax) and the

optimum temperature threshold (Topt) (Logan et al. 1976). The upper developmental threshold

Tmax is the maximum temperature at which the rate of development becomes zero and life

processes can no longer be maintained for a prolonged period. The optimum temperature Topt is

the temperature at which the maximum rate of development occurs (Walgama et al. 2006). The

Logan model does not estimate the lower developmental threshold (Tmin), because it is

asymptotic to the left of the temperature axis. The relationship between developmental rate

(1/D) and upper developmental threshold is described in the Logan 6 model as,


1/D = y exp(pT) exp(pTmax mT )


where qI is a directly measurable rate of temperature dependent physiological process at some

base temperature, p is the biochemical reaction rate and ATis the temperature range over which

'thermal breakdown' becomes the overriding influence (Logan et al. 1976). To determine the

optimum temperature (Topt) for development, the following equation (Logan et al. 1976) was

used.


T T l+ (ln(Eb
L1 Eb,









Here, e is AT/ Tmax and bois pTmax.

Statistical Analysis. The experimental design used for all experiments was completely

random. Prior to the statistical analysis, the mean of the individuals in each Petri dish/replicate

was calculated and used in the analyses. One-way analysis of variance (ANOVA) was

performed using a general linear model (GLM) for all experiments (SAS Institute, Cary, NC).

Means were compared at P = 0.05 significance level using the Tukey's HSD test. Proportions of

females (sex ratio) and survival were square-root arcsine-transformed using

p'= arcsmin

where p = proportion of female/survival, to adjust the variances (Zar 1984) prior to ANOVA, but

the untransformed data were presented in the tables.

A linear regression was performed to find the linear relationship between rate of

development and temperature and to estimate the parameters a and b (PROC REG) (SAS

Institute 1999). A non-linear regression (PROC NLIN) (SAS Institute 1999) was performed for

the non-linear section of the relationship between rate of development and temperature to find

the estimates for the parameters, q, p, Tma and AT of the Logan 6 model.

Voucher Specimens. Voucher specimens of P. marginatus were deposited in the

Entomology and Nematology Department insect collection, at the Tropical Research and

Education Center, University of Florida.

Results

Development and Survival. Eggs hatched at all temperatures except 370C (Table 3-1).

The duration of development of all stages decreased with increasing temperatures. The egg

developmental time was the same at 34 and 350C. The egg developmental time at 150C was

approximately 5 times longer than the developmental time at 350C. Eggs hatched at 15, 34, and

350C were unable to complete their first-instar development (Table 3-1). The percent survival of









eggs increased with increasing temperature until 300C, above which temperature the survival

started to decrease (Table 3-2). First-instar developmental time at 180C was more than four

times longer than the developmental time at 300C (Table 3-1). Developmental times for male

and female nymphal stages, and cumulative adult male, were not different at 25 and 300C (Table

3-1). The cumulative developmental time for the female was decreased over the temperature

range from 18-300C (Table 3-1).

A low percentage of eggs survived at 350C (Table 3-2). Survival of first and second-

instars was lowest at 180C. The cumulative adult percent survival increased with increasing

temperatures over the range from 18 to 300C (Table 3-2). Since the gender of the eggs was

difficult to differentiate, cumulative survival from egg to adult was not separately calculated for

each gender.

Reproduction. Pre-oviposition and oviposition periods decreased with increasing

temperatures with no difference at 25 and 300C (Table 3-3). Fecundity increased from 18 to

250C, and then drastically decreased at 300C (Table 3-3). Females lived longer at lower

temperatures than at higher temperatures, and with no difference at 25 and 300C (Table 3-3).

Adult male longevity was shorter at 250C than that of at 18 and 20C (Table 3-3). The

proportion of females was lowest at 250C (Table 3-3).

Thermal Requirements for Development. Between the temperatures of 18 to 250C,

there were excellent linear fits (R2 > 0.94; P=<0.0001) for developmental rate versus temperature

in the linear degree-day model for egg, male and female nymphal stages, and cumulative

numbers of adult males and females (Table 3-4). Thermal constants (K) for development rates of

egg, male and female nymphal stages were 100.0, 204.8, and 175.4 degree-days (DD),

respectively. For cumulative development of adult male and female, the thermal constants were









303.0 and 294.1 DD, respectively. The estimated lower developmental thresholds (Tmin) for egg,

male and female nymphal stages were 13.3, 14.8, and 14.30C respectively. For cumulative

development of adult males and females, the estimated lower developmental threshold, Tmin were

14.5 and 13.90C respectively.

For the non-linear section of the developmental rate against temperature, the Logan 6

model also provided excellent fits (Pseudo- R2 > 0.97; P=<0.0001) for each developmental

stadium (Table 3-4). The estimated optimum temperatures (Topt) for the developmental rates of

egg and male and female nymphal stages were 34.8, 27.9 and 28.30C, respectively. For adult

male and female, estimated optimum temperatures were 28.7 and 28.40C, respectively. The

estimated maximum temperature thresholds (Tmax) for egg, male and female nymphal stages

were 41.6, 30.5 and 31.70 C respectively. For adult male and female papaya mealybug, the

estimated Tmax were 31.9 and 32.10C, respectively.

Discussion

Insect systems function optimally within a limited range of temperatures. For a majority

of insects, enzyme activity, tissue functioning, and the behavior of the whole insect is optimal at

a relatively high temperature often in the range of 30-400C (Chapman 1998). Temperature had a

significant effect in the development of P. marginatus. Overall, the linear degree-day model and

the nonlinear Logan 6 model, which were used in predicting temperature and developmental rate

relationships in insects, estimated minimum, optimum, and maximum temperature thresholds for

P. marginatus close to results obtained in this experiment.

The development of adult female P. marginatus was arrested at an estimated minimum

temperature threshold of 14.50C and a maximum temperature threshold of 31.90C. It reached its

optimal development at about 28.70C. The cumulative developmental times of both male and

female mealybugs at 180C were three times longer than the developmental times at 300C.









Although P. marginatus was unable to develop and complete its life cycle at 150C, the Madeira

mealybug, Phenacoccus madeirensis Green, a commonly-found mealybug species in

greenhouses in Southeastern US with a worldwide distribution and with a wide host range (Ben-

Dov 1994), was able to develop, reproduce and survive well at 150C (Chong et al. 2003). At

15C, the developmental time of female P. madeirensis was 66 days and was twice as long as the

developmental time at 250C (Chong et al. 2003). The minimum, optimum, and maximum

temperature threshold for the female pink hibiscus mealybug, Maconellicoccus hirsutus (Green),

a polyphagus mealybug and a serious pest of many economically important crops, were 14.5,

29.0, and 350C respectively. At 200C, the developmental time of the female, M. hirsutus was 66

days, and was twice the developmental time at 300C (Chong et al. manuscript in review).

High fecundity in insects is an important adaptation for a successful next generation. In

nature, eggs of any insect can be exposed to natural enemies and other environmental factors

such as wind, rain, sunlight, and radiation. Although P. marginatus females were able to

develop in a shorter time and had a higher survival at 300C than at the other tested temperatures,

the fecundity at 300C was considerably lower than at 20 and 250C. The drastic drop in fecundity

at 30C suggests that even though the developmental time was shorter and survival was higher at

300C than at 250C, P. marginatus may have reached its optimal temperature for development

and reproduction between the temperatures 25 and 300C. The optimal temperature for

development estimated using the Logan 6 model, for the female nymphal stage and the

cumulative adult female was within this range, thus supporting the results obtained. Other

mealybug species such as P. madeirensis and M. hirsutus showed differences in fecundity with

increasing temperature and were able to reproduce successfully and increase their populations at

temperatures such as 250C. Compared to the fecundity at 200C, the total number of eggs laid at









250C was significantly lower for P. madeirensis (Chong et al. 2003). At 250C, a female P.

madeirensis can lay 288 eggs in 8 days and was able to emerge as an adult within 30 days

(Chong et al. 2003). Similar to P. marginatus, the fecundity at 300C was significantly lower for

M. hirsutus compared to the fecundity at 250C and adult females emerged within 31 days at 250C

and laid 300 eggs within 7 days (Chong et al. manuscript in review). At 250C, female P.

marginatus can emerge as an adult within about 26 days and can produce as many as 300 eggs in

approximately 11 days. With its short life cycle, high survival and reproductive capacity, P.

marginatus has a tremendous ability to increase its population to levels that can cause economic

damage unless suitable management practices are implemented.

Although continuous constant temperatures were used in these experiments, in nature

temperature can vary during the day and especially at night. Warmer day temperatures and

colder night temperatures in the natural environment may allow P. marginatus to develop and

survive at a higher temperature than 300C. Developing Oncopeltus eggs reared at varying

temperatures developed faster and used less metabolic energy than at the equivalent mean

constant temperatures (Gordan 1999). The living system may be better adapted to normal

environmental fluctuations than to an artificial constant state (Gordan 1999).

This information may also be helpful in monitoring the susceptible stages of P.

marginatus for application of integrated management practices including releasing of biological

control agents. The longer developmental time of eggs and immature stages, may increase the

vulnerability by prolonged exposure to natural enemies and insecticides. On the other hand, at

higher temperatures mealybugs grow quickly and become adults 2-3 times faster than at lower

temperatures, allowing them an opportunity to reduce exposure time and presumably to increase

survival and ultimately reproduce.









The estimated thermal constants for eggs and female P. marginatus were 100.0 and 294.1

DD, respectively, while those ofM. hirsutus were 101.7 and 347.2 DD, respectively (Chong et

al. manuscript in review). The estimated minimum temperature threshold for P. citri is 10.9C

and thermal constants for adult female at constant and fluctuating temperatures were 289 and 365

DD, respectively (Laflin and Parrella 2004). The thermal constant for females is considerably

lower for P. marginatus compared to that ofM. hirsutus. Although, P. marginatus and P. citri

had similar thermal constants at constant temperature, the minimum temperature threshold for P.

citri was much lower than that of P. marginatus. Planococcus citri has a wider distribution in

the US compared to currently available information on the distribution ofM. hirsutus (Ben-Dov

1994). Tropical insect species have higher values of minimum temperature thresholds than

temperate insect species, and the thermal constants decrease with the increase of minimum

temperature threshold (Trudgill et al. 2005). Considering its high minimum temperature

thresholds and the low thermal constants, P. marginatus should have a smaller distribution range

than anticipated earlier.

The developmental threshold and the thermal constant of an insect possibly are useful

indicators of its potential distribution and abundance (Messenger 1959). Estimated

developmental temperatures combined with degree-days were useful in predicting Planococcus

citri (Risso) in greenhouse cut flower production in California, in temperatures maintained at 25

to 300C (Laflin and Parrella 2004). The information on developmental temperatures combined

with degree-days from this study, should be useful in predicting possible spread of P. marginatus

in different areas in the US, the Caribbean, and the Pacific. According to the comparative

climatic data from the National Climatic Data Center (NCDC 2005), some areas in Southern

California, Southern Texas, Hawaii, and Florida have daily average temperatures that are









suitable for the development of P. marginatus. A large number of economically important fruits,

vegetables and ornamental plants are grown in Southern California including citrus, avocado,

beans, hibiscus and plumeria. Southern Texas has the third largest citrus production in the US

(CNAS 2007). In Hawaii, where P. marginatus is already established on the big island and small

islands of Maui, Oahu, and Kauai, a large number of fruits, vegetables, and ornamentals are

grown including papaya, hibiscus, and plumeria. Papaya is the second most important fruit crop

in Hawaii, after pineapple, and according to the National Agricultural Statistics Service (USDA

2007b), Hawaii currently grows 864 ha of papaya. In Florida, where approximately 100 ha of

papaya are grown (Mossler and Nesheim 2002), P. marginatus has been found in most of the

counties of Central and South Florida (Walker et al. 2003). Distribution and establishment of P.

marginatus in the areas in California and Texas that are suitable with regard to temperature can

be influenced by other factors such as type of crops grown and regulation of plant movement

from state to state.

Paracoccus marginatus has the potential to spread to Southern California and Texas.

These states produce economically important crops, which are also favored by P. marginatus. If

P. marginatus spread to California and Texas through the movement of plants or commodity, we

will expect the damage to be significant unless suitable control measures such as biological

control and restrictions of movement of susceptible plants and commodities to uninfected areas

are implemented in a timely manner to slow down the spread.










Table 3-1 Mean number of days ( SEM) for each developmental stadium ofP. marginatus reared at different constant
temperatures
T Developmental Stadia Cumulative
(C) Egg First Second Third Fourth
Male Female Male Female Male Male Female
15 27.5 0.2a -
18 23.1 0.2b 25.3 0.5a 21.1 + 1.6a 13.5 1.3a 7.0 1.8a 13.2 0.9a 11.7 1.8a 85.2 1.8a 74.4 1.4a
20 14.4 + 0.2c 14.6 + 0.5b 13.6 + 0.8b 9.3 0.7b 4.5 0.7ab 8.9 + 0.9b 8.9 + 0.7a 53.4 + 0.7b 45.9 + 0.9b
25 8.7 + 0.1d 6.5 + 0.1c 6.6 0.5c 5.5 + 0.5c 2.4 0.5b 5.2 + 0.2c 4.1 + 0.5b 28.5 + 0.3c 25.9 + 0.2c
30 7.3 + 0.2e 6.1 + 0.2c 6.3 0.4c 5.7 + 0.4c 2.6 + 0.4b 4.4 0.3c 3.6 + 0.4b 24.9 0.6c 23.2 0.3d
34 5.9 0.1f
35 5.5 0.f -
F 1922.10 400.59 57.41 17.09 5.35 15.31 15.66 725.42 521.23
df 6,212 3, 132 3,97 3, 101 3, 87 3,90 3,91 3,84 3,90
P <0.0001 <0.0001 <0.0001 <0.0001 <0.0020 <0.0001 <0.0001 <0.0001 <0.0001
n=35
Means within a column followed by the same letters are not significantly different at a = 0.05 (Tukey's HSD Test)









Table 3-2 Mean ( SEM) percent survival for each developmental stadium of P. marginatus reared at different constant
temperatures.


Egg


First


Developmental Stadia
Second Third
Male


Female


15 60.9 + 3.3cd
18 80.0 2.9b 54.1 4.7d 80.1 4.8b 96.4 1.9ab 73.2 7.3
20 90.1 + 2.1a 77.2 + 3.0c 94.1 + 1.3a 98.1 + 1.0a 83.5 + 3.8
25 83.3 + 3.4ab 83.2 4.3bc 97.5 1.3a 89.2 3.1b 79.9 4.8
30 85.9 3.3ab 90.5 2.5ab 91.0 4.0ab 96.9 1.5ab 92.6 2.9
34 73.4 5.1bc
35 33.4 6.9d
37 0.0 + 0.0
F 16.70 16.52 4.89 3.46 1.82
df 6,205 3, 131 3, 122 3,91 3,90
P <0.0001 <0.0001 <0.0030 <0.0196 <0.1494
n=35
Means within a column followed by the same letters are not significantly different at a =


Fourth
Male

98.4 1.6a
75.4 5.7c
86.7 + 2.9bc
97.8 1.6a


10.36
3, 89
<0.0001


Egg to Adult


30.5 + 4.4c
41.4 + 3.5bc
51.4 4.0b
70.8 + 4.9a


15.43
3, 121
<0.0001


0.05 (Tukey's HSD Test)


Temp.









Table 3-3 Mean ( SEM) proportion of females, adult longevity, fecundity, pre-oviposition and oviposition periods of P.
marginatus reared at four constant temperatures
Temperature Proportion of Adult Longevity (Days) Fecundity Pre-oviposition Oviposition
(C) Females Male Female Period (Days) Period (Days)
18 69.4 + 8.2a 5.5 0.5a 40.2 l1.a 160.6 13.8cd 16.7 + 0.7a 19.6 1.0a
20 81.7 3.6a 4.8 0.3a 35.7 1.0b 231.6 12.8bc 13.5 0.5b 21.4 1.la
25 42.6 5.3b 2.9 0.2b 21.1 0.7c 300.2 40.4ab 6.8 0.4c 11.4 0.8b
30 71.1 4.2a 19.2 1.4c 82.0 11.7d 7.6 0.7c 11.6 1.4b
F 10.38 14.91 93.25 15.13 70.33 24.99
df 3, 89 2,73 3,92 3,92 3,92 3,92
P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
n=35
Means within a column followed by the same letters are not significantly different at a = 0.05 (Tukey's HSD Test)









Table 3-4 Summary of statistics and the estimates ( SE) of the fitted parameters of the linear thermal summation model and the
nonlinear Logan 6 model.
Statistics Parameters Developmental Stadia


Egg
Thermal Summation Model: Y= a + bT
F 1640.79
P < 0.0001
df 1, 113
R2 0.9356
a SE -0.13 0.01
b SE 0.01 + 0.01


Logan 6 Model: 1/ D


Sexp(pT) exp(pTma


y Nymphal


1286.51
< 0.0001
1, 69
0.9491
-0.08 + 0.01
0.01 + 0.01
Tm -T
Smax T
AT


cN Nvmphal


2606.13
< 0.0001
1, 63
0.9764
-0.07 + 0.01
0.01 + 0.01


Total Y


Total c<


1653.01
< 0.0001
1, 69
0.9599
-0.05 + 0.01
0.01 + 0.01


3493.67
< 0.0001
1, 67
0.9812
-0.05 + 0.01
0.01 + 0.01


F 22354.5 6092.2 5620.06 10500.7 10252.5
P < 0.0001 <0.0001 < 0.0001 < 0.0001 <0.0001
SSR 0.00024 0.00003 0.00003 0.000009 0.000007
SScT 0.01177 0.00228 0.00221 0.00102 0.00099
Pseudo-R2 0.9796 0.9868 0.9864 0.9912 0.9929
P SE 0.01 + 0.01 0.01 + 0.01 0.01 + 0.01 0.01 0.01 0.01 0.01
p SE 0.11 + 0.02 0.15 + 0.04 0.21 0.04 0.13 + 0.03 0.15 + 0.03
Tmax SE 41.56 0.99 31.71 + 1.25 30.52 0.31 32.09 1.24 31.99 1.52
AT SE 5.24 + 0.06 1.93 + 0.46 1.62 + 0.42 2.05 0.36 1.88 + 0.47
Y= rate of development (1/days); T= ambient temperature (C); a = intercept; b = slope; K (1/b) = thermal constant; Tmin (-a/b) =
lower developmental threshold; SSR = residual sums of squares; SScT = corrected total sums of squares; pseudo-R2 = 1-SSR/SScT
q = rate of temperature dependent physiological process at some base temperature; p = biochemical reaction rate; AT= temperature
range over which 'thermal breakdown' becomes the overriding influence; Topt = optimum temperature threshold; Tmax = upper
developmental threshold









CHAPTER 4
HOST STAGE SUSCEPTIBILITY AND SEX RATIO, HOST STAGE SUITABILITY, AND
INTERSPECIFIC COMPETITION OF Acerophaguspapayae, Anagyrus loecki, AND
Pseudleptomastix mexicana: THREE INTRODUCED PARASITOIDS OF Paracoccus
marginatus WILLIAMS AND GRANARA DE WILLING

Introduction

Knowledge of host selection is indispensable for the efficient use of parasitoids, both for

mass rearing and biological control of pests. A parasitoid's biology may be greatly influenced by

the quality of the host. Host stage is an important ecological variable, which may have an

influence on a parasitoid's rate of attack, survival of its immature stages, and sex ratio of its

offspring (Waage 1986). Host selection behavior is most important in determining the sex ratio

of arrhenotokous parasitoids, which show a haplodiploid sex determination mechanism (King

1987). A female parasitoid can manipulate the offspring sex ratio at oviposition by regulating

fertilization. A particular host size may be more suitable for the development of one sex, so that,

in general, a female-biased offspring sex ratio is produced from the larger hosts and a male

biased one from the smaller hosts (King 1987).

Solitary parasitoids generally determine the host quality by the size of the host. Large

hosts are supposed to be better quality, as they are believed to contain more resources than small

ones. However, host size may not always be equated to host quality at the time of oviposition by

a parasitoid. The influence of host size on parasitoid development may differ between idiobiont

and koinobiont parasitoids (Waage 1986). Idiobiont parasitoids oviposit in host stages such as

egg and pupa, or paralyze their hosts prior to oviposition (Waage 1986) and on the other hand,

koinobiont parasitoids, which do not paralyze their hosts at the time of parasitism, and allow

hosts such as larval stages to grow, for which host size is not directly a representative of larval

resources.









Sympatric parasitoid species that share the same host species may be competitors (Van

Strien-van Liempt 1983). The greater the part of the host population that is exploited by both

species, the more they will affect each other's population density. Their competitive abilities

then, among other factors, determine their relative abundance (Van Strien-van Liempt 1983). In

solitary insect parasitoids, generally only one offspring survives in a host (Vinson 1976).

Females normally deposit one egg per host and reduce the host availability to both conspecific

and heterospecific parasitoids. Successful oviposition of a female depends on how efficient she

is in finding and parasitizing un-parasitized hosts. This leads to interspecific competition among

the parasitoids in classical biological control, where more than one parasitoid species is used

(Lawrence 1981).

Classical biological control was identified as an important pest management practice for

Paracoccus marginatus Williams and Granara de Willink (Hemiptera: Pseudococcidae), a

polyphagus mealybug species that was first identified in the US, in Florida in 1998 (Miller and

Miler 2002). Paracoccus marginatus is a pest of a large number of topical and subtropical fruits,

vegetables, and ornamentals (Miller and Miler 2002). Currently there are three parasitoid

species (Hymenoptera: Encyrtidae) used in the classical biological control of P. marginatus in

the US, the Caribbean, and the Pacific islands (Meyerdirk et al. 2004). Acerophaguspapayae

Noyes and Schauff, Anagyrus loecki Noyes and Menezes, and Pseudleptomastix mexicana

Noyes and Schauff are currently mass reared in Puerto Rico and released by the United States

Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS) in

areas infested with P. marginatus (Meyerdirk et al. 2004). These parasitoids have been released

in Guam and the Republic of Palau, and were successful in controlling P. marginatus in these

countries (Meyerdirk et al. 2004, Muniappan et al. 2006).









There is very little information available on the biology of A. papayae, A. loecki, and P.

mexicana, or on how efficient they are in the control of P. marginatus. Knowledge of host

selection and interspecific competition of parasitoids should lead to better understanding of the

population dynamics of the host and the parasitoids. Hence, they are important in evaluating and

understanding the success of biological control and integrated pest management programs. The

present study focused on the host stage susceptibility and sex ratio, host stage suitability, and

interspecific competition of A. papayae, A. loecki, and P. mexicana, three introduced koinobiont

parasitoids of P. marginatus.

Materials and Methods

Rearing Mealybugs. Paracoccus marginatus was initially collected from a papaya

(Caricapapaya L.) field in Homestead, FL. Sprouted red potatoes (Solanum tuberosum L.)

(Ryan Potato Company, East Grand Forks, MN) were used to rear a colony of P. marginatus at

the University of Florida, Tropical Research and Education Center (TREC), Homestead, FL.

Prior to sprouting, potatoes were soaked in a 1% solution of bleach (Clorox The Clorox

Company, Oakland CA; 6% sodium hypochlorite) for 15 minutes, then rinsed with water, and

dried. Potatoes were placed in black cotton cloth bags to encourage sprouting. These bags were

kept inside a dark room at 270 1IC and 65 2% R.H. Each week, 36 sprouted potatoes were

infested with ovisacs to maintain the colony. An environmental growth chamber (Percivel I-

36LL, Percival Scientific Inc. Perry, NC) was used at 250 1IC, 65 2% R.H., and a

photoperiod of 12:12 (L:D) to rear the mealybug colony.

To obtain a particular stage of mealybugs, newly laid ovisacs were selected from the

mealybug colony. A tender leaf with a 5 cm long stem was obtained from hibiscus (Hibiscus

rosa-sinensis L.) plants that were purchased from a local nursery and maintained in a shadehouse

at TREC. A 9-cm-diam Petri dish with a 0.6-cm-diam hole at the bottom was used to place the









hibiscus leaf. The stem of each hibiscus leaf was inserted through the hole in the Petri dish and

each dish was kept on a 162 ml translucent plastic souffle cup (Georgia Pacific Dixie, Atlanta,

GA) filled with water, which allowed the stem below the petiole to be in water. On each leaf, a

single ovisac was placed and the eggs were allowed to hatch and then develop into the desired

stage. The gender of mealybugs was determined during the latter part of the second instar when

males change their color from yellow to pink. Therefore, the gender was not determined for the

first and second instars, but the third instars and adults used were females. Newly molted

mealybugs, which were recognized by the size and presence of shed exuviae, were selected for

all the experiments in this study to reduce the variation in host quality.

Rearing Parasitoids. The potatoes with second and third instar P. marginatus were used

for parasitoid rearing. These potatoes were initially infested with ovisacs ofP. marginatus,

obtained from the mealybug colony. Colonies of A. papayae, A. loecki, and P. mexicana were

maintained in an insectary at TREC at 250 20C temperature, a 12:12 (L:D) photoperiod, and 65

2% R.H. Initial colonies of parasitoids were obtained from the Biological Control Laboratory,

Department of Agriculture, Puerto Rico, through USDA-APHIS. Parasitoid colonies were

established in plexiglass cages (30x30x30 cm) using sprouted red potatoes infested with P.

marginatus. In order to obtain a continuous supply of newly emerged parasitoids, mealybug-

infested potatoes were provided to each parasitoid species weekly, and potatoes with parasitized

mealybugs were moved to a new cage. A solution of honey and water (1:1) was streaked on 4

pieces (5x5 cm) of Benchkote surface protector paper (Fisherbrand Fisher Scientific,

Pittsburgh, PA) attached to the cage using labeling tape (Fisherbrand Fisher Scientific,

Pittsburgh, PA). Water was provided in two clear plastic 73.9-ml containers (Tristate Molded

Plastic Inc., North Dixon, KY) per cage. In each container, 1-cm-diam hole was made in the









center of the lid and a 7.6 cm long piece of cotton roll (TIDI Products, Neenah, WI) was

inserted through the hole to allow parasitoids to access water.

To obtain mated-female parasitoids, newly emerged females of each species were placed

singly in glass disposable culture tubes (1.2x7.5 cm) (Fisherbrand Fisher Scientific,

Pittsburgh, PA) and closed with two-ply tissue (Kimwipes EX-L, Kimberly-Clerk Global

Sales Inc. Roswell, GA) secured with a piece of rubber tubing (0.95x 2.5 cm) (Fisherbrand ,

Fisher Scientific, Pittsburgh, PA). Five newly emerged males were placed in each tube with a

female and were allowed to mate for 24 hours. A streak of honey and water (1:1) was provided

for each tube. After 24 hours, males were removed from each tube and the female was used in

the experiment. All experiments were carried out at 250 20C temperature, a 12:12 (L:D)

photoperiod, and 65 2% R.H.

Host Stage Susceptibility and Sex Ratio. For host stage susceptibility, a no-choice test

was carried out for first and second instars, third-instar nymphs (female), and for newly emerged

adult females. A hibiscus leaf with a 5 cm long stem was selected as the experimental unit. Petri

dishes were prepared as mentioned previously. The leaf was placed inside the Petri dish with the

stem inserted through the hole and then the lid was replaced. The Petri dish with the leaf was

placed on a cup filled with water with the stem inserted in the water as mentioned above. For

each mealybug stage, ten individuals were selected 24 hours before the experiment and placed on

each hibiscus leaf using a paintbrush (No.000) (American Painter 4000, Loew-Cornell Inc.,

Englewood Cliffs, NJ). These 10 individuals of each mealybug stage were considered as a single

experimental unit and a replicate. The dishes were covered with a piece of black cotton cloth,

encouraging mealybugs to settle on leaves. A streak of honey and water (1:1) was placed on the

inside of the lid. After 24 hours of placing the mealybugs in the Petri dish, a single mated female









parasitoid obtained as mentioned above was placed on each leaf and the lid was replaced. The

Petri dish was covered with a piece (15x15 cm) of chiffon cloth material (Jo-Ann Fabrics and

Crafts, Miami, FL), and secured with a rubber band to avoid parasitoid escape. Each parasitoid

was allowed to oviposit for 24 hours, and then it was removed from the Petri dish. Mummified

mealybugs were individually placed in glass culture tubes and were secured as mentioned

previously. Parasitoids emerged from these tubes were sexed and the proportion of parasitism

was estimated. There were 25 replicates for each mealybug stage and parasitoid combination.

Host Stage Suitability. To test host stage suitability, choice tests were conducted with

two host stage combinations. For mealybugs, five individuals from each stage were used in the

combinations of the second instar and third-instar female, the second instar and adult female, and

the third instar and adult female. A mated female parasitoid was introduced to each Petri dish,

and was allowed to oviposit for 24 hours and then removed. Immediately after removal of the

parasitoid, five individuals of one stage of the host in each Petri dish were moved to a new

hibiscus leaf prepared as above. The five individuals of the other host stage remained on the

hibiscus leaf. These leaves were prepared at the same time as the other leaves. This was done

for easier identification of mummified hosts for a particular stage. The choice tests were carried

out in a similar manner as the host-stage susceptibility tests. The number and the gender of

parasitoids emerging from the mummified mealybugs were recorded for each parasitoid species.

Each mealybug combination for each parasitoid species had 25 replicates.

Interspecific Competition. Interspecific competition of parasitoids was studied using 10

individuals from the second instar and third-instar females. Each host stage was separately

placed on a hibiscus leaf as prepared above. The parasitoid combinations used were A. papayae

and A. loecki, A. papayae and P. mexicana, A. loecki and P. mexicana, and A. papayae, A. loecki,









and P. mexicana. A mated female of each parasitoid species was used in all combinations. They

were allowed to parasitize for 24 hours, and were then removed. The mealybugs were allowed to

mummify on the hibiscus leaves and mummified mealybugs were treated similar to those

mentioned above. The number and the species of parasitoids, which emerged from each

combination in each mealybug instar were counted. The mean percent parasitism was calculated

from the 10 mealybugs used for each host stage in each parasitoid combination. Each parasitoid

combination for each host stage had 25 replicates.

Statistical Analysis. The experimental design was completely random for all experiments.

A two-way analysis of variance (ANOVA) was performed using a general linear model (PROC

GLM) of SAS (SAS Institute 1999) to find the interaction between parasitoids and host stages of

P. marginatus in host stage susceptibility and sex ratio experiments. Means were compared at P

= 0.05 significance level using least square means (LSMEANS) of SAS (SAS Institute 1999).

For host stage suitability tests, means between two stages ofP. marginatus for each parasitoid

species were compared at P = 0.05 significance level using a t-test (PROC TTEST) of SAS (SAS

Institute 1999). In interspecific competition studies, PROC GLM was used for significance

among the parasitoids and means were compared at P = 0.05 significance level using least square

means (LSMEANS). Proportions of females (sex ratio) and proportions of parasitism were

arcsine transformed using

p'= arcsinp

where p = proportion of female/parasitism, to adjust the variances (Zar 1984) prior to ANOVA,

but untransformed data are presented in tables.









Voucher Specimens. Voucher specimens of P. marginatus, A. papayae, A. loecki, and P.

mexicana were deposited in the Entomology and Nematology Department insect collection, at

the Tropical Research and Education Center, University of Florida.

Results

Host Stage Susceptibility and Sex Ratio. All three parasitoids were able to develop and

emerge successfully in second instar, third-instar females, and adult females of P. marginatus

(Table 4-1). No parasitoids emerged from first-instar nymphs. The mean percent parasitism

decreased with increasing host size for both A. papayae and P. mexicana.

The proportion of emerged female parasitoids increased with increasing host size (Table

4-2). Although A. papayae had a similar number of males and females that emerged from

second-instar hosts, A. loecki and P. mexicana had a lower number of females than males

emerging from second instars. In the third instar and the adult females, all three parasitoid

species had higher female than male emergence.

Host Stage Suitability. Acerophaguspapayae and P. mexicana preferred the second

instar to the third-instar female and the adult female, while A. loecki preferred the third-instar

female and the adult female to the second instar (Table 4-3). Between the third-instar female and

the adult female, A. papayae and A. loecki preferred the third-instar mealybugs while P.

mexicana had no preference.

Interspecific Competition. Acerophaguspapayae had a higher mean percent parasitism

when present with either A. loecki or P. mexicana or both in second-instar hosts (Table 4-4). In

third-instar females, A. loecki had a higher parasitism when present with either A. papayae or P.

mexicana or both. Overall, P. mexicana had a lower mean percent parasitism when present with

either A. papayae or A. loecki or both except for the presence with A. loecki in the second-instar

hosts.









Discussion

Size of the host is one of the factors that solitary endoparasitoids consider when they

select a host stage for oviposition (Vinson and Iwantsch 1980). Acerophaguspapayae, A. loecki,

and P. mexicana did not prefer first-instar nymphs as a suitable host stage for parasitoid

development. This makes the first-instar nymph ofP. marginatus, which is approximately 0.4

mm in size (Miller and Miler 2002), less vulnerable to these parasitoids.

In parasitoid behavioral studies, first-instar Rastrococcus invadens Williams were

preferred for host feeding by the parasitoid, Anagyrus mangicola Noyes (Bokonon-Ganta et al.

1995). Parasitoids such as Anagyrus kamali Moursi can oviposit in first-instar nymphs of

Maconellicoccus hirsutus Green but the percent parasitism was less than 20% (Sagarra and

Vincent 1999). In most situations, the ovipositor of A. kamali remained stuck within the first-

instar host, precluding further foraging of the parasitoid (Sagarra and Vincent 1999). The

second-instar nymphs of Planococcus citri (Risso) were often impaled on the ovipositor of

Anagyruspseudococci (Girault), thus preventing the female from further egg deposition (Islam

and Copland 1997).

By choosing a larger host, the parasitoid accessed a larger food supply and increased the

fitness of its progeny. In larger hosts, a female biased progeny was recorded for many

parasitoids (King 1987). Further, increased host size translates into both increased male and

female fitness (Charnov et al. 1981). For females this measure is the lifetime production of eggs,

and for males, it is the length of life (Charnov et al. 1981). Although, all three parasitoid species

ofP. marginatus were able to develop and complete their life cycle in second-instar hosts, only

A. papayae produced a higher proportion of female progeny. Having more males than females in

its progeny is not a desirable characteristic for a parasitoid to have as an efficient biological

control agent. The solitary endoparasitoid, Aenasius vexans Kerrich, which were able to oviposit









in second-instar nymphs of Phenacoccus herreni Cox and Williams also recorded a considerably

higher proportion of males in the second instar than in the larger instars of P. herreni (Bertschy

et al. 2000).

Except for the parasitism ofA. loecki in second-instar P. marginatus, the percent

parasitism of all three parasitoid species decreased with increasing host size. Mealybugs show

strong physical defense and escape behavior, which could be increased with the body size. The

third-instar P. citri, which was often encountered by the parasitoid A. pseudococci, showed

strong physical defense and escape behavior (Islam and Copland 1997). The higher success of

oviposition in the second-instar P. marginatus may be due to less or absence of these defense

and escape behavior in early instar mealybugs.

Interspecific competition was evident by A. papayae, A. loecki, and P. mexicana, when

competing for same host instar. Out of the three parasitoid species, A. papayae had the highest

parasitism level indicating its superior ability to compete. Intensive studies of parasitic

complexes in connection with biological control programs have shown that interspecific

competition can be extremely important (Schroder 1974). This may be one reason, why A.

papayae was well established and recovered from the field in the Republic of Palau (Muniappan

et al. 2006). In field tests conducted in Florida in 2005 and 2006, both A. papayae and A. loecki

were recovered, but P. mexicana was not (Chapter 6). Pseudleptomastix mexicana was also not

recovered in the field studies conducted in the Republic ofPalau (Muniappan et al. 2006). This

information suggests that P. mexicana may be less competitive than the other two parasitoids.

Since the preference for a host stage is similar for A. papayae and P. mexicana but

different for A. loecki, it reduces the competitiveness between A. papayae and A. loecki.

Different host stage preference of A. papayae and A. loecki also greatly reduces competition for









the same host stage. However, the developmental time of A. loecki, which is similar to the

developmental time ofA. papayae, is shorter than the developmental time of its preferred host

stage of P. marginatus (Chapter 5). Developmental times of A. papayae and A. loecki coincide

with the developmental time of the second instar P. marginatus (Chapter 5). At the beginning of

the season with the absence of overlapping generations of P. marginatus, A. loecki and A.

papayae can compete for second instars due to unavailability of preferred third instar host stages

for A. loecki. In addition to being a parasitoid ofP. marginatus, A. loecki can develop in

Dysmicoccus hurdi and Phenacoccus madeirensis Green (Hemiptera: Pseudococcidae) (Noyes

2000). Phenacoccus madeirensis is one of the commonly found mealybug species in South

Florida. Since A. loecki is not host-specific, it has the advantage of searching for other suitable

hosts such as P. madeirensis in the absence of suitable stages ofP. marginatus. This may be one

reason for the lower parasitism ofA. loecki observed in the field studies and less competitiveness

of A. loecki in the control of P. marginatus (Chapter 6). On the other hand, P. mexicana, which

also prefers the second instar P. marginatus (as does A. papayae) has longer developmental time

than the other two parasitoids (Chapter 5). Developmental time of P. mexicana does not

coincide with the developmental time of the second instar P. marginatus. This allows A.

papayae females for which developmental time of the host stage overlaps with its developmental

time, to parasitize preferred host stages when it emerges as an adult. In mass rearing of

parasitoids, second-instar P. marginatus is a suitable stage for A. papayae and P. mexicana while

third-instar females are suitable for A. loecki.

Females ofA. papayae and P. mexicana had a similar host stage preference for parasitism

while it was a different host stage preference for female A. loecki. Acerophaguspapayae shows

superior adaptability by being able to oviposit in second instar to adult-female P. marginatus as









well as causing a higher percent parasitism when present with either A. loecki or P. mexicana or

both. The information gathered from this study, will be helpful in explaining the adaptability of

these three parasitoids ofP. marginatus in the field.









Table 4-1 Mean percent parasitism ( SEM) of A. papayae, A. loecki, and P. mexicana
reared in different developmental stages of P. marginatus to evaluate host stage
susceptibility using no-choice tests.
Mean Percent Parasitism (%) for Developmental Stages of
P. marginatus
Parasitoid Second Third female Adult female
A. papayae 82.8 + 2.1aA 71.2 + 2.6bB 60.8 + 2.9bC
A. loecki 41.2 2.8cB 82.4 + 1.9aA 74.8 3.2aA
P. mexicana 70.8 + 1.9bA 50.8 + 2.5cB 40.8 + 3.6cB
ANOVA Results
Source F df P
Model 36.32 8,216 <0.0001
Parasitoid 34.06 2, 216 <0.0001
Stage 8.67 2,216 0.0002
Parasitoid* Stage 51.27 4, 216 <0.0001
n = 25
Means within a column followed by the same lowercase letters, and means within a row
followed by the same uppercase letters are not significantly different at a = 0.05 (Least Square
Means (LSMEANS) Test).









Table 4-2 Mean proportion of females (sex ratio) ( SEM) ofA. papayae, A. loecki, and P.
mexicana reared in different developmental stages of P. marginatus to evaluate host
stage susceptibility using no-choice tests.
Mean Proportion of Females (Sex Ratio) ( SEM) for Developmental
Stages of P. marginatus
Parasitoid Second Third-female Adult-female
A. papayae 0.50 + 0.0laB 0.56 0.01aA 0.57 0.01abA
A. loecki 0.40 + 0.01cC 0.51 + 0.01bB 0.54 + 0.01bA
P. mexicana 0.48 + 0.01bC 0.55 0.0laB 0.56 + 0.01aA
ANOVA
Results
Source F df P
Model 72.34 8,216 <0.0001
Parasitoid 73.68 2, 216 <0.0001
Stage 196.98 2,216 <0.0001
Parasitoid* Stage 9.35 4, 216 <0.0001
n = 25
Means within a column followed by the same lowercase letters, and means within a row
followed by the same uppercase letters are not significantly different at a = 0.05 (Least Square
Means (LSMEANS) Test).










Table 4-3 Mean percent parasitism ( SEM) of A. papayae, A. loecki, and P. mexicana reared in different stage combinations of


P. marginatus to evaluate host stage suitability using choice tests.
Host stage combination of P. marginatus Parasitoid Mean ( SEM) Percent Parasitism


Stage 1
Second


Stage 2
Third- Female


Second Adult-Female


Third Adult-Female


A. papayae
A. loecki
P. mexicana
A. papayae
A. loecki
P. mexicana
A. papayae
A. loecki
P. mexicana


Stage 1
77.6 1.8
30.4 2.9
69.6 + 2.6
76.8 1.9
32.0 + 3.1
68.8 + 2.6
60.0 3.1
79.2 + 0.8
41.6 + 3.4


Stage 2
58.4 2.6
76.0 2.0
40.8 3.6
50.4 4.2
68.8 2.6
32.0 3.3
48.0 5.0
64.8 3.1
32.8 3.2


t df
6.54 48
-13.60 48
6.73 48
-5.98 48
9.11 48
-8.78 48
-1.81 48
-4.58 48
-1.85 48


n = 25
Host stage combinations: second instar (Stage 1) and third-instar female (Stage 2), second instar (Stage 1) and adult female (Stage 2),
and third-instar female (Stage 1) and adult female (Stage 2).


T Statistics


P
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
0.0471
<0.0001
0.0691









Table 4-4 Mean percent parasitism ( SEM) of combinations of A. papayae, A. loecki, and P. mexicana reared in second and
third-instar P. marginatus to evaluate interspecific competitions of parasitoids.


Stage of P. marginatus

Second


Third


Combination of Parasitoids


Parasitoid 1
A. papayae
A. papayae

A. papayae
A. papayae
A. papayae

A. papayae
ANOVA Results


Parasitoid 2
A. loecki

A. loecki
A. loecki
A. loecki

A. loecki
A. loecki


Parasitoid 3

P. mexicana
P. mexicana
P. mexicana

P. mexicana
P. mexicana
P. mexicana


Mean ( SEM) Percent Parasitism


A. papayae
69.6 + 2.3A
78.4 1.2A

59.6 + 2.9A
42.4 3.3B
59.2 + 4.3A

38.8 + 4.4B


A. loecki
19.6 1.7B

40.4 3.5B
14.8 1.8C
54.8 3.5A

75.2 + 2.7A
47.6 + 4.5A


P. mexicana

20.0 1.3B
50.4 3.3A
23.6 + 2.1B

35.6 + 3.5B
20.4 1.4B
11.2 + 0.6C


Source F df P
Model 49.8 17,432 <0.0001
Stage 0.71 1,432 0.4887
Combination 61.67 8, 432 <0.0001
Stage*Combination 44.09 8, 432 <0.0001
N= 25
Means within a row followed by the same uppercase letters are not significantly different at a = 0.05 (Least Square Means
(LSMEANS) Test).









CHAPTER 5
DEVELOPMENTAL TIME, LONGEVITY, AND LIFETIME FERTILITY OF Acerophagus
papayae, Anagyrus loecki, AND Pseudleptomastix mexicana; THREE INTRODUCED
PARASITOIDS OF Paracoccus marginatus WILLIAMS AND GRANARA DE WILLING

Introduction

Developmental time, longevity, and lifetime fertility are important fitness parameters when

evaluating a biological control agent. Determining developmental time of a parasitoid is

necessary to determine its efficiency in controlling the host. Generally, the developmental time

of a biological control agent should be shorter than the developmental time of the host

(Greathead 1986). According to Greathead (1986), high fecundity and short generation time are

some of the desirable characters of a parasitoid. Courtship and mating are energy and time

consuming activities in insects, which can affect the outcome of the longevity, lifetime fecundity,

and progeny production of hymenopteran parasitoids (Ridley 1988). Mating is required to

achieve their full reproductive potential in some parasitoids (Ridley 1988). The progeny sex

ratio is the main fitness parameter that can be affected by mating. The majority of parasitoids

need to mate once to attain their optimal sex ratio (Ridley 1993). Some parasitoid species are

arrhenotokous, e.g. fertilized eggs lead to female progeny and unfertilized eggs give rise to

males. Lifetime fertility or progeny production of a parasitoid is important in its long-term

establishment as a biological control agent. A parasitoid with higher lifetime fertility with

female-biased progeny can parasitize a higher number of hosts. Female-biased progeny are

desirable in classical biological control (King 1987).

Classical biological control was identified as an important pest management practice for

Paracoccus marginatus Williams and Granara de Willink (Hemiptera: Pseudococcidae), a

polyphagus mealybug species that was first identified in the US in Florida in 1998 (Miller and

Miler 2002). Paracoccus marginatus is a pest of a large number of tropical and subtropical









fruits, vegetables, and ornamentals (Miller and Miler 2002). Prior to invading the US, P.

marginatus had been established in the Caribbean since 1994 (Miller et al. 1999). After the

establishment in Florida, P. marginatus was identified in the Pacific islands of Guam (Meyerdirk

et al. 2004), the Republic of Palau (Muniappan et al. 2006), and several Hawaiian islands (Heu et

al. 2007). With the joint efforts of the Dominican Republic, Puerto Rico, and the US (Walker et

al. 2003), currently there are three parasitoids (Hymenoptera: Encyrtidae) used in the classical

biological control ofP. marginatus in the US, the Caribbean, and the Pacific islands (Meyerdirk

et al. 2004). The three solitary endoparasitoid species, Acerophaguspapayae Noyes and

Schauff, Anagyrus loecki Noyes and Menezes, and Pseudleptomastix mexicana Noyes and

Schauff are currently mass reared in Puerto Rico and released in P. marginatus affected areas by

the United States Department of Agriculture (USDA), Animal and Plant Health Inspection

Service (APHIS) (Meyerdirk et al. 2004). They have been released in Guam, and the Republic

of Palau, and are successfully controlling P. marginatus (Meyerdirk et al. 2004, Muniappan et al.

2006).

No information is available on the fitness parameters of these parasitoids of P. marginatus.

Fitness parameters of parasitoids are specifically important when evaluating their efficiency and

understanding long-term effects in a system where more than one parasitoid species have been

released as classical biological control agents. This study focuses on the developmental time,

longevity, and the lifetime fertility ofA. papayae, A. loecki, and P. mexicana, three introduced

parasitoids of P. marginatus.

Materials and Methods

Rearing Mealybugs. Red potatoes (Solanum tuberosum L.) (Ryan Potato Company, East

Grand Forks, MN) were allowed to sprout and then used in rearing a colony of P. marginatus at

the University of Florida, Tropical Research and Education Center (TREC), Homestead, FL.









Initially, P. marginatus was collected from a papaya (Caricapapaya L.) field in Homestead, FL.

Prior to sprouting, potatoes were soaked in a 1% solution of bleach (Clorox The Clorox

Company, Oakland CA; 6% sodium hypochlorite) for 15 minutes, and then rinsed with water,

air-dried and placed in black cotton cloth bags to encourage sprouting. Bags were kept inside a

dark room at 270 1IC and 65 2% R.H. Each week, 36 sprouted potatoes were infested with

P. marginatus ovisacs to maintain the colony. An environmental growth chamber (Percivel I-

36LL, Percival Scientific Inc. Perry, NC) was used at 250 1IC, 65 2% R.H., and a

photoperiod of 12:12 (L:D) to rear the mealybug colony.

To obtain a particular stage of mealybugs, the newly laid ovisacs were selected and each

was reared on a hibiscus leaf inside a 9-cm-diam Petri dish with a 0.6-cm-diam hole made in the

bottom. Leaves were obtained from hibiscus (Hibiscus rosa-sinensis L.) plants maintained in a

shadehouse at TREC. A tender hibiscus leaf with a 5-cm-long stem was placed in each Petri dish

with the stem inserted through the hole at the bottom of the Petri dish. Each Petri dish was kept

on a 162 ml translucent plastic souffle cup (Georgia Pacific Dixie, Atlanta, GA) filled with

water, which allowed the stem below the petiole to be in water. A single ovisac was placed on

each leaf and the eggs were allowed to hatch and then to develop to the desired stage. It was not

possible to differentiate the sex of the mealybugs for the first and second instar while the third-

instar nymphs and the adults used were females. To reduce the variation in each mealybug instar

used, newly molted individuals recognized by their size and the presence of shed exuviae were

selected for each experiment.

Rearing Parasitoids. Colonies of A. papayae, A. loecki, and P. Mexicana were

maintained in an insectary at TREC at 250 20C temperature, a 12:12 (L:D) photoperiod, and 65

2% R.H. Initial colonies of parasitoids were obtained from the Biological Control Laboratory,









Department of Agriculture, Puerto Rico, through USDA-APHIS. Parasitoid colonies were

established in plexiglass cages (30x30x30 cm) using sprouted red potatoes with second and

third-instar P. marginatus. In order to obtain a continuous supply of newly emerged parasitoids

weekly, potatoes with second and third-instar mealybugs were provided to each parasitoid

species every week. After 7 days, potatoes were moved to a new cage for parasitoid emergence

and new mealybug-infested potatoes were provided for oviposition. A solution of honey and

water (1:1) was streaked on 4 pieces (5x5 cm) of Benchkote surface protector paper (Fisherbrand

, Fisher Scientific, Pittsburgh, PA) and attached to the cage using labeling tape, for emerging

parasitoids (Fisherbrand Fisher Scientific, Pittsburgh, PA). Water was provided in two clear

plastic 73.9 ml containers (Tristate Molded Plastic Inc., North Dixon, KY) per cage. In each of

the containers, a 1-cm-diam hole was made in the center of the lid and a 7.6-cm-long piece of

cotton roll (TIDI Products, Neenah, WI) was inserted through the hole to allow parasitoids to

access water.

To obtain mated female parasitoids for the experiments, newly emerged female parasitoids

of each species were selected, and placed singly in glass disposable culture tubes, (1.2x7.5 cm)

(Fisherbrand Fisher Scientific, Pittsburgh, PA) closed with two-ply tissue, (Kimwipes EX-

L, Kimberly-Clerk Global Sales Inc. Roswell, GA) and secured with a piece of rubber tubing

(0.95x2.5 cm) (Fisherbrand Fisher Scientific, Pittsburgh, PA). For each tube with a female,

five newly emerged males were added and allowed to mate for 24 hours. A streak of honey and

water (1:1) was provided for each tube. After 24 hours, males were removed from each tube,

and the mated female was used in the experiment. All experiments were carried out at 250 2C

temperature, a 12:12 (L:D) photoperiod, and 65 2% R.H.









Specimens of P. marginatus, A. papayae, A. loecki, and P. mexicana were sent to

Systematic Entomology Laboratory (SEL), USDA, Beltsville, MD, for species verification.

Developmental Time. To evaluate the developmental time of each parasitoid species in

different mealybug instars, 10 individuals each from second instar, third-instar females, and adult

female P. marginatus were selected and placed separately on new hibiscus leaves. The Petri

dishes with leaves were prepared 48 hours before the experiment. Mealybugs were placed on the

leaves 24 hours before the experiment to allow them to settle on the leaves. The 10 individuals

of each mealybug stage on a hibiscus leaf were considered a replicate and were used as an

experimental unit. A mated female parasitoid was placed in each Petri dish and the lid was

replaced. Each Petri dish with the lid was covered with a piece (15x15 cm) of chiffon cloth

material (Jo-Ann Fabrics and Crafts, Miami, FL) and secured with a rubber band to avoid

parasitoid escape, before it was placed on a cup of water with the stem submerged. Parasitoids

were allowed to oviposit for 24 hours and were then removed. Parasitized mealybugs became

mummified on the hibiscus leaves. Mummies were individually placed in disposable, glass

culture tubes and closed with two-ply tissue and secured with a piece of clear polyvinyl chloride

(PVC) tubing. These tubes with the parasitized mealybugs were held in the insectary until the

emergence of adults. The time for adult emergence and the sex of the adults were noted and the

mean of the 10 individuals on each hibiscus leaf was used in analyses for developmental time

and sex ratio of each parasitoid. This procedure was followed for all three parasitoid species

with 50 replicates for each species.

Longevity. Longevity was studied for three mating conditions for each female parasitoid

(unmated, mated-without oviposition, and mated-with oviposition), and two mating conditions

for each male parasitoid (unmated and mated). To collect both males and females for each









species, third-instar mealybugs on sprouted potatoes were placed in plexiglass cages and mated

females were released to each cage. After 24 hours, females were removed and mealybugs were

allowed to mummify. Mummified mealybugs were collected, and were individually placed in

glass culture tubes as above.

When parasitoids started to emerge from the mummified mealybugs, 50 newly emerged

virgin males and females were separately placed in glass culture tubes for unmated status, and

each tube was provided with a streak of honey and water, and secured with two-ply tissue. For

unmated females with oviposition, 50 newly emerged females were individually transferred to

clear plastic 500 ml deli cups (Georgia Pacific Dixie, Atlanta, GA) and provided with mealybugs

on potatoes to oviposit. Each cup was covered with a piece of chiffon cloth material before

placing the lid. A circular area of 8.5-cm-diam was removed from the 12-cm-diam lid to

facilitate air circulation. For mated males, one male was placed in a glass culture tube with a

streak of honey and five females were provided for mating for 24 hours, and then the females

were removed. The mated males were retained in the culture tube. For mated females without-

oviposition, one female was placed in a glass culture tube with a streak of honey, and five males

were provided for 24 hours and then the males were removed and the females were retained.

The same procedure was followed for the mated females with oviposition, except they were

individually transferred to clear plastic 500 ml deli cups and provided with mealybugs on

potatoes to oviposit as for unmated females with oviposition, as mentioned above. The number

of days each parasitoid lived was counted for both males and females in all the above mating

conditions. These procedures were repeated for all three parasitoid species. For each mating

condition in each sex, 50 replicates were used.









Lifetime Fertility. Lifetime fertility of mated and unmated females of each parasitoid

species was studied. A newly emerged virgin female was either held alone or allowed to mate

with five newly emerged males for 24 hours in a glass culture tube provided with a streak of

honey and water. After removing the males, the females were individually transferred to clear

plastic 3.8 liter round jars (Rubbermaid Newell Rubbermaid Inc. Atlanta, GA). Before

placing the lid, each jar was covered with a piece of chiffon cloth material. A 9-cm-diam area

was removed from the lid to allow air circulation. Unmated females were also transferred

individually to clear plastic jars as mated females. Each mated or unmated female was provided

approximately 300 third-instar female mealybugs on 1-2 infested potatoes daily for oviposition

until the death of the females. The potatoes with parasitized mealybugs were placed in clear

plastic 500 ml deli cups as above to allow mummification. When the parasitoids started to

emerge, the number of males and females were counted. For each parasitoid species, 25

replicates were used for each mating condition.

Statistical Analysis. The experimental design was completely random for all experiments.

A two-way analysis of variance (ANOVA) was performed using a general linear model (GLM)

(SAS Institute 1999) to find interaction between parasitoids and mealybug instar for

developmental time, parasitoids and mating conditions in the longevity experiment, and for

reproductive period, and male and cumulative progeny in the lifetime fertility study. Means

were compared at P = 0.05 significance level using least square means (LSMEANS) (SAS

Institute 1999). For the developmental time and the longevity studies, means were compared

within the mealybug instar and mating condition for each parasitoid, and among the parasitoids

for each mealybug instar and mating condition. One-way ANOVA was performed using a

general linear model (GLM) for number of female progeny and sex ratio. Means were compared









at P = 0.05 significance level using the Tukey's HSD test. The proportion of female (sex ratio)

was square-root arcsine-transformed by using

p'= arcsinp

where, p = proportion of female, to adjust the variances (Zar 1984) prior to ANOVA, but

untransformed data were presented in tables.

Voucher specimens. Voucher specimens of P. marginatus, A. papayae, A. loecki, and P.

mexicana were deposited in the Entomology and Nematology Department insect collection, at

the Tropical Research and Education Center, University of Florida, Homestead, FL 33031.

Results

Developmental times were shorter with increasing host age for male A. papayae and P.

mexicana, and female A. loecki (Table 5-1). Acerophaguspapayae and A. loecki had shorter

developmental times for both males and females, compared to male and female developmental

times of P. mexicana.

Longevity was highest for P. mexicana and the lowest was for A. papayae in all mating

conditions with increasing host size for both males and females (Table 5-2). There was no

difference in the longevity between unmated and mated males in all three species. The longevity

was similar for females that were unmated and mated, both without oviposition. The females

that were unmated and mated both with oviposition had similar longevity in each species but

lived a shorter time than the ones that did not oviposit.

Unmated females of all three species produced male progeny, and A. loecki and P.

mexicana produced more progeny than A. papayae (Table 5-3). In mated females, there were

more male and female progeny for A. loecki and P. mexicana than for A. papayae. The progeny

of all three species had similar sex ratios with approximately 1:1 for male:female. The









reproductive period was longest for P. mexicana, and A. papayae had the shortest reproductive

period.

Discussion

Differences in fitness parameters including developmental time, longevity, and the lifetime

fertility of A. papayae, A. loecki, and P. mexicana are useful in evaluating them as efficient

biological control agents ofP. marginatus. There were differences in developmental time,

longevity, and the lifetime fertility ofA. papayae, A. loecki, and P. mexicana.

Although increasing host size had a significant effect on developmental time of male A.

papayae, A. loecki, and P. mexicana, developmental times of female A. papayae, and P.

mexicana were not influenced by host size. Host stage had affected developmental time of other

mealybug parasitoids as well. Developmental time of Anagyrus dactylopii (Howard) was not

different among the various stages ofMaconellicoccus hirsutus (Green) (Mani and Thontadarya

1989). However, Anagyrus kamali Moursi, a parasitoid ofM. hirsutus, had shorter

developmental times when reared in the third instar and adult female M. hirsutus than when

reared in the first and second-instarMM hirsutus (Sagarra and Vincent 1999). Anagyrus kamali

had similar developmental times in the first and second instars, and in third and adult females of

M. hirsutus (Sagarra and Vincent 1999). Aenasius vexans Kerrich, an encyrtid parasitoid of

cassava mealybug, Phenacoccus herreni Cox and Williams, had a shorter developmental time in

older hosts than in early instar P. herreni (Bertschy et al. 2000). Developmental time of female

Anagyruspseudococci (Girault), a koinobiont endoparasitoid of citrus mealybug, Planococcus

citri (Risso) was similar on second and third instar, and adult P citri, but the developmental time

of male A. pseudococci was longer on second instars than on third instar and adult P. citri

(Chandler et al. 1980, Islam and Copland 1997). Gyranusoidea tebygi Noyes, a parasitoid of

mango mealybug, Rastrococcus invadens Williams, when developed on second and third instar









had similar developmental times compared to the longer developmental time in first instar R.

invadens (Bovida et al. 1995a). However, Anagyrus mangicola Noyes, the primary parasitoid of

R. invadens, had a similar developmental time on different host stages of R. invadens with no

differences in the size of emerging parasitoids (Cross and Moore 1992).

When the developmental time of a parasitoid is shorter than the developmental time of the

host, there is an advantage for the parasitoid. Later in the season with overlapping host

generations, it can produce its progeny at a faster rate than the host and can parasitize the host

populations in a shorter time. The adult female P. marginatus can develop on hibiscus in 25.9

days, and the second instar and third-instar females can emerge within 15.2 and 20.8 days

respectively (Chapter 2). Acerophagus papayae and P. mexicana prefer second instars compared

to third-instar P. marginatus (Chapter 4). Developmental time of female A. papayae overlaps

the developmental time of the second-instar P. marginatus, providing an advantage for A.

papayae over female P. mexicana, which needs a longer time to emerge as adults in second-

instar P. marginatus. Although A. loecki prefers third instars compared to second-instar P.

marginatus (Chapter 4), its developmental time is shorter than the developmental time of the

third-instar P. marginatus. Early in the season with the absence of overlapping generations of

mealybugs, the longer developmental time of the third-instar P. marginatus, which does not

overlap the emergence of A. loecki, allows A. loecki able to parasitize the available second instar

P. marginatus. When developed in second-instar P. marginatus, A. loecki produces male-biased

progeny compared to A. papayae, which produces female-biased progeny (Chapter 4). Male-

biased progeny would be less desirable than female-biased progeny in biological control (King

1987). However, because it is not a host-specific parasitoid of P. marginatus, female A. loecki









has the ability to select suitable stages of its other hosts such as Madeira mealybug, Phenacoccus

madeirensis Green (Noyes 2000), in the absence of preferred host stages of P. marginatus.

In parasitic Hymenoptera, female eggs are preferentially laid in larger hosts compared to

male eggs, which are laid in smaller hosts (King 1987). Hosts that were parasitized at different

stages may represent resources of different quality during parasitoid development and the wasp

may have adapted its sex allocation accordingly (Bertschy et al. 2000). Although both male and

female P. mexicana are larger than male and female A. papayae (Noyes and Schauff 2003), P.

mexicana females prefer to lay their eggs in the second instar rather than in third-instar P.

marginatus (Chapter 4). Since the developmental time of P. mexicana is longer than the

developmental times of A. papayae and A. loecki, a second-instar host may be more desirable

than a third instar for the development ofP. mexicana.

A fitness parameter such as the lifetime fertility of a parasitoid is important in long-term

establishment of the parasitoid as a biological control agent. A parasitoid species with more

female progeny has the ability to parasitize a higher number of hosts than one with fewer female

progeny. Body size of a parasitoid is frequently related to fecundity, longevity, and host finding

ability (Hemerik and Harvey 1999). A significant relationship between size and both longevity

and lifetime fecundity was found in fitness parameter studies in Trichogramma evanescens, a

gregarious, polyphagous egg parasitoid (Doyon and Boivin 2005). The smallest of the three

species (Noyes 2000, Noyes and Schauff 2003), A. papayae produced the least progeny

compared to A. loecki and P. mexicana, both of which produced twice the progeny of A.

papayae. In addition, A. papayae had the shortest lifespan compared to A. loecki and P.

mexicana for both males and females with different mating status. Females of all three

parasitoid species outlived males. This has been recorded in other parasitoids species as well. In









the longevity studies ofAnagyrus kamali Moursi, females lived longer than the males (Sagarra et

al. 2000b).

There are differences in the developmental time, longevity, and lifetime fertility ofA.

papayae, A. loecki, and P. mexicana. The differences in these fitness parameters are important in

evaluating their efficiency as parasitoids ofP. marginatus. This information provides the insight

needed to clarify the efficiency of A. papayae in controlling P. marginatus as well as to explain

the lower efficiency of A. loecki, and P. mexicana.









Table 5-1 Mean developmental time (egg to adult eclosion) in days ( SEM) for male and
female A. papayae, A. loecki, and P. mexicana reared in second instar, third-instar
female, and adult-female P. marginatus.
Mean Developmental Time of Parasitoids (Days)
Stage of P. marginatus
Sex Parasitoid Second-instar Third-instar female Adult-female
Male A.papayae 13.8 + 0.2bA 13.5 + 0.2bAB 13.1 + 0.2bB
A. loecki 13.7 0.2bA 13.4 0.2bAB 13.1 0.3bB
P. mexicana 21.8 + 0.2aA 21.5 + 0.2aAB 21.0 + 0.2aB
ANOVA Results


Source
Model
Parasitoid
Stage
Parasitoid* Stage
Female A. papayae
A. loecki
P. mexicana
ANOVA Results
Source
Model
Parasitoid
Stage
Parasitoid* Stage


F
339.74
1350.27
8.61
0.04
14.8 + 0.2bA
14.7 + 0.2bA
22.9 + 0.2aA

F
328.75
1306.44
8.42
0.06


df
8, 4414
2, 441
2, 441
4, 441
14.5 + 0.2bAB
14.4 + 0.2bAB
22.7 + 0.2aAB

df
8, 441
2, 441
2, 441
4, 441


P
<0.0001
<0.0001
0.0002
0.9970
14.1 + 0.2bB
14.0 + 0.2bB
22.1 + 0.3aB

P
<0.0001
<0.0001
0.0003
0.9927


n =50
Means within a column followed by the same lowercase letters, and means within a row
followed by the same uppercase letters are not significantly different at a = 0.05 (Least Square
Means (LSMEANS) Test) for males and females









Table 5-2 Mean longevity in days ( SEM) for male (unmated and mated), and female
(unmated, mated-without oviposition, and mated-with oviposition) A. papayae, A.
loecki, and P. mexicana.


Sex


Mating Condition


Male Unmated
Mated
ANOVA Results
Source
Model
Parasitoid
Mating Status
Parasitoid* Mating Status
Female Mating Status
Unmated-without oviposition
Unmated-with oviposition
Mated-without oviposition
Mated-with oviposition
ANOVA Results
Source
Model
Parasitoid
Mating Status
Parasitoid* Mating Status
n =50


Longevity (Days)
Parasitoid
A. papayae A. loecki
23.3 + 0.4C 37.3 + 0.7B
22.0 + 0.4C 36.6 + 0.5B

F df
141.97 5,294
353.58 2,294
2.41 1,294
0.14 2,294
A. papayae A. loecki
33.1 + 0.6aC 48.9+ 1.0aB
13.8 + 0.2bC 23.9 + 0.5bB
32.3 + 1.0aC 47.6 + 1.2aB
13.9 0.3bC 23.0 0.4bB

F df
322.95 11, 588
922.75 2, 588
558.98 3,588
5.01 6, 588


P. mexicana
47.5 1.8A
45.9 + 0.9A

P
<0.0001
<0.0001
0.1216
0.8722
P. mexicana
63.1 + 1.8aA
41.1 + 0.7cA
58.4 1.2bA
40.1 + 0.7cA

P
<0.0001
<0.0001
<0.0001
<0.0001


Means within a column followed by the same lowercase letters, and means within a row
followed by the same uppercase letters are not significantly different at a = 0.05 (Least Square
Means (LSMEANS) Test) for males and females.










Table 5-3 Mean (+ SEM) number of male and female progeny, cumulative progeny, sex ratio, and reproductive period of mated
and unmated A. papayae, A. loecki, and P. mexicana.
Mating Parasitoid Number of Number of Female Cumulative Sex Ratio Reproductive
Status Male Progeny Progeny Progeny (Proportion of Females) Period (Days)


Mated A.papayae
A. loecki
P. mexicana
Unmated A. papayae
A. loecki
P. mexicana
ANOVA Results
Model F


44.5 1.0c
97.8 1.3b
103.0 + 3.4b
88.0 + 2.9b
173.2 + 10.2a
159.5 + 7.7a


73.40
5, 144
<0.0001


48.3 + 1.2b
99.8 + 1.6a
105.9 3.3a




199.88
2,72
<0.0001


0.52 + 0.00
0.51 + 0.00
0.50 + 0.01


92.8 1.9c
197.6 + 2.5a
208.9 + 6.6a
88.0 + 2.9c
173.2 + 10.2b
159.5 + 7.7b


71.17
5, 144
<0.0001


n = 25
Means within a column followed by the same lowercase letters are not significantly different at a
(LSMEANS) Test) for number of male progeny, cumulative progeny, and reproductive period.
0 Means within a column followed by the same lowercase letters are not significantly different at a
number of female progeny and sex ratio.


2.9
2,7
0.056


6 13.9 +0.7c
4 20.1 0.7b
3 30.8 + 0.9a
- 11.9 0.6c
- 18.3 0.7b
- 31.6 0.9a

9 118.28
2 5, 144
6 <0.0001


0.05 (Least Square Means

0.05 (Tukey's HSD test) for









CHAPTER 6
FIELD ASSESSMENT OF THREE INTRODUCED PARASITOIDS OF Paracoccus
marginatus WILLIAMS AND GRANARA DE WILLING (HEMIPTERA:
PSEUDOCOCCIDAE)

Introduction

Paracoccus marginatus Williams and Granara de Willink is a polyphagous pest insect

that can damage fruits, vegetables and ornamentals, including Caricapapaya L. (papaya),

Hibiscus spp. L. (hibiscus), Citrus spp.(citrus), Persea americana Mill. (avocado), and Solanum

melongena L. (eggplant) (Miller and Miller 2002). This mealybug species was first described in

1992 (Williams and Granara de Willink 1992) and was re-described in 2002 (Miller and Miller

2002). Believed to be native to Mexico or Central America, P. marginatus has been established

in the Caribbean since 1994 (Miller et al. 1999). In 1998, P. marginatus was first detected in the

US, in Palm Beach County, Florida on hibiscus. Since then, it has been found on more than 25

genera of plants in the US. In recent years, P. marginatus has invaded the Pacific islands, and it

is now established in Guam (Meyerdirk et al. 2004), the Republic of Palau (Muniappan et al.

2006), and in several Hawaiian islands (Heu et al. 2007).

Paracoccus marginatus potentially poses a threat to numerous agricultural products in

the US especially in Florida and states such as California, Hawaii, and Texas, which produce

similar crops. Classical biological control was identified as an important component in the

management of P. marginatus and a program was initiated as a joint effort among the United

States Department of Agriculture, Puerto Rico Department of Agriculture, and Ministry of

Agriculture in the Dominican Republic in 1999 (Walker et al. 2003). Currently, there are three

solitary endoparasitoid hymenopterans mass reared in Puerto Rico, and released in P. marginatus

infested areas in the US, the Caribbean, and some Pacific islands (Meyerdirk et al. 2004). They

are Acerophaguspapayae Noyes and Schauff, Anagyrus loecki Noyes and Menezes, and









Pseudleptomastix mexicana Noyes and Schauff (Hymenoptera: Encyrtidae) (Noyes and Schauff

2003). In July 2003, A. papayae, A. loecki, and P. mexicana were obtained from the Biological

Control Laboratory, Department of Agriculture, Puerto Rico, and released in 21 locations in

South Florida, in Miami-Dade and Broward counties (11 locations in Miami, 5 locations in

Homestead, and 5 locations in Pembroke Pines and Miramar) (D. M. Amalin, personal

communication). A total of 6,000 parasitoids (1,400 A. papayae, 1,200 A. loecki, and 3,400 P.

mexicana) were released in South Florida in a single release attempt in July 2003 (D. M. Amalin,

personal communication). No subsequent releases have been recorded.

Information on parasitoids of P. marginatus and the field evaluation of their effectiveness

is limited in the US. Assessing the effect of a natural enemy or natural enemy complex on

its/their host populations in the field is important to evaluate the success of a biological control

project (Neuenschwander et al. 1986). This could be done by comparison of two separate pest

populations, one population with the natural enemy and the other without (Hodek et al. 1972).

Pest populations without natural enemies can be found either in pre-release situations or can be

created artificially, by using physical or chemical means to exclude the natural enemy from the

plot (Smith and DeBach 1942). Experimental exclusion methods are the fastest and most direct

way to demonstrate the effect of a natural enemy on a pest population (Smith and DeBach 1942).

In this field study, a physical exclusion method using sleeve cages was used to find the ability of

A. papayae, A. loecki, and P. mexicana to control P. marginatus in Homestead, FL.

Materials and Methods

Insect Rearing. A colony of P. marginatus was reared on sprouted red potatoes (Solanum

tuberosum L.) at the University of Florida, Tropical Research and Education Center (TREC),

Homestead, FL. Initially, P. marginatus was collected from a papaya (Caricapapaya L.) field in

Homestead, FL. Prior to sprouting, the potatoes (Ryan Potato Company, East Grand Forks, MN)









were soaked in a 1% solution of bleach (Clorox The Clorox Company, Oakland CA; 6%

sodium hypochlorite), for 15 minutes, and then rinsed with clean water and dried. Potatoes were

placed in black cotton cloth bags to encourage sprouting. The bags were kept inside a dark room

at 270 1IC. Each week, 36 sprouted potatoes were infested with P. marginatus ovisacs to

maintain the colony. Depending on the size, each potato was infested with 3 to 5 ovisacs. The

infested potatoes were kept in 3.8-L plastic containers (Rubbermaid Newell Rubbermaid Inc.

Atlanta, GA) with 12 potatoes per container. To facilitate the air circulation to developing eggs

and mealybugs, screens (Amber Lumite Bio Quip, Gardena, CA) were glued to the cut

sections of lids of these plastic containers. The mealybug colony was maintained in an

environmental growth chamber (Percivel I-36LL, Percival Scientific Inc. Perry, NC) at 250 +

1IC, 65 2% R.H., and a photoperiod of 14:10 (L:D).

Field Experiments. The research plots were selected at three homeowner locations in

Homestead, FL. The field experiments were carried out in July to August 2005 and 2006, using

the same experimental locations in both years. Paracoccus marginatus was observed in all three

locations at the time of selection. In each location, 10 hibiscus (Hibiscus rosa-sinensis L.)

plants, approximately 2.5 to 3.0 m tall, were selected. Each selected plant was considered a

replicate. The three treatments used in this experiment were closed sleeve cage, open sleeve

cage, and no cage. The sleeve cages were made of white chiffon cloth material (Jo-Ann Fabrics

and Crafts, Miami, FL), 72 cm in length and 50 cm in width. Along the length of the material, a

groove was sewn at 15 cm from each end. The piece of cloth with the groove was then folded in

half along the width, and the two ends along the width were placed together and sewn at the edge

to make a cylinder of 15 cm diameter. A piece of stainless steel (20 gauge) wire (Tower

Manufacturing Company, Madison, IN), 72 cm in length was inserted through each groove and









tied at the ends to make a ring to shape the cage into a cylindrical cage. Three branches 1-1.5 m

above ground were selected from each hibiscus plant. The branches selected were evenly

distributed among the hibiscus plants, and each branch had 7-10 leaves. All the selected

branches were cleaned with moist tissues (Kimwipes EX-L, Kimberly-Clerk Global Sales Inc.

Roswell, GA) to make them free from any insects and eggs. Each clean branch was enclosed in

a closed sleeve cage mentioned above for 7 days to observe for any insect presence or

development. To avoid the cloth material of the cage getting in contact with the leaves, a

stainless steel wire (22 gauge and 25 cm in length) was tied to the branch at the middle at each

end of the cage, and the ends were fixed to the cage along the diameter. Sleeves of the cage were

secured with a stainless steel wire tied around the enclosed branch. During this time, all

enclosed branches were checked daily for the presence of any insects by opening the sleeve at

the terminal end of the branch of each cage. If any insects were observed in a cage, the branch

was cleaned again using the above procedure.

After 7 days, five gravid females of P. marginatus collected from the mealybug colony,

were carefully placed on the terminal leaves of the branch within each sleeve cage using a paint

brush (No.000) (American Painter 4000, Loew-Cornell Inc., Englewood Cliffs, NJ).

Immediately after placing the females, the open sleeve was tied back on to the branch, closing

the cage. Approximately 21 days was allowed for the gravid females to lay eggs and the eggs to

develop into second and third-instar mealybugs. When the number of second and third instars

was at a 1:1 ratio by visual inspection, all the sleeve cages were removed, and were replaced

according to the three treatments mentioned above. Each of the three treatments was randomly

assigned among the three branches on each plant, using cages similar to those described above

and placing them over the branches infested with mealybugs. In the closed sleeve cage









treatment, cages were kept closed. The purpose of this treatment was to evaluate the

development of mealybugs in the microclimate created inside a closed cage. In the open sleeve

cage treatment, the cages were left open and the sleeves were folded back along the cylindrical

part of the cage and were fixed to the cage with four safety pins. The purpose of this treatment

was to provide the parasitoids access to the mealybugs and to provide microclimate conditions

similar to the closed sleeve cage treatment. In the no cage treatment, branches with mealybug

colonies were left un-caged. This treatment was used to assess the effect of the sleeve cages

themselves on the mealybug population growth and parasitism level. The treatments were

checked for mealybug destroyer adults and larvae (Cryptolaemus montrouzieri Mulsant), ants,

and spiders at 24, 48, and 72-hour intervals without disturbing the treatments.

At 72 hours, all treatments were covered with closed sleeve cages, and the branches were

removed from the plant. Cages were brought to the laboratory, and the number of mealybugs

was noted. The number of adults and larvae of the mealybug destroyer (coccinellid predator),

ants, and spiders was also recorded. From each replicate, 100 second and third-instar mealybugs

were randomly collected and placed on a sprouting potato for further development. These

potatoes were kept singly in 500 ml deli cups (Georgia Pacific Dixie, Atlanta, GA). Each cup

was covered with a piece of chiffon cloth held in a place with the cup lid with a circular area of

8.5 cm diam removed to facilitate air circulation. The cups were held in an insectary, maintained

at 250 1IC, 12:12 (L:D) photoperiod, and 65 2 % R.H. Mealybugs were allowed to

mummify on potatoes. Collection of mummified mealybugs was started 10 days after placing

them on potatoes. Mummified mealybugs were placed individually in disposable, glass culture

tubes of 1.2 cm diameter and 7.5 cm length (Fisherbrand Fisher Scientific, Pittsburgh, PA).

Each tube was covered with two-ply tissue (Kimwipes EX-L, Kimberly-Clerk Global Sales









Inc. Roswell, GA), secured with 2.5-cm- long piece of clear polyvinyl chloride (PVC) tubing

(Fisherbrand Fisher Scientific, Pittsburgh, PA) until the emergence of parasitoids. The

emerging parasitoids from the culture tubes were sexed and were identified as to their species.

Samples of parasitoids, mealybug destroyers, and ants were sent to the Systematic Entomology

laboratory, USDA, Beltsville, MD for verification of identification. Samples of spiders were

sent to Division of Plant Industry, Florida Department of Agriculture and Consumer Services,

Gainesville, FL for species identification.

Statistical Analysis. The experimental design was completely random with 10 replicates

at each location. A three-way analysis of variance (ANOVA) was performed using the general

linear model (PROC GLM) of SAS (SAS Institute 1999) to find the interaction among year,

location, and treatment for mealybugs, mealybug destroyers, ants, and spiders. A one-way

ANOVA was performed using the general linear model (PROC GLM) for mean number of

mealybugs collected from the three treatments. Means were compared at P = 0.05 significance

level using the Tukey's HSD test. A repeated measure ANOVA using the general linear model

(PROC GLM) was performed for mean number of adults and larvae of mealybug destroyers,

spiders, and ants collected at 24, 48, and 72-hour intervals to check the interaction between the

interval and the treatment. Means were compared between treatments using a t-test (PROC

TTEST) of SAS (SAS Institute 1999) at P = 0.05. The closed sleeve cage treatment was

excluded from the analysis since there were no natural enemies present in this treatment.

Proportions of parasitism of A. papayae and A. loecki for both open sleeve cage and no

cage treatments were arcsine-square-root transformed using,

p'= arcsin i









where, p = proportion of parasitism, to adjust the variances (Zar 1984) prior to ANOVA, but

untransformed data were presented in tables. A three-way ANOVA (PROC GLM) was

performed to find the interaction among year, location, and treatment for proportion of

parasitism for each parasitoid species. A two-way ANOVA was performed for proportions of

individual and cumulative parasitism of A. papayae and A. loecki between treatments, and means

were compared at P = 0.05 significance level using least square means (LSMEANS) of SAS

(SAS Institute 1999).

Voucher Specimens. Voucher specimens of mealybugs, mealybug destroyer adults and

larvae, ants, spiders, and parasitoids were deposited in the Entomology and Nematology

Department insect collection, at the Tropical Research and Education Center, University of

Florida.

Results

There was no interaction in the mean number of P. marginatus collected from each

treatment by location and year (F = 0.12, df = 4, 162, P = 0.9737). Therefore, the data for P.

marginatus were pooled by location, year, and treatment, and pooled data were used in the

analyses. The mean number ofP. marginatus collected from the closed sleeve cage (410.9

1.6), was higher than the numbers collected from the open sleeve cage (171.6 1.3) and the no

cage treatment (109.1 0.7) by 58.2% and 73.4% respectively (F=16800.4, df= 2, 177, P

<0.0001). There were 36.4% more mealybugs in the open sleeve cage, compared to the no cage

treatment.

Natural enemies such as mealybug destroyer adults and larvae, and spiders were observed

at all three locations used in this experiment. However, no natural enemies were present in the

closed sleeve cage treatment. There was no interaction in the mean number of individuals

collected by location, year, and treatment for mealybug destroyer adults (F = 0.01, df = 2, 346, P









= 0.9998) and larvae (F= 0.04, df = 2, 346, P = 0.9599), ants (F = 0.04, df = 2, 346, P = 0.9653),

and spiders (F= 0.14, df= 2, 346, P= 0.8733). Therefore, the pooled data for each of these

insects were used in the analyses. The repeated measures ANOVA for within subject effects

indicated that there was no interaction between the interval and the treatment (F= 0.01, df= 2,

944, P = 0.9931). There were higher mean numbers of mealybug destroyer adults and larvae

(Table 6-1), ants, and spiders (Table 6-2) in the no cage than in the open sleeve cage treatment at

24, 48, and 72-hour intervals.

The spiders collected from the treatments were comprised of Gasteracantha cancriformis

(Linnaeus), Cyclosa walckenaeri (0. P. Cambridge) (Araneae: Araneidae), Lyssomanes viridis

(Walckenaer) (Araneae: Salticidae), Misumenops sp. (Araneae: Thomisidae), Hibana sp.

(Araneae: Anyphaenidae), Theridion melanostictum 0. P.Cambridge (Araneae: Theridiidae), and

Leucauge sp. (Araneae: Tetragnathidae). None of the species of spiders collected was dominant

in any of the treatments. The ants collected form the treatments were comprised of Tapinoma

sessile Say, Pheidole sp., and Technomyrmex sp. (Hymenoptera: Formicidae). Tapinoma sessile

was the predominant ant species collected from the three locations in both 2005 and 2006, and is

a common and widely distributed North American ant species (Smith 1928).

There was no interaction in the mean proportion of parasitoids emerged from the

mealybug samples collected by treatment, location, and year for A. papayae (F = 0.86, df = 2,

108, P = 0.4260), and A. loecki (F = 0.23, df = 2, 108, P = 0.7919). Therefore, the data for

parasitoids were pooled by location and year, and pooled data were used in the analyses.

Acerophaguspapayae had higher percent parasitism in the open sleeve cage than in the no cage

treatment by 30.9% (Table 6-3). Within a treatment, A. papayae had a higher parasitism than A.

loecki by 92.6% in the open sleeve-cage and by 92.5 % in the no-cage treatment respectively.









Percent parasitism of A. loecki in the open sleeve cage was 30.8% higher than in the no cage

treatment (Table 4-3). The open sleeve cage had 30.9% higher cumulative percent parasitism

than the no cage treatment. There was no activity of P. mexicana in any of the treatments.

Discussion

In recent years, classical biological control has been used to control several invasive

mealybugs. Use ofApoanagyrus lopezi to control the cassava mealybug, Phenacoccus manihoti

Matile-Ferrero in Africa (Neuenschwander 2001), Gyranusoidea tebygi Noyes for mango

mealybug, Rastrococcus invadens (Williams) control in West Africa (Bokonon-Ganta and

Neuenschwander 1995), and the use ofAnagyrus kamali Moursi to control pink hibiscus

mealybug, Maconellicoccus hirsutus Green in the Caribbean (Kairo et al. 2000) are some of the

examples. Use of A. papayae, A. loecki, and P. mexicana to control P. marginatus in the

Caribbean, the US, and the Pacific, is another example of utilizing classical biological control to

manage an invasive mealybug species.

To determine the ecological and the economic impact of a biological control program, it

is necessary to evaluate the efficacy of the biological control agents. In order to understand this,

it is important to evaluate the pest insect population in an environment where it is not exposed to

the natural enemies (Boavida et al. 1995b). One of the principal obstacles of the host evaluation

has been the difficulty of excluding the natural enemies from the host population (Smith and

DeBach 1942). In this study, sleeve cages were used as the exclusion method to investigate the

host population without its natural enemy. A physical exclusion method using sleeve cages can

be an effective way to evaluate the effect of presence and absence of natural enemies on the

survival of their host populations (Smith and DeBach 1942). Limitations and applicability of

physical exclusion methods on different natural enemies have been evaluated (Kiritani and

Dempster 1973, Van Lenteren 1980). One limitation of this method is that it may cause









conditions within the sleeve cage to depart too far from the normal conditions outside the sleeve

cage (Smith and DeBach 1942). To overcome these limitations, open sleeve cage and no cage

treatments were included in this study. The closed sleeve cage protected the mealybugs from

natural enemies as well as from environmental factors such as the rain and the wind, while the

open sleeve cage likely provided some protection from adverse environmental conditions, and no

protection provided by the no cage treatment. The greater host population in the closed

environment indicates that when there was no outside interference from natural enemies, or no

direct impact of the wind and rain, insects survive better than in the open environment where

they are more exposed to direct environmental factors as well as their natural enemies. Similar

results have been reported for Rastrococcus invadens Williams in field assessment studies

conducted to find the impact of the introduced parasitoid, Gyranusoidea tebygi Noyes in West

Africa (Boavida et al. 1995b).

The presence of predators such as C. montrouzieri adults and larvae, and spiders may

have a negative impact on percent parasitism. Cryptolaemus montrouzieri was also collected in

relatively low numbers in field assessment studies of the parasitoids of P. marginatus, conducted

in the Republic of Palau (Muniappan et al. 2006) and in Guam in 2002 (Meyerdirk 2004). The

presence of C. montrouzieri could have had an effect on parasitism, but due to the presence of a

large number of mealybugs and the high percent parasitism observed in these areas, the effect of

C. montrouzieri may not be significant. There is a possibility that parasitized mealybugs were

preyed on by C. montrouzieri. Most coccinellid predators feed on more than one prey species;

thus, disruption of existing biological control by introduced coccinellids and the potential for

indigenous coccinellid species to disrupt introductions can happen (Rosenheim et al. 1995).

Common forms of intraguild predation include predators that attack herbivores that harbor a









developing parasitoid (Rosenheim et al. 1995). This may be one reason that higher parasitism

was observed in the open sleeve cage treatment than in the no cage treatment, because there were

more predators in the no cage treatment, and P. marginatus was directly exposed to the

environment.

Anagyrus loecki is not a host specific classical biological control agent (Noyes 2000).

The low parasitism by A. loecki in both open sleeve cage and no cage treatments was possibly

due to its multiple host preference. In addition to being a parasitoid of P. marginatus, A. loecki

can develop in Dysmicoccus hurdi and Phenacoccus madeirensis Green (Hemiptera:

Pseudococcidae) (Noyes 2000) and P. maderiensis is one of the commonly found mealybug

species in South Florida (Williams and Granara de Willink 1992, Ben- Dov 1994). Other than P.

marginatus, no other hosts have been recorded for Acerophagus papayae and P. mexicana

(Noyes and Schauff 2003).

Not recovering a single P. marginatus that was parasitized by P. mexicana or the

emergence of any hyper-parasitoids from the collected P. marginatus raises an interesting

question of whether P. mexicana successfully established in the experimental area. In July 2003,

3,400 P. mexicana were released in Florida, as a one-time release in 21 locations in Miami-Dade,

and Broward Counties including five locations in Homestead where these field studies were

conducted (D. M. Amalin, personal communication, Meyerdirk 2003). On the other hand, only

1,400 A. papayae and 1,200 A. loecki were released at the same time and in the same locations as

P. mexicana, but they both were recovered from the field. Even after several releases, P.

mexicana has not been recovered in field assessment studies conducted in the Republic of Palau

(Muniappan et al. 2006). A similar study has been conducted in Guam in 2002, although the

results were reported without the recovery data of parasitoids (Meyerdirk et al. 2004). There is









very little information on P. mexicana, and there is no information on why it was not recovered

from the field in previous studies. Further field experiments focusing on P. mexicana may be

needed to clarify why it was not recovered from the field.

In laboratory studies, P. mexicana and A. papayae showed a preference for second-instar

P. marginatus while A. loecki preferred the third instars. At 250C, the developmental time of

female P. mexicana was 22 days, and was longer than the 14-day developmental time of female

A. papayae and A. loecki (Chapter 5). The second-instar P. marginatus can emerge within 14.6

days at 250C (Chapter 5). The developmental time of second-instar P. marginatus coincides

with the developmental time of female A. papayae and A. loecki (Chapter 5). The preference for

the third-instar P. marginatus by A. loecki makes A. papayae, the dominant species in the

competition for the second-instar P. marginatus. The longer developmental time can be an

important reason for less effectiveness of P. mexicana in the field. Pseudleptomastix mexicana

also was less efficient when competing with A. papayae and A. loecki in laboratory studies

(Chapter 4).

The shorter developmental time and lack of competitors for preferred second-instar hosts

may have placed A. papayae as the dominant species over A. loecki and P. mexicana (Chapter 5).

In laboratory studies, A. papayae had better control of the host, when present singly or with A.

loecki and P. mexicana (Chapter 4). Acerophaguspapayae is also the predominant parasitoid

species recovered from field studies in both Guam (Meyerdirk et al. 2004) and the Republic of

Palau (Muniappan et al. 2006). Out of the three parasitoid species, A. papayae is the smallest

parasitoid species (Noyes and Schauff 2003). Because of its smaller size, A. papayae has the

advantage of parasitizing P. marginatus that were concealed in crevices of the host plant species.









Because of this concealed nature, there is a possibility of less predation of these mealybugs by C.

montrouzieri larvae and adults, and spiders.

Higher numbers of ants present in the no cage treatment may have affected the foraging

behavior of parasitoids. This may be one of the reasons for lower cumulative parasitism in the

no cage treatment compared to the open sleeve cage treatment. Generally, mealybugs and ants

have mutualistic relationships. Mealybugs benefit from ant association when ants promote

sanitation in mealybug populations and/or protect mealybugs from natural enemies (Gonzalez-

Hernandez 1999). It has been repeatedly observed that some pests have higher population

densities on plants where ants are active than on plants free of ants (Hodek et al. 1972). There is

considerable direct evidence of aggressive behavior toward predators or parasites in honeydew

seeking ants. Pheidole megacephala (F.) significantly decreased Dysmicoccus brevipes

(Cockerell) mortality, by Anagyrus ananatis Gahan and Nephus bilucernarius Mulsant

(Coleoptera: Coccinellidae) adults via interference with natural enemy searching behavior

(Gonzalez-Hernandez 1999). Presence of ants in both open sleeve cage and no cage treatments

may have some influence on the parasitism by A. papayae and A. loecki, although the effect of

ants on mealybugs and parasitoids was not investigated in this study.

Out of the three currently used parasitoids of P. marginatus, A. papayae is well established

in the field, and is the main contributor to the mortality of this mealybug species. Multiple host

preference may have caused the low effectiveness of A. loecki compared to A. papayae. Further

research is needed to address the ability of P. mexicana to control P. marginatus as well as its

ability to establish after release in the field.









Table 6-1 Mean ( SEM) number of mealybug destroyer (Cryptolaemus montrouzieri)
adults and larvae collected per cage from open sleeve cage and no cage treatments
using pooled data of 2005 and 2006 in three experimental locations
Treatment Mealybug-destroyer (adult)/per cage Mealybug-destroyer (larva)/per cage
Interval (Hours) Interval (Hours)
24 48 72 24 48 72
Open sleeve 2.0 +0.1 2.1 0.1 2.1 0.1 1.2 0.1 1.1 0.1 1.3 0.1
No cage 3.0 +0.1 3.0 +0.1 3.1 0.1 2.1 0.1 2.1 0.1 2.1 0.1
t -7.33 -6.79 -6.86 -8.42 -8.21 -8.42
df 118 118 118 118 118 118
P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
n = 60










Table 6-2 Mean (+ SEM) number of ants and spiders collected from open sleeve cage and
no cage treatments using pooled data of 2005 and 2006 in three experimental
locations
Treatment Ants Spiders
Interval (Hours) Interval (Hours)
24 48 72 24 48 72
Open sleeve 21.4 + 0.2 21.3 + 0.2 21.5 0.2 2.0 + 0.1 2.1 + 0.1 2.1 + 0.1
No cage 30.9 0.3 30.9 0.2 31.0 + 0.2 2.9 0.1 3.1 0.1 3.0 + 0.1
t -27.32 -30.52 -29.93 -6.88 -7.77 -7.06
df 118 118 118 118 118 118
P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
n = 60









Table 6-3 Individual and cumulative mean percent parasitism ( SEM) of P. marginatus by
A. papayae, A. loecki, and P. mexicana in open sleeve cage, and no cage treatments
using pooled data of 2005 and 2006 in three experimental locations
Percent Parasitism
Treatment A. papayae A. loecki Cumulative
Open sleeve cage 31.0 + 0.3aB 2.3 0.1aC 33.3 0.3aA
No cage 21.4 + 0.3bB 1.6 + 0.1bC 23.0 + 0.3bA
Source F df P
Model 3606.07 5, 354 <0.0001
Parasitoid 8038.28 2, 354 <0.0001
Treatment 1387.10 1,354 <0.0001
Parasitoid* Treatment 283.36 2, 354 <0.0001
n = 60
Means within a column followed by the same lowercase letters, and means within a row
followed by the same uppercase letters are not significantly different at a = 0.05 (Least Square
Means (LSMEANS) Test).









CHAPTER 7
SUMMARY AND CONCLUSIONS

Paracoccus marginatus Williams and Granara de Willink (papaya mealybug) is a

polyphagous pest insect that can damage a large number of tropical and subtropical fruits,

vegetables, and ornamental plants. A native to Mexico and/or Central America, P. marginatus is

currently established in the Caribbean, the US and several Pacific islands. Classical biological

control was identified as a suitable method for the control ofP. marginatus. Currently there are

three solitary hymenopteran endoparasitoid species mass reared in Puerto Rico and released in P.

marginatus infested areas in the Caribbean, the US and Pacific islands. They are Acerophagus

papayae, Anagyrus loecki, and Pseudleptomastix mexicana. However, there is a lack of

information about P. marginatus and its parasitoids. This dissertation focused on the life history

of P. marginatus in relation to host plant species and temperature, and evaluated the

effectiveness of the three introduced parasitoid species.

Life history studies of P. marginatus indicated that it could successfully develop,

reproduce, and survive on a wide variety of economically important ornamental plants as well as

on an aggressive weed species Parthenium hysterophorus. Egg to adult emergence occurred in

30 days or less in all plant species indicating its fast development on different host plants.

Paracoccus marginatus showed its tropical characteristics by completing its life cycle in

the temperatures ranging from 18 to 300C. Its high minimum temperature threshold of 140C and

the low thermal constant further clarified this characteristic. The optimum development of P.

marginatus can be expected around 28C, while the maximum temperature threshold can go up

as high as 320C. These characteristics may limit establishment of P. marginatus into many areas

in the US, while some areas in California, Texas, Florida and Hawaii are more vulnerable. The

ultimate movement of P. marginatus to new areas in the US that are suitable in regards to









temperature will also be influenced by other environmental factors, availability of host plant

species, plant movement from state to state, and the rules, regulations, and restrictions of plant

movement.

Of the three parasitoid species currently used in the biological control of P. marginatus, A.

papayae had higher parasitism in the field. Natural enemies including Cryptolaemus

montrouzieri, ants, and spiders were observed in the treatments exposed to the environment, and

overall their activity may have contributed to the low parasitism. Parasitism of P. mexicana was

not observed while A. loecki had a lower parasitism compared to A. papayae.

The smallest parasitoid species out of the three species, A. papayae had lower lifetime

fertility than the other two species. In addition, A. papayae and P. mexicana compete for the

second-instar P. marginatus while A. loecki prefers the third-instar hosts. At the beginning of the

season, in the absence of overlapping generations, the longer developmental time ofP. mexicana

makes it unavailable at the second-instar mealybug emergence, providing an advantage to A.

papayae in the competition for the hosts. Acerophaguspapayae had high parasitism when

present with both A. loecki and P. mexicana. The efficiency ofA. loecki may have been affected

by not being host specific, and being a parasitoid of commonly found P. madeirensis. Longer

development time of P. mexicana reduces its competitiveness with A. papayae and A. loecki.

Information gathered from these studies will provide the insight needed to understand the

life history of P. marginatus in relation to its host plants and temperature, and to explain the

effectiveness of its three introduced parasitoids, A. papayae, A. loecki and P. mexicana in the

classical biological control ofP. marginatus in the field.









REFERENCE LIST


Andrewartha, H. G., and L. C. Birch. 1954. The distribution and abundance of animals.
Chicago Press. Chicago University, Chicago, IL.

Begum, S., A. Naeed, B. S. Siddiqui, and S. Siddiqui. 1994. Chemical constituents of
the Genus Plumeria. J. Chem. Soc. Pak. 16: 280-299.

Ben-Dov, Y. 1994. A systematic Catalogue of the mealybugs of the world. Intercept, Hants,
UK.

Bertschy, C., T. C. J. Turlings, A. Bellotti, and S. Dorn. 2000. Host stage preference and sex
allocation in Aenasius vexans, an encyrtid parasitoid of the cassava mealybug. Entomol.
Exp. Appl. 95: 283-291.

Boavida, C., and P. Neuenschwander. 1995. Influence of the host plant on the mango
mealybug, Rastrococccus invadens. Entomol. Exp. Appl. 76: 179-188.

Boavida, C., M. Ahounou, M. Vos, P. Neuenschwander, and J. J. M. van Alphen. 1995a.
Host stage selection and sex allocation by Gyranusoidea tebygi (Hymenoptera:
Encyrtidae), a parasitoid of the mango mealybug, Rastrococcus invadens (Homoptera:
Pseudococcidae). Biol. Contol. 5: 487-496.

Boavida, C., P. Neuenschwander, and P. Herren. 1995b. Experimental assessment of the
impact of the introduced parasitoid Gyranusoidea tebygi Noyes on the mango mealybug
Rastrococcus invadens Williams, by physical exclusion. Biol. Control. 5: 99-103.

Bokonon-Ganta, A. H., and P. Neuenschwander. 1995a. Impact of the biological control
agent Gyranusoidea tebygi Noyes (Hymenoptera : Encyrtidae) on the mango mealybug,
Rastrococcus invadens Williams (Homoptera: Pseudococcidae), in Benin.
Biocontrol Sci. and Tech. 5: 95-107.

Bokonon-Ganta, A. H., P. Neuenschwander, J. J. M. van Alphen, and M. Vos. 1995b. Host
stage selection and sex allocation by Anagyrus mangicola (Hymenoptera: Encyrtidae), a
parasitoid of the mango mealybug, Rastrococcus invadens (Homoptera: Pseudococcidae).
Biol. Control. 5: 479-486.

Bokonon-Ganta, A. H., J. J. M. van Alphen, and P. Neuenschwander. 1996. Competition
between Gyranusoidea tebygi and Anagyrus mangicola parasitoids of mango mealybug,
Rastrococcus invadens: interspecific host discrimination and larval competition.
Entomol. Exp. Appl. 79:179-185.

Borror, D. J., C. A. Triplehorn, and N. F. Johnson. 1992. An Introduction to the Study
of Insects. 6th ed. Harcourt Brace College Publishers, Orlando, FL.









Buss, E. A., and J.C. Turner. 2006. Scale insects and mealybugs on ornamental plants.
EENY-323. Featured Creatures. Entomology and Nematology Department, Florida
Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of
Florida, Gainesville, FL.
(http://edis.ifas.ufl.edu/, October 2007)

Calatayud, P. A., B. Delobel, J. Guillaud, and Y. Rahbe. 1998. Rearing the cassava
mealybug, Phenacoccus manihoti, on a defined diet. Entomol. Exp. Appl. 86: 325-329.

Campbell, A., B. D. Frazer, N. Gilbert, A. P. Gutierrez, and M. Mackauer. 1974.
Temperature requirements of some aphids and their parasites. J. Appl. Ecol.
11:431-438.

Center for North American Studies (CNAS). 2007. Economic impact of greening on the
Texas citrus industry. Issue Brief 2007-1. Department of Agriculture Economics, Texas
Agricultural experiment Station, Texas A & M University, College Station, TX.

Chandler, L. D., D. E. Meyerdirk, W. G. Hart, and R. G. Garcia. 1980. Laboratory studies
of the development of the parasite Anagyruspseudococci (Girault) on insectary-reared
Planococcus citri (Risso). Southwest Entomol. 5: 99-103.

Chapman. R. F. 1998. The insects structure and function. 4th ed. Cambridge University Press,
New York, NY.

Charnov, E. L., R. L. L. Hartogh, W. T. Jones, and J. van den Assem. 1981. Sex ratio
evolution in a variable environment. Nature. 289: 27-33.

Chong, J-H., R. D. Getting, and M. W. van lersel. 2003. Temperature effects on the
development, survival, and reproduction of the Madeira mealybug, Phenacoccus
madeirensis Green (Hemiptera: Pseudococcidae). Ann. Entomol. Soc. Am. 96: 539-543.

Criley, R. A. 1998. Plumeria. Ornamentals and Flowers. OF-24. Cooperative Extension
Service, College of Tropical Agriculture and Human Resources, University of Hawaii,
Manoa, Hawaii.

Cross, A. E., and D. Moore. 1992. Developmental studies on Anagyrus mangicola
(Hymenoptera: Encyrtidae), a parasitoid of the mango mealybug Rastrococcus invadens
(Homoptera: Pseudococcidae). 82: 307-312.

Dent, D. 1995. Principles of integrated pest management. pp. 8-46. In D. Dent (eds.).Integrated
Pest Management. Chapman and Hall. New York. NY.

Dhileepan, K. 2001. Effectiveness of introduced biocontrol insects on the weed Parthenium
hysterophorus (Asteraceae) in Australia. Bull. Entomol. Res. 91: 167-176.









Dhileepan, K., C. J. Lockett, and R. E. McFadyen. 2005. Larval parasitism by native insects
on the introduced stem-galling moth Epiblema strenuana Walker (Lepidoptera :
Tortricidae) and its implications for biological control ofParthenium hysterophorus
(Asteraceae). Aus. J. Entomol. 44: 83-88.

Doyon, J., and G. Boivin. 2005. The effect of development time on the fitness of female
Trichogramma evanescens. J. Insect. Sci. 5: 1-5.

Ferreira de Almeida, M., A. Pires Do Prado, and C. J. Geden. 2002. Influence of
temperature on development time and longevity of Tachinaephagus zealandicus
(Hymenoptera: Encyrtidae), and effects of nutrition and emergence order on longevity.
Biol. Control. 31: 375-380.

Gauld, D. 1986. Taxonomy, its limitations and its role in understanding parasitoid biology. pp.
1-19. In J. Waage and D. Greathead. (eds.) Insect Parasitoids. Academic Press Inc.
Orlando, FL.

Gilman, E. F. 1999a. Acalypha wilkesiana. Fact Sheet. FPS-6. Florida Cooperative Extension
Service, Institute of Food and agricultural Sciences, University of Florida, Gainesville,
FL.

Gilman, E. F. 1999b. Hibiscus rosa-sinensis. Fact Sheet. FPS-254. Florida Cooperative
Extension Service, Institute of Food and agricultural Sciences, University of Florida,
Gainesville, FL.

Gonzales-Hernandez, H., M. W. Johnson, and N. J. Reimer. 1999. Impact of Pheidole
megacephala (F.) (Hymenoptera: Formicidae) on the biological control of Dysmicoccus
brevipes (Cockerell) (Homoptera: Pseudococcidae). Biol. Control. 15: 145-152.

Goolsby, J. A., A. A. Kirk, and D. E. Meyerdirk. 2002. Seasonal phenology and natural
enemies ofMaconellicoccus hirsutus (Hemiptera: Pseudococcidae) in Australia. Florida
Entomol. 85: 494- 498.

Gordan, H. T. 1999. Growth and development of insects. pp. 55-82. In C. B. Huffaker and A.
P. Gutierrez (eds.), Ecological Entomology. 2nd ed. John Wiley and Sons, Inc., New
York, NY.

Greathead, D. J. 1986. Parasitoids in classical biological control. pp. 290-315. In J. Waage and
D. Greathead. (eds.) Insect Parasitoids. Academic Press Inc., Orlando, FL.

Hansen, L. S. 2000. Development time and activity threshold of Trichogramma turkestanica
on Ephestia kuehniella in relation to temperature. Entomol. Exp. Appl. 96: 185-188.

Harborne, J. B. 2001. Twenty-five years of chemical ecology. Nat. Prod. Rep. 18: 361-379.









Harris, P. 1990. Environmental impact of introduced biological control agents. pp. 295-303. In
M. Mackauer, L. E. Ehler and J. Roland (eds.), Critical Issues in Biological Control.
Intercept Ltd, Andover, Hants, UK.

Hemerik, L., and J. A. Harvey. 1999. Flexible larval development and the timing of
destructive feeding by a solitary endoparasitoid: an optimal foraging problem in
evolutionary perspective. Ecological Entomol. 24: 308-315.

Herrera, C. J., and R. G. van Driesche, and A. C. Bellotti. 1989. Temperature-dependent
growth rates for the cassava mealybug, Phenacoccus herreni, and two of its encyrtid
parasitoids, Epidinocarsis diversicornis and Acerophagus coccois in Columbia. Entomol.
Exp. Appl. 50: 21-27.

Heu, R. A., M. T. Fukada, and P. Conant. 2007. Papaya mealybug, Paracoccus marginatus
Williams and Granara de Willink (Hemiptera: Pseudococcidae). New Pest Advisory.
4(3). State of Hawaii Department of Agriculture, Honolulu, HI.

Hodek, I., K. S. Hagen, and H. F. Van Emden. 1972. Methods for studying effectiveness of
natural enemies, pp. 147-188. In H.F. Van Emden (eds.), Aphid Technology. Academic
Press, London, England.

Hoy, M. A., A. Hamon, and R. Nguyen. 2006. Pink hibiscus mealybug, Maconellicoccus
hirsutus (Green). EENY-29. Featured Creatures. Entomology and Nematology
Department, Florida Cooperative Extension Service, Institute of Food and Agricultural
Sciences, University of Florida, Gainesville, FL.
(http://edis.ifas.ufl.edu/ October 2007)

Huffaker, C., A. Berryman, and P. Turchin. 1999. Dynamics and regulation of insect
Populations, pp. 269-305. In C. B. Huffaker and A. P. Gutierrez (eds.), Ecological
Entomology. 2nd ed. John Wiley and Sons Inc., New York, NY.

Ingram, D. L., and L. Rabinowitz. 2004. Hibiscus in Florida. ENH-44. Environmental
Horticulture Department, Florida Cooperative Extension Service, Institute of Food and
Agricultural Sciences, University of Florida, Gainesville, FL.

Islam, K. S., and M. J. W. Copland. 1997. Host preference and progeny sex ratio in a solitary
koinobiont mealybug endoparasitoid, Anagyruspseudococci (Girault), in response to its
host stage. BioControl Sci. and Tech. 7: 449-456.

Kairo, M. T. K., G. V. Pollard, D. D. Peterkin, and V. F. Lopez. 2000. Biological control of
the hibiscus mealybug, Maconellicoccus hirsutus Green (Hemiptera: Pseudococcidae) in
the Caribbean. Integrated Pest Man. Rev. 5: 241-254.

King, B. H. 1987. Offspring sex ratios in parasitoid wasps. Q. Rev. Biol. 62: 367-396.









Kiritani, K., and J. P. Dempster. 1973. Different approaches to the quantitative evaluation of
natural enemies. J. Appl. Ecol. 10: 323-329.

Laflin, H. M., and M. P. Parrella. 2004. Developmental biology of citrus mealybug under
conditions typical of California rose production. Ann. Entomol. Soc. Am. 97: 982-988.

Lawrence, P. 0. 1981. Interference competition and optimal host selection in the parasitic
wasp, Biosteres longicaudatus. Ann. Entomol. Soc. Am. 74: 540-544.

Lema. K. M., and H. R. Herren. 1985. The influence of constant temperature on population
growth rates of the cassava mealybug, Phenacoccus manihoti. Entomol Exp. Appl. 38:
165-169.

Le Ru, B., and A. Mitsipa. 2000. Influence of the host plant of the cassava mealybug
Phenacoccus manihoti on life-history parameters of the predator Exochomusflaviventris.
Entomol. Exp. Appl. 95: 209-212.

Logan, J. A., D. J. Wollkind, S. C. Hoyt, and L. K. Tanigoshi. 1976. An analytic model for
description of temperature dependent rate phenomena in arthropods. Environ. Entomol.
5: 1133-1140.

Mani, M., and T. S. Thontadarya. 1989. Development of the encyrtid parasitoid Anagyrus
dactylopii (How.) on the grape mealybug Maconellicoccus hirsutus (Green). Entomon.
14: 49-51.

Manzano, M. R., J. C. van Lenteren, C. Cardona, and Y. C. Drost. 2000. Developmental
time, sex ratio, and longevity ofAmitusfuscipennis MacGown and Nebeker
(Hymenoptera: Platygasteridae) on the greenhouse whitefly. Biol. Control. 18: 94-100.

Marohasy, J. 1997. Acceptability and suitability of seven plant species for the mealybug
Phenacoccusparvus. Entomol. Exp. Appl. 84: 239-246.

Mead, F. W., 2007.. Asian citrus psyllid, Diaphorina citri Kuwayama (Insecta: Hempitera:
Psyllidae). EENY-33. Featured Creatures. Entomology and Nematology Department,
Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences,
University of Florida, Gainesville, FL.
(http://edis.ifas.ufl.edu/ October 2007)

Mersie, W., and M. Singh. 1988. Effects of phenolic acids and ragweed parthenium
(Parthenium hysterophorus) extracts on tomato (Lycopersicon esculentum) growth and
nutrient and chlorophyll content. Weed Sci. 36: 278-281.

Messenger, P. S. 1959. Bioclimatic studies with insects. Ann. Rev. Ent. 4: 183-206.









Meyerdirk, D. E., 2003. Control of papaya mealybug, Paracoccus marginatus (Hemiptera:
Pseudococcidae). Environmental Assessment (Supplement). Center for Plant Health
Science and Technology, National Biological Control Institute, PPQ, APHIS, USDA,
Riverdale, MD.

Meyerdirk, D. E., R. Muniappan, R. Warkentin, J. Bamba, and G. V. P. Reddy. 2004.
Biological control of the papaya mealybug, Paracoccus marginatus (Hemiptera:
Pseudococcidae) in Guam. Plant Prot. Quart. 19: 110-114.

Miller, D. R., and G. L. Miller. 2002. Redescription of Paracoccus marginatus Williams and
Granara de Willink (Hemiptera: Coccoidea: Pseudococcidae) including descriptions of
the immature stages and adult male. Proc. Entomol. Soc. Wash. 104: 1-23.

Miller, D. R., D. J. Williams, and A. B. Hamon. 1999. Notes on a new mealybug (Hemiptera:
Coccoidea: Pseudococcidae) pest in Florida and the Caribbean: the papaya mealybug,
Paracoccus marginatus Williams and Granara de Willink. Insecta Mundi. 13: 179-181.

Mizell III, R. F., and T. E. Nebeker. 1978. Estimating the developmental time of the southern
pine beetle Dendroctonusfronatlis as a function of field temperatures. Environ. Entomol.
7: 592-595.

Mossler, M. A. and N. Nesheim. 2002. Florida crop/pest management profile: papaya. CIR-
1402. Pesticide Information Office, Food Science and Human Nutrition Department,
Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences,
University of Florida, Gainesville, FL.
(http://edis.ifas.ufl.edu/ October 2007)

Muniappan, R., D. E. Meyerdirk, F. M. Sengebau, D. D. Berringer, and G. V. P. Reddy.
2006. Classical biological control of the papaya mealybug, Paracoccus marginatus
(Hemiptera: Pseudococcidae) in the republic of Palau. Florida Entomol. 89: 212-217.

Narai, Y., and T. Murai. 2002. Individual rearing of the Japanese mealybug, Planococcus
kraunhiae (Kuwana) (Homoptera : Pseudococcidae) on a germinated broad bean seeds.
Appl. Entomol. Zool. 37: 295-298.

National Climatic Data Center (NCDC). 2005. Comparative Climatic Data. National
Climatic Data Center, National Oceanic and Atmospheric Administration, US
Department of Commerce, Ashville, NC.
(http://www.ncdc.noaa.gov/oa/climate/online/ccd/nrmavg.txt, October 2007)

Neuenschwander, P. 2001. Biological control of the cassava mealybug in Africa: a review.
Biol. Control. 21: 214-229.

Neuenschwander, P., F. Schulthess, and E. Madojemu. 1986. Experimental evaluation of the
efficiency ofEpidinocarsis lopezi, a parasitoid introduced into Africa against the cassava
mealybug Phenacoccus manihoti. Entomol. Exp. Appl. 42: 133-138.









Noyes, J. S. 2000. Encyrtidae of Costa Rica, I. The subfamily Tetracneminae (Hymenoptera:
Chalcidoidea), parasitoids of mealybugs (Homoptera: Pseudococcidae). Mem. Amer. Ent.
Inst. 62: 101-103.

Noyes, J. H., and M. E. Schauff. 2003. New Encyrtidae (Hymenoptera) from papaya
mealybug (Paracoccus marginatus Williams and Granara de Willink)
(Hemiptera: Stemorrhyncha: Pseudococcidae). Proc. Entomol. Soc. Wash. 105: 180-185.

Orr, D. B., and C. P.-C. Suh. 1998. Parasitoids and predators. pp. 3-34. In J. E. Rechcigl and
N. A. Rechcigl (eds.), Biological and biotechnological control of insect pests. CRC Press
LLC, Boca Raton, FL.

Picman, J., and A. K. Picman. 1984. Autotoxicity in Parthenium hysterophorus and its
possible role in control of germination. Biochem. Syst. Ecol. 12: 287-292.

Raghubanshi, A. S., L. C. Rai, J. P. Gaur, and J. S. Singh. 2005. Invasive alien species and
biodiversity in India. Current Sci. 88: 539-540.

Ridley, M., 1988. Mating frequency and fecundity in insects. Bio. Rew. 63: 509-549.

Ridley, M., 1993. A sib competitive relation between clutch size and mating frequency in
parasitic Hymenoptera. Am. Naturalist. 142: 893-910.

Rosenheim, J. A., H. K. Kaya, L. E. Ehler, J. J. Marois, and B. A. Jaffee. 1995. Intraguild
predation among biological-control agents: theory and evidence. Biol. Control. 5: 303-
335.

Sagarra, L. A., and C. Vincent. 1999. Influence of host stage on oviposition, development,
sex ratio, and survival ofAnagyrus kamali Moursi (Hymenoptera: Encyrtidae), a
parasitoid of the hibiscus mealybug, Maconellicoccus hirsutus Green (Homoptera:
Pseudococcidae). Biol. Control. 15: 51-56.

Sagarra, L. A., C. Vincent, N. F. Peters, and R. K. Stewart. 2000a. Effect of host density,
temperature, and photoperiod on the fitness ofAnagyrus kamali, a parasitoid of the
hibiscus mealybug Maconellicoccus hirsutus. Entomol. Exp. Appl. 96: 141-147.

Sagarra, L. A., C. Vincent, and R. K. Stewart. 2000b. Fecundity and survival ofAnagyrus
kamali (Hymenoptera: Encyrtidae) under different feeding and storage temperature. Eur.
J. Entomol. 97: 177-181.

Sagarra, L. A., C. Vincent, and R. K. Stewart. 2002. Impact of mating on Anagyrus kamali
Moursi (Hymenoptera: Encyrtidae) lifetime fecundity, reproductive longevity, progeny
emergence, and sex ratio. J. Appl. Entomol. 126: 400-404.

SAS Institute. 1999. SAS user's guide. Version 9.1. SAS Institute, Cary, NC.









Schroder, D. 1974. A study of the interactions between the internal larval parasites of
Rhyacionia buoliana (Lepidoptera:Olethreutidae). Entomophaga. 19: 145-171.

Sequeira, R., and M. Mackauer. 1992. Covariance of adult size and development time in the
parasitoid wasp Aphidius ervi in relation to the size of its host, Acyi itniph,, iihpisum.
Evolutionary Ecol. 6: 34-44.

Serrano, M. S., and S. L. Lapointe. 2002. Evaluation of host plants and a meridic diet for
rearing Maconellicoccus hirsutus (Hemiptera: Pseudococcidae) and its parasitoid
Anagyrus kamali (Hymenoptera: Encyrtidae). Florida Entomol. 85: 417-425.

Sinacori, A. 1995. Bio-ethological observations on Phenacoccus madeirensis Green
(Coccoidea: Pseudococcidae) in Sicily. Israel J. Entomol. XXIX: 179-182.

Sloan, S. A., J. K. Zimmerman, and A. M. Sabat. 2007. Phenology ofPlumeria alba and its
herbivores in a tropical dry forest. Biotropica. 39: 195-201.

Smith, H. S., and P. DeBach. 1942. The measurement of the effect of entomophagous insects
on population densities of the host. J. Econ. Entomol. 35: 845-849.

Smith, M. R. 1928. The biology of Tapinoma sessile Say, an important house-infesting ant.
Ann. Entomol. Soc. Am. XXI: 307-330.

Tefera, T. 2002. Allelopathic effects of Parthenium hysterophorus extracts on seed
germination and seedling growth ofEragrostis tef J. Agron. Crop Sci. 188: 306-310.

Townsend, M. L., R. D. Oetting, and J.-H. Chong. 2000. Management of the mealybug
Phenacoccus madeirensis. Proc. South. Nurs. Assoc. Res. Conf. 45: 162-166.

Trudgill, D. L., A. Honek, D. Li, and N. M. Van Straalen. 2005. Thermal time-concepts and
utility. Ann. Appl. Biol. 146: 1-14.

Uckan, F., and E. Ergin. 2002. Effect of host diet on the immature developmental time,
fecundity, sex ratio, adult longevity, and size ofApanteles galleriae (Hymenoptera:
Braconidae). Biol. Control. 31: 168-171.

USDA, NRCS. 2007a. The Plants Database. National Plant Data Center, Baton Rouge, LA.
(http://plants.usda.gov/, October 2007)

USDA, NASS. 2007b. Hawaii papayas. National Agricultural Statistics Service, Honolulu, HI.
(http://www.nass.usda.gov/hi, October 2007)

Van Driesche, R. G., and T. S. Bellows Jr. 1996. Biological Control. Kluwer
Academic Publishers, Norwell, MA.









Van Lenteren, J. C. 1980. Evaluation of control capabilities of natural enemies. Does art have
to become science? Neth. J. Zool. 30: 369-381.

Van Strien-van Liempt, W. T. F. H. 1983. The competition between Asobara tabida Nees
von Esenbeck, 1834 and Leptopilina heterotoma (Thomson, 1862) in multiparasitized
hosts. Neth. J. Zool. 33: 125-163.

Vinson, S. B. 1976. Host selection by insect parasitoids. Ann. Rev. Entomol. 21: 109-133.

Vinson, S. B., and G. F. Iwantsch. 1980. Host suitability for insect parasitoids. Ann. Rev.
Entomol. 25: 397-419.

Waage, J. K. 1986. Family planning of parasitoids: adaptive patterns of progeny and sex
allocation. pp. 63-89. In J. Waage and D. Greathead. (eds.) Insect Parasitoids. Academic
Press Inc., Orlando, FL.

Wagner, T. L., H.-I. Wu, P. J. H. Sharpe, R. M. Schoolfield, and R. N. Coulson. 1984.
Modeling insect development rates: a literature review and applications of a biophysical
model. Ann. Entomol. Soc. Am. 77: 208-225.

Walgama. R. S., and M. P. Zalucki. 2006. Evaluation of different models to describe egg and
pupal development ofXyleborusfornicatus Eichh. Coleoptera: Scolytidae), the shot-hole
borer of tea in Sri Lanka. Insect Sci. 13: 109-118.

Walker, A., M. Hoy, and D. Meyerdirk. 2003. Papaya mealybug (Paracoccus marginatus
Williams and Granara de Willink (Insecta: Hemiptera: Pseudococcidae)). EENY-302.
Featured Creatures. Entomology and Nematology Department, Florida Cooperative
Extension Service, Institute of Food and agricultural Sciences, University of Florida,
Gainesville, FL.
(http://edis.ifas.ufl.edu/, October 2007)

Williams, D. J., and M. C. Granara de Willink. 1992. Mealybugs of Central and South
America. CAB International, Wallingford, Oxon, UK.

Yang, J., and C. S. Sadof. 1995. Variegation in Coleus blumei and the life history of citrus
mealybug (Homoptera : Pseudococcidae). Environ. Entomol. 24: 1650-1655.

Zar, J. H. 1984. Biostatistical Analysis, 2nd ed. Prentice Hall, Englewood Cliffs, NJ.









BIOGRAPHICAL SKETCH

Born in Anuradhapura, Sri Lanka, Kaushalya Gunewardane Amarasekare graduated from

the University of Peradeniya, Sri Lanka with a Bachelor of Science in agriculture with honors in

October 1993. After graduation, she worked for the Department of Agriculture, Sri Lanka as a

research scientist. In 2000, she was offered an assistantship to study entomology at Oklahoma

State University, Stillwater, Oklahoma, under the guidance of Dr. Jonathan Edelson. Upon

receiving a Master of Science in December 2002, she moved to Florida to pursue her Doctor of

Philosophy degree at the University of Florida with advisor, Dr. Catharine Mannion.





PAGE 1

LIFE HISTORY OF PAPAYA MEALYBUG ( Paracoccus marginatus ), AND THE EFFECTIVENESS OF THREE INTRODUCED PARASITOIDS ( Acerophagus papayae Anagyrus loecki AND Pseudleptomastix mexicana) By KAUSHALYA GUNEWARDANE AMARASEKARE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1

PAGE 2

2007 Kaushalya Gunewardane Amarasekare 2

PAGE 3

Affectionately dedicated to my late parents 3

PAGE 4

ACKNOWLEDGMENTS The completion of this research would have been impossible but for the help of my advisors, friends, colleagues and family. Whatever cr edit this research deserves should be shared amongst all those whose help and support was in valuable throughout the course of my study. A very special word of thanks goes to Dr. Ca tharine Mannion, my major advisor, for her advice and guidance, throughout the study period. I thank my graduate advisory committee, Drs. Lance Osborne (co-chair), Robert McSorley, Wagner Vendrame, and Nancy Epsky for their advice, guidance, and encouragemen t throughout. A word of thanks must also be offered to the staff at University of Florida, Entomology and Nematology Department, Gainesville, and the Tropical Research and Education Center, Homest ead, for their assistance and support at all times. Special thanks are due to Joan Barrick and Kenneth Brown for helping me through the bad times and sharing my joy through good times. La st but not least, I thank my family and all my friends whose warmth of heart made all th is possible: their loya lty is unforgotten and unforgettable. 4

PAGE 5

TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................7 ABSTRACT .....................................................................................................................................9 CHAPTER 1 INTRODUCTION................................................................................................................. .11 Mealybugs ...............................................................................................................................11 Genus Paracoccus ..................................................................................................................13 Paracoccus marginatus Williams and Granara de Willink ....................................................13 Host Plant Species ..................................................................................................................14 Temperature ............................................................................................................................17 Chemical Control of Papaya Mealybug..................................................................................18 Biological Control ..................................................................................................................19 Classical Biological Control of Papaya Mealybug .................................................................19 Parasitoids ...............................................................................................................................20 Acerophagus papayae Noyes and Schauff ......................................................................21 Pseudleptomastix mexicana Noyes and Schauff.............................................................21 Anagyrus loecki Noyes and Menezes ..............................................................................22 Developmental Time, Longevity, and Lifetime Fertility ........................................................22 Host Stage Susceptibility, Host Stage Suitability, and Sex Ratio ..........................................24 Interspecific Competition .......................................................................................................25 Research Objectives ................................................................................................................26 2 LIFE HISTORY OF Paracoccus marginatus WILLIAMS AND GRANARA DE WILLINK (HEMIPTERA: PSEUDOCOCCIDAE) ON FOUR HOST PLANT SPECIES UNDER LABORA TORY CONDITIONS............................................................28 Introduction .............................................................................................................................28 Materials and Methods ...........................................................................................................29 Results .....................................................................................................................................32 Discussion ...............................................................................................................................34 3 EFFECT OF CONSTANT TEMPERATURE ON THE DEVELOPMENTAL BIOLOGY OF Paracoccus marginatus WILLIAMS AND GRANARA DE WILLINK (HEMIPTERA: PSEUDOCOCCIDAE).................................................................................40 Introduction .............................................................................................................................40 Materials and Methods ...........................................................................................................41 Results .....................................................................................................................................45 Discussion ...............................................................................................................................47 5

PAGE 6

4 HOST STAGE SUSCEPTIBILITY AND SEX RATIO, HOST STAGE SUITABILITY, AND INTERSPECIFIC COMPETITION OF A cerophagus papayae Anagyrus loecki AND Pseudleptomastix mexicana : THREE INTRODUCED PARASITOIDS OF Paracoccus marginatus WILLIAMS AND GRA NARA DE WILLINK..............................56 Introduction .............................................................................................................................56 Materials and Methods ...........................................................................................................58 Results .....................................................................................................................................63 Discussion ...............................................................................................................................64 5 DEVELOPMENTAL TIME, LONGEVITY, AND LIFETIME FERTILITY OF Acerophagus papayae Anagyrus loecki AND Pseudleptomastix mexicana; THREE INTRODUCED PARASITOIDS OF Paracoccus marginatus WILLIAMS AND GRANARA DE WILLINK....................................................................................................72 Introduction .............................................................................................................................72 Materials and Methods ...........................................................................................................73 Results .....................................................................................................................................79 Discussion ...............................................................................................................................80 6 FIELD ASSESSMENT OF THREE INTRODUCED PARASITOIDS OF Paracoccus marginatus WILLIAMS AND GRANARA DE WILLINK (HEMIPTERA: PSEUDOCOCCIDAE)...........................................................................................................87 Introduction .............................................................................................................................87 Materials and Methods ...........................................................................................................88 Results .....................................................................................................................................93 Discussion ...............................................................................................................................95 7 SUMMARY AND CONCLUSIONS...................................................................................103 REFERENCE LIST .....................................................................................................................105 BIOGRAPHICAL SKETCH .......................................................................................................114 6

PAGE 7

LIST OF TABLES Table page 2-1 Mean number of days ( SEM) for each developmental stadium of P marginatus reared on four host species.................................................................................................38 2-2 Mean ( SEM) percent survival for each developmental stadium of P marginatus reared on four host species. ................................................................................................39 3-1 Mean number of days ( SEM) for each developmental stadium of P. marginatus reared at different constant temperatures ...........................................................................52 3-2 Mean ( SEM) percent survival for each developmental stadium of P marginatus reared at different constant temperatures. ..........................................................................53 3-3 Mean ( SEM) proportion of females, adult longevity, fecundity, pre-oviposition and oviposition periods of P. marginatus reared at four constant temperatures ...............54 3-4 Summary of statistics and the estimates ( SE) of the fitted parameters of the linear thermal summation model and the nonlinear Logan 6 model. ...........................................55 4-1 Mean percent parasitism ( SEM) of A papayae A loecki and P mexicana reared in different developmental stages of P marginatus to evaluate host stage susceptibility using no-choice tests. ...................................................................................68 4-2 Mean proportion of females (sex ratio) ( SEM) of A papayae A loecki and P mexicana reared in different developmental stages of P marginatus to evaluate host stage susceptibility using no-choice tests. .........................................................................69 4-3 Mean percent parasitism ( SEM) of A papayae A loecki and P mexicana reared in different stage combinations of P marginatus to evaluate host stage suitability using choice tests. ..............................................................................................................70 4-4 Mean percent parasitism ( SEM) of combinations of A papayae A loecki and P mexicana reared in second and third-instar P marginatus to evaluate interspecific competitions of parasitoids. ...............................................................................................71 5-1 Mean developmental time (egg to adult ec losion) in days ( SEM) for male and female A papayae A loecki and P mexicana reared in second instar, third-instar female, and adult-female P marginatus ............................................................................84 5-2 Mean longevity in days ( SEM) for male (unmated and mated), and female (unmated, mated-without ovipositi on, and mated-with oviposition) A papayae, A loecki and P mexicana .....................................................................................................85 7

PAGE 8

5-3 Mean ( SEM) number of male and female progeny, cumulative progeny, sex ratio, and reproductive period of mated and unmated A papayae A loecki and P mexicana ............................................................................................................................86 6-1 Mean ( SEM) number of mealybug destroyer ( Cryptolaemus montrouzieri ) adults and larvae collected per cage from open sl eeve cage and no cage treatments using pooled data of 2005 and 2006 in three experimental locations .......................................100 6-2 Mean ( SEM) number of ants and spiders collected from open sleeve cage and no cage treatments using pooled data of 2005 and 2006 in three experimental locations ....101 6-3 Individual and cumulative mean percent parasitism ( SEM) of P marginatus by A papayae A loecki and P mexicana in open sleeve cage, and no cage treatments using pooled data of 2005 and 2006 in three experimental locations ..............................102 8

PAGE 9

Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy LIFE HISTORY OF PAPAYA MEALYBUG ( Paracoccus marginatus ), AND THE EFFECTIVENESS OF THREE INTRODUCED PARASITOIDS ( Acerophagus papayae Anagyrus loecki and Pseudleptomastix mexicana ) By Kaushalya Gunewardane Amarasekare December 2007 Chair: Catharine Mannion Cochair: Lance Osborne Major: Entomology and Nematology Native to Mexico and Central America, papaya mealybug ( Paracoccus marginatus ) is an adventive pest insect that can damage a large number of tr opical and subtropical fruits, vegetables, and ornamental plants in the US, the Caribbean, and th e Pacific islands. It is an important pest in Florida, and potentially poses a threat to other st ates such as California, Hawaii, and Texas. Currently, three in troduced parasitoids are used as biological control agents. Information on papaya mealybug and its parasitoids is scarce. In this dissert ation, the life history of papaya mealybug in relation to temperat ure and host plants, a nd the biology and the effectiveness of its parasitoids were investigated. Temperature is one of the important abiotic f actors that may decide the establishment and distribution of papaya mealybug in to other areas in the US. Adult males and females required 303.0 and 294.1 degree-days, and 14.5 and 13.9 C, minimum temperature threshold, respectively. In addition, papaya mealybug wa s able to complete its life cycle on three ornamental plants, hibiscus, acalypha, plumeria and the weed parthenium, which are commonly found plants in many US states. 9

PAGE 10

In the field, Acerophagus papayae provided better control than the other parasitoids. Pseudleptomastix mexicana was not observed, while Anagyrus loecki had lower parasitism. In the laboratory, all parasitoids were able to de velop and emerged successfu lly in all stages of P marginatus except for first-instar nymphs. Acerophagus papayae and P mexicana preferred second-instar P marginatus while A loecki preferred third instars. Developmental times of A papayae and A loecki were similar but P mexicana had a longer developmental time. Overall, A papayae provided better control of the host, when alone or with the other two parasitoids. Pseudleptomastix mexicana was less competitive when mixed with A papayae and A loecki. Considering its low thermal requirement and high minimum temperature threshold, papaya mealybug has a smaller distribution range than anticipated. Southern parts of Texas and California, South Florida, and Hawaii are suit able areas for its development. Its final establishment and distribution may be influenced by other factors such as host plant range, and the rules and regulations governing plan t movement from state to state. 10

PAGE 11

CHAPTER 1 INTRODUCTION Mealybugs Mealybugs are soft-bodied insects, which belong to the family Pseudococcidae in the order Hemiptera (Borror et al. 1992). The name "m ealybug" is derived from the mealy or waxy secretions that cover the bodies of these insects (Borro r et al. 1992). This layer of fine mealy wax often extends laterally to form a series of short filaments. The mealy wax covering is frequently white and the color may vary among some species (Williams and Granara de Willink 1992). The body of the adult female is normally elongate to oval, and membranous (Williams and Granara de Willink 1992). Antennae normally have 6 to 9 segments. Legs are present; each with a single tarsal segment and a single claw (Williams and Granara de Willink 1992). In common with other hemipterans, fema le mealybugs have piercing and sucking mouthparts and are generally act ive throughout their life (Ben-Dov 1994). In the tropics, their life cycle may be reduced to less than one month. They often attain hi gh numbers, killing the host plant by depleting the sap and occasionally by injecting toxins, transmitting viruses, or by excreting honeydew, which is a suitable medium for the growth of sooty mold (Ben-Dov 1994). The mold often covers the plant to such an extent that normal photosynthesis is severely reduced (Williams and Granara de Willink 1992). Alth ough some mealybugs are host plant specific, mealybugs such as Maconellicoccus hirsutus (Green), and Phenacoccus madeirensis Green are polyphagus mealybugs that can damage a large number of economically important plants (Sinacori 1995, Serrano and Lapointe 2002). Reproduction of mealybugs under greenhouse cond itions is year round, and in certain species is by the production of living nymphs or young often w ithout fertilization. Some mealybug species reproduce parthen ogenetically. The cassava mealybug, Phenacoccus manihoti 11

PAGE 12

Matile-Ferrero reprodu ces by thelytokous parthenogenesis (Calatayud et al. 1998, Le Ru and Mitsipa 2000). Many species form an ovisac in which to lay th e eggs. In sexually reproducing species, the adult males are normally minute w ithout functional mouthparts. Male mealybugs are often winged but occasionally apterous. In contrast, females are always wingless (Williams and Granara de Willink 1992). Many of the 2000 mealybug species already descri bed are important insect pests of many agricultural crops (Williams and Granara de W illink 1992). Infestations may occur within vegetative shoots or apexes and can be extremely di fficult to detect. This ability of mealybugs to form dense colonies, particularly within the shoo t and apex, often makes chemical control of this pest quite difficult. With the introduction of many new systemic insecticides, control has improved; however, with insects that are pol yphagus, and have numerous hosts, it becomes a challenge to manage them with just chemical control. Many times mealybug populations in their countries of origin are not pest problems due to their parasitoids and predators. The most serious outbreaks occur when mealybugs are accidentally introduced to new countries without th eir natural enemies. The introduction of pests on infested plant material has unfortunately b ecome fairly common. Fl orida is one of the important agricultural states in this country a nd it has weather and climatic patterns that are conducive for the establishment of many insects. In South Florida, the more subtropical climatic condition facilitates the gr owth of a variety of tropical and s ubtropical crops. This agricultural pattern, subtropical clima tic condition, increase of world tr ade, and geographic location of the state, are the main reasons for the regular invasion of insect pests to Florida. Invasive insect species such as the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae) (Mead 2007) and the pink hibiscus mealybug, Maconellicoccus hirsutus (Green) (Hemiptera: 12

PAGE 13

Pseudococcidae) (Hoy et al. 2006), which were acc identally introduced to Florida in 1998 and 2002, respectively are good examples of the pest invasions to Florida. Paracoccus marginatus Williams and Granara de Willink is one of th e mealybug species that has been accidentally introduced into the Caribbean, the US and the Pacific islands, from Central America. Genus Paracoccus The genus Paracoccus was first described by Ezzat and McConnell in 1956 by using the type species Pseudococcus burnerae Brain, by original designa tion (Ben-Dov 1994). Generic characters of Paracoccus were later described by Williams and Granara de Willink (Ben-Dov 1994). Paracoccus has a varied distribution from the "A ustro-Oriental", Et hiopian, Madagasian, Nearctic, Neotropical, New Zealand, Pacific, Pa laearctic, and Oriental regions (Ben-Dov 1994). There are about 79 species recorded from the genus Paracoccus (Ben-Dov 1994). Most of the Paracoccus species are not recognized as major econom ic pests except for two species. In South Africa, Paracoccus burnerae (Brain) is considered as a seri ous pest of citrus (Ben-Dov 1994). Paracoccus marginatus Williams and Granara de Willink (pap aya mealybug) is a pest of papaya and other economically important fruits, vegetables and ornamentals in the Caribbean, the US, and several Pacific islands. Paracoccus marginatus Williams and Granara de Willink Specimens of papaya mealybug were first collected from Mexico in 1967, which were believed to be native to Mexico and/or Central America (Miller et al. 1999). Papaya mealybug is not a serious pest in Mexico, probably because of the availability of its natural enemies (Miller et al. 1999). This species was first described by W illiams and Granara de Willink in 1992 from the specimens collected from neo-tropical regions in Belize, Costa Rica, Guatemala and Mexico (Williams and Granara de Willink 1992). In 2002, Miller and Miller re-described this mealybug species (Miller and Miller 2002). 13

PAGE 14

In early 1990, papaya mealybug invaded the Cari bbean region and became a pest of many tropical and subtropical fruits, ve getables, and ornamental plants (Miller and Miller 2002). Since 1994, it has been recorded in 14 Caribbean countries. In 2002, a heavy infestation of papaya mealybug was observed on papaya ( Carica papaya L. (Caricaceae)) in Guam (Meyerdirk et al. 2004). Subsequently, papaya mealybug infestations were reported from the Republic of Palau in 2003 and in Hawaii in 2004 (Muniappa n et al. 2006, Heu et al. 2007). The papaya mealybug is an adventive pest insect species that has been found in the US. It was first recorded on hibiscus in Palm Beach Co unty in Florida in 1998 (Miller et al. 1999) and subsequently spread into several other counties in th e state. It has been collected from more than 25 different plant genera in many counties in Florida since then (Walker et al. 2003). Paracoccus marginatus is yellow in color and has a series of short waxy filaments around the margins of the body, which are less than 1/4 th e length of the body (Miller et al. 1999). The female papaya mealybug passes through three immatu re stages (first, second, and third instar) before emerging as an adult. The ovisac produced by the adult female is on the ventral side of the body and is generally two or more times the body length (Miller et al. 1999). Generally, first instars of mealybugs are called crawlers". Ther e is no distinguishable difference between male and female crawlers, and male and female early s econd instars. In the latter part of the second instar, the color of the male changes from ye llow to pink. Later, it develops a cottony sack around itself. Male third instars are termed as prepupa. Unlike the female, the male has a fourth instar termed as "pupa", from whic h the adult male emerges (Miller et al. 1999). Host Plant Species Food is a component of the environment and may influence an animal's chance to survive and multiply by modifying its fecundity, longevit y or speed of development (Andrewartha and Birch 1954). The e conomically important host range of the papaya mealybug includes papaya, 14

PAGE 15

hibiscus, acalypha, plumeria, avo cado, citrus, cotton, tomato, eggpl ant, pepper, beans and peas, sweet potato, mango, cherry and pomegranate (M iller and Miller 2002). In addition, weed species such as Parthenium hysterophorus L. are also recorded as host plants of papaya mealybug (Miller and Miller 2002). Infestations of papaya mealybug have been observed on papaya, plumeria, hibiscus and jatropha in Hawaii with the favored hosts a ppearing to be papaya, plumeria, and hibiscus (Heu et al. 2007). However, i nsects may settle, lay eggs, and severely damage plant species that are unsuitable fo r development of immatures (Harris 1990). There is no specific information about the life history of papaya mealybug on different host plant species. Although, papaya is the dominant host plant species of papaya mealybug, it is important to find out how it can develop on popular ornamental plan ts such hibiscus, acalypha, and plumeria as well as on a commonly found invasive annual weeds such as parthenium. Hibiscus, which is believed to be native to China, is a popular ornamental and landscape shrub, and widely grown in the tropics and subt ropics (Ingram and Rabinowitz 2004). Different hibiscus species are grown in many areas of the US (USDA 2007a). Hibiscus has been grown in Florida for many years (Ingram and Rabinowitz 2004) and its potential planting range in the US includes some areas of Texas and California (Gilman 1999b). Hibiscus is widely grown in Hawaii. Hibiscus is sold nationwide as potte d flower plants, and maintained in greenhouses around the country. Pink hibiscus mealybug, Maconellicoccus hirsutus (Green) (Hemiptera: Pseudococcidae) is another important mealybug species that was introduced to Florida in 2002, and has been identified as one of the most impor tant insect pests of hibiscus (Goolsby et al. 2002, Hoy et al. 2006). Acalypha L. is a large, fast growing evergr een shrub, which can provide a continuous splash of color in the landscape with the bronze red to muted red and mottled combinations of 15

PAGE 16

green, purple, yellow, orange, pink or white (Gilman 1999a). It is believed to be native to Fiji and nearby Pacific islands. Acalypha L. is grown in many parts of the United States (USDA 2007a). Aphids, mites, scales, and mealybugs are recorded as pests of acalypha (Gilman 1999a). The genus Plumeria L. originates from Central Amer ica and its different species are popular ornamental plants that ar e widely distributed in the warm er regions of the world (Begum et al. 1994). Plumeria belongs to the fam ily Apocynaceae (dogbane) (Criley 1998) and the sap of most of the plants belonging to this family is milky, and may contain toxic alkaloids or glycosides. In Southwestern Puerto Rico, a caterpillar of the sphinx moth, Pseudosphinx tetrio L. (Lepidoptera: Sphingidae), two mealybug species ( P marginatus and Puto sp.) and one unidentified Margaroididae are the fre quently encountered herbivores of Plumeria alba (Sloan et al. 2007). The most common homopteran attacking P alba in Puerto Rico is the papaya mealybug (Sloan et al. 2007). Th ese homopterans attack the leav es, inflorescences, flowers, fruits and sometimes the stem of P alba (Sloan et al. 2007). They feed on the sap of P alba leaves when the standing crop of leaves is the greatest, causing the leaves to be frequently contorted, misshapen, and not fully expanded (Sloan, et al. 2007). Triter penoids are chemicals commonly found in plants that belong to the family Apocynaceae, and in plumeria, these compounds can be feeding deterrents to most gene ralist insects. The aposematic coloration of P tetrio suggests that it is able to detoxify and sequester secondary compounds in P alba, but these compounds can make P alba unpalatable to other generalist herbivores (Sloan et al. 2007). Parthenium hysterophorus L. is an introduced, invasive weed species, which can be found in more than 17 states in the Eastern, Southern, and South Central US (USDA 2007a). Parthenium is considered a noxious annual weed b ecause of its prolific seed production and fast spreading ability, allelopathic effect on other plants, strong competitiveness with crops and 16

PAGE 17

health hazard to humans as well as animals (Tefera 2002, Raghubanshi et al. 2005). Parthenium contains sesquiterpene lactones and phenolic acids (Picman and Picman 1984, Mersie and Singh 1988). Terpinoids, from volatile monoterpenoids to involatile triterp enoids, are broadly defensive against herbivory on plants (Harbone 2001). Parthenin is a terpinoid found in parthenium weed, which is identified as a barr ier to herbivore feedi ng (Harbone 2001). A leaf feeding beetle, Zygogramma bicolorata Pallister (Coleoptera: Chry somelidae) and a stem-galling moth, Epiblema strenuana Walker (Lepidoptera: Tortricidae) are some of the natural enemies used in the biological control of parthenium in Australia (Dhileepan 2001, Dhileepan et al. 2005). Temperature Temperature is one of the important environm ental factors that can affect the movement, establishment, and abundance of insects. Insect bi ology is influenced by various environmental factors and temperature is one of the most importa nt and critical of the abiotic factors (Huffaker et al. 1999). The rate of insect development is affected by the temperature to which the insects are exposed (Campbell et al. 1974). Insect deve lopment occurs within a definite temperature range (Wagner et al. 1984). The temperature be low which no measurable development occurs is its threshold of development. The amount of heat required over time for an insect to complete some aspect of development is considered a thermal constant (Campbell et al. 1974). The thresholds and the thermal constant are useful indicators of potential distribution and abundance of an insect (Huffaker et al. 1999). The impor tance of predicting the seasonal occurrence of insects has led to the formulation of many math ematical models that describe developmental rates as a function of temp erature (Wagner et al. 1984). The thermal summation model (Campbell et al. 1974) and Logan 6 model (Logan et al. 1976) are widely used models to explain the relationship between developmental time and temperature of arthropods. Temperature had 17

PAGE 18

pronounced effects on the development, surviv al, and reproduction of Madeira mealybug, Phenacoccus madeirensis Green (Chong et al. 2003). The female P madeirensis was able to complete its development in temperatures ra nging from 15 to 25C within 66 to 30 days respectively (Chong et al. 2003). Between 15 to 25C, survival rates of P madeirensis were not affected by temperature but the temperature had a strong influe nce on fecundity, pre-oviposition time, and the duration of reproduction (Chong et al. 2003). Between 20 and 25C, the cassava mealybug, Phenacoccus manihoti Matile-Ferrero, and Phenacoccus herreni Cox and Williams, complete development within 46 to 36 days (Lema and Herren 1985) and 91 to 41 days respectively (Herrera et al. 1989). Comparis on of whole-life developmental times of P herreni to those of P manihoti suggests that P herreni develops slower than P manihoti at cooler temperatures but faster than P manihoti at warmer temperatures (Her rera et al. 1989). This is supported by the more tropical distribution of P herreni (Columbia, The Guyana, and northern Brazil) compared to that of P manihoti which has subtropical distri bution (Herrera et al. 1989). Chemical Control of Papaya Mealybug Organophosphate and carbamate insecticides such as dimethoate, malathion, carbaryl, chlorpyrifos, diazinone, and acephate (Walker et al. 2003) were commonly used insecticides to control mealybugs. Currently neon ecotinoid insecticides such as acetamiprid, clothianidin, dinotefuran, imidacloprid, thiamethoxam, and insect growth regulators (IGR) such as pyriproxyfen are used to contro l scale insects and mealybugs (Bu ss and Turner 2006). However, there is no specific insecticide cu rrently registered for control of papaya mealybug (Walker et al. 2003). Mealybugs are generally difficult to contro l chemically due to their thick waxy secretion covering the body, and their ability to hide in the damaged buds and leaves without being exposed to the insecticide. The adult mealybugs were more difficult to control than the young and repeated applications of chemicals ta rgeting immatures were required in suppressing P 18

PAGE 19

madeirensis (Townsend et al. 2000). In addition, w ith polyphagous insects such as papaya mealybug, it would be difficult to manage it with just insecticides and to achieve long-term control with the wide variety of host plants. Development of insecticide re sistance and nontarget effects of insecticides on natural enemies make chemical control a less feasible option for the long-term control of papaya mealybug (Walke r et al. 2003). Because of these reasons, biological control was identified as a pref erred method to control the papaya mealybug. Biological Control Biological control is the use of parasitoid, predator, pathogen, antagonist, or competitor populations to suppress a pest population, making it less abundant and thus less damaging (Van Driesche and Bellows 1996). It is widely accepte d that there are three general approaches to biological control: importation, augmentation, and conservation of natural enemies. Importation biological control is often referre d to as "classical biological control" refl ecting the historical predominance of this approach (Orr and Suh 1998). Classical biological control can be defined as importation and establishment of non-native natural enemy populations for suppression of non-native or native organisms (Orr and Suh 1998) Augmentation includes activities in which natural enemy populations are incr eased through mass culture, periodi c release, and colonization. Conservation biological contro l can be defined as the st udy and modification of human influences that allow natural enemies to reali ze their potential to suppr ess pests (Orr and Suh 1998). Currently, the "classical" a pproach is probably the most r ecognized and heralded form of biological control among biologi cal control practitioners. Classical Biological Control of Papaya Mealybug Many adventive insect species become pests because they are unaccompanied by natural enemies from their native home (Orr and Suh 1998). In the classical biol ogical control of an adventive pest species, most ofte n the natural enemies of the pest are searched for in its native 19

PAGE 20

homeland by examining the pest population in its native environment (Van Driesche and Bellows 1996). These natural enemies are then co llected and shipped to the country where the pest has invaded. After being subjected to appr opriate quarantine and tes ting to ensure safety, these natural enemies are released and established. This type of introduction of natural enemies is self-maintaining and less expens ive than chemical control over the long term (Van Driesche and Bellows 1996). The United States Department of Agriculture (USDA), Animal Plant Health Inspection Service (APHIS) initia ted a classical biologi cal control program for papaya mealybug using several natural enemies in 1999. The identified natural enemies of papa ya mealybug are solitary endoparasitic wasps that belong to the family Encyrtidae in the Order Hymenoptera. These wasps were collected in Mexico as potent ial biological control agents. They were Acerophagus papayae Noyes and Schauff, Anagyrus loecki Noyes and Menezes, Anagyrus californicus Pseudophycus sp. and Pseudleptomastix mexicana Noyes and Schauff (Meyerdirk et al. 2004). Acerophagus papayae, A. loecki and P. mexicana are three parasitoid sp ecies that are currently used in the biological control of papaya mealybug. They are ma ss reared in Puerto Rico and released in papaya mealybug infested areas in th e Caribbean, the US, and the Pacific islands as needed (Meyerdirk et al. 2004). Parasitoids The term "parasitoid" embraces an exceedingl y large number of insect species (Gauld 1986). Parasitoids are arthropods that kill their ho sts and are able to complete their development on a single host (Vinson 1976). Parasitoids have been the most common type of natural enemy introduced for biological control of insects. They have been employed in the management of insect pests for centuries (Orr and Suh 1998). Th e last century, however, has seen a dramatic increase in their use as well as an understandin g of how they can be manipulated for effective, 20

PAGE 21

safe use in insect pest management systems (Orr and Suh 1998). Most parasitoids that have been used in biological control are in the orders Hymenoptera and, to a lesser degree, Diptera (Van Driesche and Bellows 1996). Of these, certain groups stand out as having more species employed in biological control projects than othe rs. The most frequently used groups in the Hymenoptera are Braconidae and Ichneumonidae in the Ichneumonoidea, and the Eulophidae, Pteromalidae, Encyrtidae, and Aphelinidae in the Chalcidoidea. In the Diptera, Tachinidae is the most frequently employed group (Greathead 1986). Although parasitoids have been recorded in the orders Strepsiptera and Coleoptera, parasi tism is not common in them (Van Driesche and Bellows 1996). Acerophagus papayae Noyes and Schauff This species of parasitoid is named for the papaya plant ( C papaya L.) on which its host feeds. It is the smallest species out of the three introduced parasitoid s of papaya mealybug. The female A papayae is 0.58 to 0.77 mm long including its ov ipositor, and males are generally 0.44 to 0.66 mm in length (Noyes and Schauff 2003). The male and female A papayae are generally pale orange in color. Other than the un-segmented clava, and gen italia, males are very similar to their females (Noyes and Schauff 2003). Acerophagus papayae was originally recorded from P marginatus in Mexico (Noyes and Schauff 2003). Pseudleptomastix mexicana Noyes and Schauff This is the second parasitoid out of the three introduced parasitoids of P marginatus ; P mexicana is named for its country of origin, Mexi co (Meyerdirk 2003, Noyes and Schauff 2003). Larger than A papayae the length of the male and female P mexicana is 0.56 to 0.84 and 0.76 to 1.03 mm, respectively. The head and thorax of the female are bl ack in color and the gaster is dark brown with a coppery a nd purple or brassy sheen. Pseudleptomastix mexicana also was originally recorded from P marginatus in Mexico (Noyes and Schauff 2003). In 2000, P 21

PAGE 22

mexicana was introduced into Puerto Rico with othe r exotic natural enem ies from Mexico to control P marginatus (Meyerdirk 2003). There are no ot her known introductions of exotic Pseudleptomastix species into various countries for the control of P marginatus or any other mealybug species (Meyerdirk 2003). Anagyrus loecki Noyes and Menezes The largest out of the three species, female A loecki is 1.45 to 1.76 mm in length, and the male is 0.94 to 1.08 mm long respectively (Noyes 2000) In the female, the head and thorax are mostly orange in color and the gaster is light br own. The male is dark brown in color and varies from the female in its size and color (Noyes 2000). This species was recorded from several mealybug species. The holotype was reared from Dysmicoccus hurdi and some of the paratypic material was laboratory reared on Phennacoccus madeirensis and P marginatus (Noyes 2000). Developmental Time, Longevit y, and Lifetime Fertility Developmental time, longevity, and lifetime fer tility are important fitness parameters when evaluating a parasitoid as a biol ogical control agent (Hemerik et al. 1999). Developmental time of a parasitoid is the durati on of time from oviposition to adult emergence. The time between adult emergence and death is termed as adult longev ity. The lifetime fertility of an insect is the total number of progeny produced during its lifetime. In koinobiont parasitoids that consume the entir e host before pupation, adult parasitoid size and developmental time are often strongly correlat ed with host size at the time when it is developmentally arrested through destructive feed ing by the parasitoid la rva (Hemerik et al. 1999). The development of Venturia canescens (Gravenhorst) (Hymenop tera: Ichneumonidae), a solitary endopa rasitoid of Plodia interpunctella (Hubner) (Lepidoptera: Pyralidae) depends on the ability of early stadia of its host to grow after pa rasitism and to reach their final stadium (Hemerik et al. 1999). The early emerging females of Trichogramma evanescens Westwood 22

PAGE 23

(Hymenoptera: Trichogrammatidae), a gregarious egg parasitoid of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) were la rger and produced more progeny a nd had higher fitness than late emerging females (Doyon and Boivin 2005). The a dult size and the developmental time of the solitary endoparasitoid, Aphidius ervi Haliday were affected by the size of its host, Acyrthosiphon pisum (Harris) (Sequeira and Mackauer 1992). The developmental time, longevity and the progeny production of parasitoids can be affected by the developmental temperature of the host (Hansen 2000). Between 15 to 30C, the developmental time of the female Trichogramma turkestanica on the host Ephestia kuehniella ranged from 32.9 to 7 days (Hansen 2000). The developmental time decreased with increasing temperature for the gregarious encyrtid endoparasitoid Tachinaephagus zealandicus reared on Chrysomya putoria (Ferreira de Almeida et al. 2002). Amitus fuscipennis MacGown and Nebeker, a potential biological control agent of Trialeurodes vaporariorum (Homoptera: Aleyrodidae), had longer developmental time a nd adult longevity at lower temperatures (Manzano et al. 2000). The lifetime fecundity and the reproductive lif e were significantly affected by temperature for Anagyrus kamali Moursi, a parasitoid of Maconellicoccus hirsutus Green reared at 26 and 32C (Sagarra et al. 2000a). Early emerged Tachinaephagus zealandicus lived longer than late emerged T zealandicus (Ferreira de Almeida et al. 2002). The host diet affected the developmenta l time, fecundity, sex ratio, and size of Apanteles galleriae Wilkinson (Hymenoptera: Braconidae), a parasitoid of Achroia grisella (F.) (Uckan and Ergin 2002). The mating status of a parasitoid can affect its fitness parameters. The mated solitary endoparasitoid female Anagyrus kamali Moursi had higher prog eny production and had a female biased sex ratio in comparison with unmated females, which had lower progeny production and male only progeny (Sagarra et al. 2002). Unmated A kamali lived longer than 23

PAGE 24

the mated ones (Sagarra et al. 2002 ). Fecundity and survival of Anagyrus kamali was also affected by higher feeding and storage temperat ures of 27C than 20C (Sagarra et al. 2000b). Host Stage Susceptibility, Host St age Suitability, and Sex Ratio Although a specific stage or stages of a mealybug are preferred by a parasitoid for oviposition, all or most of its stages can be sus ceptible to oviposition and subsequent parasitoid development. Parasitoids that develop in early instar mealybugs have a tendency to produce male progeny compared to those that develop in th e late instars, in which they can produce more female progeny (Charnov et al. 1981, Sagarra and Vincent 1999). In no choice tests, A kamali a parasitoid of the pink hibiscus mealybug, M hirsutus Green, was able to pa rasitize all nymphal stages and adult females, while choice tests indicated that A kamali prefers third instar and preoviposition adult females (Sagarra and Vincent 1999). Parasitoids emerged from hosts that were parasitized as second-instar P herreni were strongly male-biased for A vexans while apparently preferred later host stages yielded significantly more females th an males (Bertschy et al. 2000). Increased size of the host translates into bot h increased male and female fitness. For females, this measure is the lifetime production of eggs while for the male it is longevity (Charnov et al. 1981). The later the developmental stage of the host at ovi position, the faster the parasitoids develop and emerge (Bertschy et al. 2000). Within a particular host stage, the male had a shorter developmental time than the female for Aenasius vexans Kerrich, an encyrtid parasitoid of cassava mealybug, Phenacoccus herreni Cox and Williams (Bertschy et al. 2000). Depending on the instar they attack, the parasitoid progeny can be either male or female biased. The solitary endoparasito id of cassava mealybug ( Phenacoccus herreni Cox and Williams), Aenasius vexans Kerrich (Hymenoptera: Encyrtidae), shows male-biased sex ratio when it attacks second-instar P. herreni and female-biased sex ratio wh en it parasitizes third instars (Bertschy et al. 2000). 24

PAGE 25

The haplodiploid sex determination system of most parasitoid wa sps provides females a means of controlling the offspring sex ratio, beca use they can adjust the proportion of fertilized eggs at oviposition (King 1987). Parasitoid wasps provision their young with food by ovipositing in or on a host. Upon hatching the wasp larva feeds on the host, usually killing it prior to the wasp's pupation. Because a few ma les can fertilize many females, female-biased broods facilitate the use of para sitoids wasps as biological co ntrol agents (King 1987). The factors that may influence the o ffspring sex ratio are parental characteristics, environmental characteristics, host characteristics, and factors influencing local mate competition. The parental characteristics are time delay be tween emergences and insemination, number of times a female has mated, maternal and paternal age, maternal size, maternal diet, and genetics (King 1987). Photoperiod, temperature, and relative humidity ar e the environmental characteristics that can affect sex ratio. Host characte ristics such as host size, age, sex, and species can affect the progeny sex ratio of the parasitoid s. Local mate consumption th eory predicts that isolated females should produce primarily daughters with only enough sons to inseminate those daughters. Superparasitism, female density, numb er of offspring per ho st, and host density are factors affecting local mate consumption theory (King 1987). Sex ratio of the progeny can also be affected when a female hymenopteran lacks sperm and lays male eggs (Ridley 1988). Interspecific Competition According to Dent (1995), when two species compete with one a nother intensely enough over limited resources, then with time, one or the other can become extinct. When there is a dominant parasitoid, which can displace other parasitoid species, the releasing of several species might not provide the expected efficiency of a biological control program. In solitary insect parasitoids, generally only one offspring survives in a host (Vinson 1976). Females normally deposit one egg per host and this reduces the host availability to conspecific and heterospecific 25

PAGE 26

parasitoids. The successful oviposition of a female therefore, would be increased if she were the first to identify and oviposit only in hosts with no previously laid eggs (Lawrence 1981). Although, coexistence of several parasitoid specie s in the system can be more productive than a single parasitoid species, coexistence requires that some difference exist in niches among the species. When several parasitoid species attack the same host species, and one parasitoid prefers to attack early instars of the host and others prefer late instars or vice versa, there can be efficient control of the host specie s (Bokonon-Ganta et al. 1996). The pest instar they attack is the most important factor to decide the coexistence or co mpetitive exclusion of bi ological control agents when several agents are released together. The competition of parasitoids can be affected by the temperature. Some parasitoids compete more for hosts at lower temperatures and some prefer to attack hosts when temperatures are higher (Van Strien-van Liempt 1983). The parasitoids of Drosophila melanogaster Meigen and Drosophila subobscura Collin, Asobara tabida Nees von Esenbeck, and Leptopilina heterotoma (Thomson) compete differently at different temperatures. Asobara tabida is a better competitor at lower temperatures and Leptopilina heterotoma performed better at higher temperat ures (Van Strien-van Liempt 1983). Research Objectives Research studies on papaya mealybug and its parasitoids are lacking. There is no information on the life history of papaya mealybug, either in relation to its host plant species or to temperature. Understanding the life history of an insect is important in insect predictions, distribution, and its management. Determining th ermal constants and temperature thresholds is also useful in predicting insect emergence, distribution, and its ma nagement. In addition, there is very little published research on papaya mealybug parasitoid s. Information on the biology of A papayae A loecki and P mexicana and their interspecific competition, and the effectiveness in the field is scarce. It is impor tant to find out whether populations of these parasitoid species are 26

PAGE 27

established in the field, and if th ere is a need for inoculative rel eases. The goal of this study was to understand the life history of papaya meal ybug and to identify the efficient parasitoids for successful utilization of currently used biological control agents to obtain an effective and sustainable biological control pr ogram for papaya mealybug infestation in the US. Therefore, research was conducted to determine the life hi story of papaya mealybug, and then to evaluate the effectiveness of three introdu ced parasitoids of papaya meal ybug. There were five objectives for this study. The first objective was to define the life hist ory of papaya mealybug using four host plant species commonly found in Florida. The sec ond objective was to understand the effect of constant temperature on development, reproduc tion and survival of papaya mealybug, and then to estimate its thermal constants and temperat ure thresholds for development. The third objective was to evaluate the eff ectiveness of currently released parasitoids of papaya mealybug, A papayae A loecki and P mexicana in the field. The fourth objective was to study the developmental time, longevity and the lifetime fertility of A papayae A loecki and P mexicana. The fifth and final objective was to investigate the host stage susceptibi lity and suitability, sex ratio, and interspecific competition of A papayae A loecki and P mexicana 27

PAGE 28

CHAPTER 2 LIFE HISTORY OF Paracoccus marginatus WILLIAMS AND GRANARA DE WILLINK (HEMIPTERA: PSEUDOCOCCIDAE) ON FOUR HOST PLANT SPECIES UNDER LABORATORY CONDITIONS Introduction Paracoccus marginatus Williams and Granara de Willink (Hemiptera: Pseudococcidae) is a polyphagus insect and a pest of various tropical fruits, ve getables and ornamental plants (Miller and Miller 2002). Its host range includes Carica papaya L. (papaya), Citrus spp. L. (citrus), Persea americana P. Mill. (avocado), Solanum melongena L. (eggplant), Hibiscus spp. L. (hibiscus), Plumeria spp. L. (plumeria), and Acalypha spp. L. (acalypha) (Miller and Miller 2002). Paracoccus marginatus was first described by Williams and Granara de Willink (1992) and re-described by Miller and Miller (2002). Paracoccus marginatus was originally reported from the neotropical regions in Belize, Cost a Rica, Guatemala, and Mexico (Williams and Granara de Willink 1992). This species was introdu ced to the Caribbean in the early 1990's, and spread among many of the Caribbean islands by 1994 (Walker et al. 2003). In 1998, P marginatus was first reported in the US in Florida, in Palm Beach County on hibiscus (Miller et al. 1999). Thereafter, it was recorded in several ot her counties in Florida from more than 25 genera of plants (Walker et al 2003). Heavy infestations of P marginatus on C papaya were recorded in Guam in 2002 (Walker et al. 2003, Me yerdirk et al. 2004) and in the Republic of Palau in 2003 (Walker et al. 2003, Muniappan et al. 2006). In 2004, P marginatus was reported in Hawaii on papaya, plumeria, hibiscus, and Jatropha sp. L. (Heu et al. 2007). Since its introduction to the Caribbea n, the US, and the Pacific islands, P marginatus has established in most of the Caribbean islands, Fl orida, Guam, the Republic of Palau, and Hawaii. Paracoccus marginatus potentially poses a threat to numer ous agricultural products in the US especially in Florida, and states such as California and Hawaii, which produce similar crops. In 28

PAGE 29

southern parts of Texas, where the country's th ird largest citrus produc tion exists (CNAS 2007) is also a susceptible area for P marginatus The potential planting range of hibiscus includes Southern Texas (Gilman 1999b). Life history of P marginatus has not been investigated. U nderstanding the life history of a pest insect is important in pr edicting its development, emergen ce, distribution, and abundance. Life history information also plays an important role in pest management, especially when applying chemical and biological control methods. Since there is a high po ssibility of spreading P marginatus into other areas in the US, it is important to study its life hi story using host plant species that are either widely grown in the sus ceptible areas, or potted plant species that are commonly transported to these areas. In this study, three ornamental plants Hibiscus rosasinensis L (hibiscus), Plumeria rubra L. ( plumeria), Acalypha amentacea Roxb. ssp. wilkesiana (Muell.-Arg.) cutivar Marginata ( acalypha), and one weed species, Parthenium hysterophorus L. (parthenium) were selected to study the life history of P marginatus These four plant species were previously record ed as host plants of P marginatus (Miller and Miller 2002) and are widely grown in many areas in the US. Materials and Methods Rearing Mealybugs. Paracoccus marginatus was initially collected from a papaya ( Carica papaya L.) field in Homestead, FL. Red potatoes ( Solanum tuberosum L.) (Ryan Potato Company, East Grand Forks, MN) were allowed to sprout and then used in rearing a colony of P marginatus Potatoes were soaked in 1% solution of bleach (Clorox The Clorox Company, Oakland, CA; 6% sodium hypochlorite) for 15 minutes, and then rinsed with water, air-dried and placed in bags made from black cotton cloth to encourage sprouti ng. Bags were kept inside a dark room at 27 1C and 65% 2 R.H. Each week, 30 newly sprouted potatoes were infested with ovisacs of P marginatus to maintain the colony. Each spr outed potato was infested with 3 29

PAGE 30

to 5 ovisacs depending on the size of the potato a nd ovisacs. Infested potat oes were kept in 3.8L plastic containers at the rate of 10 per container (Rubbermaid Newell Rubbermaid Inc. Atlanta, GA). Prior to placing the infested potatoes, screens (Amber Lumite Bio Quip, Gardena, CA) were glued to cut se ctions of lids in thes e containers to facili tate air circulation. The mealybug colony was held in an environmenta l growth chamber (Percivel I-36LL, Percival Scientific Inc. Perry, NC) at 25 1C, 65 2% R.H., and a photoperiod of 12:12 (L:D). Eggs to be used in the studies were obtained from gravid females identified by a body length (2-2.5 mm) which is approximately twice the size of newly emerged virgin females (1.11.3 mm). To obtain eggs, gravid females from th e colony (each from a different infested potato) were placed individually on newly sprouted potatoes. Development and Survival. All plant material was collected and prepared 24 hours before the experiment. Hibiscus cuttings were obtained from 1-yr old container-grown hibiscus and maintained in a shadehouse. Acalypha and plum eria cuttings were obtained from plants in the landscape on TREC premises. Parthenium seedlings were collected from the field. A fully expanded young leaf with a stem 4-cm long was us ed for each replicate of hibiscus and acalypha. For parthenium, a whole plant approximately 8-cm in height with an intact root system was used as each replicate. A tender leaf was selected from each parthenium plant and the remaining leaves were removed. For plumeria, a 5-cm long terminal shoot with one tender leaf was selected as each replicate. Host tissue was placed in arenas (9-cm-diam Petri dish with a 0.6-cm-diam hole in the bottom for hibiscus, acalypha, and parthenium; 18-c m-diam Petri dish for plumeria). The stem of each leaf of hibiscus and acalypha was insert ed through the hole and the lid was placed on the Petri dish. For parthenium, the main stem of the plant was insert ed through the hole in the Petri 30

PAGE 31

dish until the leaf was completely placed inside the Petri dish. Each Petri dish was kept on a 162 ml translucent plastic souffl cup (Georgia Pacific Dixie, Atlanta, GA) filled with distilled water into which the stem was submerged. For plumer ia, each terminal shoot was hydrated using a ball of cotton tied to the cut end of the shoot, and moistened daily with distilled water. Eggs collected from a single female were plac ed on the leaves of all four hosts with 10 eggs per leaf using a pain tbrush (No.000) (American Pa inter 4000, Loew-Cornell Inc., Englewood Cliffs, NJ). Eggs were collected wi thin 24 h of oviposition. Dishes were checked daily for egg hatch and shed exuviae. The number of days to egg hatch, and emergence and survival of each instar, and number of emerging adult males and females were recorded. The developmental time and the survival of eggs and first instars were not separated by gender. The gender of each individual mealybug was determined during the latter part of the second instar when males change their color from yellow to pi nk. At this point, the developmental times of males and females were counted separately. Fo r each plant species, 35 Pe tri dishes (replicates) each with 10 eggs were used. This experiment was repeated twice at the end of the preceding experiment. All experiments were carried out inside an environmenta l growth chamber as above. Reproduction. Newly emerged virgin females obtained from the developmental study of each plant species were used to assess reproducti on. Virgin females were placed individually in Petri dishes with either a leaf or a terminal shoot of each plant species prepared as mentioned above. Females were held alone to assess asexual reproduction or were provided with three newly emerged males from the same plant species for sexual reproduction. Petri dishes were kept in an environmental growth chamber as ab ove. The date oviposition began, the number of eggs laid, and adult mortality were recorded. Fo r each of the two treatments (sexual and asexual) 31

PAGE 32

35 females were used, and each female was cons idered a replicate. This experiment was repeated twice using newly emerged males a nd females collected from developmental time experiments. Statistical Analysis. The experimental design was completely random for all experiments. The 10 eggs or mealybugs in each Petri dish were considered as a single unit/replicate and the mean of the response variable was calculated and used in subsequent analyses in all experiments. Data of the initial and repeated experiments we re pooled together after a two-way analysis of variance (ANOVA) indicated no interaction among the experiments (F = 0.69, df = 6, 408, P = <0.6539). One-way ANOVA was pe rformed using a general linear model (GLM) for all experiments (SAS Institute 1999) Means were compared at P = 0.05 significance level using the Tukey's HSD test. Data for proportions of fe males (sex ratio) and survival were square-root arcsine-transformed, when nece ssary prior to ANOVA (Zar 1984). Voucher Specimens. Voucher specimens of P marginatus were deposited in the Entomology and Nematology Department insect collection, at Tropical Research and Education Center, University of Florida. Results Preliminary studies demonstrated that it takes approximately one month for eggs of P marginatus to hatch and develop into adults. Use of tender leaves could avoid leaf senescence during this time. Hibiscus cutti ngs can root within two to three weeks time in water. Even after 30 days, acalypha cuttings were not rooted. Use of rooting hormones could have accelerated the process of rooting, however the im pact of rooting hormones on the development of insects is not known. Therefore, the fresh cuttings were use d. Cuttings obtained from parthenium, a soft herbaceous plant, were unable to survive 30 days in water. When parthenium plants with intact root system were used, the leaves were able to withstand this period. Plumeria cuttings were 32

PAGE 33

able to survive more than 30 days without leaf senescence with the provision of daily hydration through a ball of cotton tied around the cut end of the plumeria terminal. During this time, new leaves grew from the shoots indi cating that these shoots were c ontinuously growing and alive. Use of hard water in the contai ners to which the stems of the cuttings were submerged, could stain the bottom of the Perti dish and disturb the checking procedures for the mealybugs, which were dislodged from the leaf into the Petri dish. Use of distilled water did not significantly affect the development, reproduction, and survival of P marginatus compared to hard water. Therefore, distilled water was used instead of hard water. Development. There were differences in the developmental times of P marginatus reared on four host species (Table 2-1). Male s had longer developmental time than females. Adult females emerged earlier from the eggs on acalypha and parthenium than from the eggs on hibiscus and plumeria. Adult males had longer developmental time on acalypha and plumeria than on parthenium and hi biscus (Table 2-1). Survival. Eggs survived similarly on all four plants (Table 2-2). The lo wer survival of the first and second instars on plumeria was reflected in the cumulative adult survival on plumeria. Survival for the third-instar males and females, and the fourth-instar males were not affected by the host species (Table 2-2). Proportion of Females and Adult Longevity. Adults emerged on plumeria with a higher proportion of females than on the other three host species (F = 8.15, df = 3, 416, P <0.0001). The mean proportion of adult females ranged from 53-59 % (acalypha: 53.9 1.3, hibiscus: 53.7 1.1, parthenium: 53.4 1.0, and plumeria: 58.9 1.7) No difference in adult longevity of males (F = 0.69, df = 3, 416, P = 0.5562) and females (F = 0.52, df = 3, 416, P = 0.6659) 33

PAGE 34

occurred among the hosts. Mean longevity of adult males and females was 2.3 0.1 and 21.2 0.1 d, respectively. Reproduction. Virgin females did not lay any eggs on any of the four plant species. Mated females reared on plumeria laid a lower nu mber of eggs (186.3 1.8) than the number of eggs laid by females reared on hibiscus (244.4 6.8), acalypha (235.2 3.5), and parthenium (230.2 5.3) (F = 29.9, df = 3, 416, P = <0.0001). The mean pre-oviposition (6.3 0.1) and oviposition periods (11.2 0.1) were not affected by the plant species (F = 0.23, df = 3, 416, P = 0.8739, F = 0.12, df = 3, 416, P = 0.9496). Discussion Determining the life history of an insect is important to understand its development, distribution and abundance. In pol yphagus insects, life history can vary with the plant species it feeds on. There were differences in the life history parameters of P marginatus reared on four plant species, however, P marginatus was able to develop, surviv e and reproduce on all four plants. Different plant species provide differe nt nutritional quality and chemical constituents, which can affect the development, reproduction an d survival of an insect. The differences observed in the life history of P marginatus may be due to nutritive factors, allelochemical compounds, and physical differences in leaf structur es, which may be involved in the variation in plant suitability, althoug h these factors were not investigated for P marginatus in this study. Use of different presentations may have conf ounded the results but preliminary studies found that these were the best ways to maintain th ese hosts in a condition suitable for the tests. Different host plant species have affected the life history parame ters of other mealybug species. Longer pre-reproductive period and a higher progeny production were observed for Rastrococcus invadens Williams reared on different varieties of Mangifera indica L. (Bovida and Neuenschwander 1995). Mortality of the of citrus mealybug Planococcus citri (Risso) was 34

PAGE 35

higher on green than on red or yellow variegated Coleus blumei "Bellevue" (Bentham) plants, and developed faster and had a higher fecundity when developed on red-variegated plants (Yang and Sadof 1995). The developmental time of female Planococcus kraunhiae (Kuwana) was shorter when reared on germinated Vicia faba L. seeds than on leaves of a Citrus sp. L. and on Cucurbita maxima Duchesne, and it survived better when reared on germinated V. faba seeds than on citrus leaves (Narai and Murai 2002). The pink hibiscus mealybug, Maconellicoccus hirsutus (Green), was able to develop equally well on Cucurbita pepo L. as on C maxima (Serrano and Lapointe 2002). Ther e was no difference in survival, development, and fecundity of cohorts of the mealybug, Phenacoccus parvus Morrison when reared on Lantana camara L. Lycopersicon esculentum Miller, and Solanum melongena L (Marohasy 1997). However, Gossypium hirsutum L., Ageratum houstonianum Miller, and Clerodendrum cunninghamii Benth were identified as less suitable host plants for the development of P parvus compared to L camara (Marohasy 1997). Although the eggs of P marginatus on plumeria hatched in a si milar manner to the eggs on other three plant species, there was less survival of the first a nd second instars on plumeria. Stickiness observed on plumeria leaves may have contributed to this low survival. This stickiness may have resulted from the experimental conditions such as the hydration method used in this experiment. In the Republic of Palau, P marginatus has caused serious damage to plumeria (Muniappan et al. 2006), and it is f ound to be the most co mmon homopteran found on Plumeria alba L. in Puerto Rico (Sloan et al. 2007) indicating its ability to develop well on this plant species. A loss of 17 to 18% of the first instars was also observed on hibiscus, acalypha, and parthenium. A low survival rate of first-instar mealybugs was also observed when P kraunhiae were reared on V faba seeds (Narai and Murai 2002). The loss of first instar P 35

PAGE 36

marginatus may be due to the movement of crawlers (first instars) away from the leaf tissues and they falling off the plants. This movement was observed on all plant species, although it was more evident on plumeria. Crawlers have a tendency to move toward light so the 12-h photoperiod used in this experiment may have cau sed them to move toward light and dislodge from the leaves or the shoots. Preliminary studies demonstrated that the crawlers of P marginatus which were dislodged from the leaf, were not be able to survive, unless they moved back or were placed back on the leaf. Ultimately, the low percent survival of eggs and first instars was reflected in the low egg to adult survival of P marginatus I nsects may settle, lay eggs, and severely da mage plant species that are unsuitable for development of immatures (Harris 1990). However, m ales and females that emerged from hibiscus, acalypha, plumeria, and parthenium were able to mate and reproduce successfully. Under experimental conditions, th e mean number of eggs produced by an insect could be lower than its actual capacity due to restricted condi tions and the experimental arena used. With a female developmental time of 24 to 25 d, even with the lowest fecundity observed from the females reared on plumeria, the number of e ggs obtained was large enough to build up a substantial population in the field in a short time. Although some mealybugs such as the cassava mealybug, Phenacoccus manihoti MatileFerrero can reproduce by thelytokou s parthenogenesis (Calatayud et al. 1998, Le Ru and Mitsipa 2000), no virgin females produced eggs in the curre nt study. The sex ratio was slightly female biased, thus there is no evidence for parthe nogenetic reproduction in this species. The ability of P marginatus to develop on these plant species demonstrates the possibility of movement, distri bution, and esta blishment of P marginatus into new areas in the US. Hibiscus, acalypha, and plumeria are popular ornamental plants widely grown in Florida, 36

PAGE 37

37 California, and Hawaii (Criley 1998, Gilman 1999a USDA 2007a). Different hibiscus species are grown in many US states (Gilman 1999b, USDA 2007a), and potted hibiscus plants are transported to other parts of the US and Ca nada. Parthenium is a noxious annual weed commonly found among the ornament al plants in the landscape of urban areas, agricultural lands, and in disturbed soil in mo re than 17 states in the Easter n, Southern, and South central US (USDA 2007a). There is a possibility that P marginatus can spread from weeds such as parthenium to economically important fruits, vege tables and ornamental plants. However, the ultimate movement, distribution, and establishment of P marginatus in the other areas in the US could be decided by the other abiotic and biotic factors, such as temperature, availability of host plants, and the rules and regulations governing the movement of plant material from one state to the other. Life history of P marginatus is affected by host plant. However, it has the ability to develop, survive, and reproduce on a variety of host plant species. The information gathered from this study will be important in the management of P marginatus by providing a better understanding of its life cy cle, and its ability to survive on different host plant species. This information is needed in the development of integrated pest management of this pest.

PAGE 38

Table 2-1 Mean number of days ( SEM) for each developmental stadium of P marginatus reared on four host species (gender could not be determined before the second instar). Host Stadia Cumulative Egg First Second Third Fourth Male Female Male Female Male Male Female Acalypha 8.6 0.1b 5.9 0.1c 6.5 0.1c 3.8 0.1c 2.8 0.1b 6.3 0.1a 4.5 0.1a 28.4 0.1b 24.5 0.1b Hibiscus 8.4 0.1c 6.2 0.1b 6.8 0.1bc 5.0 0.1b 2.3 0.1c 5.9 0.1b 3.9 0.1b 27.6 0.1c 25.5 0.1a Parthenium 8.8 0.1a 5.8 0.1c 5.6 0.1d 5.2 0.1a b 3.4 0.1a 4.7 0.1d 4.1 0.1b 27.7 0.1c 24.4 0.1b Plumeria 8.5 0.1b 6.6 0.2a 9.6 0.1a 5.3 0.1a 2.7 0.1b 5.1 0.1c 2.6 0.1c 30.0 0.1a 25.5 0.1a F 25.44 63.78 358.88 122.56 32.37 109.51 128.68 239.96 74.78 df 3, 416 3, 416 3, 413 3, 416 3, 415 3, 416 3, 415 3, 415 3, 416 P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 n = 105 Means within a column followed by the same letters are not significantly different at = 0.05 (Tukey's HSD test). 38

PAGE 39

39 Table 2-2 Mean ( SEM) percent surviv al for each developmental stadium of P marginatus reared on four host species. Host Egg First Second Third Fourth Egg to Adult Male Female Male Acalypha 82.8 0.7 83.2 0.9a 89.4 1.1a 89.6 1.4 89.3 1.4 89.7 1.6 49.9 0.8a Hibiscus 83.3 0.6 82.7 0.9a 89.4 1.0a 89.8 1.5 89.0 1.5 89.7 1.6 50.4 0.8a Parthenium 83.5 0.6 82.7 0.9a 89.1 0.9a 89.5 1.4 89.7 1.4 89.6 1.5 50.1 0.7a Plumeria 82.2 0.7 58.4 1.4b 64.9 1.7b 84.5 2.3 81.8 2.4 81.4 3.5 20.0 0.5b F 0.89 73.44 58.67 0.42 1.55 0.48 379.44 df 3, 416 3, 416 3, 416 3, 416 3, 416 3, 416 3, 416 P 0.4475 <0.0001 <0.0001 0.7398 0.1998 0.6955 <0.0001 n = 105 Means within a column followed by the same letters are not significantly different at = 0.05 (Tukey's HSD test).

PAGE 40

CHAPTER 3 EFFECT OF CONSTANT TEMPERATURE ON THE DEVELOPMENTAL BIOLOGY OF Paracoccus marginatus WILLIAMS AND GRANARA DE WILLINK (HEMIPTERA: PSEUDOCOCCIDAE) Introduction Understanding the developmental biology of an insect can provide useful information for pest management. Developmental biology, however, can be influenced by various environmental factors. Temperature is one of the most important and critical of the abiotic factors that can affect insect development. Insects requir e a certain amount of heat units to develop from one stage of their life cycle to another, which can be measured in degree-days (Gordan 1999). The ability of an insect to deve lop at different temperatures is an important adaptation to survive varying climatic condi tions, and is important in insect population predictions and control strategi es (Mizell et al. 1978). Temp erature also influences the population dynamics of insect pests and thei r natural enemies (Huffaker et al. 1999). Temperature range and climatic condition of an ar ea determine the ability of an adventive insect species to invade that area. There is no in formation on the effect of temperature on the development and survival of one such adventive pest species, Paracoccus marginatus Williams and Granara de Willink (Hemiptera: Pseudococcidae) which has been recently introduced in to the US. Paracoccus marginatus is a polyphagus insect that has been recognized as a significant pest of a large number of tropi cal and subtropical fruits, vege tables, and ornamental plants (Miller and Miller 2002). First described by Williams and Granara de W illink in (1992) and re-described by Miller and Miller (2002), P. marginatus is believed to be native to Mexico and Central America (Miller et al. 1999). Its economically important hosts include papaya, hibiscus avocado, citrus, cotton, tomato, egg plant, beans and peas, sweet potat o, mango, cherry, and pomegranate (Walker et al. 40

PAGE 41

2003). It is an important pest in the Caribbean, the US (Mill er and Miller 2002) and some Pacific islands such as the Republic of Palau (Muniappan et al. 2006), Guam (Meyerdirk et al. 2004) and Hawaii (Heu et al. 2007). Since 1994, P. marginatus has been recorded in 14 Caribbean countries (Walke r et al. 2003). In 1998, P marginatus was first discovered in the US in Palm Beach County, Florida, on hibiscus plan ts, and since then has been recorded on more than 25 genera of hosts (M iller and Miller 2002). Paracoccus marginatus was subsequently found in several other counties in Florida, and potentially poses a threat to numerous agricultural products in Florida as well as to the other US states producing si milar crops (Walker et al. 2003). The ability of P marginatus to spread into other states in the US may depend on its ability to develop and survive at different temperatures. Determining the effect of temperature on the life history of P marginatus and estimating its thermal requirements will be useful in predicting where this pest can pot entially spread in the US. Th is study focuses on the effect of constant temperature on the developmental biology and thermal requirements of P marginatus Materials and Methods Insect Rearing. Paracoccus marginatus was initially collected from a papaya ( Carica papaya L.) field in Homestead, FL Sprouted red potatoes ( Solanum tuberosum L.) (Ryan Potato Company, East Grand Forks, MN) we re used to rear a colony of P marginatus at the University of Florida, Tropical Research and Education Cent er (TREC), Homestead, FL. Prior to sprouting, potatoes were soaked in a 1% solution of bl each (Clorox The Clorox Company, Oakland CA; 6% sodium hypochlorite) for 15 minutes, and then rinsed with clean water and dried. Potatoes were placed in black cotton clot h bags, and kept inside a dark room at 27 1C to encourage sprouting. Each week, 30 sprouted potatoes were infested with P marginatus ovisacs collected from the previously infested potatoes selected from the colony to maintain the mealybug population. The mealybug colony wa s held in an environmental growth chamber (Percivel I41

PAGE 42

36LL, Percival Scientific Inc. Perry, NC) at 25 1C, 65 2% R.H., and a photoperiod of 14:10 (L:D). Development and Survival. Hibiscus ( Hibiscus rosasinensis L.) leaves were used as the host tissues. These leaves were taken from hibiscus plants that were obtained from a local nursery and maintained outdoors. The experimental arena consisted of a 9-cm-diam Petri dish. A 0.6-cm-diam hole was made in the bottom of th e Petri dish using a heated cork borer. A tender hibiscus leaf with a 4 cm long stem was placed in each Petri dish with the stem inserted through the hole at the bottom of the Petri dish a nd the lid was replaced. Each Petri dish with a hibiscus leaf was placed on a cup of water so th at the stem below the petiole was immersed in water. The development and survival of P marginatus was initially evaluate d at five constant temperatures of 15, 20, 25, 30, and 35C, all 1C. Eggs maintained at 15 and 35C hatched but these nymphs were unable to complete their first in star development. Three new temperatures of 18, 34, and 37C, all 1C were later included in the experiment to find out more about the egg and the first instar development For each temperature, 35 gravid females were collected from the colony to obtain eggs. To acclimatize them for each temperature, gravid females were kept individually on hibiscus leaves prepared as above, and were tran sferred to environmental growth chambers (TCI model, Environmental Growth Cham bers, Chagrin Falls, OH) at the experimental temperatures, 65 2% R.H., a nd a photoperiod of 14:10 (L:D), 48 hours before the experiment. Eggs were collected within 24 hours of ovipositi on. Ten eggs collected from a single female were placed on each hibiscus leaf arranged in a Petri dish prepared as above. There were 35 replicates for each temperature. Immediately after placing the eggs, the Petri dishes were transferred to each environmental growth chamber with the specific temperature. Petri dishes were checked daily for egg hatch and molting. Wh en eggs started to hatch, chambers were 42

PAGE 43

maintained in complete darkness for 72 hours to en courage the first-instar nymphs (crawlers) to settle on the leaves. The gender of each individual was determined during the latter part of the second instar when the males change their co lor from yellow to pink. At this point, the developmental times of males and females were counted separately. The developmental time and the survival of eggs and firs t instars were not separated by gender. The number of surviving individuals at each stage was counted. Reproduction. To determine the effect of consta nt temperatures on reproduction of P marginatus newly emerged males and females were separated as soon as they emerged as adults at 18, 20, 25, and 30 C Each adult female was placed on a hibiscus leaf, which was arranged in a Petri dish prepar ed as above. Each female was provided with 2-3 newly emerged adult males to ensure mating. Each female repres ented a replicate. There were 35 replicates for each temperature. The number of eggs laid by each female was counted daily. The number of days for the pre-oviposition period (number of days from adult em ergence to oviposition) and the oviposition period (time between beginning and end of oviposition) were also counted. Adult Longevity. The number of days from adult emergence to death was evaluated at four constant temperatures (18, 20, 25, and 30 C ) for both males and females. Each individual was placed in a Petr i dish prepared as mentioned a bove. For each temperature, 35 individuals (replicates) e ach of both males and females were evaluated. Developmental Thresholds and Thermal Constant. A linear regression analysis (PROC REG) (SAS Institute 1999) wa s carried out to calculate th e thermal constant and lower developmental threshold (T min ) for P marginatus using rate of development (reciprocal of development) from egg to adult against the cons tant temperatures used. The linear degree-day model (thermal summation model) estimates the re lationship between temperature and the rate of 43

PAGE 44

development in a linear relationship (Campbell et al. 1974). This linear relationship is Y = a + bT where Y is the rate of development (1/days), T ambient temperature (C), and the regression parameters intercept ( a ) and slope ( b ). The thermal constant K (1/ b) is the number of degreedays above the threshold summed over the developmental period. Lower developmental threshold T min (a / b) is the minimum temperature at which the rate of development is zero or no measurable development occurs. To describe the developmental rate over a wider temperature range, a nonlinear model (Logan 6 model) was used to calculat e the upper developmental threshold (T max ) and the optimum temperature threshold (T opt ) (Logan et al. 1976). The upper developmental threshold T max is the maximum temperature at which the rate of development becomes zero and life processes can no longer be maintained for a prolonged period. The optimum temperature T opt is the temperature at which the maximum rate of development occurs (Walgama et al. 2006). The Logan model does not estimate the lower developmental threshold (T min ), because it is asymptotic to the left of the temperature axis The relationship between developmental rate (1/D) and upper developmental threshold is described in the Logan 6 model as, ) exp()exp( /1max maxT TT TT D where is a directly measurable rate of temper ature dependent physiological process at some base temperature, is the biochemical reaction rate and T is the temperature range over which 'thermal breakdown' becomes the overriding infl uence (Logan et al. 1976). To determine the optimum temperature (T opt ) for development, the following equation (Logan et al. 1976) was used. ) 1 )ln( (1max o o optb b TT 44

PAGE 45

Here, is T / T max and b 0 is T max Statistical Analysis. The experimental design used for all experiments was completely random. Prior to the statistical analysis, the mean of the individuals in each Petri dish/replicate was calculated and used in the analyses. One-way analysis of variance (ANOVA) was performed using a general linear model (GLM) for all experiments (SAS Institute, Cary, NC). Means were compared at P = 0.05 significance level using the Tukey's HSD test. Proportions of females (sex ratio) and survival were square-root arcsine-transformed using p p arcsin' where p = proportion of female/survival, to adjust the variances (Zar 1984) prior to ANOVA, but the untransformed data were presented in the tables. A linear regression was performed to find the linear relationship between rate of development and temperature and to estimate the parameters a and b (PROC REG) (SAS Institute 1999). A non-linear re gression (PROC NLIN) (SAS Inst itute 1999) was performed for the non-linear section of the rela tionship between rate of development and temperature to find the estimates for the parameters, T max and T of the Logan 6 model. Voucher Specimens. Voucher specimens of P marginatus were deposited in the Entomology and Nematology Department insect collection, at the Tr opical Research and Education Center, University of Florida. Results Development and Survival. Eggs hatched at all temperatures except 37C (Table 3-1). The duration of development of all stages decrea sed with increasing temperatures. The egg developmental time was the same at 34 and 35C. The egg developmental time at 15C was approximately 5 times longer than the developmen tal time at 35C. Eggs hatched at 15, 34, and 35C were unable to complete their first-instar de velopment (Table 3-1). The percent survival of 45

PAGE 46

eggs increased with increasing temperature until 30C, above which temperature the survival started to decrease (Table 3-2). First-instar developmental time at 18C was more than four times longer than the developmental time at 30C (Table 3-1). Developmental times for male and female nymphal stages, and cumulative adult male, were not different at 25 and 30C (Table 3-1). The cumulative developmental time for the female was decreased over the temperature range from 18-30C (Table 3-1). A low percentage of eggs survived at 35C (Table 3-2). Survival of first and secondinstars was lowest at 18C. The cumulative a dult percent survival increased with increasing temperatures over the range from 18 to 30C (Table 3-2). Since the gender of the eggs was difficult to differentiate, cumulative survival from egg to adult was not separately calculated for each gender. Reproduction. Pre-oviposition and oviposition peri ods decreased with increasing temperatures with no difference at 25 and 30C (Table 3-3). Fecundity increased from 18 to 25C, and then drastically decreased at 30C (Table 3-3). Females lived longer at lower temperatures than at higher temperatures, and with no difference at 25 and 30C (Table 3-3). Adult male longevity was shorter at 25C than that of at 18 and 20C (Table 3-3). The proportion of females was lowest at 25C (Table 3-3). Thermal Requirements for Development. Between the temperatures of 18 to 25C, there were excellent linear fits (R 2 0.94; P =<0.0001) for developmental rate versus temperature in the linear degree-day mode l for egg, male and female nymphal stages, and cumulative numbers of adult males and females (Table 3-4). Thermal constants (K) for development rates of egg, male and female nymphal stages were 100.0, 204.8, and 175.4 degree-days (DD), respectively. For cumulative development of adult male and female, the thermal constants were 46

PAGE 47

303.0 and 294.1 DD, respectively. The estimated lower developmental thresholds (T min ) for egg, male and female nymphal stages were 13.3, 14.8, and 14.3C respectively. For cumulative development of adult males and females, th e estimated lower developmental threshold, T min were 14.5 and 13.9C respectively. For the non-linear section of the developmental rate ag ainst temperature, the Logan 6 model also provided exce llent fits (PseudoR 2 0.97; P =<0.0001) for each developmental stadium (Table 3-4). The estimated optimum temperatures (T opt ) for the developmental rates of egg and male and female nymphal stages we re 34.8, 27.9 and 28.3C, respectively. For adult male and female, estimated optimum temperat ures were 28.7 and 28.4C, respectively. The estimated maximum temperature thresholds (T max ) for egg, male and female nymphal stages were 41.6, 30.5 and 31.7 C respectively. For adult male and female papaya mealybug, the estimated T max were 31.9 and 32.1C, respectively. Discussion Insect systems function optimally within a lim ited range of temperatures. For a majority of insects, enzyme activity, tissue functioning, and th e behavior of the whole insect is optimal at a relatively high temperature often in the rang e of 30-40C (Chapman 1998). Temperature had a significant effect in the development of P. marginatus Overall, the linear degree-day model and the nonlinear Logan 6 model, which were used in predicting temperature and developmental rate relationships in insects, estimated minimum, optimum, and maximum temperature thresholds for P. marginatus close to results obtained in this experiment. The development of adult female P. marginatus was arrested at an estimated minimum temperature threshold of 14.5C and a maximum temperature threshold of 31.9C. It reached its optimal development at about 28.7C. The cumu lative developmental times of both male and female mealybugs at 18C were three times longer than the developmental times at 30C. 47

PAGE 48

Although P marginatus was unable to develop and complete its life cycle at 15C, the Madeira mealybug, Phenacoccus madeirensis Green, a commonly-fou nd mealybug species in greenhouses in Southeastern US with a worldwide dist ribution and with a wide host range (BenDov 1994), was able to develop, reproduce and su rvive well at 15C (Chong et al. 2003). At 15C, the developmental time of female P madeirensis was 66 days and was twice as long as the developmental time at 25C (Chong et al. 2003 ). The minimum, optimum, and maximum temperature threshold for the female pink hibiscus mealybug, Maconellicoccus hirsutus (Green), a polyphagus mealybug and a serious pest of many economically important crops, were 14.5, 29.0, and 35C respectively. At 20C, the developmental time of the female, M hirsutus was 66 days, and was twice the developmental time at 30C (Chong et al. manuscript in review). High fecundity in insects is an important ad aptation for a successful next generation. In nature, eggs of any insect can be exposed to natural enemies and other environmental factors such as wind, rain, sunlight, and radiation. Alt hough P. marginatus females were able to develop in a shorter time and had a higher survival at 30C than at the other tested temperatures, the fecundity at 30C was considerably lower than at 20 and 25 C. The drastic drop in fecundity at 30C suggests that even though the developmental time was shorter and survival was higher at 30C than at 25C P. marginatus may have reached its optimal temperature for development and reproduction between the temperatures 25 and 30C The optimal temperature for development estimated using the Logan 6 model, for the female nymphal stage and the cumulative adult female was within this range thus supporting the results obtained. Other mealybug species such as P madeirensis and M hirsutus showed differences in fecundity with increasing temperature and were ab le to reproduce successfully and increase their populations at temperatures such as 25C. Compared to the fecundity at 20C, the total nu mber of eggs laid at 48

PAGE 49

25C was significantly lower for P madeirensis (Chong et al. 2003). At 25C, a female P madeirensis can lay 288 eggs in 8 days and was able to emerge as an adult within 30 days (Chong et al. 2003). Similar to P marginatus the fecundity at 30C was significantly lower for M hirsutus compared to the fecundity at 25C and a dult females emerged within 31 days at 25C and laid 300 eggs within 7 days (Chong et al manuscript in review). At 25C, female P marginatus can emerge as an adult within about 26 days and can produce as many as 300 eggs in approximately 11 days. With its short life cycle, high survival a nd reproductive capacity, P marginatus has a tremendous ability to increase its population to leve ls that can cause economic damage unless suitable management practices are implemented. Although continuous constant temp eratures were used in thes e experiments, in nature temperature can vary during the day and especial ly at night. Warmer day temperatures and colder night temperatures in the natural environment may allow P. marginatus to develop and survive at a higher temperat ure than 30C. Developing Oncopeltus eggs reared at varying temperatures developed faster and used less me tabolic energy than at the equivalent mean constant temperatures (Gordan 1999). The living system may be better adapted to normal environmental fluctuations than to an artificial constant state (Gordan 1999). This information may also be helpful in monitoring the susceptible stages of P. marginatus for application of integrated management practices including re leasing of biological control agents. The longer developmental time of eggs and immature stages, may increase the vulnerability by prolonged exposure to natural enemies and insecticides. On the other hand, at higher temperatures mealybugs grow quickly and b ecome adults 2-3 times faster than at lower temperatures, allowing them an opp ortunity to reduce exposure time and presumably to increase survival and ultimately reproduce. 49

PAGE 50

The estimated thermal constants for eggs and female P marginatus were 100.0 and 294.1 DD, respectively, while those of M hirsutus were 101.7 and 347.2 DD, respectively (Chong et al. manuscript in review). The estimat ed minimum temperature threshold for P citri is 10.9C and thermal constants for adult female at consta nt and fluctuating temperatures were 289 and 365 DD, respectively (Laflin and Parrella 2004). The th ermal constant for females is considerably lower for P marginatus compared to that of M hirsutus. Although, P marginatus and P citri had similar thermal constants at constant temp erature, the minimum temperature threshold for P citri was much lower than that of P marginatus Planococcus citri has a wider distribution in the US compared to currently availa ble information on the distribution of M hirsutus (Ben-Dov 1994). Tropical insect species have higher values of minimum temperature thresholds than temperate insect species, and the thermal constants decrease with the increase of minimum temperature threshold (Trudgill et al. 2005). Considering its high minimum temperature thresholds and the low thermal constants, P marginatus should have a smaller distribution range than anticipated earlier. The developmental threshold and the thermal c onstant of an insect possibly are useful indicators of its potential distribution and abundance (Messenger 1959). Estimated developmental temperatures combined with degree-days were useful in predicting Planococcus citri (Risso) in greenhouse cut flower production in California, in temperatures maintained at 25 to 30C (Laflin and Parrella 2004). The inform ation on developmental temperatures combined with degree-days from this study, should be useful in predictin g possible spread of P. marginatus in different areas in the US, th e Caribbean, and the Pacific. According to the comparative climatic data from the National Climatic Data Center (NCDC 2005), some areas in Southern California, Southern Texas, Hawaii, and Florid a have daily average temperatures that are 50

PAGE 51

51 suitable for the development of P marginatus A large number of econo mically important fruits, vegetables and ornamental plants are grown in Southern California including citrus, avocado, beans, hibiscus and plumeria. Southern Texas has the third largest citrus production in the US (CNAS 2007). In Hawaii, where P marginatus is already established on the big island and small islands of Maui, Oahu, and Kauai, a large numbe r of fruits, vegetables, and ornamentals are grown including papaya, hibiscus, and plumeria. Papaya is the second most important fruit crop in Hawaii, after pineapple, and according to th e National Agricultural Statistics Service (USDA 2007b), Hawaii currently grows 864 ha of papaya. In Florida, where approximately 100 ha of papaya are grown (Mossler and Nesheim 2002), P marginatus has been found in most of the counties of Central and South Florida (Walker et al. 2003). Dist ribution and esta blishment of P marginatus in the areas in California and Texas that ar e suitable with regard to temperature can be influenced by other factors such as type of crops grown and regulation of plant movement from state to state. Paracoccus marginatus has the potential to spread to Southern California and Texas. These states produce economically importa nt crops, which are also favored by P marginatus If P marginatus spread to California and Texas through th e movement of plants or commodity, we will expect the damage to be significant unless suitable control measures such as biological control and restrictions of movement of suscep tible plants and commodities to uninfected areas are implemented in a timely manner to slow down the spread.

PAGE 52

Table 3-1 Mean number of days ( SEM) for each developmental stadium of P. marginatus reared at diffe rent constant temperatures T Developmental Stadia Cumulative (C) Egg First Second Third Fourth Male Female Male Female Male Male Female 15 27.5 0.2a 18 23.1 0.2b 25.3 0.5a 21.1 1.6a 13.5 1.3a 7.0 1.8a 13.2 0.9a 11.7 1.8a 85.2 1.8a 74.4 1.4a 20 14.4 0.2c 14.6 0.5b 13.6 0.8b 9.3 0.7b 4.5 0.7ab 8.9 0.9b 8.9 0.7a 53.4 0.7b 45.9 0.9b 25 8.7 0.1d 6.5 0.1c 6.6 0.5c 5.5 0.5c 2.4 0.5b 5.2 0.2c 4.1 0.5b 28.5 0.3c 25.9 0.2c 30 7.3 0.2e 6.1 0.2c 6.3 0.4c 5.7 0.4c 2. 6 0.4b 4.4 0.3c 3.6 0.4b 24.9 0.6c 23.2 0.3d 34 5.9 0.1f 35 5.5 0.1f F 1922.10 400.59 57.41 17.09 5.35 15.31 15.66 725.42 521.23 df 6, 212 3, 132 3, 97 3, 101 3, 87 3, 90 3, 91 3, 84 3, 90 P <0.0001 <0.0001 <0.0001 <0.0001 <0.0020 <0.0001 <0.0001 <0.0001 <0.0001 n = 35 Means within a column followed by the same letters are not significantly different at = 0.05 (Tukey's HSD Test) 52

PAGE 53

Table 3-2 Mean ( SEM) percent surviv al for each developmental stadium of P marginatus reared at diffe rent constant temperatures. Temp. Developmental Stadia Third Egg First Second Male Female Fourth Male Egg to Adult 15 60.9 3.3cd 18 80.0 2.9b 54.1 4.7d 80.1 4.8b 96.4 1.9ab 73.2 .3 98.4 1.6a 30.5 4.4c 20 90.1 2.1a 77.2 3.0c 94.1 1.3a 98.1 1.0a 83.5 3.8 75.4 5.7c 41.4 3.5bc 25 83.3 3.4ab 83.2 4.3bc 97.5 1.3a 89.2 3.1b 79.9 4.8 86.7 2.9bc 51.4 4.0b 30 85.9 3.3ab 90.5 2.5ab 91.0 4.0ab 96.9 1.5ab 92.6 2.9 97.8 1.6a 70.8 4.9a 34 73.4 5.1bc 35 33.4 6.9d 37 0.0 0.0 F 16.70 16.52 4.89 3.46 1.82 10.36 15.43 df 6, 205 3, 131 3, 122 3, 91 3, 90 3, 89 3, 121 P <0.0001 <0.0001 <0.0030 <0.0196 <0.1494 <0.0001 <0.0001 n = 35 53 Means within a column followed by the same letters are not significantly different at = 0.05 (Tukey's HSD Test)

PAGE 54

Table 3-3 Mean ( SEM) proportion of females, adult l ongevity, fecundity, pre-oviposition and oviposition periods of P. marginatus reared at four constant temperatures Adult Longevity (Days) Temperature (C) Proportion of Females Male Female Fecundity Pre-oviposition Period (Days) Oviposition Period (Days) 18 69.4 8.2a 5.5 0.5a 40.2 1.1a 160.6 13.8cd 16.7 0.7a 19.6 1.0a 20 81.7 3.6a 4.8 0.3a 35.7 1.0b 231.6 12.8bc 13.5 0.5b 21.4 1.1a 25 42.6 5.3b 2.9 0.2b 21.1 0.7c 300.2 40.4ab 6.8 0.4c 11.4 0.8b 30 71.1 4.2a 19.2 1.4c 82.0 11.7d 7.6 0.7c 11.6 1.4b F 10.38 14.91 93.25 15.13 70.33 24.99 df 3, 89 2, 73 3, 92 3, 92 3, 92 3, 92 P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 n = 35 Means within a column followed by the same letters are not significantly different at = 0.05 (Tukey's HSD Test) 54

PAGE 55

55 Table 3-4 Summary of statistics and the estimates ( SE) of th e fitted parameters of the linear thermal summation model and th e nonlinear Logan 6 model. Developmental Stadia Statistics Parameters Egg Nymphal Nymphal Total Total Thermal Summation Model: Y = a + bT F 1640.79 1286.51 2606.13 1653.01 3493.67 P < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 df 1, 113 1, 69 1, 63 1, 69 1, 67 R 2 0.9356 0.9491 0.9764 0.9599 0.9812 a SE -0.13 0.01 -0.08 0.01 -0.07 0.01 -0.05 0.01 -0.05 0.01 b SE 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 Logan 6 Model: ) exp()exp( /1max maxT TT TT D F 22354.5 6092.2 5620.06 10500.7 10252.5 P < 0.0001 <0.0001 < 0.0001 < 0.0001 <0.0001 SS R 0.00024 0.00003 0.00003 0.000009 0.000007 SS CT 0.01177 0.00228 0.00221 0.00102 0.00099 Pseudo-R 2 0.9796 0.9868 0.9864 0.9912 0.9929 SE 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 SE 0.11 0.02 0.15 0.04 0.21 0.04 0.13 0.03 0.15 0.03 T max SE 41.56 0.99 31.71 1.25 30.52 0.31 32.09 1.24 31.99 1.52 T SE 5.24 0.06 1.93 0.46 1.62 0.42 2.05 0.36 1.88 0.47 Y = rate of development (1/days); T = ambient temperature (C); a = intercept; b = slope; K (1/ b ) = thermal constant; T min (a/ b ) = lower developmental threshold; SS R = residual sums of squares; SS CT = corrected total sums of squares; pseudo-R 2 = 1-SS R /SS CT = rate of temperature dependent physiol ogical process at some base temperature; = biochemical reaction rate; T = temperature range over which 'thermal breakdown' becomes the overriding influence; T opt = optimum temperature threshold; T max = upper developmental threshold

PAGE 56

CHAPTER 4 HOST STAGE SUSCEPTIBILITY AND SEX RA TIO, HOST STAGE SUITABILITY, AND INTERSPECIFIC COMPETITION OF A cerophagus papayae Anagyrus loecki, AND Pseudleptomastix mexicana: THREE INTRODUCED PARASITOIDS OF Paracoccus marginatus WILLIAMS AND GRA NARA DE WILLINK Introduction Knowledge of host selection is indispensable fo r the efficient use of parasitoids, both for mass rearing and biological control of pests. A parasitoid's biology may be greatly influenced by the quality of the host. Host stage is an im portant ecological variab le, which may have an influence on a parasitoid's rate of attack, survival of its immatu re stages, and sex ratio of its offspring (Waage 1986). Host selection behavior is most important in de termining the sex ratio of arrhenotokous parasitoids, which show a haplodiploid sex determination mechanism (King 1987). A female parasitoid can manipulate the offspring sex ratio at oviposition by regulating fertilization. A particular host size may be more suitable for the development of one sex, so that, in general, a female-biased offspring sex ratio is produced from the larger hosts and a male biased one from the smaller hosts (King 1987). Solitary parasitoids generally determine the host quality by the size of the host. Large hosts are supposed to be better quality, as they ar e believed to contain more resources than small ones. However, host size may not always be equated to host quality at the time of oviposition by a parasitoid. The influence of host size on para sitoid development may differ between idiobiont and koinobiont parasitoids (Waage 1986). Idiobiont parasitoids oviposit in host stages such as egg and pupa, or paralyze their hosts prior to oviposition (Waage 1986) and on the other hand, koinobiont parasitoids, which do not paralyze thei r hosts at the time of parasitism, and allow hosts such as larval stages to grow, for which host size is not directly a representative of larval resources. 56

PAGE 57

Sympatric parasitoid species that share the same host species may be competitors (Van Strien-van Liempt 1983). The greater the part of the host population that is exploited by both species, the more they will a ffect each other's population density. Their competitive abilities then, among other factors, determine their relati ve abundance (Van Strien-van Liempt 1983). In solitary insect parasitoids, ge nerally only one offspring surviv es in a host (Vinson 1976). Females normally deposit one egg per host and redu ce the host availability to both conspecific and heterospecific parasitoids. Successful oviposition of a fema le depends on how efficient she is in finding and parasitizing un-parasitized hosts This leads to interspecific competition among the parasitoids in classical biological control, wh ere more than one parasitoid species is used (Lawrence 1981). Classical biological control was identified as an important pest management practice for Paracoccus marginatus Williams and Granara de Willink (Hemiptera: Pseudococcidae), a polyphagus mealybug species that was first identified in the US, in Florida in 1998 (Miller and Miler 2002). Paracoccus marginatus is a pest of a large number of topical and subtropical fruits, vegetables, and ornamentals (Mi ller and Miler 2002). Currently there are three parasitoid species (Hymenoptera: Encyrtidae) used in the classical biological control of P marginatus in the US, the Caribbean, and the Pacifi c islands (Meyerdirk et al. 2004). Acerophagus papayae Noyes and Schauff Anagyrus loecki Noyes and Menezes, and Pseudleptomastix mexicana Noyes and Schauff are currently mass reared in Puerto Rico and released by the United States Department of Agriculture (USDA), Animal and Plant Health Inspecti on Service (APHIS) in areas infested with P marginatus (Meyerdirk et al. 2004). These parasitoids have been released in Guam and the Republic of Palau, and were successful in controlling P marginatus in these countries (Meyerdirk et al 2004, Muniappan et al. 2006). 57

PAGE 58

There is very little informa tion available on the biology of A. papayae A loecki and P mexicana or on how efficient they are in the control of P marginatus Knowledge of host selection and interspecific competition of parasi toids should lead to be tter understanding of the population dynamics of the host and the parasitoids. Hence, they are important in evaluating and understanding the success of biol ogical control and integrated pe st management programs. The present study focused on the host stage susceptib ility and sex ratio, host stage suitability, and interspecific competition of A. papayae A loecki and P mexicana, three introduced koinobiont parasitoids of P marginatus. Materials and Methods Rearing Mealybugs. Paracoccus marginatus was initially collected from a papaya ( Carica papaya L.) field in Homestead, FL. Sprouted red potatoes ( Solanum tuberosum L.) (Ryan Potato Company, East Grand Forks, MN) were used to rear a colony of P marginatus at the University of Florida, Tropical Research and Education Center (T REC), Homestead, FL. Prior to sprouting, potatoes were soaked in a 1% solution of bleach (Clorox The Clorox Company, Oakland CA; 6% sodium hypochlorite) for 15 minutes, then rinsed with water, and dried. Potatoes were placed in black cotton clot h bags to encourage sprouting. These bags were kept inside a dark room at 27 1C and 65 2% R.H. Each week, 36 sprouted potatoes were infested with ovisacs to maintain the colony. An environmental growth chamber (Percivel I36LL, Percival Scientific Inc. Perry, NC) was used at 25 1C, 65 2% R.H., and a photoperiod of 12:12 (L:D) to rear the mealybug colony. To obtain a particular stage of mealybugs, newly laid ovisacs were selected from the mealybug colony. A tender leaf with a 5 cm long stem was obtained from hibiscus ( Hibiscus rosasinensis L.) plants that were purchased from a lo cal nursery and maintained in a shadehouse at TREC. A 9-cm-diam Petri dish with a 0.6-cm -diam hole at the bottom was used to place the 58

PAGE 59

hibiscus leaf. The stem of each hibiscus leaf was inserted through the hole in the Petri dish and each dish was kept on a 162 ml translucent plastic souffl cup (Georgia Pacific Dixie, Atlanta, GA) filled with water, which allowed the stem below the petiole to be in water. On each leaf, a single ovisac was placed and the eggs were allowe d to hatch and then develop into the desired stage. The gender of mealybugs was determined dur ing the latter part of the second instar when males change their color from yellow to pink. Th erefore, the gender was not determined for the first and second instars, but th e third instars and adults used were females. Newly molted mealybugs, which were recognized by the size and pr esence of shed exuviae, were selected for all the experiments in this study to reduce the variation in host quality. Rearing Parasitoids. The potatoes with second and third instar P. marginatus were used for parasitoid rearing. These potatoes we re initially infested with ovisacs of P. marginatus obtained from the mealy bug colony. Colonies of A. papayae A loecki, and P mexicana were maintained in an insectary at TREC at 25 2C temperature, a 12:12 (L:D) photoperiod, and 65 2% R.H. Initial colonies of parasitoids were obtained from the Biol ogical Control Laboratory, Department of Agriculture, Puerto Rico, thr ough USDA-APHIS. Parasi toid colonies were established in plexiglass cages (30x30x30 cm) using sprouted red potatoes infested with P. marginatus In order to obtain a co ntinuous supply of newly em erged parasitoids, mealybuginfested potatoes were provided to each parasitoid species weekl y, and potatoes with parasitized mealybugs were moved to a new cage. A soluti on of honey and water (1:1) was streaked on 4 pieces (5x5 cm) of Benchkote surface protector paper (Fisherbrand Fisher Scientific, Pittsburgh, PA) attached to the cage using labe ling tape (Fisherbrand Fisher Scientific, Pittsburgh, PA). Water was provided in two clea r plastic 73.9-ml containers (Tristate Molded Plastic Inc., North Dixon, KY) per cage. In each container, 1-cm-diam hole was made in the 59

PAGE 60

center of the lid and a 7.6 cm long piece of cotton roll (TIDI Products, Neenah, WI) was inserted through the hole to allow parasitoids to access water. To obtain mated-female parasitoids, newly em erged females of each species were placed singly in glass disposable culture tubes ( 1.2x7.5 cm) (Fisherbrand Fisher Scientific, Pittsburgh, PA) and closed with two-ply tissue (Kimwipes EX-L, Kimberly-Clerk Global Sales Inc. Roswell, GA) secured with a piece of rubber tubing (0.95x 2.5 cm) (Fisherbrand Fisher Scientific, Pittsburgh, PA). Five newly emerged males were placed in each tube with a female and were allowed to mate for 24 hours. A streak of honey and water (1:1) was provided for each tube. After 24 hours, males were removed from each tube and the female was used in the experiment. All experiments were carried out at 25 2C temperature, a 12:12 (L:D) photoperiod, and 65 2% R.H. Host Stage Susceptibility and Sex Ratio. For host stage susceptibi lity, a no-choice test was carried out for first and sec ond instars, third-instar nymphs (female), and for newly emerged adult females. A hibiscus leaf with a 5 cm long st em was selected as the experimental unit. Petri dishes were prepared as mentioned previously. The leaf was placed inside the Petri dish with the stem inserted through the hole and then the lid wa s replaced. The Petri dish with the leaf was placed on a cup filled with water with the stem in serted in the water as mentioned above. For each mealybug stage, ten individuals were selected 24 hours before the experiment and placed on each hibiscus leaf using a paintbrush (N o.000) (American Painter 4000, Loew-Cornell Inc., Englewood Cliffs, NJ). These 10 individuals of each mealybug stage were considered as a single experimental unit and a replicate. The dishes were covered with a pie ce of black cotton cloth, encouraging mealybugs to settle on leaves. A st reak of honey and water (1:1) was placed on the inside of the lid. After 24 hours of placing the mealybugs in the Petri dish, a single mated female 60

PAGE 61

parasitoid obtained as mentioned above was pla ced on each leaf and the lid was replaced. The Petri dish was covered with a piece (15x15 cm) of chiffon cloth material (Jo-Ann Fabrics and Crafts, Miami, FL), and secured with a rubber band to avoid parasitoid escape. Each parasitoid was allowed to oviposit for 24 hours, and then it was removed from the Petri dish. Mummified mealybugs were individually placed in glass culture tubes and were secured as mentioned previously. Parasitoids emerged from these tubes were sexed a nd the proportion of parasitism was estimated. There were 25 replicates for ea ch mealybug stage and parasitoid combination. Host Stage Suitability. To test host stage suitability, choice tests were conducted with two host stage combinations. For mealybugs, five i ndividuals from each stage were used in the combinations of the second instar and third-instar female, the second instar and adult female, and the third instar and adult female. A mated female parasitoid was introduced to each Petri dish, and was allowed to oviposit for 24 hours and then removed. Immediately after removal of the parasitoid, five individuals of one stage of th e host in each Petri dish were moved to a new hibiscus leaf prepared as above The five individuals of the other host stage remained on the hibiscus leaf. These leaves were prepared at th e same time as the other leaves. This was done for easier identification of mummified hosts for a pa rticular stage. The c hoice tests were carried out in a similar manner as the host-stage suscepti bility tests. The num ber and the gender of parasitoids emerging from the mummified mealybugs were recorded for each parasitoid species. Each mealybug combination for each para sitoid species had 25 replicates. Interspecific Competition. Interspecific competition of parasitoids was studied using 10 individuals from the second instar and third-instar females. Each host stage was separately placed on a hibiscus leaf as prepared above. The parasitoid combinations used were A papayae and A loecki A papayae and P mexicana A loecki and P mexicana, and A papayae A loecki, 61

PAGE 62

and P mexicana. A mated female of each parasitoid species was used in all combinations. They were allowed to parasitize for 24 hours, and were then removed. The mealybugs were allowed to mummify on the hibiscus leav es and mummified mealybugs were treated similar to those mentioned above. The number and the species of parasitoids, which emerged from each combination in each mealybug instar were counte d. The mean percent parasitism was calculated from the 10 mealybugs used for each host stage in each parasitoid combination. Each parasitoid combination for each host stage had 25 replicates. Statistical Analysis. The experimental design was completely random for all experiments. A two-way analysis of variance (ANOVA) was pe rformed using a general linear model (PROC GLM) of SAS (SAS Institute 1999) to find the inte raction between parasitoids and host stages of P marginatus in host stage susceptibility and sex rati o experiments. Means were compared at P = 0.05 significance level using least square mean s (LSMEANS) of SAS (SAS Institute 1999). For host stage suitability tests, means between two stages of P marginatus for each parasitoid species were compared at P = 0.05 significance level using a ttest (PROC TTEST) of SAS (SAS Institute 1999). In interspeci fic competition studies, PROC GLM was used for significance among the parasitoids and m eans were compared at P = 0.05 significance level using least square means (LSMEANS). Proportions of females (sex ratio) and proportions of parasitism were arcsine transformed using p p arcsin' where p = proportion of female/parasitism, to adju st the variances (Zar 1984) prior to ANOVA, but untransformed data are presented in tables. 62

PAGE 63

Voucher Specimens. Voucher specimens of P marginatus A papayae A loecki, and P mexicana were deposited in the Entomology and Nematology Department insect collection, at the Tropical Research and Education Center, University of Florida. Results Host Stage Susceptibility and Sex Ratio. All three parasitoids were able to develop and emerge successfully in second instar, thir d-instar females, and adult females of P marginatus (Table 4-1). No parasitoids emerged from first-instar nymphs. The mean percent parasitism decreased with increasing host size for both A papayae and P mexicana The proportion of emerged female parasitoids in creased with increasing host size (Table 4-2). Although A papayae had a similar number of males and females that emerged from second-instar hosts, A loecki and P mexicana had a lower number of females than males emerging from second instars. In the third inst ar and the adult females, all three parasitoid species had higher female than male emergence. Host Stage Suitability. Acerophagus papayae and P mexicana preferred the second instar to the third-instar female and the adult female, while A loecki preferred the third-instar female and the adult female to the second instar (Table 4-3). Between the third-instar female and the adult female, A papayae and A loecki preferred the third-instar mealybugs while P mexicana had no preference. Interspecific Competition. Acerophagus papayae had a higher mean percent parasitism when present with either A loecki or P mexicana or both in second-instar hosts (Table 4-4). In third-instar females, A loecki had a higher parasitism when present with either A papayae or P mexicana or both. Overall, P mexicana had a lower mean percent parasitism when present with either A papayae or A loecki or both except for the presence with A loecki in the second-instar hosts. 63

PAGE 64

Discussion Size of the host is one of the factors that solitary endoparasitoids consider when they select a host stage for ovipositi on (Vinson and Iwantsch 1980). Acerophagus papayae A loecki, and P mexicana did not prefer first-instar nymphs as a suitable host stage for parasitoid development. This makes the first-instar nymph of P marginatus which is approximately 0.4 mm in size ( Miller and Miler 2002), less vulne rable to these parasitoids. In parasitoid behavioral studies, first-instar Rastrococcus invadens Williams were preferred for host feeding by the parasitoid, Anagyrus mangicola Noyes (Bokonon-Ganta et al. 1995). Parasitoids such as Anagyrus kamali Moursi can oviposit in first-instar nymphs of Maconellicoccus hirsutus Green but the percent parasitism was less than 20% (Sagarra and Vincent 1999). In most situations, the ovipositor of A kamali remained stuck within the firstinstar host, precluding further foraging of th e parasitoid (Sagarra and Vincent 1999). The second-instar nymphs of Planococcus citri (Risso) were often impaled on the ovipositor of Anagyrus pseudococci (Girault), thus preventing the fe male from further egg deposition (Islam and Copland 1997). By choosing a larger host, the parasitoid accessed a larger food supply and increased the fitness of its progeny. In larger hosts, a fe male biased progeny was recorded for many parasitoids (King 1987). Further, increased host size translates into both increased male and female fitness (Charnov et al. 1981). For female s this measure is the lifetime production of eggs, and for males, it is the length of life (Charnov et al. 1981). Although, all three parasitoid species of P marginatus were able to develop and complete thei r life cycle in secondinstar hosts, only A papayae produced a higher proportion of female pr ogeny. Having more males than females in its progeny is not a desirable characteristic for a parasitoid to have as an efficient biological control agent. The so litary endoparasitoid, Aenasius vexans Kerrich, which were able to oviposit 64

PAGE 65

in second-instar nymphs of Phenacoccus herreni Cox and Williams also recorded a considerably higher proportion of males in the second instar than in the larger instars of P herreni (Bertschy et al. 2000). Except for the parasitism of A loecki in second-instar P marginatus the percent parasitism of all three parasitoid species decr eased with increasing host size. Mealybugs show strong physical defense and escape behavior, which could be incr eased with the body size. The third-instar P citri which was often encountered by the parasitoid A pseudococci, showed strong physical defense and escape behavior (Is lam and Copland 1997). The higher success of oviposition in the second-instar P marginatus may be due to less or absence of these defense and escape behavior in early instar mealybugs. Interspecific competition was evident by A papayae A loecki and P mexicana when competing for same host instar. Out of the three parasitoid species, A papayae had the highest parasitism level indicating its superior ability to compete. Intensive studies of parasitic complexes in connection with biological cont rol programs have shown that interspecific competition can be extremely important (Sch roder 1974). This may be one reason, why A papayae was well established and recovered from th e field in the Republic of Palau (Muniappan et al. 2006). In field tests c onducted in Florida in 2005 and 2006, both A papayae and A loecki were recovered, but P mexicana was not (Chapter 6). Pseudleptomastix mexicana was also not recovered in the field studies conducted in the Republic of Palau (Muniappan et al. 2006). This information suggests that P mexicana may be less competitive than the other two parasitoids. Since the preference for a host stage is similar for A papayae and P mexicana but different for A loecki, it reduces the competitiveness between A papayae and A loecki. Different host stage preference of A papayae and A loecki also greatly reduces competition for 65

PAGE 66

the same host stage. However, the developmental time of A loecki, which is similar to the developmental time of A papayae is shorter than the developm ental time of its preferred host stage of P marginatus (Chapter 5). Developmental times of A papayae and A loecki coincide with the developmental time of the second instar P marginatus (Chapter 5). At the beginning of the season with the absence of overlapping generations of P marginatus A loecki and A papayae can compete for second instars due to unavaila bility of preferred third instar host stages for A loecki In addition to being a parasitoid of P marginatus A loecki can develop in Dysmicoccus hurdi and Phenacoccus madeirensis Green (Hemiptera: Pseudococcidae) (Noyes 2000). Phenacoccus madeirensis is one of the commonly fo und mealybug species in South Florida. Since A loecki is not host-specific, it has the adva ntage of searching for other suitable hosts such as P madeirensis in the absence of suitable stages of P marginatus This may be one reason for the lower parasitism of A loecki observed in the field studi es and less competitiveness of A loecki in the control of P marginatus (Chapter 6). On the other hand, P mexicana which also prefers the second instar P marginatus (as does A papayae ) has longer developmental time than the other two parasitoids (Chapter 5). Developmental time of P mexicana does not coincide with the developmental time of the second instar P marginatus This allows A papayae females for which developmental time of th e host stage overlaps with its developmental time, to parasitize preferred host stages when it emerges as an adult. In mass rearing of parasitoids, second-instar P marginatus is a suitable stage for A papayae and P mexicana while third-instar females are suitable for A loecki Females of A papayae and P mexicana had a similar host stage preference for parasitism while it was a different host stage preference for female A l oecki. Acerophagus papayae shows superior adaptability by being able to oviposit in second instar to adult-female P marginatus as 66

PAGE 67

well as causing a higher percent para sitism when present with either A loecki or P mexicana or both. The information gathered from this study, w ill be helpful in explaining the adaptability of these three parasitoids of P marginatus in the field. 67

PAGE 68

Table 4-1 Mean percent parasitism ( SEM) of A papayae A loecki and P mexicana reared in different developmental stages of P marginatus to evaluate host stage susceptibility using no-choice tests. Mean Percent Parasitism (%) for Developmental Stages of P marginatus Parasitoid Second Third female Adult female A papayae 82.8 2.1aA 71.2 2.6bB 60.8 2.9bC A loecki 41.2 2.8cB 82.4 1.9aA 74.8 3.2aA P mexicana 70.8 1.9bA 50.8 2.5cB 40.8 3.6cB ANOVA Results Source F df P Model 36.32 8, 216 <0.0001 Parasitoid 34.06 2, 216 <0.0001 Stage 8.67 2, 216 0.0002 Parasitoid*Stage 51.27 4, 216 <0.0001 n = 25 Means within a column followed by the same lowercase letters, and means within a row followed by the same uppercase letters are not significantly different at = 0.05 (Least Square Means (LSMEANS) Test). 68

PAGE 69

69 Table 4-2 Mean proportion of fe males (sex ratio) ( SEM) of A papayae A loecki and P mexicana reared in different developmental stages of P marginatus to evaluate host stage susceptibility using no-choice tests. Mean Proportion of Females (Sex Ratio) ( SEM) for Developmental Stages of P marginatus Parasitoid Second Third-female Adult-female A papayae 0.50 0.01aB 0.56 0.01aA 0.57 0.01abA A loecki 0.40 0.01cC 0.51 0.01bB 0.54 0.01bA P mexicana 0.48 0.01bC 0.55 0.01aB 0.56 0.01aA ANOVA Results Source F df P Model 72.34 8, 216 <0.0001 Parasitoid 73.68 2, 216 <0.0001 Stage 196.98 2, 216 <0.0001 Parasitoid*Stage 9.35 4, 216 <0.0001 n = 25 Means within a column followed by the same lowercase letters, and means within a row followed by the same uppercase letters are not significantly different at = 0.05 (Least Square Means (LSMEANS) Test).

PAGE 70

Table 4-3 Mean percent parasitism ( SEM) of A papayae A loecki and P mexicana reared in different stage combinations of P marginatus to evaluate host stage suitability using choice tests. Host stage combination of P marginatus Parasitoid Mean ( SEM) Percent Parasitism T Statistics Stage 1 Stage 2 Stage 1 Stage 2 t df. P Second ThirdFemale A papayae 77.6 1.8 58.4 2.6 6.54 48 <0.0001 A loecki 30.4 2.9 76.0 2.0 -13.60 48 <0.0001 P mexicana 69.6 2.6 40.8 3.6 6.73 48 <0.0001 Second Adult-Female A papayae 76.8 1.9 50.4 4.2 -5.98 48 <0.0001 A loecki 32.0 3.1 68.8 2.6 9.11 48 <0.0001 P mexicana 68.8 2.6 32.0 3.3 -8.78 48 <0.0001 Third Adult-Female A papayae 60.0 3.1 48.0 5.0 -1.81 48 0.0471 A loecki 79.2 0.8 64.8 3.1 -4.58 48 <0.0001 P mexicana 41.6 3.4 32.8 3.2 -1.85 48 0.0691 n = 25 Host stage combinations: second instar (Stage 1) and third-instar female (Stage 2), s econd instar (Stage 1) and adult female (S tage 2), and third-instar female (Stage 1) and adult female (Stage 2). 70

PAGE 71

71 Table 4-4 Mean percent parasitism ( SEM) of combinations of A papayae A loecki and P mexicana reared in second and third-instar P marginatus to evaluate interspecific competitions of parasitoids. Stage of P marginatus Combination of Parasitoids Mean ( SEM) Percent Parasitism Second Parasitoid 1 Parasitoid 2 Parasitoid 3 A papayae A loecki P mexicana A papayae A loecki 69.6 2.3A 19.6 1.7B A papayae P mexicana 78.4 1.2A 20.0 1.3B A loecki P mexicana 40.4 3.5B 50.4 3.3A A papayae A loecki P mexicana 59.6 2.9A 14.8 1.8C 23.6 2.1B Third A papayae A loecki 42.4 3.3B 54.8 3.5A A papayae P mexicana 59.2 4.3A 35.6 3.5B A loecki P mexicana 75.2 2.7A 20.4 1.4B A papayae A loecki P mexicana 38.8 4.4B 47.6 4.5A 11.2 0.6C ANOVA Results Source F df P Model 49.8 17, 432 <0.0001 Stage 0.71 1, 432 0.4887 Combination 61.67 8, 432 <0.0001 Stage*Combination 44.09 8, 432 <0.0001 N = 25 Means within a row followed by the same uppercas e letters are not significantly different at = 0.05 (Least Square Means (LSMEANS) Test).

PAGE 72

CHAPTER 5 DEVELOPMENTAL TIME, LONGEVITY, AND LIFETIME FERTILITY OF Acerophagus papayae Anagyrus loecki AND Pseudleptomastix mexicana ; THREE INTRODUCED PARASITOIDS OF Paracoccus marginatus WILLIAMS AND GRANARA DE WILLINK Introduction Developmental time, longevity, and lifetime fer tility are important fitness parameters when evaluating a biological control ag ent. Determining developmental time of a parasitoid is necessary to determine its efficiency in contro lling the host. Generally, the developmental time of a biological control agent should be shorte r than the developmental time of the host (Greathead 1986). According to Greathead (19 86), high fecundity and short generation time are some of the desirable characters of a parasi toid. Courtship and mating are energy and time consuming activities in insects, which can affect the outcome of the longevity, lifetime fecundity, and progeny production of hyme nopteran parasitoids (Ridley 1988). Mating is required to achieve their full reproductive po tential in some parasitoids (Ridley 1988). The progeny sex ratio is the main fitness parameter that can be affected by mating. The majority of parasitoids need to mate once to attain their optimal sex ra tio (Ridley 1993). Some parasitoid species are arrhenotokous, e.g. fertilized eggs lead to fema le progeny and unfertilized eggs give rise to males. Lifetime fertility or progeny production of a parasitoid is important in its long-term establishment as a biological control agent. A parasitoid with higher lifetime fertility with female-biased progeny can parasitize a higher nu mber of hosts. Female-biased progeny are desirable in classical biol ogical control (King 1987). Classical biological control was identified as an important pest management practice for Paracoccus marginatus Williams and Granara de Willink (Hemiptera: Pseudococcidae), a polyphagus mealybug species that was first identified in the US in Florida in 1998 (Miller and Miler 2002). Paracoccus marginatus is a pest of a large numbe r of tropical and subtropical 72

PAGE 73

fruits, vegetables, and ornamentals (Miller and Miler 2002). Prior to invading the US, P marginatus had been established in the Caribbean since 1994 (Miller et al. 1999). After the establishment in Florida, P marginatus was identified in the Pacific islands of Guam (Meyerdirk et al. 2004), the Republic of Pa lau (Muniappan et al. 2006), and se veral Hawaiian islands (Heu et al. 2007). With the joint efforts of the Dominican Republic, Puerto Rico, and the US (Walker et al. 2003), currently there are thr ee parasitoids (Hymenoptera: Ency rtidae) used in the classical biological control of P marginatus in the US, the Caribbean, and the Pacific islands (Meyerdirk et al. 2004). The three solit ary endoparasitoid species, Acerophagus papayae Noyes and Schauff, Anagyrus loecki Noyes and Menezes, and Pseudleptomastix mexicana Noyes and Schauff are currently mass reared in Puerto Rico and released in P marginatus affected areas by the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS) (Meyerdirk et al. 2004). They have been released in Guam, and the Republic of Palau, and are suc cessfully controlling P marginatus (Meyerdirk et al. 2004, Muniappan et al. 2006). No information is available on the fitne ss parameters of th ese parasitoids of P marginatus Fitness parameters of parasitoids are specifically important when evaluating their efficiency and understanding long-term effects in a system where more than one parasitoid species have been released as classical biological control agents. This study fo cuses on the developmental time, longevity, and the lifetime fertility of A. papayae A loecki and P mexicana, three introduced parasitoids of P marginatus Materials and Methods Rearing Mealybugs. Red potatoes ( Solanum tuberosum L.) (Ryan Potato Company, East Grand Forks, MN) were allowed to sprout and then used in rearing a colony of P marginatus at the University of Florida, Tropical Research and Education Center (T REC), Homestead, FL. 73

PAGE 74

Initially, P marginatus was collected from a papaya ( Carica papaya L.) field in Homestead, FL. Prior to sprouting, potatoes were soaked in a 1% solution of bleach (Clorox The Clorox Company, Oakland CA; 6% sodium hypochlorite) for 15 minutes, and then rinsed with water, air-dried and placed in black cotton cloth bags to encourage sprouting. Bags were kept inside a dark room at 27 1C and 65 2% R.H. Each week, 36 sprouted potatoes were infested with P marginatus ovisacs to maintain the colony. An e nvironmental growth chamber (Percivel I36LL, Percival Scientific Inc. Perry, NC) was used at 25 1C, 65 2% R.H., and a photoperiod of 12:12 (L:D) to rear the mealybug colony. To obtain a particular stage of mealybugs, the newly laid ovisacs were selected and each was reared on a hibiscus leaf inside a 9-cm-diam Petri dish wi th a 0.6-cm-diam hole made in the bottom. Leaves were obtained from hibiscus ( Hibiscus rosa sinensis L.) plants maintained in a shadehouse at TREC. A tender hibiscus leaf with a 5-cm-long stem was placed in each Petri dish with the stem inserted through the hole at the botto m of the Petri dish. Each Petri dish was kept on a 162 ml translucent plastic souffl cup (Georg ia Pacific Dixie, Atlanta, GA) filled with water, which allowed the stem below the petiole to be in water. A single ovisac was placed on each leaf and the eggs were allowe d to hatch and then to develop to the desired stage. It was not possible to differentiate the sex of the mealybugs for the first and second instar while the thirdinstar nymphs and the adults used were females. To reduce the variation in each mealybug instar used, newly molted individuals r ecognized by their size and the presence of shed exuviae were selected for each experiment. Rearing Parasitoids. Colonies of A. papayae A loecki and P Mexicana were maintained in an insectary at TREC at 25 2C temperature, a 12:12 (L:D) photoperiod, and 65 2% R.H. Initial colonies of parasitoids were obtained from the Biol ogical Control Laboratory, 74

PAGE 75

Department of Agriculture, Puerto Rico, thr ough USDA-APHIS. Parasi toid colonies were established in plexiglass cages (30x30x30 cm) using sprouted red potatoes with second and third-instar P. marginatus In order to obtain a continuous supply of newly emerged parasitoids weekly, potatoes with second and third-instar mealybugs were provided to each parasitoid species every week. After 7 days, potatoes were moved to a new cage fo r parasitoid emergence and new mealybug-infested potatoes were provi ded for oviposition. A solution of honey and water (1:1) was streaked on 4 pieces (5x5 cm) of Benchkote surface protector paper (Fisherbrand Fisher Scientific, Pittsburgh, PA) and attached to the cage using labeling tape, for emerging parasitoids (Fisherbrand Fisher Scientific, Pittsburgh, PA). Water was provided in two clear plastic 73.9 ml containers (Tristat e Molded Plastic Inc., North Di xon, KY) per cage. In each of the containers, a 1-cm-diam hole was made in th e center of the lid and a 7.6-cm-long piece of cotton roll (TIDI Products, Neena h, WI) was inserted through the hole to allow parasitoids to access water. To obtain mated female parasitoids for the experiments, newly emerged female parasitoids of each species were selected, and placed singly in glass disposable culture tubes, (1.2x7.5 cm) (Fisherbrand Fisher Scientific, Pittsburgh, PA ) closed with two-ply tissue, (Kimwipes EXL, Kimberly-Clerk Global Sales Inc. Roswell, GA) and secured with a piece of rubber tubing (0.95x2.5 cm) (Fisherbrand Fisher Scientific, Pittsburgh, PA). For each tube with a female, five newly emerged males were added and allowed to mate for 24 hours. A streak of honey and water (1:1) was provided for each tube. After 24 hours, males were removed from each tube, and the mated female was used in the experiment All experiments were ca rried out at 25 2C temperature, a 12:12 (L:D) photoperiod, and 65 2% R.H. 75

PAGE 76

Specimens of P marginatus A papayae A loecki and P mexicana were sent to Systematic Entomology Laboratory (SEL), USDA Beltsville, MD, for species verification. Developmental Time. To evaluate the developmental tim e of each parasito id species in different mealybug instars, 10 individuals each from second instar, third-instar females, and adult female P marginatus were selected and placed separately on new hibiscus leaves. The Petri dishes with leaves were prepared 48 hours before the experiment. Mealybugs were placed on the leaves 24 hours before the experiment to allow them to settle on the leaves. The 10 individuals of each mealybug stage on a hibiscus leaf were considered a replicate and were used as an experimental unit. A mated female parasitoid was placed in each Petri dish and the lid was replaced. Each Petri dish with the lid was covered with a piece (15x15 cm) of chiffon cloth material (Jo-Ann Fabrics and Crafts, Miami, FL) and secured with a rubber band to avoid parasitoid escape, before it was placed on a cup of water with th e stem submerged. Parasitoids were allowed to oviposit for 24 hours and were then removed. Parasitized mealybugs became mummified on the hibiscus leaves. Mummies we re individually placed in disposable, glass culture tubes and closed with two-ply tissue and secured with a piece of clear polyvinyl chloride (PVC) tubing. These tubes with the parasitized mealybugs were held in the insectary until the emergence of adults. The time for adult emergen ce and the sex of the adu lts were noted and the mean of the 10 individuals on each hibiscus leaf was used in analyses for developmental time and sex ratio of each parasitoid. This procedur e was followed for all three parasitoid species with 50 replicates for each species. Longevity. Longevity was studied for three mating conditions for each female parasitoid (unmated, mated-without oviposition, and mate d-with oviposition), and two mating conditions for each male parasitoid (unmated and mated) To collect both males and females for each 76

PAGE 77

species, third-instar mealybugs on sprouted potatoes were placed in plexiglass cages and mated females were released to each cage. After 24 hours, females were removed and mealybugs were allowed to mummify. Mummified mealybugs were collected, and were individually placed in glass culture tubes as above. When parasitoids started to emerge from the mummified mealybugs, 50 newly emerged virgin males and females were separately placed in glass culture tubes for unmated status, and each tube was provided with a streak of honey and water, and secured with two-ply tissue. For unmated females with oviposition, 50 newly emerged females were individually transferred to clear plastic 500 ml deli cups (G eorgia Pacific Dixie, Atlanta, GA) and provided with mealybugs on potatoes to oviposit. Each cup was covered wi th a piece of chiffon cloth material before placing the lid. A circular area of 8.5-cm-diam was removed from the 12-cm-diam lid to facilitate air circulation. For mated males, one male was placed in a glass culture tube with a streak of honey and five females were provided for mating for 24 hours, and then the females were removed. The mated males were retained in the culture tube. For mated females withoutoviposition, one female was placed in a glass culture tube with a streak of honey, and five males were provided for 24 hours and then the males were removed and the females were retained. The same procedure was followed for the mated females with oviposition, except they were individually transferred to clear plastic 500 ml deli cups and provided with mealybugs on potatoes to oviposit as for unmated females with oviposition, as mentioned above. The number of days each parasitoid lived was counted for both males and females in all the above mating conditions. These procedures were repeated for all three paras itoid species. For each mating condition in each sex, 50 replicates were used. 77

PAGE 78

Lifetime Fertility. Lifetime fertility of mated and unmated females of each parasitoid species was studied. A newly emerged virgin female was eith er held alone or allowed to mate with five newly emerged males for 24 hours in a glass culture tube provi ded with a streak of honey and water. After removing the males, the fe males were individually transferred to clear plastic 3.8 liter round jars (Rubbermaid Newe ll Rubbermaid Inc. Atlanta, GA). Before placing the lid, each jar was covere d with a piece of chiffon cloth material. A 9-cm-diam area was removed from the lid to allow air circula tion. Unmated females were also transferred individually to clear plastic jars as mated fema les. Each mated or unmated female was provided approximately 300 third-instar female mealybugs on 1-2 infested potatoes daily for oviposition until the death of the females. The potatoes wi th parasitized mealybugs were placed in clear plastic 500 ml deli cups as above to allow mumm ification. When the parasitoids started to emerge, the number of males and females were counted. For each parasitoid species, 25 replicates were used for each mating condition. Statistical Analysis. The experimental design was completely random for all experiments. A two-way analysis of variance (ANOVA) was pe rformed using a general linear model (GLM) (SAS Institute 1999) to find interaction be tween parasitoids and mealybug instar for developmental time, parasitoids and mating cond itions in the longevity experiment, and for reproductive period, and male and cumulative prog eny in the lifetime fertility study. Means were compared at P = 0.05 significance level using l east square means (LSMEANS) (SAS Institute 1999). For the developmental time a nd the longevity studies, means were compared within the mealybug instar and mating condition for each parasitoid, a nd among the parasitoids for each mealybug instar and mating conditi on. One-way ANOVA was performed using a general linear model (GLM) for number of female progeny and sex ratio. Means were compared 78

PAGE 79

at P = 0.05 significance level using the Tukey's HSD te st. The proportion of female (sex ratio) was square-root arcsin e-transformed by using p p arcsin' where, p = proportion of female, to adjust the variances (Zar 1984) prior to ANOVA, but untransformed data were presented in tables. Voucher specimens. Voucher specimens of P marginatus A papayae A loecki, and P mexicana were deposited in the Entomology and Nematology Department insect collection, at the Tropical Research and Education Center, University of Florida, Homestead, FL 33031. Results D evelopmental times were shorter with increasing host age for male A papayae and P mexicana and female A loecki (Table 5-1) Acerophagus papayae and A loecki had shorter developmental times for both males and females, compared to male and female developmental times of P mexicana Longevity was highest for P mexicana and the lowest was for A papayae in all mating conditions with increasing host size for both ma les and females (Table 5-2). There was no difference in the longevity between unmated and ma ted males in all three species. The longevity was similar for females that were unmated a nd mated, both without oviposition. The females that were unmated and mated both with oviposition had similar longevity in each species but lived a shorter time than the ones that did not oviposit. Unmated females of all three species produced male progeny, and A loecki and P mexicana produced more progeny than A papayae (Table 5-3). In mated females, there were more male and female progeny for A loecki and P mexicana than for A papayae The progeny of all three species had similar sex ratios with approximately 1:1 for male:female. The 79

PAGE 80

reproductive period was longest for P mexicana and A papayae had the shortest reproductive period. Discussion Differences in fitness parameters including developmental time, longevity, and the lifetime fertility of A papayae A loecki and P mexicana are useful in evaluating them as efficient biological control agents of P marginatus There were differences in developmental time, longevity, and the lifetime fertility of A papayae A loecki and P mexicana Although increasing host size ha d a significant effect on deve lopmental time of male A papayae A loecki and P mexicana developmental times of female A papayae and P mexicana were not influenced by host size. Host st age had affected developmental time of other mealybug parasitoids as well. Developmental time of Anagyrus dactylopii (Howard) was not different among the various stages of Maconellicoccus hirsutus (Green) (Mani and Thontadarya 1989). However, Anagyrus kamali Moursi, a parasitoid of M hirsutus, had shorter developmental times when reared in the third instar and adult female M hirsutus than when reared in the first and second-instar M hirsutus (Sagarra and Vincent 1999). Anagyrus kamali had similar developmental times in the first and s econd instars, and in third and adult females of M hirsutus (Sagarra and Vincent 1999). Aenasius vexans Kerrich, an encyrt id parasitoid of cassava mealybug, Phenacoccus herreni Cox and Williams, had a shorter developmental time in older hosts than in early instar P herreni (Bertschy et al. 2000). De velopmental time of female Anagyrus pseudococci (Girault), a koinobiont endopa rasitoid of citrus mealybug, Planococcus citri (Risso) was similar on second and third instar, and adult P citri, but the developmental time of male A pseudococci was longer on second instars th an on third instar and adult P citri (Chandler et al. 1980, Islam and Copland 1997). Gyranusoidea tebygi Noyes, a parasitoid of mango mealybug, Rastrococcus invadens Williams, when developed on second and third instar 80

PAGE 81

had similar developmental times compared to th e longer developmental time in first instar R invadens (Bovida et al. 1995a). However, Anagyrus mangicola Noyes, the primary parasitoid of R invadens, had a similar developmental tim e on different host stages of R invadens with no differences in the size of emerging parasitoids (Cross and Moore 1992). When the developmental time of a parasitoid is shorter than the developmental time of the host, there is an advantage for the parasito id. Later in the season with overlapping host generations, it can produce its progeny at a faster rate than the host and can parasitize the host populations in a shorter time. The adult female P. marginatus can develop on hibiscus in 25.9 days, and the second instar and third-instar females can emerge within 15.2 and 20.8 days respectively (Chapter 2). Acerophagus papayae and P mexicana prefer second instars compared to third-instar P marginatus (Chapter 4). Developmental time of female A papayae overlaps the developmental time of the second-instar P marginatus providing an advantage for A papayae over female P mexicana, which needs a longer time to emerge as adults in secondinstar P marginatus Although A loecki prefers third instars co mpared to second-instar P marginatus (Chapter 4), its developmental time is shorter than the developmental time of the third-instar P marginatus Early in the season with the ab sence of overlapping generations of mealybugs, the longer developmental time of the third-instar P marginatus which does not overlap the emergence of A loecki, allows A loecki able to parasitize the available second instar P marginatus When developed in second-instar P marginatus A loecki produces male-biased progeny compared to A papayae which produces female-biased progeny (Chapter 4). Malebiased progeny would be less desirable than female-biased progeny in biological control (King 1987). However, because it is not a host-specific parasitoid of P marginatus female A loecki 81

PAGE 82

has the ability to select suitable stages of its other hosts such as Madeira mealybug, Phenacoccus madeirensis Green (Noyes 2000), in the absen ce of preferred host stages of P marginatus In parasitic Hymenoptera, female eggs are pref erentially laid in larg er hosts compared to male eggs, which are laid in smaller hosts (King 1987). Hosts that were pa rasitized at different stages may represent resources of different quality during parasitoid development and the wasp may have adapted its sex allocation accordingl y (Bertschy et al. 2000). Although both male and female P mexicana are larger than male and female A papayae (Noyes and Schauff 2003), P mexicana females prefer to lay their eggs in the second instar ra ther than in third-instar P marginatus (Chapter 4). Since the developmental time of P mexicana is longer than the developmental times of A papayae and A loecki a second-instar host may be more desirable than a third instar fo r the development of P mexicana A fitness parameter such as the lifetime fertility of a parasitoid is important in long-term establishment of the parasitoid as a biological control agent. A parasitoid species with more female progeny has the ability to parasitize a high er number of hosts than one with fewer female progeny. Body size of a parasitoid is frequently related to fecundity, longevity, and host finding ability (Hemerik and Harvey 1999). A significan t relationship between si ze and both longevity and lifetime fecundity was found in fitness parameter studies in Trichogramma evanescens, a gregarious, polyphagous egg paras itoid (Doyon and Boivin 2005). The smallest of the three species (Noyes 2000, Noyes and Schauff 2003), A papayae produced the least progeny compared to A loecki and P mexicana both of which produced twice the progeny of A papayae In addition, A papayae had the shortest lifespan compared to A. loecki and P mexicana for both males and females with different mating status. Females of all three parasitoid species outlived males. This has been recorded in other parasitoids species as well. In 82

PAGE 83

the longevity studies of Anagyrus kamali Moursi, females lived longer than the males (Sagarra et al. 2000b). There are differences in the developmental time, longevity, and lif etime fertility of A papayae A loecki and P mexicana The differences in these fitn ess parameters are important in evaluating their efficiency as parasitoids of P. marginatus This information provides the insight needed to clarify the efficiency of A papayae in controlling P. marginatus as well as to explain the lower efficiency of A loecki and P mexicana 83

PAGE 84

Table 5-1 Mean developmental time (egg to adu lt eclosion) in days ( SEM) for male and female A papayae A loecki and P mexicana reared in second instar, third-instar female, and adult-female P marginatus Mean Developmental Time of Parasitoids (Days) Stage of P marginatus Sex Parasitoid Second-instar Third-instar female Adult-female Male A papayae 13.8 0.2bA 13.5 0.2bAB 13.1 0.2bB A loecki 13.7 0.2bA 13.4 0.2bAB 13.1 0.3bB P mexicana 21.8 0.2aA 21.5 0.2aAB 21.0 0.2aB ANOVA Results Source F df P Model 339.74 8, 4414 <0.0001 Parasitoid 1350.27 2, 441 <0.0001 Stage 8.61 2, 441 0.0002 Parasitoid*Stage 0.04 4, 441 0.9970 Female A papayae 14.8 0.2bA 14.5 0.2bAB 14.1 0.2bB A loecki 14.7 0.2bA 14.4 0.2bAB 14.0 0.2bB P mexicana 22.9 0.2aA 22.7 0.2aAB 22.1 0.3aB ANOVA Results Source F df P Model 328.75 8, 441 <0.0001 Parasitoid 1306.44 2, 441 <0.0001 Stage 8.42 2, 441 0.0003 Parasitoid*Stage 0.06 4, 441 0.9927 n = 50 Means within a column followed by the same lowercase letters, and means within a row followed by the same uppercase letters are not significantly different at = 0.05 (Least Square Means (LSMEANS) Test) for males and females 84

PAGE 85

85 Table 5-2 Mean longevity in days ( SEM) for male (unmated and mated), and female (unmated, mated-without ovipositi on, and mated-with oviposition) A papayae, A loecki and P mexicana Longevity (Days) Sex Mating Condition Parasitoid A papayae A loecki P mexicana Male Unmated 23.3 0.4C 37.3 0.7B 47.5 1.8A Mated 22.0 0.4C 36.6 0.5B 45.9 0.9A ANOVA Results Source F df P Model 141.97 5, 294 <0.0001 Parasitoid 353.58 2, 294 <0.0001 Mating Status 2.41 1, 294 0.1216 Parasitoid* Mating Status 0.14 2, 294 0.8722 Female Mating Status A papayae A loecki P mexicana Unmated-without oviposition 33.1 0.6aC 48.9 1.0aB 63.1 1.8aA Unm ated-with oviposition 13.8 0.2bC 23.9 0.5bB 41.1 0.7cA Mated-without oviposition 32.3 1.0aC 47.6 1.2aB 58.4 1.2bA Mated-with oviposition 13.9 0.3bC 23.0 0.4bB 40.1 0.7cA ANOVA Results Source F df P Model 322.95 11, 588 <0.0001 Parasitoid 922.75 2, 588 <0.0001 Mating Status 558.98 3, 588 <0.0001 Parasitoid* Mating Status 5.01 6, 588 <0.0001 n = 50 Means within a column followed by the same lowercase letters, and means within a row followed by the same uppercase letters are not significantly different at = 0.05 (Least Square Means (LSMEANS) Test) for males and females.

PAGE 86

86 Table 5-3 Mean ( SEM) number of male and female progeny, cumulative progeny, sex ratio, a nd reproductive period of mated and unmated A papayae A loecki and P mexicana. Mating Status Parasitoid Number of Male Progeny Number of Female Progeny Cumulative Progeny Sex Ratio (Proportion of Females) Reproductive Period (Days) A papayae 44.5 1.0c 48.3 1.2b 92.8 1.9c 0.52 0.006 13.9 0.7c A loecki 97.8 1.3b 99.8 1.6a 197.6 2.5a 0.51 0.004 20.1 0.7b Mated P mexicana 103.0 3.4b 105.9 3.3a 208.9 6.6a 0.50 0.013 30.8 0.9a A papayae 88.0 2.9b 88.0 2.9c 11.9 0.6c A loecki 173.2 10.2a 173.2 10.2b 18.3 0.7b Unmated P mexicana 159.5 7.7a 159.5 7.7b 31.6 0.9a ANOVA Results Model F 73.40 199.88 71.17 2.99 118.28 df 5, 144 2, 72 5, 144 2, 72 5, 144 P <0.0001 <0.0001 <0.0001 0.0566 <0.0001 n = 25 Means within a column followed by the same lower case letters are not significantly different at = 0.05 (Least Square Means (LSMEANS) Test) for number of male proge ny, cumulative progeny, and reproductive period. Means within a column followed by the same lower case letters are not significantly different at = 0.05 (Tukey's HSD test) for number of female progeny and sex ratio.

PAGE 87

CHAPTER 6 FIELD ASSESSMENT OF THREE IN TRODUCED PARASITOIDS OF Paracoccus marginatus WILLIAMS AND GRANARA DE WILLINK (HEMIPTERA: PSEUDOCOCCIDAE) Introduction Paracoccus marginatus Williams and Granara de Willink is a polyphagous pest insect that can damage fruits, vegetabl es and ornamentals, including Carica papaya L. (papaya), Hibiscus spp. L. (hibiscus), Citrus spp.(citrus), Persea americana Mill. (avocado), and Solanum melongena L. (eggplant) (Miller and Miller 2002). This mealybug species was first described in 1992 (Williams and Granara de Willink 1992) and was re-described in 2002 (Miller and Miller 2002). Believed to be native to Mexico or Central America, P. marginatus has been established in the Caribbean since 1994 (M iller et al. 1999). In 1998, P. marginatus was first detected in the US, in Palm Beach County, Florida on hibiscus. Since then, it has been found on more than 25 genera of plants in the US. In recent years, P. marginatus has invaded the Pacific islands, and it is now established in Guam (Meyerdirk et al. 2004), the Republic of Palau (Muniappan et al. 2006), and in several Hawaiian islands (Heu et al. 2007). Paracoccus marginatus potentially poses a threat to numerous agricultu ral products in the US especially in Florida and states such as California, Hawaii, and Texas, which produce similar crops. Classical b iological control was identified as an important component in the management of P. marginatus and a program was initiated as a joint effort among the United States Department of Agriculture Puerto Rico Department of Agriculture, and Ministry of Agriculture in the Dominican Republic in 1999 (W alker et al. 2003). Currently, there are three solitary endoparasitoid hyme nopterans mass reared in Puerto Rico, and released in P marginatus infested areas in the US, the Ca ribbean, and some Pacific islands (Meyerdirk et al. 2004). They are Acerophagus papayae Noyes and Schauff, Anagyrus loecki Noyes and Menezes, and 87

PAGE 88

Pseudleptomastix mexicana Noyes and Schauff (Hymenoptera: Encyrtidae) (Noyes and Schauff 2003). In July 2003, A papayae A loecki and P mexicana were obtained from the Biological Control Laboratory, Department of Agriculture, Puerto Rico, and releas ed in 21 locations in South Florida, in Miami-Dade and Broward count ies (11 locations in Miami, 5 locations in Homestead, and 5 locations in Pembroke Pi nes and Miramar) (D. M. Amalin, personal communication). A total of 6,000 parasitoids (1,400 A papayae 1,200 A loecki and 3,400 P mexicana ) were released in South Florida in a singl e release attempt in July 2003 (D. M. Amalin, personal communication). No subsequent releases have been recorded. Information on parasitoids of P marginatus and the field evaluation of their effectiveness is limited in the US. Assessing the effect of a natural enemy or natural enemy complex on its/their host populations in the field is important to evaluate the success of a biological control project (Neuenschwander et al. 1986). This could be done by comparison of two separate pest populations, one population with th e natural enemy and the other without (Hodek et al. 1972). Pest populations without natural enemies can be f ound either in pre-release situations or can be created artificially, by using physic al or chemical means to excl ude the natural enemy from the plot (Smith and DeBach 1942). Experimental excl usion methods are the fa stest and most direct way to demonstrate the effect of a natural enemy on a pest population (Smith and DeBach 1942). In this field study, a physical excl usion method using sleeve cages wa s used to find the ability of A papayae A loecki, and P mexicana to control P marginatus in Homestead, FL. Materials and Methods Insect Rearing. A colony of P marginatus was reared on sprouted red potatoes (Solanum tuberosum L.) at the University of Florida, Tropi cal Research and Education Center (TREC), Homestead, FL. Initially, P marginatus was collected from a papaya (Carica papaya L.) field in Homestead, FL. Prior to sprou ting, the potatoes (Ryan Potato Co mpany, East Grand Forks, MN) 88

PAGE 89

were soaked in a 1% solution of bleach (C lorox The Clorox Co mpany, Oakland CA; 6% sodium hypochlorite), for 15 minutes, and then rinsed with clean water and dried. Potatoes were placed in black cotton cloth bags to encourage spr outing. The bags were kept inside a dark room at 27 1C. Each week, 36 spr outed potatoes were infested with P marginatus ovisacs to maintain the colony. Depending on the size, each pot ato was infested with 3 to 5 ovisacs. The infested potatoes were kept in 3.8-L plastic containers (Rubbermaid Newell Rubbermaid Inc. Atlanta, GA) with 12 potatoes per container. To facilitate the air circulation to developing eggs and mealybugs, screens (Amber Lumite Bio Quip, Gardena, CA) were glued to the cut sections of lids of these plastic containe rs. The mealybug colony was maintained in an environmental growth chamber (P ercivel I-36LL, Percival Scien tific Inc. Perry, NC) at 25 1C, 65 2% R.H., and a photoperiod of 14:10 (L:D). Field Experiments. The research plots were selected at three homeowner locations in Homestead, FL. The field experiments were ca rried out in July to August 2005 and 2006, using the same experimental locations in both years. Paracoccus marginatus was observed in all three locations at the time of selecti on. In each location, 10 hibiscus ( Hibiscus rosasinensis L.) plants, approximately 2.5 to 3.0 m tall, were sele cted. Each selected plant was considered a replicate. The three tr eatments used in this experiment were closed sleeve cage, open sleeve cage, and no cage. The sleeve cages were made of white chiffon cloth material (Jo-Ann Fabrics and Crafts, Miami, FL), 72 cm in length and 50 cm in width. Along the length of the material, a groove was sewn at 15 cm from each end. The piece of cloth with the groove was then folded in half along the width, and the two ends along the widt h were placed together and sewn at the edge to make a cylinder of 15 cm diameter. A piece of stainless steel (20 gauge) wire (Tower Manufacturing Company, Madison, IN), 72 cm in length was inserted through each groove and 89

PAGE 90

tied at the ends to make a ring to shape the cage into a cylindri cal cage. Three branches 1-1.5 m above ground were selected from each hibiscus plant. The branches selected were evenly distributed among the hibiscus plants, and each branch had 7-10 leaves. All the selected branches were cleaned with moist tissues (Kim wipes EX-L, Kimberly-Clerk Global Sales Inc. Roswell, GA) to make them free from any insects and eggs. Each clean branch was enclosed in a closed sleeve cage mentioned above for 7 days to observe for any insect presence or development. To avoid the cloth material of the cage getting in contact with the leaves, a stainless steel wire (22 gauge and 25 cm in length) was tied to the branch at the middle at each end of the cage, and the ends were fixed to the cage along the diameter. Sleeves of the cage were secured with a stainless steel wire tied around the enclosed branch. During this time, all enclosed branches were checked daily for the pr esence of any insects by opening the sleeve at the terminal end of the branch of each cage. If any insects were observed in a cage, the branch was cleaned again using the above procedure. After 7 days, five gravid females of P marginatus collected from the mealybug colony, were carefully placed on the terminal leaves of the branch within each sleeve cage using a paint brush (No.000) (American Painter 4000, Loew -Cornell Inc., Englewood Cliffs, NJ). Immediately after placing the females, the open sleeve was tied back on to the branch, closing the cage. Approximately 21 days was allowed for th e gravid females to lay eggs and the eggs to develop into second and third-in star mealybugs. When the number of second and third instars was at a 1:1 ratio by visual inspection, all th e sleeve cages were removed, and were replaced according to the three treatments mentioned above. Each of the three treatments was randomly assigned among the three branches on each plant, using cages similar to those described above and placing them over the branches infested with mealybugs. In the closed sleeve cage 90

PAGE 91

treatment, cages were kept closed. The pur pose of this treatment was to evaluate the development of mealybugs in the microclimate created inside a closed cage. In the open sleeve cage treatment, the cages were left open and the sleeves were folded back along the cylindrical part of the cage and were fixed to the cage with four safety pins. The purpose of this treatment was to provide the parasitoids access to the mealybugs and to provide microclimate conditions similar to the closed sleeve cage treatment. In the no cage treatment, branches with mealybug colonies were left un-caged. This treatment wa s used to assess the effect of the sleeve cages themselves on the mealybug population growth and parasitism level. The treatments were checked for mealybug destroye r adults and larvae ( Cryptolaemus montrouzieri Mulsant), ants, and spiders at 24, 48, and 72-hour intervals without disturbing the treatments. At 72 hours, all treatments were covered with closed sleeve cages, and the branches were removed from the plant. Cages were brought to the laboratory, and the number of mealybugs was noted. The number of adults and larvae of the mealybug destroyer (coccinellid predator), ants, and spiders was also recorded. From each replicate, 100 second and third-instar mealybugs were randomly collected and placed on a sprouting potato for further development. These potatoes were kept singly in 500 ml deli cups (Georgia Pacific Dixie, Atlanta, GA). Each cup was covered with a piece of chiffon cloth held in a place with the cup lid w ith a circular area of 8.5 cm diam removed to facilitate air circulation. The cups were held in an insectary, maintained at 25 1C, 12:12 (L:D) photoperiod, and 65 2 % R.H. Mealybugs were allowed to mummify on potatoes. Collect ion of mummified mealybugs was started 10 days after placing them on potatoes. Mummified mealybugs were pl aced individually in disposable, glass culture tubes of 1.2 cm diameter and 7.5 cm length (Fishe rbrand Fisher Scien tific, Pittsburgh, PA). Each tube was covered with two-ply tissue (K imwipes EX-L, Kimberly-Clerk Global Sales 91

PAGE 92

Inc. Roswell, GA), secured with 2.5-cmlong pi ece of clear polyvinyl chloride (PVC) tubing (Fisherbrand Fisher Scientific, Pittsburgh, PA) until the emergence of parasitoids. The emerging parasitoids from the culture tubes were sexed and were identified as to their species. Samples of parasitoids, mealybug destroyers, and ants were sent to the Systematic Entomology laboratory, USDA, Beltsville, MD for verification of identification. Samples of spiders were sent to Division of Plant Industry, Florida Depa rtment of Agriculture and Consumer Services, Gainesville, FL for sp ecies identification. Statistical Analysis. The experimental design was completely random with 10 replicates at each location. A three-way analysis of vari ance (ANOVA) was perfor med using the general linear model (PROC GLM) of SAS (SAS Institute 1999) to find the interaction among year, location, and treatment for mealybugs, mealybug de stroyers, ants, and spiders. A one-way ANOVA was performed using the general linear model (PROC GL M) for mean number of mealybugs collected from the three treat ments. Means were compared at P = 0.05 significance level using the Tukey's HSD test. A repeated measure ANOVA using the general linear model (PROC GLM) was performed for mean number of adults and larvae of mealybug destroyers, spiders, and ants collected at 24, 48, and 72-hour intervals to ch eck the interaction between the interval and the treatment. Means were compar ed between treatments using a t-test (PROC TTEST) of SAS (SAS Institute 1999) at P = 0.05. The closed sleeve cage treatment was excluded from the analysis since there were no natural enemies present in this treatment. Proportions of parasitism of A papayae and A loecki for both open sleeve cage and no cage treatments were arcsine-square-root transformed using, p p arcsin' 92

PAGE 93

where, p = proportion of parasitism, to adjust the variances (Zar 1984) prior to ANOVA, but untransformed data were presented in tables. A three-way ANOVA (PROC GLM) was performed to find the intera ction among year, location, and treatment for proportion of parasitism for each para sitoid species. A two-way ANOVA was performed for proportions of individual and cumulative parasitism of A papayae and A loecki between treatments, and means were compared at P = 0.05 significance level using leas t square means (LSMEANS) of SAS (SAS Institute 1999). Voucher Specimens. Voucher specimens of mealybugs, mealybug destroyer adults and larvae, ants, spiders, and parasitoids were deposited in the Entomology and Nematology Department insect collection, at the Tropical Re search and Education Center, University of Florida. Results There was no interaction in the mean number of P marginatus collected from each treatment by location and year (F = 0.12, df = 4, 162, P = 0.9737). Therefore, the data for P marginatus were pooled by location, y ear, and treatment, and pooled data were used in the analyses. The mean number of P marginatus collected from the cl osed sleeve cage (410.9 1.6), was higher than the numbers collected fr om the open sleeve cage (171.6 1.3) and the no cage treatment (109.1 0.7) by 58.2% and 73.4% respectively (F=16800.4, df= 2, 177, P <0.0001). There were 36.4% more mealybugs in th e open sleeve cage, compared to the no cage treatment. Natural enemies such as mealybug destroyer ad ults and larvae, and spiders were observed at all three locations used in this experiment. However, no natural enemies were present in the closed sleeve cage treatment. There was no inte raction in the mean number of individuals collected by location, year, and treatment for mealybug destroyer adults (F = 0.01, df = 2, 346, P 93

PAGE 94

= 0.9998) and larvae (F= 0.04, df = 2, 346, P = 0.9599), ants (F = 0.04, df = 2, 346, P = 0.9653), and spiders (F= 0.14, df = 2, 346, P = 0.8733). Therefore, the pooled data for each of these insects were used in the analyses. The repeat ed measures ANOVA for within subject effects indicated that there was no inte raction between the in terval and the treatme nt (F= 0.01, df = 2, 944, P = 0.9931). There were higher mean numbers of mealybug destroyer adults and larvae (Table 6-1), ants, and spiders (Tab le 6-2) in the no cage than in the open sleeve cage treatment at 24, 48, and 72-hour intervals. The spiders collected from the treatments were comprised of Gasteracantha cancriformis (Linnaeus), Cyclosa walckenaeri (O. P. Cambridge) (Araneae: Araneidae), Lyssomanes viridis (Walckenaer) (Araneae: Salticidae), Misumenops sp. (Araneae: Thomisidae), Hibana sp. (Araneae: Anyphaenidae), Theridion melanostictum O. P.Cambridge (Araneae: Theridiidae), and Leucauge sp. (Araneae: Tetragnathidae). None of the species of spiders collected was dominant in any of the treatments. The ants collected form the treatments were comprised of Tapinoma sessile Say, Pheidole sp., and Technomyrmex sp. (Hymenoptera: Formicidae). Tapinoma sessile was the predominant ant species collected from the three locations in both 2005 and 2006, and is a common and widely distributed Nort h American ant species (Smith 1928). There was no interaction in the mean pr oportion of parasitoids emerged from the mealybug samples collected by treatment, location, and year for A papayae (F = 0.86, df = 2, 108, P = 0.4260), and A loecki (F = 0.23, df = 2, 108, P = 0.7919). Therefore, the data for parasitoids were pooled by location and year, and pooled data were used in the analyses. Acerophagus papayae had higher percent parasitism in th e open sleeve cage than in the no cage treatment by 30.9% (Table 6-3). Within a treatment, A papayae had a higher parasitism than A loecki by 92.6% in the open sleeve-cage and by 9 2.5 % in the no-cage trea tment respectively. 94

PAGE 95

Percent parasitism of A loecki in the open sleeve cage was 30.8% higher than in the no cage treatment (Table 4-3). The open sleeve cage had 30.9% higher cumulative percent parasitism than the no cage treatment. There was no activity of P mexicana in any of the treatments. Discussion In recent years, classical biological control has been used to control several invasive mealybugs. Use of Apoanagyrus lopezi to control the cassava mealybug, Phenacoccus manihoti Matile-Ferrero in Africa (Neuenschwander 2001), Gyranusoidea tebygi Noyes for mango mealybug, Rastrococcus invadens (Williams) control in West Africa (Bokonon-Ganta and Neuenschwander 1995), and the use of Anagyrus kamali Moursi to control pink hibiscus mealybug, Maconellicoccus hirsutus Green in the Caribbean (Kairo et al. 2000) are some of the examples. Use of A papayae A loecki and P mexicana to control P marginatus in the Caribbean, the US, and the Pacific, is another ex ample of utilizing classical biological control to manage an invasive mealybug species. To determine the ecological and the economic impact of a biological control program, it is necessary to evaluate the efficacy of the biolog ical control agents. In order to understand this, it is important to evaluate the pest insect population in an environment where it is not exposed to the natural enemies (Boavida et al. 1995b). One of the principal obstacles of the host evaluation has been the difficulty of excluding the natura l enemies from the host population (Smith and DeBach 1942). In this study, sleeve cages were us ed as the exclusion method to investigate the host population without its natura l enemy. A physical exclusion method using sleeve cages can be an effective way to evaluate the effect of presence and absence of natural enemies on the survival of their host populations (Smith and DeBach 1942). Limitations and applicability of physical exclusion methods on different natural enemies have been evaluated (Kiritani and Dempster 1973, Van Lenteren 1980). One limitati on of this method is that it may cause 95

PAGE 96

conditions within the sleeve cage to depart too fa r from the normal conditions outside the sleeve cage (Smith and DeBach 1942). To overcome these limitations, open sleeve cage and no cage treatments were included in th is study. The closed sleeve cage protected the mealybugs from natural enemies as well as from environmental factors such as the rain and the wind, while the open sleeve cage likely provided some protection from adverse environmental conditions, and no protection provided by the no cage treatment. The greater host population in the closed environment indicates that when there was no out side interference from natural enemies, or no direct impact of the wind and rain, insects su rvive better than in th e open environment where they are more exposed to direct environmental fact ors as well as their natural enemies. Similar results have been reported for Rastrococcus invadens Williams in field assessment studies conducted to find the impact of the introduced parasitoid, Gyranusoidea tebygi Noyes in West Africa (Boavida et al. 1995b). The presence of predators such as C montrouzieri adults and larvae, and spiders may have a negative impact on percent parasitism. Cryptolaemus montrouzieri was also collected in relatively low numbers in field assess ment studies of the parasitoids of P marginatus conducted in the Republic of Palau (Muniappan et al. 2006 ) and in Guam in 2002 (Meyerdirk 2004). The presence of C montrouzieri could have had an effect on parasitism, but due to the presence of a large number of mealybugs and the high percent parasi tism observed in these areas, the effect of C montrouzieri may not be significant. There is a po ssibility that paras itized mealybugs were preyed on by C montrouzieri Most coccinellid pr edators feed on more than one prey species; thus, disruption of existing biological contro l by introduced coccinellids and the potential for indigenous coccinellid species to disrupt intr oductions can happen (Rosenheim et al. 1995). Common forms of intraguild predation include pred ators that attack herb ivores that harbor a 96

PAGE 97

developing parasitoid (Rosenheim et al. 1995). This may be one reason that higher parasitism was observed in the open sleeve cage treatment than in the no cage treatment, because there were more predators in the no cage treatment, and P marginatus was directly exposed to the environment. Anagyrus loecki is not a host specific classical biological co ntrol agent (Noyes 2000). The low parasitism by A loecki in both open sleeve cage and no cage treatments was possibly due to its multiple host preference. In addition to being a parasitoid of P marginatus A loecki can develop in Dysmicoccus hurdi and Phenacoccus madeirensis Green (Hemiptera: Pseudococcidae) (Noyes 2000) and P maderiensis is one of the commonly found mealybug species in South Florida (Williams and Granar a de Willink 1992, BenDov 1994). Other than P marginatus no other hosts have been recorded for Acerophagus papayae and P mexicana (Noyes and Schauff 2003). Not recovering a single P marginatus that was parasitized by P mexicana or the emergence of any hyper-parasitoids from the collected P marginatus raises an interesting question of whether P mexicana successfully establishe d in the experimental area. In July 2003, 3,400 P mexicana were released in Florida, as a one-ti me release in 21 locations in Miami-Dade, and Broward Counties including fi ve locations in Homestead where these field studies were conducted (D. M. Amalin, pers onal communication, Meyerdirk 2003). On the other hand, only 1,400 A papayae and 1,200 A loecki were released at the same time and in the same locations as P mexicana but they both were recovered from th e field. Even after several releases, P. mexicana has not been recovered in field assessmen t studies conducted in the Republic of Palau (Muniappan et al. 2006). A similar study has been conducted in Guam in 2002, although the results were reported without the re covery data of parasitoids (Mey erdirk et al. 2004). There is 97

PAGE 98

very little information on P mexicana and there is no information on why it was not recovered from the field in previous studies. Further field experiments focusing on P mexicana may be needed to clarify why it was not recovered from the field. In laboratory studies, P mexicana and A papayae showed a preference for second-instar P marginatus while A loecki preferred the third instars. At 25C, the developmental time of female P mexicana was 22 days, and was longer than the 14-day developmental time of female A papayae and A loecki (Chapter 5). The second-instar P marginatus can emerge within 14.6 days at 25C (Chapter 5). The de velopmental time of second-instar P marginatus coincides with the developmental time of female A papayae and A loecki (Chapter 5). The preference for the third-instar P marginatus by A loecki makes A papayae the dominant species in the competition for the second-instar P marginatus The longer developmental time can be an important reason for le ss effectiveness of P mexicana in the field. Pseudleptomastix mexicana also was less efficient when competing with A papayae and A loecki in laboratory studies (Chapter 4). The shorter developmental time and lack of co mpetitors for preferre d second-instar hosts may have placed A papayae as the dominant species over A loecki and P mexicana (Chapter 5). In laboratory studies, A papayae had better control of the host, when present singly or with A loecki and P mexicana (Chapter 4). Acerophagus papayae is also the predominant parasitoid species recovered from field st udies in both Guam (Meyerdirk et al. 2004) and the Republic of Palau (Muniappan et al. 2006). Out of the three parasitoid species, A papayae is the smallest parasitoid species (Noyes and Schauff 2003). Because of its smaller size, A papayae has the advantage of parasitizing P marginatus that were concealed in crevices of the host plant species. 98

PAGE 99

Because of this concealed nature, there is a po ssibility of less predat ion of these mealybugs by C montrouzieri larvae and adults, and spiders. Higher numbers of ants presen t in the no cage treatment may have affected the foraging behavior of parasitoids. This may be one of the reasons for lower cumulative parasitism in the no cage treatment compared to th e open sleeve cage treatment. Generally, mealybugs and ants have mutualistic relationships. Mealybugs bene fit from ant association when ants promote sanitation in mealybug populations and/or protect mealybugs from natural enemies (GonzalezHernandez 1999). It has been repeatedly obs erved that some pests have higher population densities on plants where ants are active than on pl ants free of ants (Hodek et al. 1972). There is considerable direct evidence of aggressive beha vior toward predators or parasites in honeydew seeking ants. Pheidole megacephala (F.) signific antly decreased Dysmicoccus brevipes (Cockerell) mortality, by Anagyrus ananatis Gahan and Nephus bilucernarius Mulsant (Coleoptera: Coccinellidae) adults via interfer ence with natural enemy searching behavior (Gonzalez-Hernandez 1999). Presence of ants in both open sleeve cage and no cage treatments may have some influence on the parasitism by A papayae and A loecki, although the effect of ants on mealybugs and parasitoids wa s not investigated in this study. Out of the three currently used parasitoids of P marginatus A papayae is well established in the field, and is the main contributor to the mortality of this mealybug species. Multiple host preference may have caused the low effectiveness of A loecki compared to A papayae Further research is needed to address the ability of P mexicana to control P marginatus as well as its ability to establish after release in the field. 99

PAGE 100

Table 6-1 Mean ( SEM) number of mealybug destroyer ( Cryptolaemus montrouzieri ) adults and larvae collected per cage from open sleeve cage and no cage treatments using pooled data of 2005 and 2006 in three experimental locations Mealybug-destroyer (adult)/per cage Mealybug-destroyer (larva)/per cage Interval (Hours) Interval (Hours) Treatment 24 48 72 24 48 72 Open sleeve 2.0 0.1 2.1 0.1 2.1 0.1 1.2 0.1 1.1 0.1 1.3 0.1 No cage 3.0 0.1 3.0 0.1 3.1 0.1 2.1 0.1 2.1 0.1 2.1 0.1 t -7.33 -6.79 -6.86 -8.42 -8.21 -8.42 df 118 118 118 118 118 118 P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 n = 60 100

PAGE 101

Table 6-2 Mean ( SEM) number of ants a nd spiders collected from open sleeve cage and no cage treatments using pooled data of 2005 and 2006 in three experimental locations Ants Spiders Interval (Hours) Interval (Hours) Treatment 24 48 72 24 48 72 Open sleeve 21.4 0.2 21.3 0.2 21.5 0.2 2.0 0.1 2.1 0.1 2.1 0.1 No cage 30.9 0.3 30.9 0.2 31.0 0.2 2.9 0.1 3.1 0.1 3.0 0.1 t -27.32 -30.52 -29.93 -6.88 -7.77 -7.06 df 118 118 118 118 118 118 P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 n = 60 101

PAGE 102

Table 6-3 Individual and cumulative mean percent parasitism ( SEM) of P marginatus by A papayae A loecki, and P mexicana in open sleeve cage, and no cage treatments using pooled data of 2005 and 2006 in three experimental locations Percent Parasitism Treatment A papayae A loecki Cumulative Open sleeve cage 31.0 0.3aB 2.3 0.1aC 33.3 0.3aA No cage 21.4 0.3bB 1.6 0.1bC 23.0 0.3bA Source F df P Model 3606.07 5, 354 <0.0001 Parasitoid 8038.28 2, 354 <0.0001 Treatment 1387.10 1, 354 <0.0001 Parasitoid* Treatment 283.36 2, 354 <0.0001 n = 60 Means within a column followed by the same lowercase letters, and means within a row followed by the same uppercase letters are not significantly different at = 0.05 (Least Square Means (LSMEANS) Test). 102

PAGE 103

CHAPTER 7 SUMMARY AND CONCLUSIONS Paracoccus marginatus Williams and Granara de Willink (papaya mealybug) is a polyphagous pest insect that can damage a larg e number of tropical a nd subtropical fruits, vegetables, and ornamental plants. A native to Mexico and/or Central America, P marginatus is currently established in the Caribbean, the US and several Pacific islands. Classical biological control was identified as a suit able method for the control of P marginatus Currently there are three solitary hymenopteran endopara sitoid species mass reared in Puerto Rico and released in P marginatus infested areas in the Caribbean, the US and Pacific islands. They are Acerophagus papayae Anagyrus loecki and Pseudleptomastix mexicana However, there is a lack of information about P marginatus and its parasitoids. This disser tation focused on the life history of P marginatus in relation to host plant species and temperature, and evaluated the effectiveness of the three in troduced parasitoid species. Life history studies of P marginatus indicated that it c ould successfully develop, reproduce, and survive on a wide variety of econo mically important ornamental plants as well as on an aggressive weed species Parthenium hysterophorus. Egg to adult emergence occurred in 30 days or less in all plant species indicating it s fast development on different host plants. Paracoccus marginatus showed its tropical characterist ics by completing its life cycle in the temperatures ranging from 18 to 30C. Its high minimum temperature threshold of 14C and the low thermal constant further clarified this characteristic. The optimum development of P marginatus can be expected around 28C, while the maximum temperature threshold can go up as high as 32C. These characteristics may limit establishment of P marginatus into many areas in the US, while some areas in California, Texa s, Florida and Hawaii ar e more vulnerable. The ultimate movement of P marginatus to new areas in the US that are suitable in regards to 103

PAGE 104

temperature will also be influenced by other en vironmental factors, availability of host plant species, plant movement from state to state, and the rules, regulations, an d restrictions of plant movement. Of the three parasitoid species currently used in the biol ogical control of P marginatus A papayae had higher parasitism in the field. Natural enemies including Cryptolaemus montrouzieri ants, and spiders were observed in the tr eatments exposed to the environment, and overall their activity may have contributed to the low parasitism. Parasitism of P mexicana was not observed while A loecki had a lower parasitism compared to A papayae. The smallest parasitoid species out of the three species, A papayae had lower lifetime fertility than the other two species. In addition, A papayae and P mexicana compete for the second-instar P marginatus while A loecki prefers the third-instar hosts At the beginning of the season, in the absence of overlapping gene rations, the longer developmental time of P mexicana makes it unavailable at the second-instar meal ybug emergence, providing an advantage to A papayae in the competition for the hosts. Acerophagus papayae had high parasitism when present with both A loecki and P mexicana. The efficiency of A loecki may have been affected by not being host specific, and bei ng a parasitoid of commonly found P madeirensis. Longer development time of P mexicana reduces its competitiveness with A papayae and A loecki. Information gathered from these studies will provide the insight n eeded to understand the life history of P marginatus in relation to its host plants a nd temperature, and to explain the effectiveness of its three introduced parasitoids, A papayae A loecki and P mexicana in the classical biological control of P marginatus in the field. 104

PAGE 105

REFERENCE LIST Andrewartha, H. G., and L. C. Birch. 1954. The distribution and abundance of animals. Chicago Press. Chicago University, Chicago, IL. Begum, S., A. Naeed, B. S. Siddiqui, and S. Siddiqui. 1994. Chemical constituents of the Genus Plumeria J. Chem. Soc. Pak. 16: 280-299. Ben-Dov, Y. 1994. A systematic Catalogue of the meal ybugs of the world. Intercept, Hants, UK. Bertschy, C., T. C. J. Turlings, A. Bellotti, and S. Dorn. 2000. Host stage preference and sex allocation in Aenasius vexans, an encyrtid parasitoid of the cassava mealybug. Entomol. Exp. Appl. 95: 283-291. Boavida, C., and P. Neuenschwander. 1995. Influence of the host plant on the mango mealybug, Rastrococccus invadens. Entomol. Exp. Appl. 76: 179-188. Boavida, C., M. Ahounou, M. Vos, P. Ne uenschwander, and J. J. M. van Alphen. 1995a Host stage selection and sex allocation by Gyranusoidea tebygi (Hymenoptera: Encyrtidae), a parasitoid of the mango mealybug, Rastrococcus invadens (Homoptera: Pseudococcidae). Biol. Contol. 5: 487-496. Boavida, C., P. Neuenschwander, and P. Herren. 1995b. Experimental assessment of the impact of the introduced parasitoid Gyranusoidea tebygi Noyes on the mango mealybug Rastrococcus invadens Williams, by physical exclus ion. Biol. Control. 5: 99-103. Bokonon-Ganta, A. H., and P. Neuenschwander. 1995a. Impact of the biological control agent Gyranusoidea tebygi Noyes (Hymenoptera : Ency rtidae) on the mango mealybug, Rastrococcus invadens Williams (Homoptera: Pseudococcidae), in Benin. Biocontrol Sci. and Tech. 5: 95-107. Bokonon-Ganta, A. H., P. Neuenschwander, J. J. M. van Alphen, and M. Vos. 1995b. Host stage selection and sex allocation by Anagyrus mangicola (Hymenoptera: Encyrtidae), a parasitoid of the mango mealybug, Rastrococcus invadens (Homoptera: Pseudococcidae). Biol. Control. 5: 479-486. Bokonon-Ganta, A. H., J. J. M. van Alphen, and P. Neuenschwander. 1996. Competition between Gyranusoidea tebygi and Anagyrus mangicola parasitoids of mango mealybug, Rastrococcus invadens : interspecific host discrimina tion and larval competition. Entomol. Exp. Appl. 79:179-185. Borror, D. J., C. A. Triplehorn, and N. F. Johnson. 1992. An Introduction to the Study of Insects. 6th ed. Harcourt Brac e College Publishers, Orlando, FL. 105

PAGE 106

Buss, E. A., and J.C. Turner. 2006. Scale insects and mealybugs on ornamental plants. EENY-323. Featured Creatures. Entomology and Nematology Department, Florida Cooperative Extension Service, Institute of Food and Agricultu ral Sciences, University of Florida, Gainesville, FL. ( http://edis.ifas.ufl.edu/ October 2007) Calatayud, P. A., B. Delobel, J. Guillaud, and Y. Rahbe. 1998. Rearing the cassava mealybug, Phenacoccus manihoti on a defined diet. Entomol. Exp. Appl. 86: 325-329. Campbell, A., B. D. Frazer, N. Gilbert, A. P. Gutierrez, and M. Mackauer. 1974. Temperature requirements of some aphids and their parasites. J. Appl. Ecol. 11: 431-438. Center for North American Studies (CNAS). 2007. Economic impact of greening on the Texas citrus industry. Issue Brief 2007-1. Department of Agriculture Economics, Texas Agricultural experiment Station, Texas A & M University, College Station, TX. Chandler, L. D., D. E. Meyerdirk, W. G. Hart, and R. G. Garcia. 1980. Laboratory studies of the development of the parasite Anagyrus pseudococci (Girault) on insectary-reared Planococcus citri (Risso). Southwest Entomol. 5: 99-103. Chapman. R. F. 1998. The insects structure and function. 4 th ed. Cambridge University Press, New York, NY. Charnov, E. L., R. L. L. Hartogh, W. T. Jones, and J. van den Assem. 1981. Sex ratio evolution in a variable envi ronment. Nature. 289: 27-33. Chong, J-H., R. D. Oetting, and M. W. van Iersel. 2003. Temperature effects on the development, survival, and re production of the Madeira mealybug, Phenacoccus madeirensis Green (Hemiptera: Pseu dococcidae). Ann. Entomol. Soc. Am. 96: 539-543. Criley, R. A. 1998. Plumeria. Ornamentals and Flow ers. OF-24. Cooperative Extension Service, College of Tropical Agriculture and Human Resources, University of Hawaii, Manoa, Hawaii. Cross, A. E., and D. Moore. 1992. Developmental studies on Anagyrus mangicola (Hymenoptera: Encyrtidae), a pa rasitoid of the mango mealybug Rastrococcus invadens (Homoptera: Pseudococcidae). 82: 307-312. Dent, D. 1995. Principles of integrated pest management pp. 8-46. In D. Dent (eds.).Integrated Pest Management. Chapma n and Hall. New York. NY. Dhileepan, K. 2001. Effectiveness of introduced biocontrol insects on the weed Parthenium hysterophorus (Asteraceae) in Australia. Bull. Entomol. Res. 91: 167-176. 106

PAGE 107

Dhileepan, K., C. J. Lockett, and R. E. McFadyen. 2005. Larval parasitism by native insects on the introduced stem-galling moth Epiblema strenuana Walker (Lepidoptera : Tortricidae) and its implicatio ns for biological control of Parthenium hysterophorus (Asteraceae). Aus. J. Entomol. 44: 83-88. Doyon, J., and G. Boivin. 2005. The effect of development time on the fitness of female Trichogramma evanescens. J. Insect. Sci. 5: 1-5. Ferreira de Almeida, M., A. Pires Do Prado, and C. J. Geden. 2002. Influence of temperature on development time and longevity of Tachinaephagus zealandicus (Hymenoptera: Encyrtidae), a nd effects of nutrition and em ergence order on longevity. Biol. Control. 31: 375-380. Gauld, D. 1986. Taxonomy, its limitations and its role in understanding parasitoid biology. pp. 1-19. In J. Waage and D. Greathead. (eds.) Insect Parasitoids. Academic Press Inc. Orlando, FL. Gilman, E. F. 1999a. Acalypha wilkesiana Fact Sheet. FPS-6. Florid a Cooperative Extension Service, Institute of Food and agricultural Sciences, Universi ty of Florida, Gainesville, FL. Gilman, E. F. 1999b. Hibiscus rosa-sinensis Fact Sheet. FPS-254. Florida Cooperative Extension Service, Institute of Food and agri cultural Sciences, University of Florida, Gainesville, FL. Gonzales-Hernandez, H., M. W. Johnson, and N. J. Reimer. 1999. Impact of Pheidole megacephala (F.) (Hymenoptera: Formicidae) on the biological control of Dysmicoccus brevipes (Cockerell) (Homoptera: Pseudococ cidae). Biol. Control. 15: 145-152. Goolsby, J. A., A. A. Kirk, and D. E. Meyerdirk. 2002. Seasonal phenology and natural enemies of Maconellicoccus hirsutus (Hemiptera: Pseudococcidae) in Australia. Florida Entomol. 85: 494498. Gordan, H. T. 1999. Growth and development of insects. pp. 55-82. In C. B. Huffaker and A. P. Gutierrez (eds.), Ecological Entomology. 2 nd ed. John Wiley and Sons, Inc., New York, NY. Greathead, D. J. 1986. Parasitoids in classical biological control. pp. 290-315. In J. Waage and D. Greathead. (eds.) Insect Parasitoid s. Academic Press Inc., Orlando, FL. Hansen, L. S. 2000. Development time and activity threshold of Trichogramma turkestanica on Ephestia kuehniella in relation to temperature. Entomol. Exp. Appl. 96: 185-188. Harborne, J. B. 2001. Twenty-five years of chemical ecology. Nat. Prod. Rep. 18: 361-379. 107

PAGE 108

Harris, P. 1990. Environmental impact of introduced biological control agents. pp. 295-303. In M. Mackauer, L. E. Ehler and J. Roland (eds .), Critical Issues in Biological Control. Intercept Ltd, Andover, Hants, UK. Hemerik, L., and J. A. Harvey. 1999. Flexible larval development and the timing of destructive feeding by a solit ary endoparasitoid: an op timal foraging problem in evolutionary perspective. Ecological Entomol. 24: 308-315. Herrera, C. J., and R. G. van Driesche, and A. C. Bellotti. 1989. Temperature-dependent growth rates for the cassava mealybug, Phenacoccus herreni and two of its encyrtid parasitoids, Epidinocarsis diversicornis and Acerophagus coccois in Columbia. Entomol. Exp. Appl. 50: 21-27. Heu, R. A., M. T. Fukada, and P. Conant. 2007. Papaya mealybug, Paracoccus marginatus Williams and Granara de Willink (Hemiptera : Pseudococcidae). New Pest Advisory. 4(3). State of Hawaii Department of Agriculture, Honolulu, HI. Hodek, I., K. S. Hagen, and H. F. Van Emden. 1972. Methods for studying effectiveness of natural enemies, pp. 147-188. In H.F. Van Emden (eds.), Aphid Technology. Academic Press, London, England. Hoy, M. A., A. Hamon, and R. Nguyen. 2006. Pink hibiscus mealybug, Maconellicoccus hirsutus (Green). EENY-29. Featured Creat ures. Entomology and Nematology Department, Florida Cooperative Extension Se rvice, Institute of Food and Agricultural Sciences, University of Fl orida, Gainesville, FL. ( http://edis.ifas.ufl.edu/ October 2007) Huffaker, C., A. Berryman, and P. Turchin. 1999. Dynamics and regulation of insect Populations, pp. 269-305. In C. B. Huffaker a nd A. P. Gutierrez (eds.), Ecological Entomology. 2 nd ed. John Wiley and Sons Inc., New York, NY. Ingram, D. L., and L. Rabinowitz. 2004. Hibiscus in Florida. ENH-44. Environmental Horticulture Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL. Islam, K. S., and M. J. W. Copland. 1997. Host preference and progeny sex ratio in a solitary koinobiont mealybug endoparasitoid, Anagyrus pseudococci (Girault), in response to its host stage. BioControl Sc i. and Tech. 7: 449-456. Kairo, M. T. K., G. V. Pollard, D. D. Peterkin, and V. F. Lopez. 2000. Biological control of the hibiscus mealybug, Maconellicoccus hirsutus Green (Hemiptera: Pseudococcidae) in the Caribbean. Integrated Pest Man. Rev. 5: 241-254. King, B. H. 1987. Offspring sex ratios in parasito id wasps. Q. Rev. Biol. 62: 367-396. 108

PAGE 109

Kiritani, K., and J. P. Dempster. 1973. Different approaches to the quantitative evaluation of natural enemies. J. Appl. Ecol. 10: 323-329. Laflin, H. M., and M. P. Parrella. 2004. Developmental biology of citrus mealybug under conditions typical of California rose production. Ann. Ento mol. Soc. Am. 97: 982-988. Lawrence, P. O. 1981. Interference competition and optimal host selection in the parasitic wasp, Biosteres longicaudatus. Ann. Entomol. Soc. Am. 74: 540-544. Lema. K. M., and H. R. Herren. 1985. The influence of consta nt temperature on population growth rates of the cassava mealybug, Phenacoccus manihoti Entomol Exp. Appl. 38: 165-169. Le Ru, B., and A. Mitsipa. 2000. Influence of the host plant of the cassava mealybug Phenacoccus manihoti on life-history parameters of the predator Exochomus flaviventris Entomol. Exp. Appl. 95: 209-212. Logan, J. A., D. J. Wollkind, S. C. Hoyt, and L. K. Tanigoshi. 1976. An analytic model for description of temperature dependent rate phenomena in arthropods. Environ. Entomol. 5: 1133-1140. Mani, M., and T. S. Thontadarya. 1989 Development of the encyrtid parasitoid Anagyrus dactylopii (How.) on the grape mealybug Maconellicoccus hirsutus (Green). Entomon. 14: 49-51. Manzano, M. R., J. C. van Lenteren, C. Cardona, and Y. C. Drost. 2000. Developmental time, sex ratio, and longevity of Amitus fuscipennis MacGown and Nebeker (Hymenoptera: Platygasteridae) on the gree nhouse whitefly. Biol. C ontrol. 18: 94-100. Marohasy, J. 1997. Acceptability and suitability of seven plant species for the mealybug Phenacoccus parvus Entomol. Exp. Appl. 84: 239-246. Mead, F. W., 2007.. Asian citrus psyllid, Diaphorina ci tri Kuwayama (Insecta: Hempitera: Psyllidae). EENY-33. Featured Creatures. Ento mology and Nematology Department, Florida Cooperative Extension Service, Inst itute of Food and Agricultural Sciences, University of Florida, Gainesville, FL. ( http://edis.ifas.ufl.edu/ October 2007) Mersie, W., and M. Singh. 1988. Effects of phenolic acids and ragweed parthenium ( Parthenium hysterophorus) extracts on tomato ( Lycopersicon esculentum ) growth and nutrient and chlorophyll conten t. Weed Sci. 36: 278-281. Messenger, P. S. 1959. Bioclimatic studies with insects. Ann. Rev. Ent. 4: 183-206. 109

PAGE 110

Meyerdirk, D. E., 2003. Control of papaya mealybug, Paracoccus marginatus (Hemiptera: Pseudococcidae). Environmental Assessment (Supplement). Center for Plant Health Science and Technology, National Biologica l Control Institute, PPQ, APHIS, USDA, Riverdale, MD. Meyerdirk, D. E., R. Muniappan, R. Warke ntin, J. Bamba, and G. V. P. Reddy. 2004. Biological control of the papaya mealybug, Paracoccus marginatus (Hemiptera: Pseudococcidae) in Guam. Plant Prot. Quart. 19: 110-114. Miller, D. R., and G. L. Miller. 2002. Redescription of Paracoccus marginatus Williams and Granara de Willink (Hemiptera: Coccoidea: Ps eudococcidae) including descriptions of the immature stages and adult male. Proc. Entomol. Soc. Wash. 104: 1-23. Miller, D. R., D. J. Williams, and A. B. Hamon. 1999. Notes on a new mealybug (Hemiptera: Coccoidea: Pseudococcidae) pest in Flor ida and the Caribbean: the papaya mealybug, Paracoccus marginatus Williams and Granara de Willi nk. Insecta Mundi. 13: 179-181. Mizell III, R. F., and T. E. Nebeker. 1978. Estimating the developmental time of the southern pine beetle Dendroctonus fronatlis as a function of field temp eratures. Environ. Entomol. 7: 592-595. Mossler, M. A. and N. Nesheim. 2002. Florida crop/pest manageme nt profile: papaya. CIR1402. Pesticide Information Office, Food Sc ience and Human Nutrition Department, Florida Cooperative Extension Service, Inst itute of Food and Agricultural Sciences, University of Florida, Gainesville, FL. ( http://edis.ifas.ufl.edu/ October 2007) Muniappan, R., D. E. Meyerdirk, F. M. Sengeb au, D. D. Berringer, and G. V. P. Reddy. 2006. Classical biological c ontrol of the papaya mealybug, Paracoccus marginatus (Hemiptera: Pseudococcidae) in the republic of Palau. Florida Entomol. 89: 212-217. Narai, Y., and T. Murai. 2002. Individual rearing of the Japanese mealybug, Planococcus kraunhiae (Kuwana) (Homoptera : Pseudococcidae) on a germinated broad bean seeds. Appl. Entomol. Zool. 37: 295-298. National Climatic Data Center (NCDC). 2005. Comparative Climatic Data. National Climatic Data Center, National Oceani c and Atmospheric Administration, US Department of Commerce, Ashville, NC. ( http://www.ncdc.noaa.gov/oa/c limate/online/ccd/nrmavg.txt October 2007) Neuenschwander, P. 2001. Biological control of the cassava mealybug in Africa: a review. Biol. Control. 21: 214-229. Neuenschwander, P., F. Schulthess, and E. Madojemu. 1986. Experimental evaluation of the efficiency of Epidinocarsis lopezi a parasitoid introduced into Africa against the cassava mealybug Phenacoccus manihoti Entomol. Exp. Appl. 42: 133-138. 110

PAGE 111

Noyes, J. S. 2000. Encyrtidae of Costa Rica, I. The subfamily Tetracneminae (Hymenoptera: Chalcidoidea), parasitoids of mealybugs (Hom optera: Pseudococcidae). Mem. Amer. Ent. Inst. 62: 101-103. Noyes, J. H., and M. E. Schauff. 2003. New Encyrtidae (Hymenoptera) from papaya mealybug ( Paracoccus marginatus Williams and Granara de Willink) (Hemiptera: Sternorrhyncha: Pseudococcidae). Proc. Entomol. Soc. Wash. 105: 180-185. Orr, D. B., and C. P.-C. Suh. 1998. Parasitoids and predators. pp. 3-34. In J. E. Rechcigl and N. A. Rechcigl (eds.), Biological and biotec hnological control of in sect pests. CRC Press LLC, Boca Raton, FL. Picman, J., and A. K. Picman. 1984. Autotoxicity in Parthenium hysterophorus and its possible role in control of germin ation. Biochem. Syst. Ecol. 12: 287-292. Raghubanshi, A. S., L. C. Rai, J. P. Gaur, and J. S. Singh. 2005. Invasive alien species and biodiversity in India. Current Sci. 88: 539-540. Ridley, M., 1988. Mating frequency and fecundity in insects. Bio. Rew. 63: 509-549. Ridley, M., 1993. A sib competitive relation between clutch size and mating frequency in parasitic Hymenoptera. Am. Naturalist. 142: 893-910. Rosenheim, J. A., H. K. Kaya, L. E. Ehler, J. J. Marois, and B. A. Jaffee. 1995. Intraguild predation among biological-contro l agents: theory and eviden ce. Biol. Control. 5: 303335. Sagarra, L. A., and C. Vincent. 1999. Influence of host stage on oviposition, development, sex ratio, and survival of Anagyrus kamali Moursi (Hymenopte ra: Encyrtidae), a parasitoid of the hibiscus mealybug, Maconellicoccus hirsutus Green (Homoptera: Pseudococcidae). Biol. Control. 15: 51-56. Sagarra, L. A., C. Vincent, N. F. Peters, and R. K. Stewart. 2000a. Effect of host density, temperature, and photoper iod on the fitness of Anagyrus kamali a parasitoid of the hibiscus mealybug Maconellicoccus hirsutus. Entomol. Exp. Appl. 96: 141-147. Sagarra, L. A., C. Vincent, and R. K. Stewart. 2000b. Fecundity and survival of Anagyrus kamali (Hymenoptera: Encyrtidae) under different feeding and storage temperature. Eur. J. Entomol. 97: 177-181. Sagarra, L. A., C. Vincent, and R. K. Stewart. 2002. Impact of mating on Anagyrus kamali Moursi (Hymenoptera: Encyrtidae) lifetime fecundity, reproduc tive longevity, progeny emergence, and sex ratio. J. Appl. Entomol. 126: 400-404. SAS Institute. 1999. SAS user's guide. Version 9.1. SAS Institute, Cary, NC. 111

PAGE 112

Schroder, D. 1974. A study of the interactions between the internal larval parasites of Rhyacionia buoliana (Lepidoptera:Olethreutidae). Entomophaga. 19: 145-171. Sequeira, R., and M. Mackauer. 1992. Covariance of adult size a nd development time in the parasitoid wasp Aphidius ervi in relation to the size of its host, Acyrthosiphon pisum Evolutionary Ecol. 6: 34-44. Serrano, M. S., and S. L. Lapointe. 2002. Evaluation of host plants and a meridic diet for rearing Maconellicoccus hirsutus (Hemiptera: Pseudococcidae) and its parasitoid Anagyrus kamali (Hymenoptera: Encyrtidae). Florida Entomol. 85: 417-425. Sinacori, A. 1995. Bio-ethological observations on Phenacoccus madeirensis Green (Coccoidea: Pseudococcidae) in Sic ily. Israel J. Entomol. XXIX: 179-182. Sloan, S. A., J. K. Zimmerman, and A. M. Sabat. 2007. Phenology of Plumeria alba and its herbivores in a tropical dr y forest. Biotropica. 39: 195-201. Smith, H. S., and P. DeBach. 1942. The measurement of the effect of entomophagous insects on population densities of the host. J. Econ. Entomol. 35: 845-849. Smith, M. R. 1928. The biology of Tapinoma sessile Say, an important house-infesting ant. Ann. Entomol. Soc. Am. XXI: 307-330. Tefera, T. 2002. Allelopathic effects of Parthenium hysterophorus extracts on seed germination and seedling growth of Eragrostis tef J. Agron. Crop Sci. 188: 306-310. Townsend, M. L., R. D. Oetting, and J.-H. Chong. 2000. Management of the mealybug Phenacoccus madeirensis Proc. South. Nurs. Assoc. Res. Conf. 45: 162-166. Trudgill, D. L., A. Honek, D. Li, and N. M. Van Straalen. 2005. Thermal time-concepts and utility. Ann. Appl. Biol. 146: 1-14. Uckan, F., and E. Ergin. 2002. Effect of host diet on the immature developmental time, fecundity, sex ratio, adult longevity, and size of Apanteles galleriae (Hymenoptera: Braconidae). Biol. Control. 31: 168-171. USDA, NRCS. 2007a. The Plants Database. National Plant Data Center, Baton Rouge, LA. ( http://plants.usda.gov/ October 2007) USDA, NASS. 2007b. Hawaii papayas. National Agricultural St atistics Service, Honolulu, HI. ( http://www.nass.usda.gov/hi October 2007) Van Driesche, R. G., and T. S. Bellows Jr. 1996. Biological Control. Kluwer Academic Publishers, Norwell, MA. 112

PAGE 113

Van Lenteren, J. C. 1980. Evaluation of control capabilities of natural enemies. Does art have to become science? Neth. J. Zool. 30: 369-381. Van Strien-van Liempt, W. T. F. H. 1983. The competition between Asobara tabida Nees von Esenbeck, 1834 and Leptopilina heterotoma (Thomson, 1862) in multiparasitized hosts. Neth. J. Zool. 33: 125-163. Vinson, S. B. 1976. Host selection by insect parasitoids. Ann. Rev. Entomol. 21: 109-133. Vinson, S. B., and G. F. Iwantsch. 1980. Host suitability for insect parasitoids. Ann. Rev. Entomol. 25: 397-419. Waage, J. K. 1986. Family planning of parasitoids: ad aptive patterns of progeny and sex allocation. pp. 63-89. In J. Waage and D. Greathead. (eds.) Insect Parasitoids. Academic Press Inc., Orlando, FL. Wagner, T. L., H.-I. Wu, P. J. H. Sharpe, R. M. Schoolfield, and R. N. Coulson. 1984. Modeling insect development rates: a literatur e review and applica tions of a biophysical model. Ann. Entomol. Soc. Am. 77: 208-225. Walgama. R. S., and M. P. Zalucki. 2006. Evaluation of different mo dels to describe egg and pupal development of Xyleborus fornicatus Eichh. Coleoptera: Scolytidae), the shot-hole borer of tea in Sri Lanka Insect Sci. 13: 109-118. Walker, A., M. Hoy, and D. Meyerdirk. 2003. Papaya mealybug ( Paracoccus marginatus Williams and Granara de Willink (Insecta: Hemiptera: Pseudococcidae)). EENY-302. Featured Creatures. Entomology and Nematology Department, Florida Cooperative Extension Service, Institute of Food and agri cultural Sciences, University of Florida, Gainesville, FL. ( http://edis.ifas.ufl.edu/ October 2007) Williams, D. J., and M. C. Granara de Willink. 1992. Mealybugs of Central and South America. CAB International, Wallingford, Oxon, UK. Yang, J., and C. S. Sadof. 1995. Variegation in Coleus blumei and the life history of citrus mealybug (Homoptera : Pseudococcidae). Environ. Entomol. 24: 1650-1655. Zar, J. H. 1984. Biostatistical Analysis, 2 nd ed. Prentice Hall, Englewood Cliffs, NJ. 113

PAGE 114

BIOGRAPHICAL SKETCH Born in Anuradhapura, Sri Lanka, Kaushaly a Gunewardane Amarasekare graduated from the University of Peradeniya, Sri Lanka with a B achelor of Science in agriculture with honors in October 1993. After graduation, sh e worked for the Department of Agriculture, Sri Lanka as a research scientist. In 2000, she was offered an assistantship to st udy entomology at Oklahoma State University, Stillwater, Oklahoma, under the guidance of Dr. J onathan Edelson. Upon receiving a Master of Science in December 2002, she moved to Fl orida to pursue her Doctor of Philosophy degree at the University of Flor ida with advisor, Dr. Catharine Mannion. 114


xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20101118_AAAAAK INGEST_TIME 2010-11-18T08:20:04Z PACKAGE UFE0021652_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 9387 DFID F20101118_AAAHXM ORIGIN DEPOSITOR PATH amarasekare_k_Page_111thm.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
fd1473de3066527e7f8066857c8241cb
SHA-1
aec099ec1a8a39ecb5eebb0845be8d9ac680b705
25271604 F20101118_AAAGUK amarasekare_k_Page_008.tif
7fe89eab41160ed7ab74ea663a96a447
f4c42cb2cfbcfff191e37fb01cdc8ff0b483564c
2097 F20101118_AAAGTW amarasekare_k_Page_065.txt
2a2513a4ccfd4b39b4a225ac90b7fdcb
15164db085cab016f6d263eac138ff49dbb5e59d
9252 F20101118_AAAHWY amarasekare_k_Page_096thm.jpg
c10e77839f36c9e546105dc35d107ca1
2a8344b2da9a723d70e840a7e5778d6aec559d61
104067 F20101118_AAAHAE amarasekare_k_Page_046.jpg
d5aadf2b2f97a25fc3adab31acb21d43
54292570200ab06109c0d445debaaaf6ba7facf3
37942 F20101118_AAAHXN amarasekare_k_Page_111.QC.jpg
333994c1dd7635fa2de2de825201121d
c02a135802a767565debbd65b6d1accc3317ec7d
1053954 F20101118_AAAGUL amarasekare_k_Page_101.tif
de3379b5c4f1ebf9ede758eacc85ee56
8879ff633d3df85a7716ea2b3825ca36bf234936
2201 F20101118_AAAGTX amarasekare_k_Page_091.txt
318d89634ecac0aa1eea2a3264bdd0df
bf1100b9692227dac28e646f8dc26fa2e0701c6f
8822 F20101118_AAAHWZ amarasekare_k_Page_098thm.jpg
cc0673a470e5458432246e760391819c
6ef64298a11b489cabc3ac90b40597c31ef730db
112041 F20101118_AAAHAF amarasekare_k_Page_047.jpg
2c71e6fd4d2452e57ff65351c57f8f80
c263ae62a70c6a9a22bc50ec4230ce14880b3fcf
37321 F20101118_AAAHXO amarasekare_k_Page_112.QC.jpg
d46fa4273c7c0090b1015d8ce8575db2
3ceff76ae6bb1fc125177ada9b5d74c04458d35b
1252 F20101118_AAAGVA amarasekare_k_Page_039.txt
62cf570ac350f7681d39e296bbd0da0f
75c2bbf5efc86ff4a95fa574f47bf2d9263968eb
1644 F20101118_AAAGUM amarasekare_k_Page_067thm.jpg
58f124e9ac36686a980e20574a932c1d
82dec916bb4a07ab64a598c067ab4ee2f3a9c437
118783 F20101118_AAAHAG amarasekare_k_Page_048.jpg
13de5d01cb4f61b0da109abbf21a0c09
c011ad85cc79a28b731b2e4ca330e50a91852558
32109 F20101118_AAAHXP amarasekare_k_Page_113.QC.jpg
5ca6824b7217ae598de928811db84411
b9eb9f3ea47a9f717e6c5a883d4b02846d19e832
113676 F20101118_AAAGVB amarasekare_k_Page_042.jpg
148beac75d7e39cbf0175e07d6aa524e
362bb26fe756cb60dbc48ac0809bc7016de240c7
56603 F20101118_AAAGUN amarasekare_k_Page_088.pro
d889c7a3ea27b2ef260f9b4965c20cad
b2041700b194fa25cdf51ae3dc99c0c98b1c9e42
9236 F20101118_AAAGTY amarasekare_k_Page_107thm.jpg
f8735a8ccd86f2a134387c324323886c
ec2b5eb31bf50f16247e858407dcaa7730b2bb6b
115687 F20101118_AAAHAH amarasekare_k_Page_050.jpg
b5af3049cd257e1008a6d1d4aecbf014
c6febf5199b400394461134c735c82bc45ddf9b0
3346 F20101118_AAAHXQ amarasekare_k_Page_114thm.jpg
3e2394b7aa92fc05ffe2dc4ba1fb1344
6f807f1caf08e66932f7146764d1d5b1e491f1a6
51588 F20101118_AAAGVC amarasekare_k_Page_040.pro
5fca1ef66563268bd83fe442bf293457
e13105ed921b66bc4e70f3e2cb99127773ae25db
1051974 F20101118_AAAGUO amarasekare_k_Page_113.jp2
9ba247fc5640df79665cd609b6826605
8ce1e08f6963b49d192e1aeb907756f06e926b73
56645 F20101118_AAAGTZ amarasekare_k_Page_061.pro
3884446dfbfd244c61dcbd1004ae23f3
4d8f01af8f8d2f8c06c8d54f642c093f81bffef8
96938 F20101118_AAAHAI amarasekare_k_Page_051.jpg
6c124c62d97cf15333ee959ae4fe8e7c
f2c37bad045ef4407cf1b7ea30ab1cede91d7991
8994 F20101118_AAAGVD amarasekare_k_Page_076thm.jpg
0a20c629465bf60f77684944fbebcef9
b3fab4cdea549d38116d80a76a594c17bd526927
2892 F20101118_AAAGUP amarasekare_k_Page_100thm.jpg
2a5125ddc2ed870fa8ed1b4c119f8b7e
07365aba2d345b6c39eb567c39df8baa15f0b5e1
37738 F20101118_AAAHAJ amarasekare_k_Page_052.jpg
04d3d6e093ae99d1ce6780783a273c50
f09c6006c3f0f4147ab58d37ec21c6ed2fcd5c2d
F20101118_AAAGVE amarasekare_k_Page_110.tif
85b8ebb63837980784ce2226a65d1b10
674efb8dccfcf59c184c66a25fb96f6599d9882a
1054428 F20101118_AAAGUQ amarasekare_k_Page_054.tif
56d495d191cc2654af29762be8ab52d7
0e42805a02a290cc67360c00eb43f90b6853ffe6
36753 F20101118_AAAHAK amarasekare_k_Page_053.jpg
ea472bd569e39e64e6349342816c890b
81393526ffb519b51ce64faec216451db100dafb
2037 F20101118_AAAGVF amarasekare_k_Page_075.txt
50608ab53ecc3518a1dd5507758a5e61
89b65212f98e1d075dd224810150a6277f4c8db0
2801 F20101118_AAAGUR amarasekare_k_Page_083thm.jpg
59f6d4110537744c134849d6c672e1c5
28bcc967914873c89dbe8f016c55c7521bdab6d4
114497 F20101118_AAAHBA amarasekare_k_Page_072.jpg
a19610a5060b725097fd7e8b9a5ed83a
7e8afb8a5fe3f671b7cdb2a50d66485cc4b581cd
115561 F20101118_AAAHAL amarasekare_k_Page_056.jpg
61f400598c577a4b27c1bd6ce2117d55
8b6c058487a0caecb285e432eefabf363041fb30
38220 F20101118_AAAGVG amarasekare_k_Page_058.QC.jpg
13468b067de15616935b1042980e1548
ffe300d03fb014f45a89e7a4d64a5dfaa1db697c
37283 F20101118_AAAGUS amarasekare_k_Page_022.QC.jpg
a48615e69e262eb65030a99ed17add1d
e35624556efcf6dcc0c97f7d506eeb7f82e99748
114156 F20101118_AAAHBB amarasekare_k_Page_074.jpg
af4ae2fbcd6772f437f3e33754cb2a7f
cfe4ad3861895c91a77475d8e06029a207be307b
118470 F20101118_AAAHAM amarasekare_k_Page_057.jpg
3ce92d6e9606d2a7e08770b8e18c0a66
2fe367eb8b6008471c8bb380f123d607416badcd
2149 F20101118_AAAGVH amarasekare_k_Page_056.txt
30d5834cf58dd17fd630f25afe45c6d8
d8b29792c06cd16790ef4b49d7ed636a7ad54630
26985 F20101118_AAAGUT amarasekare_k_Page_068.pro
91f4acb232e77cfb571945dca4de5d31
ff537c74f00878abd2516d2a7b91b88074553fda
106901 F20101118_AAAHBC amarasekare_k_Page_075.jpg
821b45d58d7aef81870f077441aa470d
2b8c5ee99c15f871c16b6663595aedd571d6ba50
116185 F20101118_AAAHAN amarasekare_k_Page_058.jpg
37d2141ed33e0d85946b000774a2a9d7
2877df695a1bfdaacd967826ebadc96a147bf6cf
59208 F20101118_AAAGVI amarasekare_k_Page_055.jpg
564d4351ddce7a837ec59cf0fcd23ab2
f62f43723076c5378d2a1d7408182561c851dd94
1051979 F20101118_AAAGUU amarasekare_k_Page_106.jp2
8304a96b3f981695e44e6b0deaddacd8
b4f3a0c2fecf9650d70337875e01062bbdbed377
112026 F20101118_AAAHBD amarasekare_k_Page_076.jpg
76aef0e2080639f09de50f1ae6d17ffb
ea37765f4c229d0e97d1b3c68668c3a79e50169a
115541 F20101118_AAAHAO amarasekare_k_Page_059.jpg
a57aabed304551288d41a963bd37f534
e87fa5498c3768f0f14ba6f1737a8deb46f90a44
F20101118_AAAGVJ amarasekare_k_Page_099.tif
18661c52b2899735980efc0e497ce44c
26c694726d21768a969f4d626f51ce2a6ccf5564
53664 F20101118_AAAGUV amarasekare_k_Page_081.pro
18fcf00046f56a1e3088cf4f74e73ef1
de2ab0921b455c7e553f373ac29d7ca5be193aa2
104884 F20101118_AAAHBE amarasekare_k_Page_077.jpg
0cc78b07bc15f3d664e69f9cb13a38d1
c06bd71d9e1f6b6fb624ff6e2d44ade2bebb9097
114925 F20101118_AAAHAP amarasekare_k_Page_061.jpg
cf0ab3364fa94ab01a6dbd0053bf5cce
d2ab7856729b809739e9f19fb4e68049df01ed76
1051913 F20101118_AAAGVK amarasekare_k_Page_063.jp2
3200c7c90ac61a43c4aa2b78e8eacdbf
a8436ce07cc86ea8d1d447bd3d4edc541994d94c
8531 F20101118_AAAGUW amarasekare_k_Page_077thm.jpg
059974904d12fdf556e2e9eba160e9db
78895c8aca5bbc4ae8b7120b524ba51a28315425
95219 F20101118_AAAHAQ amarasekare_k_Page_062.jpg
cc63553aa5ca8c466b852868cda72fa9
92629d26044f4b9c7fb567491e31c7b599659f45
24366 F20101118_AAAGVL amarasekare_k_Page_006.QC.jpg
0f4227de6b4bd8332b7e80af92d24842
c46add632aa6a8ca6a8590367316b3626a9deea6
111396 F20101118_AAAGUX amarasekare_k_Page_011.jp2
32949b05230439352410334b7d63ffaf
e891dde20655af32bedeb84ac4fcd1a59ea8efd4
112371 F20101118_AAAHBF amarasekare_k_Page_078.jpg
ee3c4780806d04bad018c6f2dc170c04
b637edb26bf66318a24683f7b5d65009162ab0e6
105493 F20101118_AAAHAR amarasekare_k_Page_063.jpg
619b85b32ecbade7527157375508f232
e5d83c2cad7003cb8fbc057edac7a1a884d04165
37548 F20101118_AAAGVM amarasekare_k_Page_091.QC.jpg
84f3648627877b81d89ada4913fa1d48
7e6591ba34b397c7401f1f725acbe8007bb7acb9
33590 F20101118_AAAGUY amarasekare_k_Page_084.pro
60ded335780d90e9138c8cfc7f62288d
2acbcc9d00d7f8fff0f92214aeb6fbda23d2d200
98503 F20101118_AAAHBG amarasekare_k_Page_079.jpg
f6f927793ff4d4ddfaa29dc82f697d67
5328077748763790160682cead8cda5b5dd0c39c
9315 F20101118_AAAGWA amarasekare_k_Page_059thm.jpg
3a01d41b34493dab0f5e0af0796a7f96
9af116f5bb405daf2f3017bc3cc992e4b8f60453
115730 F20101118_AAAHAS amarasekare_k_Page_064.jpg
29e927c53538ac08654297ec21629565
10f14860c23b6b79337e4248635b8ad669b04051
2610 F20101118_AAAGVN amarasekare_k_Page_055.txt
4474f09a4f087af7f80dc9bcba05b72b
30f32938c6fb7dfe47c3355d0acb9fb868fb2f35
113493 F20101118_AAAHBH amarasekare_k_Page_080.jpg
35c615b5dc87986ed0c0c7e6ac2c15d6
b9a40965b7a2fcf6d4fabd94205de7c50641ffee
119532 F20101118_AAAGWB amarasekare_k_Page_014.jpg
a6ed0e576b4c820b50349bfd9300ab91
80e264100eae0bf822ec933e334caad67d763298
111324 F20101118_AAAHAT amarasekare_k_Page_065.jpg
6813bcca6f73f096364fc3e1cea8437d
196b0edf2906e98ea178c9890c7ca99a8b5e4704
1051929 F20101118_AAAGVO amarasekare_k_Page_065.jp2
a3e177f2995362590b20344f682a9e55
04434eee72344c7912522a62b250fc1d45816eaf
8979 F20101118_AAAGUZ amarasekare_k_Page_078thm.jpg
1e33571e83299728042a252ee0cad4c9
019cf6f145fcc76cb6af5639fd43b09910fbfd12
110079 F20101118_AAAHBI amarasekare_k_Page_081.jpg
6be2684ad21c8fcea3d49c9f20d1c706
f545c5c6ce5985fac29151c0078de426cccf284d
1051978 F20101118_AAAGWC amarasekare_k_Page_034.jp2
ceb654b98727103ebe2881808fc7024a
16aab82c489bb832f611d835d0e79301e4144c25
113321 F20101118_AAAHAU amarasekare_k_Page_066.jpg
7a44caba06a937344ee674bbf9913a69
6b41d36ce96ccd591944a1b7fea29cd6fe0877a8
F20101118_AAAGVP amarasekare_k_Page_043.tif
d13bc1cd0072e21bb39e841cf4ffe067
cc85650ab2434a074a848d529b9b14edf7eceeb1
112646 F20101118_AAAHBJ amarasekare_k_Page_082.jpg
bcf5a10d53c0076b629d06da5a8cca2b
23b2900ec24b3c6794e4d4e0bcd4894189b1e2ab
1051950 F20101118_AAAGWD amarasekare_k_Page_056.jp2
c8f6bfb2c4fa040814871fa0ee911f6c
d63710819fb4891f61132162d04ba55fb2944b0c
16557 F20101118_AAAHAV amarasekare_k_Page_067.jpg
f2794366c4b826844a91ddd934d3b14d
83ff6c65ca95995a8865e5af676ab3c8feaceef0
94549 F20101118_AAAGVQ amarasekare_k_Page_055.jp2
aafd01a8c7e48d726477c04266abb3c9
9ccbceae4528f674a11f847b23fd4f984cfeadcd
72105 F20101118_AAAHBK amarasekare_k_Page_084.jpg
82ac56014ecb492fd9568145adea5128
6f7e570440f27bf693bd1fdc4bc6d428c225c667
28623 F20101118_AAAGWE amarasekare_k_Page_005.QC.jpg
1e4860bbaca8d425467e7420ad907f3a
8e82383f2193aef7edcfd39909c5ae28366c9744
51729 F20101118_AAAHAW amarasekare_k_Page_068.jpg
3604708320553b31697c549e45a5da28
606b6a5d756034f567ff838136b45fa3c56eccfd
37151 F20101118_AAAGVR amarasekare_k_Page_096.QC.jpg
4a132e7fa94e638eda7bc7d76789be65
e6d85f588fde49f91484a9b3c73735979f304616
119479 F20101118_AAAHCA amarasekare_k_Page_105.jpg
2e3069299dc9528d04ef669aa20e0ae1
df0784f5935872af258e10670a8f3b1d53a967bb
74576 F20101118_AAAHBL amarasekare_k_Page_085.jpg
90e6511d1688d8f4d45b14dd9b7bb641
06dd063c2084a6d5cf6a543820741eed1f500697
19146 F20101118_AAAGWF amarasekare_k_Page_100.pro
8bdd08e78833ad54f3ea8b058ed49312
41d3b0521ff1961bbef10a3cfad70f28928713db
52991 F20101118_AAAHAX amarasekare_k_Page_069.jpg
55d6e1a38304fc939d346f699d7b214f
8776c261eafdeeec3920bcbc5ef0fc9ffea5c7e0
115784 F20101118_AAAGVS amarasekare_k_Page_089.jpg
0a80617cb837fc14bafceb56eeba4f48
0c4d0728bcff3a4ee31bf08c64ab9a60af50d051
131178 F20101118_AAAHCB amarasekare_k_Page_106.jpg
354b435528415f15b81522c9de169234
d66603d562c5f534aba950b41bb5eaebd6c5d851
114683 F20101118_AAAHBM amarasekare_k_Page_087.jpg
2c834e383765acbd9a8ea81295709c0a
42b15f885b02f1d9cb15b66a870216d026f5a39d
9112 F20101118_AAAGWG amarasekare_k_Page_043thm.jpg
0790ca5a8c7d19fd0b02800ecfeb1e36
5b1e44b1204b519ed5ca536b1fecad2cd37b4be9
39092 F20101118_AAAHAY amarasekare_k_Page_070.jpg
39abb46a029f184e329a115d50a64cc4
c452acd01d355e5e972607778d3b9d93bfaa851a
F20101118_AAAGVT amarasekare_k_Page_006.tif
818fb8b7a79ddf4e6e478de7103116e9
2f0c5993e3da0695ba864834c2eff87859b55756
123938 F20101118_AAAHCC amarasekare_k_Page_107.jpg
dff35801bf4102b21ac460092a8c9389
385c6191933472c38a64a6700374d069f386f224
117207 F20101118_AAAHBN amarasekare_k_Page_088.jpg
ee912df16bcbe6306ba59826232b4435
c132a6499b1bd459ec3b813643eef3265bab3713
1353 F20101118_AAAGWH amarasekare_k_Page_053.txt
9bb59ce643b4603d4f44b915942357b1
477f2d2834ce63581a05bef9029ac822ced4cec4
43387 F20101118_AAAHAZ amarasekare_k_Page_071.jpg
9673a705b3d98c56eb0a0cf5254f30a9
2076ded79ed9fec6294312968e7f756b66b46495
116318 F20101118_AAAGVU amarasekare_k_Page_024.jpg
26857a3d7e72ba57eab48c776b662f5e
c1f51b051fd4c1e82fc5daf8996d799d539be978
137535 F20101118_AAAHCD amarasekare_k_Page_108.jpg
40f0714dacbb05c43dfb4b76d98057ed
b9ff5235709008dd0ed05c47edeb9361fc336346
112870 F20101118_AAAHBO amarasekare_k_Page_090.jpg
6a3c9d830eb19c0655ff32f5db7a642c
ddb0aed78b6443099b40cbc0cc61c6545e06d741
8210 F20101118_AAAGWI amarasekare_k_Page_045thm.jpg
dbdc257c699e78885e0d0385dad0f2fa
7f42da4ed071fefbc70b4d16a4fb66948654ba2c
9311 F20101118_AAAGVV amarasekare_k_Page_015thm.jpg
5db347169995243b95c0760de7a05df9
c798757e9c656c68c9f1f6485dee3fff6ee48541
121802 F20101118_AAAHCE amarasekare_k_Page_109.jpg
ed4d7cd1e0302b9fd48dcce88626fd43
446538bb2ebc94c0ecbd149bec74c58d863fc0a5
102225 F20101118_AAAHBP amarasekare_k_Page_092.jpg
a453095f529c9e54392bca133217f5c8
0309042343a442ef9d7db776d798caa819f9ea8e
919 F20101118_AAAGWJ amarasekare_k_Page_101.txt
35ab901ad99fcece7b3d023f6d3cd011
900553cbf493a317fbeb5ae95a7fd56179e8cb4a
F20101118_AAAGVW amarasekare_k_Page_112thm.jpg
b09cc95003d7d92fbeb4b8593daf3b06
170e3c346b0f5a7cd7a296e7f9029f9abb1cd3cd
147021 F20101118_AAAHCF amarasekare_k_Page_110.jpg
02bd476be779d9ab7fa1fcc40d74855d
dad060b15c51e940f9459c4eebdb17f6f3f9fd59
103604 F20101118_AAAHBQ amarasekare_k_Page_093.jpg
154bf7894455509a05bb7e7b2035c025
1ebd846c9c55219f0e1a6ec194f2482c418d63ea
53071 F20101118_AAAGWK amarasekare_k_Page_068.jp2
943c6be3c2ecf9cfab41fca6cb0de36f
bda449d251026ff5627e30c723ebaedd76d8e05d
2126 F20101118_AAAGVX amarasekare_k_Page_030.txt
36905bd7ca4c7b4f136ed20ed3efb73f
4dfcd0157b7b3acd66a7e53649ccf559aedd85b8
111472 F20101118_AAAHBR amarasekare_k_Page_094.jpg
0f030e5aec5ecd139f32d721c54c27d5
a396e9b932f92af0bcd96fc32b542882fc9ad582
1805 F20101118_AAAGWL amarasekare_k_Page_062.txt
61393b5fca0e8b4ede918a8f70107c2a
3b9cde6e18daf0af109a4e44d01ed2a3d08fa8a2
1051986 F20101118_AAAGVY amarasekare_k_Page_014.jp2
64c063ebf7780933b9ca231f543d9b58
521ac1048cdf2c5ce6fced7c0ed4461eba5ae88a
123503 F20101118_AAAHCG amarasekare_k_Page_112.jpg
af72283adc38bf55834619ef78aaac4b
45b9fb798d98aea0bf2804678e63adb91a4f942f
9260 F20101118_AAAGXA amarasekare_k_Page_017thm.jpg
62832114e70405d16930b50a44f87718
2c1c024bf5c179bcd6b5ade46126f288e9b8c48a
112360 F20101118_AAAHBS amarasekare_k_Page_095.jpg
44f7e15b2b333d88a2660add265b1f82
77de5edaa5befa9ee2b32c674e2fa41d35f7a425
2222 F20101118_AAAGWM amarasekare_k_Page_061.txt
c9adc80c63f74c23ea96692cdd543eb4
8ac5596fe2e2fe915c742f3074344bb9779e50f2
118027 F20101118_AAAGVZ amarasekare_k_Page_031.jp2
a3721783c6074118d84e5c08ac7375d4
7e5f41f0149ca23ec18a8ac72530e8fcd098d9b3
106199 F20101118_AAAHCH amarasekare_k_Page_113.jpg
10ac5e39fa6799ea8f7468fe97ea1fd7
8a9e98a60546777aca5ee4a512a179191acd76d5
36532 F20101118_AAAGXB amarasekare_k_Page_049.QC.jpg
0a76559301364442e4d1bc4a79dacca8
e3c8a53cf5e38693f4080e4af583909dd7ff66f3
107553 F20101118_AAAHBT amarasekare_k_Page_097.jpg
c22d84effaf64549844018e229267e43
2dcd39e7ca612357a26218d0a94d9ee21bacb4e6
9142 F20101118_AAAGWN amarasekare_k_Page_050thm.jpg
b0d13517d145d01514d9e1fcbcfbdf00
fcd467f56add52a42a2fd0767bd0b29a2b08cada
40430 F20101118_AAAHCI amarasekare_k_Page_114.jpg
acc70a3745b86dc2ec5ea8b4d68edb55
2f6c475f651269dcba699f9109df49fa621cf927
3754 F20101118_AAAGXC amarasekare_k_Page_068thm.jpg
33e432ae89cc9457cf9cc7d39910273b
a473597a4923f33cdbe08a06e37ee44cbc48a585
108757 F20101118_AAAHBU amarasekare_k_Page_098.jpg
9d5a5ffc855187a39a54a59fdf1213d7
e9f3f16bc61fe1f6a0b70e9f5e89d42d97b77c55
F20101118_AAAGWO amarasekare_k_Page_059.tif
b27387008366b79cf08d5b25ce4952ca
01ed4af36b05c8d45d7cda2c97ac40d4a3e05fd9
33210 F20101118_AAAHCJ amarasekare_k_Page_001.jp2
b06d095d1e07083159bf215e1f386411
4755bf2c5d94c7b6ae99928eaa1a0f531e7a1ab3
F20101118_AAAGXD amarasekare_k_Page_013.tif
4be3a97c85c6f8c0057037a90031d325
3033512f5b4beed6fa17f4b3470fa4250cbba7f7
99374 F20101118_AAAHBV amarasekare_k_Page_099.jpg
90b69b2ce39357c51ff4caf5226585a4
c656f78b068fda1c286a6827039e83e2185b53f8
F20101118_AAAGWP amarasekare_k_Page_018.tif
5111afee9f61fc9eb5717a947596fbc7
15e1cd537ff77faca81aa21bb67185547ee8a83e
6718 F20101118_AAAHCK amarasekare_k_Page_002.jp2
63385b37954312b6d8db64e971a46daa
756636b2a48dac33a1d131ff63fea74f25b592f2
2138 F20101118_AAAGXE amarasekare_k_Page_073.txt
7d05baf79218af58d8929425d2a6df9a
176a492ed9e27002a36a1fd8467da1767d17e894
39925 F20101118_AAAHBW amarasekare_k_Page_100.jpg
883c2caa2888e70aca31555a713e2a4f
76d52ca67d009f70dd30130d6e53ea3b028812a5
1051962 F20101118_AAAGWQ amarasekare_k_Page_087.jp2
762aec6dfd519e7dd03091b3d295687b
6edc3d7e8d1713755fe297f892a54d550d93d02d
6456 F20101118_AAAHCL amarasekare_k_Page_003.jp2
01034ccd5cd355716ef643f6a6bd9892
439253cd485fb31cf6fe0fb04c24b8c67c738824
36957 F20101118_AAAGXF amarasekare_k_Page_078.QC.jpg
7fed79f21dd061bdc687b5da1d6fd2c5
c0e46ab3ce38b051f09ce7ce41e523877e858021
46694 F20101118_AAAHBX amarasekare_k_Page_102.jpg
abab76c870d44965bd290068fbd599a1
caa07d95c712bd8f40824bcd06cb736db87daf09
8105 F20101118_AAAGWR amarasekare_k_Page_092thm.jpg
d3bfb7b2591e03fdfaebe8bbbc6a4532
2c2584fb0afaedbb82cbea23450de836b06a7636
124921 F20101118_AAAHDA amarasekare_k_Page_025.jp2
68f875be758ddd8f26eee941018d7b78
7fe0784ee8ededa7e2abc982ced98fa8523200ac
67890 F20101118_AAAHCM amarasekare_k_Page_004.jp2
bbeb1c343af0cdbca498471b73b91d57
9534505c63a6e43a36a42b1069864b2533c463e7
53617 F20101118_AAAGXG amarasekare_k_Page_043.pro
573590c6c539173287b70794c937d117
33c01df99066bbab24982ee2a883b917cfe501f1
109994 F20101118_AAAHBY amarasekare_k_Page_103.jpg
7b9f90bb3c9d949ac0602277b401fcdf
3df28714c87c7db266d938e81f99def4166ff7c1
101467 F20101118_AAAGWS amarasekare_k_Page_044.jp2
2866350fa0dc6c1afb5484c6ea655b97
aa39f7a1d2a38daeec29dce6ab9794c96533fb1d
126130 F20101118_AAAHDB amarasekare_k_Page_026.jp2
3f715af17852b69ef0b46432ff6b1c81
a92eb11e4b62ef558f9e613c2b086388191e8b6e
F20101118_AAAHCN amarasekare_k_Page_007.jp2
28e068a98660146ad7dd9eb70ebddf9d
aa5e81b527501a9dc62e0ce58140b15ec4113fce
F20101118_AAAGXH amarasekare_k_Page_084.tif
655d8375cde9aaf40c4a8d2c69a1fc8d
9d5c6ae552c9cfb7210bd180ce79f77f5bd4f22f
99352 F20101118_AAAHBZ amarasekare_k_Page_104.jpg
439ef6a6b400446339f0d0110c38de0c
15a0c7f0f4b1c47df61197189dd05616bc25f92a
2182 F20101118_AAAGWT amarasekare_k_Page_017.txt
f569ba4743873b82964846603598f2f7
4d6c0b6331f6cb28ece6ed4f4c747ec0e5275ce2
83315 F20101118_AAAHDC amarasekare_k_Page_027.jp2
8218e7da1b222721b7fbd430cd5e5211
35de2e89585545dfe17a34b5a53769ad1d3e9697
1051965 F20101118_AAAHCO amarasekare_k_Page_008.jp2
c090ce10f104c40c198ffa801587b99e
e276e2b18764ff5a366713cf94642eed49a8235b
F20101118_AAAGXI amarasekare_k_Page_057.tif
0aa5932f34c2bc55d40d33fb782c9562
447900e32931bf837af32938a11b70822be17d81
F20101118_AAAGWU amarasekare_k_Page_035.tif
3d198043b21cb2f4eaae1f7cbd7e5b32
a8b150addbd8a2771aa0aae797703c4a2697462d
1051977 F20101118_AAAHDD amarasekare_k_Page_028.jp2
d96eb1e8bf9fcf4f634bab65cb4c9dfb
6b202569c4894f7f6fa2ed67e3e16c94fedd8481
98779 F20101118_AAAHCP amarasekare_k_Page_009.jp2
103bccc98f5b499117fff968a1ceb450
2630e826c04092c355865dd7452c6010e935426b
8896 F20101118_AAAGXJ amarasekare_k_Page_022thm.jpg
3e040bd25fd3b321dd22181734869aa2
a715fb998d29f106e385e5c3b01576a48aec6299
112419 F20101118_AAAGWV amarasekare_k_Page_060.jpg
84590e871764840141d2cb5083d17d79
0af2fae6174e3e4847fd15d93cb23fa8e0269326
1051954 F20101118_AAAHDE amarasekare_k_Page_029.jp2
aefbeb7e515f3b6448eb43a4abf6f52b
1cc5f57b0b2d46a3544e4a18d74ccb333fcd7d13
70604 F20101118_AAAHCQ amarasekare_k_Page_010.jp2
0e9ddcc008192fcd0f7ffd5017c8d53f
0fb9ed9e0f293c53b8db2b9d35c0fac3af8e736f
103677 F20101118_AAAGXK amarasekare_k_Page_006.jpg
fde514b84c6ee5b86c1799f6bb667f3d
5fbdc79c6a4e904bbe674d68caebdefd43a7e3bd
35465 F20101118_AAAGWW amarasekare_k_Page_109.QC.jpg
4e5428033e3537526da70c73373c6dfe
329af344bc8d5ce884992e0f7809757e433e1713
115444 F20101118_AAAHDF amarasekare_k_Page_030.jp2
df45082e5d85793a7a3f9bcd35fe3a41
41c5a42908e7463979ef0fedfcd9e3b4264152e8
118602 F20101118_AAAHCR amarasekare_k_Page_013.jp2
ff5311425841c47d7c846384e7131858
707550affbbb81805119a07db7c4bfacf784f74e
115796 F20101118_AAAGXL amarasekare_k_Page_096.jpg
c9dde51aa864120a1a12c6d4a618555a
87863753496ff5c4ac16cce03ba2a3ad165f3b08
117217 F20101118_AAAGWX amarasekare_k_Page_012.jp2
d385c27836518e5b491560a16f47fd43
b4a269645e2f8455b9fa80a8b9f44676ac7e8f7f
113947 F20101118_AAAHDG amarasekare_k_Page_032.jp2
3ae4ded99eb1730c76f6646cc097dfc4
52167dc57d2c14496069dc02812e588bb1859a37
1051984 F20101118_AAAHCS amarasekare_k_Page_015.jp2
7607f198d40d8da1e8ee58ee02d10095
eabe02fd09873cdc1d4e64302ee14ca30b9c348b
31708 F20101118_AAAGXM amarasekare_k_Page_038.pro
869639b35e6268132f013954e7d91c8d
847c21cc0adc1347be093fdb627c4450c1ee0a46
F20101118_AAAGWY amarasekare_k_Page_093.tif
6ac6ab3c2539f34f93c06a4fd6de00c9
374eda2234b7bc13d355244a841f0cb98aec7f93
49170 F20101118_AAAGYA amarasekare_k_Page_093.pro
b19a125bf2d116a2341c83fffa6b70b8
8ae5e1ad1dd1b1f3d5f161c67b868c060c8185d0
F20101118_AAAHCT amarasekare_k_Page_016.jp2
3d53a4d75b5007c6d4fb4a5ea131f67f
c19bd61fb519a01c28f0cb72056c971b12d66a2b
1051947 F20101118_AAAGXN amarasekare_k_Page_006.jp2
4fbdb539615664b2d79004f44016cd97
7d1edbc028858754807285186d2a5ff7727e3b28
2093 F20101118_AAAGWZ amarasekare_k_Page_049.txt
bf0a1e4620da9538adb097053d5b3eb7
48924b05ac20ddf2abe35748defbd252dcd06529
107115 F20101118_AAAHDH amarasekare_k_Page_033.jp2
b1aebaa66dcc2df12e95d2c46f9bffa0
f76eebc46956248714536267b642221859d16fb0
36143 F20101118_AAAGYB amarasekare_k_Page_103.QC.jpg
40835917753fe39a0b71d15b636509a1
049ff23466cd178d3b6225a2383f7b78cf608ba5
F20101118_AAAHCU amarasekare_k_Page_017.jp2
e5e1a2c4b0821dee5d78d98802b9f8bc
26e95356fdd181609604f39d5ff84ec166b65ef8
32225 F20101118_AAAGXO amarasekare_k_Page_101.jpg
f88d3069caa6ea2442d4cd8ca0b70bc4
bf47995792e7d1aa26065e112a84cc1455aa6734
1051982 F20101118_AAAHDI amarasekare_k_Page_035.jp2
e9b1241a8d2a36564a90fb68911cd60f
2a7b1d70957a2b5f0b7bf10a036038822e1527f0
F20101118_AAAGYC amarasekare_k_Page_096.tif
c1eb61adb653f6f0dd8a16651730c55b
cb5e5600b78f55c359ffbf6162e4b0d9ac846668
125902 F20101118_AAAHCV amarasekare_k_Page_018.jp2
084c0f9001e4670ad1fd10b4a988fb89
dfbb4292713af02e4928d0c2ff83a324930a943e
114770 F20101118_AAAGXP amarasekare_k_Page_073.jpg
aa3a79d5e42fc3b13389c84e4436a37e
500d78868ef39719d4ca569d483f33033770cbda
F20101118_AAAHDJ amarasekare_k_Page_036.jp2
0a84a409767228e968f0752b9814ba80
12d1857432987eadc7ab44f1f8e6c0ffe8ebb450
36401 F20101118_AAAGYD amarasekare_k_Page_066.QC.jpg
b49a3f3c725196a6d1dd6ca99b788f4b
d7bb5862f77d655aabc6ddf4c96bca15e85ff771
120229 F20101118_AAAHCW amarasekare_k_Page_019.jp2
22b4d372f3b5bd6c29b24ff2ef66d4a3
84658e9f38c48a4e78b1f12e6aabdcdb7813aca1
9298 F20101118_AAAGXQ amarasekare_k_Page_060thm.jpg
89255e5cfa0cdb4ded104b48bee0c2d5
4d33f1d4e092528a49f27a10e6abadad38b278e0
1051981 F20101118_AAAHDK amarasekare_k_Page_037.jp2
4a74d736b66d13aa22ca6fe16116110c
47bc582f7534a0ddee0a51ba2a6dacabb3e2dbeb
1876 F20101118_AAAGYE amarasekare_k_Page_079.txt
9f291b574e5bb8c1fc3f33c1a870a8e6
4ade1b4e7058c06bfd2b4fab38cb125a95651fc6
1277 F20101118_AAAGXR amarasekare_k_Page_070.txt
3b446f43085afd5b717b10816744a6e9
a2253d5ddce8be7d8fc256ab843036d8ff097989
F20101118_AAAHEA amarasekare_k_Page_057.jp2
778e75c85d5ad51eade1e7aaac70265d
83ec750d0166367a9388c4002f7fb1ebbdeede6f
55570 F20101118_AAAHDL amarasekare_k_Page_038.jp2
70dbd30f2b2e08660c117850e3b047e7
20e7603f82dd51c9ccdfc21011d3c824b8382b79
37309 F20101118_AAAGYF amarasekare_k_Page_007.QC.jpg
c37fc4e94e4e790b31dd8cdc4b1d5b68
9f23f1376d7b190b70569f0aeac44c545f2914f2
119596 F20101118_AAAHCX amarasekare_k_Page_021.jp2
e95a7cc4e1baf46accdb507d5f351fed
74c349f58868ac7c15568486b8473930ae7b6f33
7470 F20101118_AAAGXS amarasekare_k_Page_009thm.jpg
4d2f5d93a8ee988164fb6728df206a1a
bd2f98651bd5edc7f1c742af161727b07c52d3fa
F20101118_AAAHEB amarasekare_k_Page_058.jp2
d9f6b38353920a558929f447e45dce09
58f50ff517cd94d8fa988cae8d73400510d9629e
48034 F20101118_AAAHDM amarasekare_k_Page_039.jp2
310a378224e9b05459b5d1a324c179aa
415d8fc9380ae1e531ed6d532e9c5204d891a1a7
124150 F20101118_AAAGYG amarasekare_k_Page_023.jp2
cc66c3e46e4ebc2126eb5f5a0d361251
457912da7e713c6434a29ac3d14008005a2b0891
119253 F20101118_AAAHCY amarasekare_k_Page_022.jp2
6548dd2ac5d2d36fd1ed7a27e965ec4b
921451b867f022d23e40944b253bd2af3dceb758
109084 F20101118_AAAGXT amarasekare_k_Page_020.jpg
69a0970cd57ee85a0fdb933543914a5f
7d6d71ab77122c788d07dc9fb7186732aaf00f48
122454 F20101118_AAAHEC amarasekare_k_Page_059.jp2
c2c6a66f53d70403f272bf7007af08cf
847df2375d3eb94ea194bcde94fcdb364bdb4f53
1051980 F20101118_AAAHDN amarasekare_k_Page_041.jp2
2cf9ad6289d01530b32aaf141d600d81
ef3a85f85bd79acb8c94a988e2656efa25080a83
118820 F20101118_AAAGYH amarasekare_k_Page_103.jp2
e9c525a99907c680860b234cecfdc75d
26f85a971417944bcf629fd55adbc8906ec98b49
125656 F20101118_AAAHCZ amarasekare_k_Page_024.jp2
cc72059dcbcb79590407073b1d48b3bc
81f2d9bbff7f49444a0148e01dbe7e6d3d122b63
8413 F20101118_AAAGXU amarasekare_k_Page_113thm.jpg
7a0d9683f567adf6c2c5fe8d7d670072
45cdffcb1029ba96073715915de4b975cf02a800
118268 F20101118_AAAHED amarasekare_k_Page_060.jp2
df9a9cc9816839769cdfaa9ad53ae24b
3f70249182eb1b7c61a0f2d84de6a8d0b1e4f2a2
1051944 F20101118_AAAHDO amarasekare_k_Page_042.jp2
6da78074cb82227dd7854f33acbc056f
75991680e76536218ead36e08ae101d17a65579b
F20101118_AAAGYI amarasekare_k_Page_021.tif
b812311a32ca013788168274107e7393
7a08749f41643a8082e2618a89655a3d24ba5037
54815 F20101118_AAAGXV amarasekare_k_Page_042.pro
3af58e01f6eae76dc30081eb7c005929
67fc52ad68388a477e5b688ab4a63ff08e15e6a7
122659 F20101118_AAAHEE amarasekare_k_Page_061.jp2
b721630af188291a9e58926424d9dc16
22d13c648b83dd8a9469c47f1f7baea2e055869d
F20101118_AAAHDP amarasekare_k_Page_043.jp2
35e42e421f49a9ac931066880e8760f6
f7310e4248b6a3a65e22a496a4c2d31598740323
108111 F20101118_AAAGYJ amarasekare_k_Page_092.jp2
b523958d2788d9aff6eeb097b0e15166
c754cc586a31bbc1aae3b91d96b1c0c30a64d0ea
2192 F20101118_AAAGXW amarasekare_k_Page_103.txt
f882b45edacc018a4619d239c15d635c
5e9fc742aebc56f2eecc1a1d73aee056581542ca
102165 F20101118_AAAHEF amarasekare_k_Page_062.jp2
496665a5b1fd07ebef8702b8e519f5bf
29cb13b8378ebd3d16142880cd75b4e8df7c9f44
107352 F20101118_AAAHDQ amarasekare_k_Page_045.jp2
e82f161dfab6df844b24596d75e3d0fa
fb510f81ed92d2598da4c4b8f14786b8d99bfdd4
35764 F20101118_AAAGYK amarasekare_k_Page_056.QC.jpg
b9b08a1c2a060e00574ea79f4547594a
12858427f323af82f13a4cd144ba9842b8875275
53829 F20101118_AAAGXX amarasekare_k_Page_057.pro
d3661b2bb71f718f8630d593d27930aa
f93d64fe5a2f7eb16b307d4f6844610960aee6f4
1051985 F20101118_AAAHEG amarasekare_k_Page_064.jp2
057b9b2329a417286ba609e8f9afe3a4
76692f30cfe9b2d9522faa8307c6c1c5913fe8e9
111124 F20101118_AAAHDR amarasekare_k_Page_046.jp2
2530f765a9d8c26cc45cd5e69ce55c75
c91c8a7494e92f3f1f242cd9d9a9e8edae5823a0
F20101118_AAAGYL amarasekare_k_Page_028.tif
34512040bddb5fbc74251f77e5d4c9e9
ea3b709c0e1f7abc49d42304eb3cd1fe6b2ee479
F20101118_AAAGXY amarasekare_k_Page_040.jp2
022af668a690e7520f3a4f649c070702
eefecd300aa85756469e3b2b5398184a11edde03
123466 F20101118_AAAHEH amarasekare_k_Page_066.jp2
802c35b6499bd5b1325bdbadd2a319b0
1c3c482f1a2a76b2264c50c5fed3b4778c9138ee
65575 F20101118_AAAGZA amarasekare_k_Page_010.jpg
837e2d12f7c61823d4cfd41cadfd985e
f7cbb27531a2fb582647b54053404c9d94528ba0
F20101118_AAAHDS amarasekare_k_Page_047.jp2
ead1d05e8c7f1bae24005bb79c0d6194
aee0e95ff9aa1fc1ffc0c7fa06cd39a025dd4d05
F20101118_AAAGYM amarasekare_k_Page_092.tif
9782562115a4a8e56cda9fd5bcfa0985
21b01ef39e78a9614b87fe494c9fa6ee81cb1238
43035 F20101118_AAAGXZ amarasekare_k_Page_086.jpg
ba19beb3f8d44caad834c5739ad22e67
209eaf0c8082e73cf8917fa33807f0b43feddf27
106233 F20101118_AAAGZB amarasekare_k_Page_011.jpg
c0cdb8ed518f43cb88bee530f06fff5a
0fad165762c058c3a3081c7d363eea9b91ad93c7
F20101118_AAAHDT amarasekare_k_Page_048.jp2
dd6eeff8f3aeacba9ce156c0d03440f6
e55af163e248463382545999649e781ab671de54
F20101118_AAAGYN amarasekare_k_Page_015.tif
49c1fd7955c463c29fe18483d5ee7d6d
09e147060c79853b027493968206e1e62cb41a52
17910 F20101118_AAAHEI amarasekare_k_Page_067.jp2
023eb0d855d4b94c6dd01f206cbfdf50
8a8b658b2c844106856c8b1c5529c07dbd54da8c
111150 F20101118_AAAGZC amarasekare_k_Page_012.jpg
bdfdf068da4b5002564d7096bfce8b16
b1435a8ba3d4471539d0dad429abf7532f511420
F20101118_AAAHDU amarasekare_k_Page_049.jp2
a813da78b75f452c0a2d073ece19ed08
11863a0e061a1a5a1ec1de9e4ac4eac8861bb985
2215 F20101118_AAAGYO amarasekare_k_Page_016.txt
382939ede0c855b57acf13f077eba07a
80bd31303924775f7216de2d943544061e2b5f47
55327 F20101118_AAAHEJ amarasekare_k_Page_069.jp2
a976ba2f7003cf015b2877ef299c6b66
b047a2dffb8c33b3b994e0ffd10b0463f3ba2f1d
110822 F20101118_AAAGZD amarasekare_k_Page_013.jpg
6ab1b449d8d072c8510b08664112bf55
f70f87272bf785c700994b9871d0e96c719c1178
F20101118_AAAHDV amarasekare_k_Page_050.jp2
4f614b5ab8d9e39b427534d76e9ec350
240c23babad2f65187a0e71f5040a74d49f86d87
115096 F20101118_AAAGYP amarasekare_k_Page_091.jpg
daa828c9c9ba9516fd208c87aac53367
39876584ab09498d9b5a96a9b1e040c3b2fbbdf4
63301 F20101118_AAAHEK amarasekare_k_Page_070.jp2
7ad898051b01549de927e4e55cb5c91e
2ccd63e4ec57acaf2787daa02b1d4ebfd667303d
116664 F20101118_AAAGZE amarasekare_k_Page_015.jpg
53bf82f6bc184d941707aa723a33d3b7
5a5da729ea630993c9167e159c52e1eeb45b0531
1051932 F20101118_AAAHDW amarasekare_k_Page_051.jp2
efe301a9a8ffed84687afebaa9e97f0a
0c037ff61ff31eeb207a8214ae245db288626eb0
135625 F20101118_AAAGYQ UFE0021652_00001.mets FULL
c4e5e6a938ee1119103844ed57ee252f
2c08982a53b65ffa41109145922db606af6f60d5
67598 F20101118_AAAHEL amarasekare_k_Page_071.jp2
4a194d3513d6fb82cf53eaf377852ccb
91d4ff56bf2fd4cb6ffaf5456166e9ad470aaf0f
123030 F20101118_AAAGZF amarasekare_k_Page_016.jpg
ff8d920970be48479a9ef52764d5d347
c943f64bb4f6f163d1e9a5fb9e6ad10aaf4bb6b9
57172 F20101118_AAAHDX amarasekare_k_Page_052.jp2
14080ed692d7526d7fec83fdf74ef18b
92a1cad12b4e18c20eeeaee57ae57ff3d3b9f99c
70111 F20101118_AAAHFA amarasekare_k_Page_086.jp2
36f50543032420f1328663ace896b089
497ef5fef6d5d2de6349e22fdb8544aca90253be
120938 F20101118_AAAHEM amarasekare_k_Page_072.jp2
2515cf2e316cf2c04649d5c61cdf1091
69144f0e490dfeb85bcaaa3d358b95734246fa10
116799 F20101118_AAAGZG amarasekare_k_Page_017.jpg
296c1f605cf555fcf21dfb0d6c06df5b
7eb5fe5e9ab2e29a46dfcb647192e8bc19ad7cb2
55592 F20101118_AAAHDY amarasekare_k_Page_053.jp2
9723f6c48a1a6716c6dc095ca26d480a
dbe2a7e57d8add35407dc4a3086f9b746d86c4d7
122870 F20101118_AAAHFB amarasekare_k_Page_088.jp2
51222cec2b9328962d169b3821ae7ff3
a4e22513b83da87e0d1c5734b9a21ac1fcfb0973
1051921 F20101118_AAAHEN amarasekare_k_Page_073.jp2
7c50139077c79435cd44e928f7b58618
d47788cb8f0ce923cc3191021e49a8e7ea8dfaf8
117800 F20101118_AAAGZH amarasekare_k_Page_018.jpg
a7601589ad47c74db5efc1edb02439a7
1b630a300c2130d4ab9740ca14fa52ec757b6d2c
51403 F20101118_AAAHDZ amarasekare_k_Page_054.jp2
053c58bab4243614cddbf1a8b8e71e23
542b9d6ab1dc552f6687cb5356f4ebf802f1c492
33452 F20101118_AAAGYT amarasekare_k_Page_001.jpg
9752be14995cbf357abc500ea0f651c8
515098ae75278f641da8b58c06d7cd9540f45284
1051938 F20101118_AAAHFC amarasekare_k_Page_089.jp2
cdbf27c54330e18c61b58afc635bd9a1
308bf1df0c1c655ce9366250041099e36b6e6be4
F20101118_AAAHEO amarasekare_k_Page_074.jp2
85d771c6d1e103ed13eec8de74e5046a
6ad76346ef4da42afe0a4283dca0b0cf6ad54bec
111198 F20101118_AAAGZI amarasekare_k_Page_019.jpg
b60134cc01f9475c867f5f598a451693
6e6f93105067c10f3fba96365502c982e288fe28
5327 F20101118_AAAGYU amarasekare_k_Page_002.jpg
3fee57a2ee73dbf99181b547b81158c3
9d283e1f870c74d07fc320805448a4611c345e38
120174 F20101118_AAAHFD amarasekare_k_Page_090.jp2
cbdf61cfcc57c98f7e61d63fe75ef4ee
197cdf55b577460de039152cef88cf2e33369394
113648 F20101118_AAAHEP amarasekare_k_Page_075.jp2
5977310bbd276033f12efe97ea022c04
3dd25485cfa0d0b45aa9d5b57cd2ae225c6e6f8d
111473 F20101118_AAAGZJ amarasekare_k_Page_021.jpg
0c9ffc42cd15226da2648b45fb1ad00e
56b0566115084d128f0796f8512a7ebd05a5bcd3
5047 F20101118_AAAGYV amarasekare_k_Page_003.jpg
33427eee7f653ed4703542dfd74a0464
72d12a8bc7b8beeb63c9182df20a31bb1adaaa37
121850 F20101118_AAAHFE amarasekare_k_Page_091.jp2
96257d018c4671c4f69a13e1ec870a99
feb5feb58edb1136ff781d55d3112ab1e48bcdd0
118372 F20101118_AAAHEQ amarasekare_k_Page_076.jp2
9d849d2ab4f54f2f5cb8203531aa9031
3f5d40ecf12e0fe5115959eada11b77092f02aa7
109335 F20101118_AAAGZK amarasekare_k_Page_022.jpg
16d1052d71d36943847f434539cc8eb8
6b256b6d5b31362df56b46e1d26e295f8c59bee7
65633 F20101118_AAAGYW amarasekare_k_Page_004.jpg
df508f13e2782ab1286db45e8a4d5faa
04114986ed6c447106a76bb7962a70de54c1d6a9
108365 F20101118_AAAHFF amarasekare_k_Page_093.jp2
404d61cbcbaa76594a960410354fbfd0
6581bdb71a8ca5e52f51cbee4a2f1cac9065a75b
111926 F20101118_AAAHER amarasekare_k_Page_077.jp2
5c2c5eb44d55bbe67dd329a3f2eed333
59a87722bb8f34af749a2609328e6723d4d47d9f
113792 F20101118_AAAGZL amarasekare_k_Page_023.jpg
bff1ecd2b4f6ec6842c8a322c16afded
e4c207ef1d195405e9807718dbe98f3935405dbf
141209 F20101118_AAAGYX amarasekare_k_Page_007.jpg
1e76003024f7d260deccf3140ff6059b
7455ab46f38acdcc3d0e372e6a8e1b10b27b04f4
119798 F20101118_AAAHFG amarasekare_k_Page_094.jp2
a0e6220882ee6e99f7166d2490700dc6
40624615645054dd9bd15a2b9d3f0cf6dc53edca
119080 F20101118_AAAHES amarasekare_k_Page_078.jp2
c756b333aa210818fbc874e850392232
27bf0e1a9d3a316556c6c88ad2ff786a3f67d606
115862 F20101118_AAAGZM amarasekare_k_Page_025.jpg
25c9844247eff814d26986f126767dcb
d8dec053c4c8c3526d38f84577fe49081b0b6b15
62311 F20101118_AAAGYY amarasekare_k_Page_008.jpg
3ab7f99337f28883383df4ce9a305953
ff1744f6351972e43b620b65d233d0938b8d99a5
121242 F20101118_AAAHFH amarasekare_k_Page_095.jp2
800e21905468080a46c557c414e34eca
4579e57085efc71e3fb8d6b3fcfd5b0dc2b8d19a
F20101118_AAAHET amarasekare_k_Page_079.jp2
08a7e396e2e30fb8aa9a2c0c0c05b15a
fadfc352aecbeed7b4d94a34ef7da09c0b00735a
116798 F20101118_AAAGZN amarasekare_k_Page_026.jpg
0c9a0483c7736d4f2acbf1da484c4afd
b71871c835cfeda6314ac6b0935d5eb59b74d033
95252 F20101118_AAAGYZ amarasekare_k_Page_009.jpg
582e4f88003c1802ac0e11288b131456
2fc76d784eef7105c6e5d954c6252f28bcad6e35
1051959 F20101118_AAAHFI amarasekare_k_Page_096.jp2
13864bf528bba38c63a44e3415df4b2b
f58cb8600c4cf04a93550d4f70d89fccb6ed85ae
1051933 F20101118_AAAHEU amarasekare_k_Page_080.jp2
2137427f055a9e8a74955ab5d18e6cb7
d0a92f5201d3352758166247f2c0643ecdb46820
76557 F20101118_AAAGZO amarasekare_k_Page_027.jpg
665dc11cbe3a7e7673e57f72bebbc8e1
ddd42ce4e91b3a1a58ba98e012a10e6d9d0b13e3
118569 F20101118_AAAHEV amarasekare_k_Page_081.jp2
d6b21e5132bddd12d48fc7d9e4a3b25f
ebf3fd81d0fd1510ed9bcf0ea23ef3645d88ba41
122927 F20101118_AAAGZP amarasekare_k_Page_028.jpg
86d99fa7394cdcf20fff980ffb12f033
d4d9dfa241d026a2d0b7dcc529db6ffa994caab9
115706 F20101118_AAAHFJ amarasekare_k_Page_097.jp2
62b4bd35fcb646ae4e12fdec6e8572b1
a9deda3295c494b8326720274681af1ebc341361
122003 F20101118_AAAHEW amarasekare_k_Page_082.jp2
2c393f586e6c28670c09d4cb444a7948
b30ecb421ad25a962373a90e00c2518125017bbf
110387 F20101118_AAAGZQ amarasekare_k_Page_030.jpg
d897a7454752e25dad55649ac68b3b5b
e809109376cd25151616939aeac219ed3095aaa4
F20101118_AAAHFK amarasekare_k_Page_098.jp2
3c5d812f92e96df7183218d477d85c9d
ecc0c00449590eaf12b6f24bcaa0a9f839d63de7
35325 F20101118_AAAHEX amarasekare_k_Page_083.jp2
30c71965986a42e1a51cb99173eda7f6
d267e53106e300c552f996db11f0dd681ec33247
111579 F20101118_AAAGZR amarasekare_k_Page_031.jpg
73341480b351a9d9b74f0c88afb86021
fe9612e90b46a3e90c18b6f5def6e14ef145ee19
F20101118_AAAHGA amarasekare_k_Page_004.tif
3972ec011cf3d10cf70ded4cae83777e
6d253f4ae75015e9fd33e95154a80c7baa4fcb2a
107516 F20101118_AAAHFL amarasekare_k_Page_099.jp2
d0ab48aaf6b83ec0ef765f5aff58d694
86802d8683072479bfe1429a2bad640e3a15287f
73615 F20101118_AAAHEY amarasekare_k_Page_084.jp2
61fc3d9b4e89d60ef658542401145225
daf6c5436d0baaf945b1a448ba6e730b31ec0ff5
106613 F20101118_AAAGZS amarasekare_k_Page_032.jpg
db7e27a4279198b4c06cc4e2ceb96fc8
f1d24643daff4c05eb7fcb06579fda9cb6bb9d9b
F20101118_AAAHGB amarasekare_k_Page_005.tif
b3ed39d74ba3a29b0973f624cf951c8a
f2bfe5147931dfcb60d3ca0bffd878d77e8395f6
38343 F20101118_AAAHFM amarasekare_k_Page_100.jp2
a492b0b861bff64fcafdf6e39d15dfaa
211673168708dcdea52d2a0c404601301366ea79
75963 F20101118_AAAHEZ amarasekare_k_Page_085.jp2
9f539ad185403bea3cebacee1e62cb57
d090407172c8df2d6e821b9359c1730089ed092b
102508 F20101118_AAAGZT amarasekare_k_Page_033.jpg
83704436bffbef260a0c108a0d433633
7ca1f6f4ff9464c12f02aed31c7a53ddf3635a06
F20101118_AAAHGC amarasekare_k_Page_007.tif
a5b7dd68eace91a686e9b2c522a25f9a
db1686ef5923f9acc77b83d4af0cb64567b1cebb
31145 F20101118_AAAHFN amarasekare_k_Page_101.jp2
47e50999fc2fb12dfd4526ab6e5effb7
aeaa7c2422b498f04c9eaa0ad996734e0455cb9c
115268 F20101118_AAAGZU amarasekare_k_Page_034.jpg
e3bea632b3837733d14934b27931387d
387453bf149ffd97d84b0ec08f19ea5e2e4a690f
F20101118_AAAHGD amarasekare_k_Page_009.tif
b62069200ccff3e6cd6505517cd49dfd
411492ba5a6f2163a23ba65a2694e16b73680dd4
48762 F20101118_AAAHFO amarasekare_k_Page_102.jp2
18d188fb21bb0eade27b3da312d94fbb
6c6ce39f10783334268f49ca71edfa4ef54da73e
113088 F20101118_AAAGZV amarasekare_k_Page_036.jpg
c63977c329d6490e806bb58ab302a378
41adf5c9354896ade61f13d1f39fc00b393c7aa4
F20101118_AAAHGE amarasekare_k_Page_010.tif
63b2e70e3ac2a2a91c1a464ba2a19920
af354cad3691cad6808376a0f488f0dad431cd83
128763 F20101118_AAAHFP amarasekare_k_Page_105.jp2
00a13e3dc08c5e8a95c31e4fe33049db
b4cc2f3da67bb250aa633bfc28f488d620084d08
80181 F20101118_AAAGZW amarasekare_k_Page_037.jpg
34d2803c7e488803ae03cfd13f2f6f90
2ed14b4bbf2acbb42b8f50822a94a708f2b12906
F20101118_AAAHGF amarasekare_k_Page_011.tif
236f1e25a32ffd782df4b7d3da322d10
6e4fc61fabf0aea4fbe3976f202054cc247909ed
129676 F20101118_AAAHFQ amarasekare_k_Page_107.jp2
cc7087fc35edf7e789ea19c4bb8b7b78
0a03a4aa907f7e68080a3ee67d361ad9c56ecc88
37060 F20101118_AAAGZX amarasekare_k_Page_038.jpg
97db589dd02d111a6b669d1605d6e1b3
bb9ba51d8c620def298d1203118998be80caf675
F20101118_AAAHGG amarasekare_k_Page_012.tif
02fa58d008ea3540bdf0c2da4ebeda18
20897d5f58b58f4739abcd86957ad2adf3e35a96
1051971 F20101118_AAAHFR amarasekare_k_Page_108.jp2
11d0cc1b1eb6ade05ba941f811c20ad0
2c254a6a571b66b639d401bfa36333e13cd6aea8
32148 F20101118_AAAGZY amarasekare_k_Page_039.jpg
0c35ec88e55356fa2b2ed2421044d448
254937dd37099b107220e5179a5fc3ec8d086662
F20101118_AAAHGH amarasekare_k_Page_014.tif
6e4211c1e7b055090b117f0917166638
ef453e1ae64ff99e6fbfbd72e9513896c5cc7307
F20101118_AAAHFS amarasekare_k_Page_109.jp2
7033b1035b375cfcfdd4dc106380a7cf
bcc7ae9177e5e1f1fd31d6cbcdfcdce2f05f8480
111281 F20101118_AAAGZZ amarasekare_k_Page_040.jpg
b28f419be533f83c496ea629c9e74f20
1d7f28b37b956e95d047f6c609efd0b4d6ded262
F20101118_AAAHGI amarasekare_k_Page_016.tif
91f2f45e4ac388ebaf6cced6c0593118
f01856b5d933c7fe896de571515ba85b4de78b05
1051975 F20101118_AAAHFT amarasekare_k_Page_110.jp2
4b3e6d338039bce52676b9e301239f5f
a6a063496be152f91d4bb8685a8c42bcaae54c56
F20101118_AAAHGJ amarasekare_k_Page_017.tif
89089de8d299a2e3cbfad0872899c0ac
8d036d1942c4c18e77f58e1e65e7a9055a1b06b6
133574 F20101118_AAAHFU amarasekare_k_Page_111.jp2
e344745bf4b8a527be65fddc489fd5b6
606981c9ada95137eefef7fe96ece83234d40ba1
1051970 F20101118_AAAHFV amarasekare_k_Page_112.jp2
18c6c9dc219a440c17ba03fa4f9a5cf4
012599111fa348ad3f8667d22e9d67735d0a24d8
F20101118_AAAHGK amarasekare_k_Page_019.tif
cb2a8fde8ef6984898e261af09e3ec94
535412e186af10248dc5109953952e650370cb19
42106 F20101118_AAAHFW amarasekare_k_Page_114.jp2
3f637431170a1d4f97d42cf740bdb373
18bd0757756e2c8ade01276376d0bae98dd95200
F20101118_AAAHHA amarasekare_k_Page_039.tif
65f962c5ecd00cd36f44fd455cf94990
7153f232240189077e6507b7933d4b26cb39b79f
F20101118_AAAHGL amarasekare_k_Page_020.tif
5cdbbbd3002e9dbbf6a5523e4ec5a7e9
8eecaa42a4f853be8eb7674801c7f4cf1d809726
F20101118_AAAHFX amarasekare_k_Page_001.tif
c5843b4bcaad65cd4c2280aa80b73d88
f1ca3946389e738bbc40f75e7d284d444ee8a523
F20101118_AAAHHB amarasekare_k_Page_040.tif
8aa6de1038f09a2d6f3d4e9cc0d4f946
e570abd06b1fdb7d8f4a9f31c47ff49d7ad83965
F20101118_AAAHGM amarasekare_k_Page_022.tif
abbf36c8e5936df57f53ab5badef76d7
a60801d17ccfc55b3e42140c4abbe141b13278e2
F20101118_AAAHFY amarasekare_k_Page_002.tif
5c4392e345d53166178b400115deb04e
aedf82183fd6e088c550a73c0e0bf96b4a3daf9c
F20101118_AAAHHC amarasekare_k_Page_041.tif
3f67d50ab5bc2aa90492fa78890aa3c6
31a0a82e395eb86e59ba5a2c8713caaa5a56f9b5
F20101118_AAAHGN amarasekare_k_Page_023.tif
92a6611aa899b63543c8cabdace08b65
1a5ca8dd62750a557e2e5a61a12ccfc5cf59b432
F20101118_AAAHFZ amarasekare_k_Page_003.tif
1c9b4bdeab74b020a2f0eefbbff6937c
98ff35a3772249dd9a4c48c67af8f2882f068270
F20101118_AAAHHD amarasekare_k_Page_044.tif
c62c2ec428c9afc00c35b56813f4196b
fb9fdc8fec3d64f344bc745769f730f350207c6b
F20101118_AAAHGO amarasekare_k_Page_024.tif
a921f463ef1df052f41f0127805bc589
417bc0d996830460446719cada2f47c41da4b721
F20101118_AAAHHE amarasekare_k_Page_045.tif
e7d94db724ee005ef5d156b888987dfd
2ec3c00b0c0b2839b7eb47f8330e4a1ea2415ac5
F20101118_AAAHGP amarasekare_k_Page_025.tif
a5ce2f260897e724dfac91aef419d6bf
e95b707f9bb7589ad8a1e15b2261d31e122318ba
F20101118_AAAHHF amarasekare_k_Page_046.tif
79916ccbe6b95301a26187a9b1ea2d32
7703232afd0b51df2167e09c245ddc58861fbb51
F20101118_AAAHGQ amarasekare_k_Page_026.tif
8d5fbe9da9a78c83b13fc0960dc016a4
6eac83186a413bdf21ae1ee977a401f5ca1a13c5
F20101118_AAAHHG amarasekare_k_Page_047.tif
e06bba4b48476d3422b5deee143f63c1
94e44de53a2fe2b6282df9443a770888d0b087ac
F20101118_AAAHGR amarasekare_k_Page_027.tif
6007e3dd1ab6c761bb22784c8760626c
f3d032c2a214aba70f65f435e1086bf18f787c48
F20101118_AAAHHH amarasekare_k_Page_048.tif
cf3be43baa0d58ba4cbbf8675734a7bc
fc8fece5374d0dae0b5fb9eece5e3b5d6bbe86e0
F20101118_AAAHGS amarasekare_k_Page_029.tif
5a3f6abdab30fa94a4a1b87361e5585a
4a301febfefd39696ac749b2cb3a5079ec70dafe
F20101118_AAAHHI amarasekare_k_Page_049.tif
66053f5b1b9854af56aef21669bd8643
5c21a49c1c3410a1ced554859be36820d176750a
F20101118_AAAHGT amarasekare_k_Page_030.tif
18fcb2a84e2f4e5b5b613dee463c50df
4aaa8de934f532cdc1e3b405c6ce23d83e1c466d
F20101118_AAAHHJ amarasekare_k_Page_050.tif
3cf4a87db2e25d09a81d22dab2f4a7b2
ab7fa748a4f47b0f240a9e444bd3e52a481f0afc
F20101118_AAAHGU amarasekare_k_Page_031.tif
afa6aeed2bf16e96c280763d061dbe69
ea3d329d80d60786a16c9f4d0e51c610e24276e1
F20101118_AAAHHK amarasekare_k_Page_051.tif
888c254a468e9cdfb0ba9a705b1b4dd8
a0b33ac1f55fa21c450acfd5d7b7b5e8d23e503e
F20101118_AAAHGV amarasekare_k_Page_033.tif
324ed149dc556753c38e6e52223d718a
c78cd117d2e8a4031e198f180e970a382ae5bbdc
F20101118_AAAHGW amarasekare_k_Page_034.tif
ebd62592268b63b5399f9addfe557ee8
4e95ec3c57dd8ea92b1fc09d7cc28d0c58f5a1b6
F20101118_AAAHHL amarasekare_k_Page_052.tif
fd1682543c79a44ab5fa6ad77c78a833
9a2f7bcf97fb98f8fce737aa7c3b8e192f863905
F20101118_AAAHGX amarasekare_k_Page_036.tif
53262d0f4951a2732cd8d9f3395ea5db
8943e161afabde682e713aad79f49d35cce51a7c
F20101118_AAAHIA amarasekare_k_Page_070.tif
5f6fa6b941f0ec720c0119886167098c
58e4bf17b9e8d5e1eb6bb17268dfc0fbd0bfa794
F20101118_AAAHHM amarasekare_k_Page_053.tif
3267dac02a4304047f4779a337a669c2
14194ef621c89983cca3d6e54aee5fe5e1a3c3a6
F20101118_AAAHGY amarasekare_k_Page_037.tif
ae1cfacc7a892c46d810f2cfd27b9aa8
26846511f2732b29977a76e9c78a695ee4be5eef
F20101118_AAAHIB amarasekare_k_Page_071.tif
75ee01c2f51e62b4a62d367fa348cc43
fb1abb3f83b21be61a43511919a681b89719daf0
F20101118_AAAHHN amarasekare_k_Page_055.tif
4d6fda13df2d9412a2ccdab1e8e2f993
4c593866c87af858f0d52809a1eeff2454f66af2
F20101118_AAAHGZ amarasekare_k_Page_038.tif
7e8e79525319c1facde762d7fb3f2a27
d452c46032772115c1e3eda0d737c303af57d7ba
F20101118_AAAHIC amarasekare_k_Page_072.tif
670885a5133fc935dc344505f1662839
3bafdd4b452f7b59938e41fad2692061e8da2316
F20101118_AAAHHO amarasekare_k_Page_056.tif
1718e5b76eb677f14ad73c3600a6d730
24cc22cae932348f78468094ad6bf444a1e01578
F20101118_AAAHID amarasekare_k_Page_073.tif
3f1b4e302622d6499cb253400743a3a5
824e7e7105dd83978511ac3376d898ad3f91e587
F20101118_AAAHHP amarasekare_k_Page_058.tif
d0cbdc0d0bd0cbdaba64766d718b6be1
ae0e4e259a165a3f50cb702f3e683edcceaa5a92
F20101118_AAAHIE amarasekare_k_Page_074.tif
307bbd8ceb7c5cda78d94a0e91ce2042
55ea3c5596986433e09a15982ff2b9e44c919fc0
F20101118_AAAHHQ amarasekare_k_Page_060.tif
eb638dd435af97dad254f3d428a36b31
b4b2e456f819f68c5da5879226b443db88befeb5
F20101118_AAAHIF amarasekare_k_Page_075.tif
447a2b1c4c81c2259bcedecc2d312c51
1c72badc7e670d55f60b98e24bd083b8b5c2b441
F20101118_AAAHHR amarasekare_k_Page_061.tif
af3c159bc9f8a9f684d6b2bf663589be
74421c66dfac9d2e68fc305d29bd22234b326648
F20101118_AAAHIG amarasekare_k_Page_076.tif
c5d3c9d7d38defcdf0f9eee280b47113
19d0dd3643b3e12eb275738eff0c2187bf34cb46
F20101118_AAAHHS amarasekare_k_Page_062.tif
3f6d19d63e62bb23b0cf49aa03896676
7f0bbb9a626273dcaacb211c1c4b7b3b40c33c60
F20101118_AAAHIH amarasekare_k_Page_077.tif
e3eef5a8d17590b78a9535dc23e2fe7e
661de37425e2a6e05393ee1a6f9b41b3ed508d1b
F20101118_AAAHHT amarasekare_k_Page_063.tif
7e7a70966eb7bb34f980b3ca276bada1
074a8cfe34c321c1934ab2e5b5907302b1d9dad0
F20101118_AAAHII amarasekare_k_Page_078.tif
0e593b256622b00bf2c082961b731964
a2945744420e597b53cfae8b29498fde3b69ec88
F20101118_AAAHIJ amarasekare_k_Page_079.tif
c42b8a311456bcd6bbb9b9a6cc749de9
5eec09e4a38e7a63030df07dee7b967ff42bf3f0
F20101118_AAAHHU amarasekare_k_Page_064.tif
861fa7e10fa92d45058a9ee23693e270
60cd7f3ee6413327ae20e7fe0593e0f7b7e2fc63
F20101118_AAAHIK amarasekare_k_Page_080.tif
0285fcc6e4ac772d44789eb271e2cccb
a4084fe885f48ec13300aa1a24420166e601c28d
F20101118_AAAHHV amarasekare_k_Page_065.tif
070fa2f0fc00bcf532a3712983355498
416dea1e720c604ee33ccdd6f7242f2a057031e2
F20101118_AAAHIL amarasekare_k_Page_081.tif
edc625c08537c06ac45e0eacdbd40c4c
30be3c43ed45f7c28ad530e5e2f4ce9edbfa1248
F20101118_AAAHHW amarasekare_k_Page_066.tif
6478946a28281b557c344f1f504feaff
2a8b805cd22e145f54a7deb04c4b169823120e18
F20101118_AAAHJA amarasekare_k_Page_105.tif
0fc2ae814ac8b303a3114d03c0f199ff
9bab77212e2daa7609dbd4f745e5b10c4d9ed4f8
F20101118_AAAHHX amarasekare_k_Page_067.tif
4142758d4bfb3d1a276edc82c991de22
3f96c8a066fa7ae04d9ec623c2289a9bccb7807e
F20101118_AAAHJB amarasekare_k_Page_106.tif
b0f0d064b62c1440849f185bc626de5a
937f2b98bbca33e9340474e2ab514f7a8ba56ae2
F20101118_AAAHIM amarasekare_k_Page_082.tif
deedd292975e550a20fecd6bf4cff547
ab98f1a450f532cc5e29e31f565cde2606809798
F20101118_AAAHHY amarasekare_k_Page_068.tif
fc6d45ead0489fc7fdcd7630c441f02d
895eeb8ece6516832ac166cd4b50aeb0affd6739
F20101118_AAAHJC amarasekare_k_Page_107.tif
56ed5b66e472698f9d3e4311ff254f04
89efa051ff6b2f948d5f866da5f4c02cf6380292
F20101118_AAAHIN amarasekare_k_Page_083.tif
f8e3371026f58593636c487d8d39078a
1db67f4b60af215c87d20e7993eebc121edbe640
F20101118_AAAHHZ amarasekare_k_Page_069.tif
8161a2989b6254df503d5098966abec3
612e265cee9ec1bc546937f7676b8162bc871994
F20101118_AAAHJD amarasekare_k_Page_108.tif
23c384550ba73e2c47e17cb983325958
9277220dbfd1e86d53c71925d594803e4fef4df0
F20101118_AAAHIO amarasekare_k_Page_086.tif
ef120de4757d33d91afa28381c5325ba
8f767bcb5cf3cf3d7006af58743316f744769fb6
F20101118_AAAHJE amarasekare_k_Page_109.tif
49a0db1134b393a6665cbe480d82410e
c3ef52f3b9494bf5cc3e1ef1734c26f8ebfa97d3
F20101118_AAAHIP amarasekare_k_Page_087.tif
4bd11e54cc86de1af448cfc6c0fbb3be
bdbfb74af97ceab9b193ba20d015f85ee4b3f8ab
F20101118_AAAHJF amarasekare_k_Page_111.tif
b6056099112d4873d36919f314fa2539
602eb5ae856c4cdbf37dec93b05149e7cc133e6a
F20101118_AAAHIQ amarasekare_k_Page_088.tif
080e00f2d4a1adc984f12cade8fe5bf4
bc78352a06c0765b6db59baccf7e5aa7edd02646
F20101118_AAAHJG amarasekare_k_Page_112.tif
b9da1d9f0b90919191230db1eaa0c7bf
543ef0cba932689f5ed474c8bb7413c6f94e26fb
F20101118_AAAHIR amarasekare_k_Page_089.tif
8187ec2c2bc932931963d8bafdc00e5d
7c0223a0d26197157918b7a33acab49254ccc474
F20101118_AAAHJH amarasekare_k_Page_113.tif
9d886878dc2d3994dbde40c207357854
9864a08dbdec4088f974926d6d8f9b443d7f5326
F20101118_AAAHIS amarasekare_k_Page_090.tif
450dc855e43ecd1f907aa0c32e3dd1ba
fa0428293a597b0610684132914e0eadfdc9a5c2
F20101118_AAAHJI amarasekare_k_Page_114.tif
e93ad30bbf1ee6fa313fe83ba7b0fd1b
c2bd9c66830e5677061469b54a3bc44a638a20ec
F20101118_AAAHIT amarasekare_k_Page_091.tif
5b1ca175cb335e1bd1bc891b469d038d
36c6cf08395effa0d0139aa3ee1a42365d4c8074
11317 F20101118_AAAHJJ amarasekare_k_Page_001.pro
2ad31e47a0e42f291345919c1e67c6ec
9f1940ae840c66f3913dfb1949ec85d7c05a4e14
F20101118_AAAHIU amarasekare_k_Page_094.tif
eee4b602bc659ddc51c2871fc1bd000a
3069904846b0c5581abe89e3a5eaf75aef4710f4
1334 F20101118_AAAHJK amarasekare_k_Page_002.pro
e8aa27606c302c5c521a76ed2fc5ea8c
16ad6f748f044d34a780d4d2c742da0f74a1d998
F20101118_AAAHIV amarasekare_k_Page_097.tif
a0a189b9189463cdb65629fe519130bc
42602a9c6bfb41e5a588d16025ab7a45b1d22fcb
1410 F20101118_AAAHJL amarasekare_k_Page_003.pro
05baf004edd9704da3368ab980e32506
20f406c44743a148933b8da98eb266ae3bfe6897
F20101118_AAAHIW amarasekare_k_Page_098.tif
27a4af16e3705ca574545fc58a88d255
dde2d451d6cca45db545d4361a32ffa064d07c47
29608 F20101118_AAAHJM amarasekare_k_Page_004.pro
0a07a55144ff6c1e36ef83beb8f8ab6f
cc2dbf257a5b3b7d1f6fd78629ed4b76eba77a81
F20101118_AAAHIX amarasekare_k_Page_102.tif
d5ffac19e17e2acd5bda0b785d70c734
218c6fb5ab603be256d63bd64fea9d466927c231
53334 F20101118_AAAHKA amarasekare_k_Page_020.pro
90f8b4603b30d8b49bb6096d8478f0d8
44781600ff6972f4baff5c4c01b3318787233774
F20101118_AAAHIY amarasekare_k_Page_103.tif
975a1b5e3ae2f819494209e80e2c9086
f8b73fd90ca5366416b9734130ea6fd8d7c571a4
53423 F20101118_AAAHKB amarasekare_k_Page_021.pro
be0752a76eaa5311863e7826cd2b09c8
8bb2a358dfa2bb5487e620db8358078de27b027d
93593 F20101118_AAAHJN amarasekare_k_Page_005.pro
d9cd11c82ac89b432e5fcb6f6517b519
f5a9f99252e5dca548ac48b03fb3a4b9b2916670
F20101118_AAAHIZ amarasekare_k_Page_104.tif
75d378ea94964dd3bf63a33174d49a21
8196d0643b622276ee0225f5eb727c0e3dfe9dc2
54328 F20101118_AAAHKC amarasekare_k_Page_022.pro
b3b1ce58ac2ae265b9a55cdce7645b00
e90980ae5dd5ea20685a8140dda26147700b87ea
64288 F20101118_AAAHJO amarasekare_k_Page_006.pro
b85ff9ba8a5e67e7c935de6a3b8353a4
6b1ae61f4423575689a1b4d5d289dfd0e6a9bd24
54273 F20101118_AAAHKD amarasekare_k_Page_023.pro
ae6ab96eac9117c45ce0a4821afb19db
079a2565a459f8e6dee63cc0bc61eaaea2b11630
75853 F20101118_AAAHJP amarasekare_k_Page_007.pro
ae2756393c95c93b7b08050fac65e018
73e0388825f03181c94b033c8343c1e168f7f3bc
57240 F20101118_AAAHKE amarasekare_k_Page_024.pro
9d954c85bfda058f851f1c6860c0b6d0
647d187233ca702aa00f96cd45efc508c6e449bc
25345 F20101118_AAAHJQ amarasekare_k_Page_008.pro
82a862d3f0dfe21daa8cf166cd1a13a5
c90fb80e821ad643c77eab5bd38075db31d118fb
57752 F20101118_AAAHKF amarasekare_k_Page_025.pro
67a75a30f2f94a65815dbbb58e57ca18
9a4feb6f570250c32ba5b744a34a6d4bbf5fb82c
44305 F20101118_AAAHJR amarasekare_k_Page_009.pro
4bdd7e71edd9f2257fe6bacea3480759
93561b32eeeed740d71255a8401d5ee285c9fe1c
58395 F20101118_AAAHKG amarasekare_k_Page_026.pro
f18760b63e58b4ea011d16a89adca5d3
8acc46dd7f17bbd66db94b581f75b76abeb83434
30763 F20101118_AAAHJS amarasekare_k_Page_010.pro
6ea3a2f79d3ccbb7395b5b7ad3d8b492
31f722f47328fd69b731dff9fb4d19cf6452594b
37308 F20101118_AAAHKH amarasekare_k_Page_027.pro
cce472da3566c6bf7996ed1935716312
cdf2036f98d51cf5f66e925c10461db344346691
50265 F20101118_AAAHJT amarasekare_k_Page_011.pro
957a1bd93c32d265c7297dc787a3d4b2
3f1c419878fc49f47e4cfc46225a76abb148d5ab
53722 F20101118_AAAHKI amarasekare_k_Page_028.pro
2f2f9728f9c748e4165318116e2808bd
7592f2ac6872523ce8c9cea63b40e1156f421ea3
54256 F20101118_AAAHJU amarasekare_k_Page_012.pro
8c1c8302db212ec5dd97f4f94688e08d
a1e52cd6be99fb8f85105fa456fb4ed051e50987
54653 F20101118_AAAHKJ amarasekare_k_Page_029.pro
fb0543b5f63e2e1a7b4f8a22aaf93853
bdab63394fafa4804c3a97bf2beaf7adb1b45e4b
52669 F20101118_AAAHJV amarasekare_k_Page_013.pro
ef2bba09eae2cf7801f3d5588fd2b4d9
5e9940d6d66ecc8122c8c5008e95d2b8e6e9ba8b
53773 F20101118_AAAHKK amarasekare_k_Page_030.pro
7ecc3d5dd47a3ae386336ad5fe831944
3bc21b21c648a868be150ab98a4ad43e161ca462
56396 F20101118_AAAHJW amarasekare_k_Page_016.pro
dd1dbc42efa4c48eda282e58a45a8fad
3361c52eccd1fe15395fda9396561ef006275f10
F20101118_AAAHKL amarasekare_k_Page_031.pro
dbd6a5dd9430827be2b8a7ab00c4a768
7ad34571b7d2daae2c91ea60c657e17a855e8781
54867 F20101118_AAAHJX amarasekare_k_Page_017.pro
47772c9b76f7246af21b7e1b5360979f
9f93aef09f86d3160ed833e1223f974d8e91a579
27388 F20101118_AAAHLA amarasekare_k_Page_053.pro
c35d44e6eb2f696fa775733af614ccc3
7a5100455a5b5f4102f2acae0d38f73db77c6d56
50328 F20101118_AAAHKM amarasekare_k_Page_033.pro
a42accb2fa8207ca48d83b2ad8931ce0
d2b0ec0ac837c5294fb8c8bec052bd14018ad37b
57410 F20101118_AAAHJY amarasekare_k_Page_018.pro
3c6baeadc5985c5008cde31575c9134b
b96746dd5e9e47e0ade203e0ee80071dfdfeb289
27423 F20101118_AAAHLB amarasekare_k_Page_054.pro
2f0eedf0e646f3e05ebd82fb71a0075f
66af62241630243347110918cdc41421414a0625
54244 F20101118_AAAHKN amarasekare_k_Page_034.pro
044acdaea976a1a40a785fcf9e1ee7fa
cc8654457058b127e7d39575b680f84a2c525f3b
55515 F20101118_AAAHJZ amarasekare_k_Page_019.pro
67399b052ed755e44c8f230ccd3ac9f7
81e1456073e42458d9c34139205ca8fd564723f8
53764 F20101118_AAAHLC amarasekare_k_Page_055.pro
b1e2100b79962b19579c0b718dd454ad
38b374807dad5a20d2d686f304d5f76cabc0bf78
51864 F20101118_AAAHLD amarasekare_k_Page_056.pro
f9cbd5eb169367bc948ad6bf117230a5
2ef35586bdfb95383635c6ad8b95996368629e66
53936 F20101118_AAAHKO amarasekare_k_Page_035.pro
a2209791b15fbb3b0724f9c12f651cd8
ee14888620b111a1d07222b8301e613dd9d08cc8
54036 F20101118_AAAHLE amarasekare_k_Page_058.pro
d1e9e5c6fc3e40550400e8316b70388a
bf3c16a96914da8a7e0b7b7785e252d0c75067b9
25416 F20101118_AAAHKP amarasekare_k_Page_039.pro
6ffca108faa236f1404c2b5129ca7b73
a749e82efe458c73d186e5f3e1c69288ccf0a171
56241 F20101118_AAAHLF amarasekare_k_Page_059.pro
9bcafca95432b9e9d98e239933ae2f1b
f95640d78c176030c6460d813c37de655cf8a7e8
57340 F20101118_AAAHKQ amarasekare_k_Page_041.pro
66a029668601625058647568efee50f5
e606e5382a72ba3e33a0651161cc5610cefc7a67
54621 F20101118_AAAHLG amarasekare_k_Page_060.pro
6912e33061e41d55ee0e97c26280cd40
4af994094cfcbabe7dcea1f6734b743114634fba
45851 F20101118_AAAHKR amarasekare_k_Page_044.pro
94e3521d91540dd91ccbdd376c05b383
c2be99fb3e419f787733e02c6ae27bd750dd8662
44895 F20101118_AAAHLH amarasekare_k_Page_062.pro
3b5ee8f0c5690b934063d65cd69fa348
8e4156bd19eaab70d26c3d97366b55d21aa400b7
48636 F20101118_AAAHKS amarasekare_k_Page_045.pro
1ab9cd60237be0359af7ffea22bf481e
366815bdb3ec821880bfb3d616edbab6534cca94
49986 F20101118_AAAHLI amarasekare_k_Page_063.pro
6e343ca6ec7c65032b3dc768fb4b5561
7eee59c9e8b8a2efab1d4d15b3625bab6bdd820e
50664 F20101118_AAAHKT amarasekare_k_Page_046.pro
be0b927933bf7d3b9819230305cad9b4
073fa5f2ef825f7682cc4985c81b447fd22961b3
54813 F20101118_AAAHLJ amarasekare_k_Page_064.pro
c5ecb2478a75862149e0186160cbfe71
702190e15b8a66934d27514ed4bd01a9fc2606b8
52747 F20101118_AAAHKU amarasekare_k_Page_047.pro
1f654528bec72c8c2bf6c6bb8001b9b2
ee6d71949223d62df1f05712e50004570aa5de6d
53156 F20101118_AAAHLK amarasekare_k_Page_065.pro
dea73414ac1143bdea2089ee1df19359
ed5a0a8ff475432c9e4edd7df996831a3ac8c929
55876 F20101118_AAAHKV amarasekare_k_Page_048.pro
174f2148e7961270e0ba2e5cd640b834
c4f7990f73e620e474fc9e52fa2324a24991d74c
55868 F20101118_AAAHLL amarasekare_k_Page_066.pro
e7ed2f36f2592fa24777d7bcaa419075
a1c66104c0acfdc5cdad1ca6f38a65169e3a9b75
53125 F20101118_AAAHKW amarasekare_k_Page_049.pro
c3e779187b93bca2dc0ffa258f25fd41
6ee524913d3facd308bf23e2b18a8b2707007dd9
34970 F20101118_AAAHMA amarasekare_k_Page_085.pro
732ac89eb52b00e678f425a58b032fee
21e1e4f6ada1191b3a3c7c2babd6506bd2ff2c21
28468 F20101118_AAAHLM amarasekare_k_Page_069.pro
ab45e6234957d3a12c9b87b2c58a1a37
a18592407d9aeb77324ccc8c8ca5253134adac59
54526 F20101118_AAAHKX amarasekare_k_Page_050.pro
201a7599d3126fe9a1879591b0efb9d3
8714999af488be8d3b9fdfa9ab7d5e8a30f3eef1
36061 F20101118_AAAHMB amarasekare_k_Page_086.pro
1811feb1ec5d044df9ff931acf1023f0
a8239745b5428cb369868814980a886f1a63925e
29345 F20101118_AAAHLN amarasekare_k_Page_070.pro
cc4107628b13b788b9bf192595a9957e
969ab5ed9dedd8d1b3e0e80a07fdf31fd002aba4
43931 F20101118_AAAHKY amarasekare_k_Page_051.pro
e9e13f08093249e9c937b758bc70d04e
9ca6c11ee41597b2dcaaeae5a58b541cce479783
52454 F20101118_AAAHMC amarasekare_k_Page_087.pro
f241dedaaf7191441e8fce459932173d
06ccf8261d98fd05aca1a646709855cd817e3c66
32206 F20101118_AAAHLO amarasekare_k_Page_071.pro
cb2f2319293141a7b37dd81ef6f4f9f9
abe77b67e34899fe1a037e727e95345359f09ca1
33849 F20101118_AAAHKZ amarasekare_k_Page_052.pro
c3b5d94f1fc456f022f6116bce2eded1
aba522badb1566ddfbdd6138ca07bb0977d7ab1b
55398 F20101118_AAAHMD amarasekare_k_Page_089.pro
0a8965ceb241dea63009cb20c31166f5
f32318cd7b02be90ec1a7cdf4bff83f14bc25354
55134 F20101118_AAAHME amarasekare_k_Page_090.pro
a32378648abd971ff3f12cd047c78621
f1dc71b5a17fb2d615df6d02ffa600fc8c32c625
54492 F20101118_AAAHLP amarasekare_k_Page_072.pro
587f8db4b5f5026a73be31617095faf2
af2a0226a33fac6daa4071b4cb5f31648ade62fa
56038 F20101118_AAAHMF amarasekare_k_Page_091.pro
12035a722d091f09fb2fc0857facc1b4
fbeeb1b2df318907d803864fa2111cb4d146af8c
53474 F20101118_AAAHLQ amarasekare_k_Page_073.pro
96b5c5f43e9dfd78eb56c9874a1fc590
cc3e34c31274409175fb9c0a7bc771595cef6bf6
48384 F20101118_AAAHMG amarasekare_k_Page_092.pro
d0fb0cf2ea88d92696251d72ecf5d97a
0c41605ba8d0c6aeed200bd519b8230b2a5bab86
54185 F20101118_AAAHLR amarasekare_k_Page_074.pro
2a7320a29d5323680deb31262bf9f2f0
f800791ab33fe7e523072aa68794bbf1946076fd
54252 F20101118_AAAHMH amarasekare_k_Page_094.pro
00757e943f396f27f86cef018d66c903
398e9b72e09ba4ee3bc20972f8da21716b6c5bb0
51347 F20101118_AAAHLS amarasekare_k_Page_075.pro
5e8825d41e05a4aad2201c5fe8bc5978
8f40b53beac68c7d8a14e194df9244bdfd3353fe
55589 F20101118_AAAHMI amarasekare_k_Page_096.pro
20fa48fb2fa052190a5c2f486f8d67c9
2324821d67a5d4b282e9a5f16167418a570d12bc
54472 F20101118_AAAHLT amarasekare_k_Page_076.pro
c5ee146296f4436e9e8d5a8b94c10f1a
4607d3d25ee4243e8996008fab49dce0d183ad99
52150 F20101118_AAAHMJ amarasekare_k_Page_097.pro
a371de2f7e42ad9afecb0aac60bc7f69
df2b2d0f0f6331360c71dff3f05d473f1d4f7aca
50888 F20101118_AAAHLU amarasekare_k_Page_077.pro
5fd6ae0e46fccf89bcbe1513580bdfb2
c7cd7aa0f0c8c3f05c35eb81358078702a6d36df
51310 F20101118_AAAHMK amarasekare_k_Page_098.pro
b2d731070ca2aa362acf34b7a1b7ae8b
f146b41cc9a500211f1e8e8f78fc3f21fec51321
54208 F20101118_AAAHLV amarasekare_k_Page_078.pro
3a5ebe954d3998563fa0672d375698ea
3dcc48f61e4b35bb0f80171253045c249b74c189
48363 F20101118_AAAHML amarasekare_k_Page_099.pro
a17167967eef89b1bfde1780ba82b33a
ee755963eee0443af217371f928459c911066b47
45626 F20101118_AAAHLW amarasekare_k_Page_079.pro
50a88e5814457583d04474bbdfac1918
c2c8fc7952b61bc26e50db6b11fbd2edb9030a16
17205 F20101118_AAAHMM amarasekare_k_Page_101.pro
08e1db4ed07c37a5e1136e08802722a1
32c555b206f4017b1fd809a5f03f932573b660c6
54187 F20101118_AAAHLX amarasekare_k_Page_080.pro
bfbc876a1d35c16080bb4f19333a5ed2
570988b9f55169d1f9ebc50e6d143ccd0724129c
594 F20101118_AAAHNA amarasekare_k_Page_001.txt
b78f18ad628b83746c63085c19e6e781
f1f40f14641c9b752abe2d75eb83b81952060457
24546 F20101118_AAAHMN amarasekare_k_Page_102.pro
78778e3a3ab4d705c5a51424a6db77b1
38a9f60df51916cc85ca7b7a880025046619ddd1
55578 F20101118_AAAHLY amarasekare_k_Page_082.pro
e61a1bb32421248eb29768b8f386ea3d
5960ddb28bc304f2ea210fa8ff143579f2cf1f68
107 F20101118_AAAHNB amarasekare_k_Page_002.txt
9de9c62704d0ccd091ef1a2afb4ef83c
3c80c000d52968158194ca0feb82d9bafc6ce2af
53925 F20101118_AAAHMO amarasekare_k_Page_103.pro
6da399c1667148a27b0df5fcdcaa7c3b
b1f8f6d5ae9fe69fe2d01372fcd7276052b6a5dc
14448 F20101118_AAAHLZ amarasekare_k_Page_083.pro
6d2cbca1785d882b7c69a118ffc61d67
c292847ee592a874f316b58eceeda4bdecb7437b
108 F20101118_AAAHNC amarasekare_k_Page_003.txt
675a339b7c919a232834e339cf37b149
6bd9323fd0292190c8f6384ef741a1c9a957caee
48276 F20101118_AAAHMP amarasekare_k_Page_104.pro
75ea55ab348b0b1c6628d02bf12c724d
646dbc256254d1b52c4f451652583ceca9048d02
3827 F20101118_AAAHND amarasekare_k_Page_005.txt
921bb66bd13dabb1f1309c77911bd501
ac0f5f806cdb4b0b1bafdb8f2402ae0fe36f2ff9
2563 F20101118_AAAHNE amarasekare_k_Page_006.txt
6f3dd2d31fa2b6dae4687be6058a19c0
658916b99e58f16e063478761148e735b3298d3f
60405 F20101118_AAAHMQ amarasekare_k_Page_105.pro
e22c842e4c3283aeb2d87538ad500b49
ab68c69fb535ffec627e7cefb7aeb634ffca2636
3131 F20101118_AAAHNF amarasekare_k_Page_007.txt
6579fb86ae926873acceb2358f95d3de
74e276d8c40d65f2aba7c1fd602b431edaacb682
1051 F20101118_AAAHNG amarasekare_k_Page_008.txt
c28ea6299cdb94e3a2873dcf8309556c
da28a26e8ebc002ad6df85963d44a0b22c112827
61711 F20101118_AAAHMR amarasekare_k_Page_106.pro
fcc4f7a2a2d5842ab6d0713d8d57ea10
7022a5e9b5f7eab156bc7f66622a4e28d4d4dc7c
1935 F20101118_AAAHNH amarasekare_k_Page_009.txt
571d8865abce20cc8528be53385ac757
4e8a8729b1990c85ee87d3a26f57b51f9ee9bd84
61875 F20101118_AAAHMS amarasekare_k_Page_107.pro
29a78757f58aab01285dc3655b63406c
3dc674cd5e468e14a30e7a2164fa271de7b9ff09
1230 F20101118_AAAHNI amarasekare_k_Page_010.txt
d83522500675f2c8c9b42b5eb18c12bc
3d9fa5b8e00631b2cf9036546d4e58d5d6553bc6
65981 F20101118_AAAHMT amarasekare_k_Page_108.pro
55be8577be57f27607a19322c13cb59b
2c962357901352e66487db6c8bee406de1448b75
2099 F20101118_AAAHNJ amarasekare_k_Page_011.txt
f59a406b07dddfb1658b4fae10633a7c
4bbfb007985db4220268a391c7a05a5aed437c49
59814 F20101118_AAAHMU amarasekare_k_Page_109.pro
31724ac099dc3d943f14d392c7c58bbd
4a5a884d7534cc26a5370608d6c8c984675d8d9c
2130 F20101118_AAAHNK amarasekare_k_Page_012.txt
fc0b289a8a91a409107356ae6d55ae25
6f7699c2289e24f194f1644982c16e35dec5e7ec
69033 F20101118_AAAHMV amarasekare_k_Page_110.pro
77268fd8d47fa4e23d479cd182b62700
144ec6f799d708204affeb4629d2e92630e3c8ca
2122 F20101118_AAAHNL amarasekare_k_Page_013.txt
68152c17088b3729b8bcf2625e7080d0
986d4f16be4f92fde26f9fd5885f2084b7bf924e
63649 F20101118_AAAHMW amarasekare_k_Page_111.pro
e556ecbcc733096ac4392be3d0a658a5
2add85fef28e0f1eb42d22803e50d8f9b004db90
2131 F20101118_AAAHOA amarasekare_k_Page_031.txt
4db0f4aaed956905588fb18d83f52711
bcec14c72b8beb0f55c8247a189c754d4e9bc850
2274 F20101118_AAAHNM amarasekare_k_Page_014.txt
643436fdd8712be9ae401bb9e3d85b8c
a9e1b730d3f43fb13ea503919f081bceba858d7e
59398 F20101118_AAAHMX amarasekare_k_Page_112.pro
869cb1c38c6a0e338860a6314a2e011c
3743e58b2d9eeb3f574e750b505515db956d8da7
2076 F20101118_AAAHOB amarasekare_k_Page_032.txt
1f0047f83fa4b65becfc00574c396718
15fdd0ad3d701c27601d0baf1d97196b1044d8aa
F20101118_AAAHNN amarasekare_k_Page_015.txt
5554e9eb870e2f5f7ac1e69fbc67045d
d3d1d242601aff762a05dcbec5074b74cbdb2caa
51968 F20101118_AAAHMY amarasekare_k_Page_113.pro
bf6c2c7a1c61db60f796ca9f64077f42
d81858c68819ab0ba1736bcc15ac6ab966065ea4
1997 F20101118_AAAHOC amarasekare_k_Page_033.txt
1fff79f212f8a06e2e3345473b459428
86ca60b9c05393723f57f0a693987e6875582df9
2267 F20101118_AAAHNO amarasekare_k_Page_018.txt
017f006614e26c4b032d954673d1dfe3
19b5a557b58e7527d668a4f31f117f3dddcf5d77
17323 F20101118_AAAHMZ amarasekare_k_Page_114.pro
c5a90ab4cd846c3d350d2d862b9fd902
f227b28ebead6078e075e9716e28ad487c27fe33
2184 F20101118_AAAHOD amarasekare_k_Page_034.txt
30f7ce215fd0c59f6c42ebde4c324ecc
849cecb105597551cac40f1275d138e97102097f
2237 F20101118_AAAHNP amarasekare_k_Page_019.txt
537a40ba0bc1e1334df129889c3b7e87
0516be922c64e492eceddafa82a423d842f0f4e6
2113 F20101118_AAAHOE amarasekare_k_Page_035.txt
7db90fc2ec6d7181ed68e953aa0620aa
796119c9adb9715116482e192bf10d354b2f2830
2135 F20101118_AAAHNQ amarasekare_k_Page_020.txt
117a1c76419af0e1e778534d6cc63591
d08abad43f00cbbf99135b29e74ff8b073f99a1f
F20101118_AAAHOF amarasekare_k_Page_036.txt
0b723ebbe7efa53802873a75683e115c
9745cc961a8d7acb1ead2b01d0186b7c1a3e0f6d
1456 F20101118_AAAHOG amarasekare_k_Page_038.txt
fb95ad842f571db59cd415d5ebb72e3e
a5a9fb79b8810e21c0510272fda379bb494862e1
2098 F20101118_AAAHNR amarasekare_k_Page_021.txt
8e06116e0d069a0e458288db7fc580ca
eca74d427d1f387be168182e61e0e0aef6a15dcf
2141 F20101118_AAAHOH amarasekare_k_Page_040.txt
e1f733b54c09e19f086e2aa305c86d98
5299f680ec656c8a2eaa37ef9fadf14bad987ad3
2155 F20101118_AAAHNS amarasekare_k_Page_022.txt
7d6432791df4de9245534ddf92a8c32c
2d31e2721ffe7e5a6f02c6a5bf064e1f535b8d27
2283 F20101118_AAAHOI amarasekare_k_Page_041.txt
cdf949ea423bda90b05e648b540cb971
f26df9ffca425f62506e0bb480d6a18bfeb33344
2133 F20101118_AAAHNT amarasekare_k_Page_023.txt
ba9b47ee27cd7566ccf28d471895f459
8261090c1a360ef103e45c5fe509df917db525c3
2156 F20101118_AAAHOJ amarasekare_k_Page_042.txt
2ef8a4eae46c25240c1c423158e8c75c
a504532a81260e4fef5f969a4a3f6c9c01bdaeb7
2260 F20101118_AAAHNU amarasekare_k_Page_024.txt
51bd1df5c59d6b3de249d266a1a8806e
7460afa62db88671cbb653a3fb6c8c2d2e47329b
2117 F20101118_AAAHOK amarasekare_k_Page_043.txt
5fa9158e9a3ffa3571006289eb1c0a15
ec30de8273b7399849efa1bac6ce624a87994641
2292 F20101118_AAAHNV amarasekare_k_Page_025.txt
e9a26065a60f3db82923acf62ca63cfe
d933c1b38ff05e53c4670d45a35b1303f0b44c7b
1857 F20101118_AAAHOL amarasekare_k_Page_044.txt
ff13327a2b5e846f1ac03cdf6029f24d
2e93eaba5a82fb505e13d6bb095b6a1f7fcfaa62
2314 F20101118_AAAHNW amarasekare_k_Page_026.txt
e05d0fb40a573fb386b119b9c4eadf0f
47ffb427c1e952237f8331ca16fb7305f2909b17
1976 F20101118_AAAHOM amarasekare_k_Page_045.txt
ea3ca75bf5b33b0b3855f32b40d8091a
ad66e9fc60d10e60add9bc161c93251d5319d918
1484 F20101118_AAAHNX amarasekare_k_Page_027.txt
fe1c602ec530361ab8a7aa6ffafd6624
78f69040934a6389f23365a0dfd8530765c71040
271 F20101118_AAAHPA amarasekare_k_Page_067.txt
b973332b07b3ea53fa7350dbdc620504
f7ee6d599130f35f656be10bed7e4b45507a1410
2000 F20101118_AAAHON amarasekare_k_Page_046.txt
70f5268a8070549677b5730b4e818e61
4ab8bce08f58782e69119a4d6479fd04d7210d9a
2218 F20101118_AAAHNY amarasekare_k_Page_028.txt
3c91537a188ca991c6369655f129cc91
cfe3c093ff28d3ff3bc72c1a26a009cc2bc417eb
1384 F20101118_AAAHPB amarasekare_k_Page_068.txt
2af97597c6c776b6a0f9b47204ff9f4b
34c128dbe6bb500a44e1682af89ec5bbec3026cd
2120 F20101118_AAAHOO amarasekare_k_Page_047.txt
71960d9e3ef5c846d69c83669cfa3187
07c769fced38c5d92b6c2b755ac3cc49869e2750
2179 F20101118_AAAHNZ amarasekare_k_Page_029.txt
58758992028352245a8bcc775fba980f
761ba297340e3bdaeb6ffdc39b4c13993846aae8
1471 F20101118_AAAHPC amarasekare_k_Page_069.txt
6917ed1372c4208bf0698238a784a40c
2a6213311ae52974e51140534267461277fc3ca1
2191 F20101118_AAAHOP amarasekare_k_Page_048.txt
97c2828188f2c35d2c1cdd8e485a24b9
1f103b2efdb5c383e482e15c3e64b0abfb27cd3f
1454 F20101118_AAAHPD amarasekare_k_Page_071.txt
023307a609c57078c45249c33159fd50
d31e36da995f03dbef132905317439a62f9d29d5
2145 F20101118_AAAHOQ amarasekare_k_Page_050.txt
8290272735fcf8151b535db7f42e9570
05ae77f70c4e39739bb5663e2233425a123b6780
2223 F20101118_AAAHPE amarasekare_k_Page_072.txt
8ede52611bceff60c2694f84cf3e25f3
541e715de124af661007f85d00ce2663816debaf
1744 F20101118_AAAHOR amarasekare_k_Page_051.txt
7cdb8c9b7326932c501415d404bb2702
7ee19efa90152aab6a2e1309254f4196c9a98a68
2146 F20101118_AAAHPF amarasekare_k_Page_074.txt
0271e41d93d62e8c47d4f00e0ad047aa
b5089908a9d172feff4af92fc1eccf58adbc5c70
F20101118_AAAHPG amarasekare_k_Page_076.txt
2ed8cd6c09f0ad5ec16d908f8b61dc30
f40764d4ba98e21296e65732c348bf629c393904
1677 F20101118_AAAHOS amarasekare_k_Page_052.txt
4ea5877a44c600d2bb1daae93327ec13
0192dd16178965d4c743022a3f01bf55c17d8407
2002 F20101118_AAAHPH amarasekare_k_Page_077.txt
ad121355c9874ed7622cdff0cfbf191f
80d657b1cf1f55abee992847348d649d8b83da7b
1367 F20101118_AAAHOT amarasekare_k_Page_054.txt
26c783d4c5cede69c83131a55e61762f
019ee04012b60bb9e63f6c558e2fd3e377e65dc9
F20101118_AAAHPI amarasekare_k_Page_078.txt
a4f58bfaad6d9c2c82f3e02002502f6c
a3d6bf991189586e4a227437351a2020f16399f8
2119 F20101118_AAAHOU amarasekare_k_Page_057.txt
0ef9cbeec131f23ea00ede265b41e11d
407a069759949bb1debfcb7de1fbb2c1c770f6cd
2163 F20101118_AAAHPJ amarasekare_k_Page_080.txt
0209d22e3245acd796e05ab89694dfcb
9c7e606e6916398f1cf2d14fc373547ad6107ed0
2171 F20101118_AAAHOV amarasekare_k_Page_058.txt
a2966ca0ba0ad119f319c147509e0832
7179dda719e854b4b6a050f54e05512a0f470d7a
2106 F20101118_AAAHPK amarasekare_k_Page_081.txt
6c0574a888b2a1b509b67d5918c86616
5bd77f6dcdedd42f735019e0fc8bb247be579fd2
2209 F20101118_AAAHOW amarasekare_k_Page_059.txt
ac6d2853505881f0e2138811d76e028b
9de91c8753d6ea35667844b0fba753fe05e4e66f
F20101118_AAAHPL amarasekare_k_Page_082.txt
3632192c5fb2869826815f391334b40c
aaa192afe90d0b77debb1a85a2f6341914711fa0
F20101118_AAAHOX amarasekare_k_Page_060.txt
f98ab22ad8ce0b80b10f6c27da753cfe
a28b19af0934875575e917d72cb6a499edb62d4f
951 F20101118_AAAHQA amarasekare_k_Page_100.txt
3d9cd31899b081192800c681c9e316dc
549e869dacfe013f8d422084a9c7fc72dbce6218
582 F20101118_AAAHPM amarasekare_k_Page_083.txt
9b62698539265973be5dee948f955248
e54af1c8f55e22e37b886fb76dfa4d002e484f37
2027 F20101118_AAAHOY amarasekare_k_Page_063.txt
d9f27d1158f1fd11ec5e3f5acdb6ab1d
1b93e978ecf0c13b3d96d1d790398000beb6e67b
1227 F20101118_AAAHQB amarasekare_k_Page_102.txt
308d02ffa01bbd73172e04737b5db724
2261e99947bb1d21a1325c73495b1dcbe04ccbcf
1831 F20101118_AAAHPN amarasekare_k_Page_084.txt
d1538250db2113d1aa3a92f96b108689
3552b6fd9314375136aba408053a615c181e889f
2195 F20101118_AAAHOZ amarasekare_k_Page_064.txt
3242db861216e9e869a9a96fae57da6f
2099fbd4568ce3ac8cf9f2ab1c16388059d62eb3
1918 F20101118_AAAHQC amarasekare_k_Page_104.txt
4e0abdc0fafab73b2a72fbdee196b0f2
085dee1843c35027537b512592894a99619b737f
1855 F20101118_AAAHPO amarasekare_k_Page_085.txt
d32c2937de7d12c3df421c2154decce5
9100845200938463d4b4c26dee6f8d0d2dcb679b
2467 F20101118_AAAHQD amarasekare_k_Page_105.txt
2bf648be4ec9bc3a4e05f8578d7a7a02
a1e9f54396c75aab00a24b831120e57b7a0eb278
1650 F20101118_AAAHPP amarasekare_k_Page_086.txt
5df2176bbd833cbcf5ab00175798a8a0
2cc8e037679f67fe5117549ea25ecbbec0cf2294
2533 F20101118_AAAHQE amarasekare_k_Page_106.txt
62e0e869a699ad278158ba07d2ca91f4
b9a39b850c637d155b8b7a98823423faac5b384e
2183 F20101118_AAAHPQ amarasekare_k_Page_087.txt
225a1b0f274b0731c82c8df056c321a5
5da00beb9c876500a79260e1002f7202bd3ac280
2543 F20101118_AAAHQF amarasekare_k_Page_107.txt
7a80b8522f29fda919da90153957133c
590ebf9c50f32388eee8ddd6b9c29df14fafa041
2255 F20101118_AAAHPR amarasekare_k_Page_088.txt
9a28f5d8d9b9a7570dda5ea33ebac074
45caed18842471f719799edadfdf56020754cf8e
2714 F20101118_AAAHQG amarasekare_k_Page_108.txt
855f07d0880893ed416bb39771a3a4cb
2e893c763e6e4038e9dc14e7c49d643112d1a732
2178 F20101118_AAAHPS amarasekare_k_Page_089.txt
c8663425ccfc570c5bae865b0238bcfa
f1ff93415fb7e222abc5fa0f3635e373091458fc
2456 F20101118_AAAHQH amarasekare_k_Page_109.txt
d99be0a76063baf05532b165c4e90d9a
ac1f0bfd82f974e284fa52aabdfeff87c0614d2a
2597 F20101118_AAAHQI amarasekare_k_Page_111.txt
7e2bcc00e1f2e57e33f7175d727c9f72
10b9274a7264733374d7ff0374cf0ad174997edc
2160 F20101118_AAAHPT amarasekare_k_Page_090.txt
b2eab6a6d721e2c96dc9427deb58729e
f4b44d5d127cfa62c08a655dea112d20172db7e0
2426 F20101118_AAAHQJ amarasekare_k_Page_112.txt
a47ccf479a538d8df0db73ac8d1011d7
f704be4d286455daa110e72fa4e18bbc4a7a01dc
F20101118_AAAHPU amarasekare_k_Page_092.txt
06004c533d21a6af86996b1e2d9677b4
90a90c6d564c6f38a0f5ad9ef8aa1279b7d82c62
F20101118_AAAHQK amarasekare_k_Page_113.txt
efd722740841b59b179ecf71b6cdd9a5
316beccba8efe0731714a63adb72c2a6fd8822e4
1988 F20101118_AAAHPV amarasekare_k_Page_093.txt
a84401d195e6565385e8be8dc48f0f7a
3fd52fd8329fd5aa0ab7509a9ff9313e35e97b98
724 F20101118_AAAHQL amarasekare_k_Page_114.txt
4c0ad0ce433c586e1e36ff7879c2bc68
3c457c7731f1abece833cddb2f0b1499446efd9d
2205 F20101118_AAAHPW amarasekare_k_Page_095.txt
2bade546505116e62daaf130a28bd31a
8f933060428ba06f13dfcbc21114a223418e6fee
7660 F20101118_AAAHRA amarasekare_k_Page_062thm.jpg
eeecb33eb5ea626b8a19a83ffdb9105f
ab9b69810afd3e98426f02364b424e1283ef17be
2490 F20101118_AAAHQM amarasekare_k_Page_001thm.jpg
e793de0d5f91612601908effdc6e1756
14febf01232c3ef723043609f026d5d53275fc98
2051 F20101118_AAAHPX amarasekare_k_Page_097.txt
7858bae2eec9621c762084ecb403ee77
bfc4c2ee9e0956db0d86b56bf064c7844edeee8e
35671 F20101118_AAAHRB amarasekare_k_Page_030.QC.jpg
5ea335fc9c95c6af877e726fe0b81186
c5400fa4fdd50379efe690a52572acc9f2e99ba1
970679 F20101118_AAAHQN amarasekare_k.pdf
aeb775afca10a1e2466d19ecfc944fb3
6aaca3e51cfad9c86fe0198fb01420bbe740729f
2023 F20101118_AAAHPY amarasekare_k_Page_098.txt
42f9d4a2ef805dd9616d942365da242b
72760097265dbe098df59856c1f8775d5594eb25
8895 F20101118_AAAHRC amarasekare_k_Page_105thm.jpg
5b2288e1934b4a919514460cd3928dbc
663be52db94da6ceac4711b1d9577e3096b74588
6269 F20101118_AAAHQO amarasekare_k_Page_027thm.jpg
f4989da99ec609d957adbfa8546e0095
fc4e67e2b10ef9b589b73dd4621ce1785b500566
1917 F20101118_AAAHPZ amarasekare_k_Page_099.txt
d4139ac2bef82e6c8275927b5f36c908
0f217cd09416b3de0bfe22c45be99300a5d58b40
6791 F20101118_AAAHRD amarasekare_k_Page_037thm.jpg
ad060ad61e0d3dc356eecdb4912562c2
655d7f908af7c968d44a26e25724c132ecad31fa
2983 F20101118_AAAHQP amarasekare_k_Page_052thm.jpg
6cf22243ec1f2fa973feaa4bbdb3bda5
77f7e4824ebe9a9739524abceb18276f7b36b68a
34675 F20101118_AAAHRE amarasekare_k_Page_077.QC.jpg
f48695652c85157938cb990b7c1f525c
7ed9963a43a7fda676e741798c001fcc4a62f477
37456 F20101118_AAAHQQ amarasekare_k_Page_061.QC.jpg
04df48c3a1c3b8101d29e6e454017732
1d3dec2fcca28ff6c23a6f5be478a8ad6a591089
37698 F20101118_AAAHRF amarasekare_k_Page_034.QC.jpg
3372f0d0cb4ae651ea213d08e0c26837
bd292842370340f7ef1d2a1ea15396aa39f2d6c2
36161 F20101118_AAAHQR amarasekare_k_Page_065.QC.jpg
5d39de64a2ab382dbc58f91904d594e9
6fe6c49afbbe73a3a3d85a0ad73f2611225a5785
5957 F20101118_AAAHRG amarasekare_k_Page_067.QC.jpg
e24776f182e9d40a310ef93ffa4d6ca7
784c19799ff0faf4cde310f32b51dc06f88a32a1
39184 F20101118_AAAHQS amarasekare_k_Page_028.QC.jpg
250b3bdae02436e903254fc5003201d6
020ae989e0ed12d580438184d2c96b94eb2b4038
8888 F20101118_AAAHRH amarasekare_k_Page_097thm.jpg
814bb43757d10978ff2091510c843d53
183fcd40a3aadef212c6392aca79b6cf715ddb1a
9093 F20101118_AAAHQT amarasekare_k_Page_064thm.jpg
2b4d2dfffc44ae5d497747c75a1e91fd
0616380d814b595c81fb4bc97dbcdb8cde0fe24d
10069 F20101118_AAAHRI amarasekare_k_Page_101.QC.jpg
498ff4a13fbe1dcc3b467e8200052752
2a5dd2f960b83b3cbd6b974ba4fa638a9a650411
37132 F20101118_AAAHRJ amarasekare_k_Page_072.QC.jpg
2daabe562fa10cf77661709aa4014bdb
a75a7fea6b84e3d956a274c1a89c6d85e162e010
9156 F20101118_AAAHQU amarasekare_k_Page_025thm.jpg
7f551c6a2744d2329e4361a08c1bf93a
7b3ac47076a24d7a7b0e6065ff882d46029956c4
1985 F20101118_AAAHRK amarasekare_k_Page_002.QC.jpg
af7c64e5d8ec664782075b064b891909
bd98972e30eac660a073e6572bc0d7318c32fe3d
9310 F20101118_AAAHQV amarasekare_k_Page_035thm.jpg
2c58f8d6f5d7779d52a95d92eded24a5
81ece06ab90af640dd1bf1ad588b52f02b64de71
29504 F20101118_AAAHRL amarasekare_k_Page_009.QC.jpg
558ffc0f9c18a0d5b49e6ca04887ab6d
46881bdbdbf2cee1685aedbf4a963e79a99d4a68
11461 F20101118_AAAHQW amarasekare_k_Page_100.QC.jpg
37f8e7928a65780f7d792af582eb6003
d2a8c74ea3e5d3314e21cc48a93e642d8c71cc99
39464 F20101118_AAAHRM amarasekare_k_Page_014.QC.jpg
f4f5417f09d6bf0089653ae420ae444f
95fda08b727d6e82794289b4983ec010a01f0803
36649 F20101118_AAAHQX amarasekare_k_Page_082.QC.jpg
d8965d38109b27b8ed0d76f6cd3c73b8
6c288b4bf38afa1cd4f6f1f95539d7e49da63fdb
38580 F20101118_AAAHSA amarasekare_k_Page_029.QC.jpg
40eaf6128488855212d6bf30e3300875
66b0ad733f1b5e1ef338ac4fe7275c9fad78e04c
33997 F20101118_AAAHRN amarasekare_k_Page_046.QC.jpg
a19ff454fa206b0b348a5a45afd74f3c
820fb6c2c98bd4226f415a2f1addf07a50c78862
3069 F20101118_AAAHQY amarasekare_k_Page_053thm.jpg
571496258cd99a874e5e9e5a98dcfb35
80f1eec584b5659866f260cd23a651c35cab3da8
8738 F20101118_AAAHSB amarasekare_k_Page_019thm.jpg
4b6a2a7998dd25b1dc11e57612962d8b
258ddedaeea41856d8c19a49aaf970ef522e5b35
37419 F20101118_AAAHRO amarasekare_k_Page_047.QC.jpg
9af8400343a0404818198d7f46cbb8fb
6850f18cbafe3848822a230f9d67e3214f4fbac1
4346 F20101118_AAAHQZ amarasekare_k_Page_055thm.jpg
4e48393cfb7062a1678f2ca2e9d26f9d
9224e8e0efb37e2b1d7f45c06b5cd23908443ace
9213 F20101118_AAAHSC amarasekare_k_Page_061thm.jpg
2bb0051fe85a267e6c0f9e6922959d5a
b37acf6b52d394672a6b1ec944c5ac7ffaa8301e
19997 F20101118_AAAHSD amarasekare_k_Page_084.QC.jpg
b18854af60a7f79899a436d7331dabc2
f2fb13003bf223b960e038d04b57e787972f7340
8515 F20101118_AAAHRP amarasekare_k_Page_033thm.jpg
77afe94e7c7723d65916fa58ec71d754
183ea3d49376b652fcba009c34a962bdfaea27f4
36650 F20101118_AAAHSE amarasekare_k_Page_043.QC.jpg
011073fd03e6b591a6ad7b3ea35b35c1
9c111524ee46431a6a6083337f87dfc5ab60a2c3
38459 F20101118_AAAHRQ amarasekare_k_Page_018.QC.jpg
4e677d1f47b91257f783ad83aac3dbc5
054e1b69ece0fe706566ed1f72e9799d8e2414fe
36851 F20101118_AAAHSF amarasekare_k_Page_023.QC.jpg
6df66b865641630f3b49d80b819c6f8b
73ef78a39e703964929e484a0b22267e01f95ee7
9055 F20101118_AAAHRR amarasekare_k_Page_090thm.jpg
6abc62c20a767465c4f69df66182f762
5d9ceb8cdbf893697903ee5c5d558a44ee56e7ef
3712 F20101118_AAAHSG amarasekare_k_Page_008thm.jpg
49510a6a341015e59bb05040b197d8d9
d1f7c809f7a693725aaa714cf1153b99fd585504
40633 F20101118_AAAHRS amarasekare_k_Page_110.QC.jpg
3402222e6a10e0e3811d1de5197521b9
8a954f276305282c096f332bf08c13a6c3bb29a5
8999 F20101118_AAAHSH amarasekare_k_Page_094thm.jpg
e0723c8250a36743513e21feb283c4d2
6a7e00db736cab0a1a41c89d038becae2fdba4c5
33157 F20101118_AAAHRT amarasekare_k_Page_045.QC.jpg
278bc8d11d2009581280c333e1e5a53d
c1c6a30c1d28879766f61159ccda239da0a0b092
9399 F20101118_AAAHSI amarasekare_k_Page_054.QC.jpg
fc2a9f062304fb5baee2807bae29de9a
b48db762c5760260a339624c1b266749368f972c
35138 F20101118_AAAHRU amarasekare_k_Page_098.QC.jpg
a1ee5eda6f64b829ae522a41897ed478
dea63c04622c61aaed44b904c1b67b4851a29c04
3849 F20101118_AAAHSJ amarasekare_k_Page_069thm.jpg
5e653870218a9f98a22b91bf4838522c
553d097fb55db1161e2cd4a13f02d1444fd89f3f
9128 F20101118_AAAHSK amarasekare_k_Page_088thm.jpg
be3954642c605955f2e2c4b24d03df9e
17619ba02222c1ceceb50fece0ce8b6028918913
5169 F20101118_AAAHRV amarasekare_k_Page_010thm.jpg
09355ba35ce904a953e3b20ca3eca17b
c2b11d04d434762df89c3982b86e0fe8f22b079f
13802 F20101118_AAAHSL amarasekare_k_Page_102.QC.jpg
b3ab19bdf51587e56c2f93fa2f2e908f
59551c3e2a9aa45f9f737809d94ab7a07e1983b6
38348 F20101118_AAAHRW amarasekare_k_Page_024.QC.jpg
e9c22835803631da66f16bcc1925a40c
145bf2778a2461f86ec6638c27794cab3251c21f
5196 F20101118_AAAHTA amarasekare_k_Page_084thm.jpg
8c3f8e04937877d7d21751466a91032e
525aac083a1ad8574e4599e421b1301304f2ed49
9129 F20101118_AAAHSM amarasekare_k_Page_018thm.jpg
56506f0853a4025c2ba1c0979e9ad8c0
f7179a9cff805998d21e01f23668e7143563396f
34697 F20101118_AAAHRX amarasekare_k_Page_105.QC.jpg
fe73306499a7d2e892ab9bf8fb658c05
12fefedadba0cbc008e5a70ba868fac35748e161
36382 F20101118_AAAHTB amarasekare_k_Page_076.QC.jpg
9515fb1c1e929d50088e22335bd15923
75aec74de2d33063eb191fb09a37b63124c5cb2a
9501 F20101118_AAAHSN amarasekare_k_Page_039.QC.jpg
d04730325b935199dfe166364e97f06c
d108590eaf0e6e9c99ea3f05e1d3afa7533866c3
9281 F20101118_AAAHRY amarasekare_k_Page_109thm.jpg
91601a207abb3af7e9b0e84579201be7
9adfb05bde545edb8c7f397d7138aa100569a4e4
35073 F20101118_AAAHTC amarasekare_k_Page_011.QC.jpg
e2601671601201103b1f98572ca6fc02
d7401054be7aa9cc175f2f07ba3d4572f6de3885
5252 F20101118_AAAHSO amarasekare_k_Page_004thm.jpg
f056137a3050a83084516b119a1f9acb
7d14ddc756ecaca62947ac35e0ed9412dcc3194d
11725 F20101118_AAAHRZ amarasekare_k_Page_053.QC.jpg
332dcd3e0c20c53136d5e9554e0b6400
b10356a28716e688616e2fe6315571f230657509
11443 F20101118_AAAHTD amarasekare_k_Page_052.QC.jpg
51206dc475a104dc9fe3b9632ebd4403
5306c7329e8a86c8d9f9cca2ff99161a950d465a
6089 F20101118_AAAHSP amarasekare_k_Page_006thm.jpg
35566333ae71fd3fc047e8682474fc30
717741c6b6f365e7e78191bd1cce3f14ee90532a
33932 F20101118_AAAHTE amarasekare_k_Page_093.QC.jpg
513a0dd9b7bbfcfe84a7748861ff77a3
c887042bc8a7b6f3b9e21da142f4cba3642a30df
8989 F20101118_AAAHSQ amarasekare_k_Page_056thm.jpg
f88c4ebe1ae792fdb63d76f0949eb31d
a13b1bfe892950a1cd997b607eeffd8826eb4254
36853 F20101118_AAAHTF amarasekare_k_Page_090.QC.jpg
19bc618edf9e7f6414ded5b17d19c125
d18a9871bdcbbdbc7d021f3b209e454e4b7477bd
12644 F20101118_AAAHSR amarasekare_k_Page_114.QC.jpg
cd3e8d02cbc0b81960a8646a37eed6be
0050983715f7784c4d5a6a2f7beb623ccfe5fbcf
8919 F20101118_AAAHTG amarasekare_k_Page_012thm.jpg
5bab44592ea14d7671ab973d0d05287e
b5d8330b1b3a22ed3d50a197499e794634861649
9427 F20101118_AAAHSS amarasekare_k_Page_014thm.jpg
c41ef5a1d89f89779739c3102fb2d081
427b524f88a4d65ed40216f20ab61a83fb34b2ce
31372 F20101118_AAAHTH amarasekare_k_Page_044.QC.jpg
1828b632a3b749f9f9c3bf3ef0201b36
ec19c8bd6aa65b69e56d2520e840a324c70f4c47
36590 F20101118_AAAHST amarasekare_k_Page_036.QC.jpg
da451cc08738b446a43e6bba5d51d3c9
1588f11730effd8d34bdcd61e76bde1f60db67e1
37138 F20101118_AAAHTI amarasekare_k_Page_106.QC.jpg
4b8781050ab6114f39f1d12eebbd0f13
d05197268ba6f11b44f3a7fe1b0d51594fcdeb8c
35301 F20101118_AAAHSU amarasekare_k_Page_075.QC.jpg
a65686c1340ce5558a0a412d0d9d0c26
ee74dae786de16b67aac08732f4812d400ef8f45
21277 F20101118_AAAHTJ amarasekare_k_Page_010.QC.jpg
d36bcf7d09d5ff6d309c90846a773f37
6416a2b63b181fb7e33766a4d4734320d331d4ae
15244 F20101118_AAAHSV amarasekare_k_Page_069.QC.jpg
4a41d65fdc3d747e42021afcc3e12145
5326776ee42b3a2295675727d541498bdb672e6a
8737 F20101118_AAAHTK amarasekare_k_Page_020thm.jpg
221263252660f81cb2813cc18efb750b
c72073f01076ba0560bf9ed34cc83e1df5943541
9649 F20101118_AAAHTL amarasekare_k_Page_001.QC.jpg
0c2ea4e7e333b70c20af363712a2eb84
ba6468069cc8bd9730f9f43fff9d84b37f83e4a0
37459 F20101118_AAAHSW amarasekare_k_Page_015.QC.jpg
1c5f1d22866669598d56c149b8a5c389
a98f314db015442599622d1880aa2cf628eabc9e
10611 F20101118_AAAHTM amarasekare_k_Page_038.QC.jpg
bccb38caab715475a684491dbdc5e13a
3bcbeaa2c6c541ed65577e4729c35a85057faf2a
37337 F20101118_AAAHSX amarasekare_k_Page_019.QC.jpg
a16cbcb2560654059e7dfec5c23ff8d9
8e9b543bc41667c374bc4b8d3a3bbc78aa9b2b90
F20101118_AAAHUA amarasekare_k_Page_013thm.jpg
2ef8e874754ecba021fabef60b2cc512
f785ebcd1ed58f1b17b25c0e41cf0e4feaac7760
9246 F20101118_AAAHTN amarasekare_k_Page_036thm.jpg
25968f5bc181acbe6ade1e69ab973074
d9601474c3c1c91b27080099c7dae8b1dd4f753a
8824 F20101118_AAAHSY amarasekare_k_Page_032thm.jpg
99d16274ef86699cd806bcb1799e45c1
1ea10406f7c9a0d931c6e0e69d78851ba546a022
36740 F20101118_AAAHUB amarasekare_k_Page_013.QC.jpg
665c1d9643077d39d255156934dd5a1e
ca284eefcadadf2750372071ce095b7fe4b765c5
8165 F20101118_AAAHTO amarasekare_k_Page_093thm.jpg
6459c2393ea23deac715832be7dc0469
fef3971256341e2a1c43114b87ea9c36b63ebb75
9105 F20101118_AAAHSZ amarasekare_k_Page_080thm.jpg
ffd1cbeaf134ea9031574f35ccf9f2b6
f5e59fd424645404db4c5d212dd32a263b2af169
9613 F20101118_AAAHUC amarasekare_k_Page_016thm.jpg
e77667fa7eae7ce498c5f86d80541e0f
600ea6dd7b18f3cc1edb62c820abf9c4b744850c
35606 F20101118_AAAHTP amarasekare_k_Page_097.QC.jpg
920971b9845849eefe0c5c571377fcd0
ca90eaf5ed771ecbb923fcef44bd1e07005c21ec
38842 F20101118_AAAHUD amarasekare_k_Page_016.QC.jpg
4556f1e622f99e65be7e8f0d3ff1468c
7f591f518b512eaf105f7e1e9ea03382f9bbae5a
175704 F20101118_AAAHTQ UFE0021652_00001.xml
dfdb9d3d9f0d57bd0d5f289b83b728f7
bbeade6091395f615fbc9bc6468f7bbe40b1c9fb
38258 F20101118_AAAHUE amarasekare_k_Page_017.QC.jpg
742698978ef8cc77355040361b49c779
f32c834ed8a4d4fe6c5c8ff341495e5fdcd731de
698 F20101118_AAAHTR amarasekare_k_Page_002thm.jpg
7ff88e3018585110bb8de7c7f00699cc
88714576bd5a013d9573d0a0a4f99cf447422be3
36437 F20101118_AAAHUF amarasekare_k_Page_020.QC.jpg
6a9507c29796f6471a598f709271d4bf
3c00c97f99c6c57777be1060bef41b4dbdb2e6a3
671 F20101118_AAAHTS amarasekare_k_Page_003thm.jpg
10103d171866ca56c6c9a7442db142d3
fafcf325f68765c5a9b3b011a01a0a2692945046
8959 F20101118_AAAHUG amarasekare_k_Page_021thm.jpg
e42acca564090ebfba7c45d77f6b2e53
7d943a7702f04d129189467dad671f673fc1825b
1885 F20101118_AAAHTT amarasekare_k_Page_003.QC.jpg
aa57f93a4e87521c110e5274201172cb
6dde796375362ba374a32ae80dc0aeec70e8c7a9
36867 F20101118_AAAHUH amarasekare_k_Page_021.QC.jpg
5b676fd0929df746b3fba79e9a13951c
ceeb703d03b4fc2a8dc8dd996378c584ebbf9d03
20633 F20101118_AAAHTU amarasekare_k_Page_004.QC.jpg
f898292e2232c47f05146e5d1b96be7e
86bcf8895d79586847f555ae98832a7d1a9612a7
9216 F20101118_AAAHUI amarasekare_k_Page_024thm.jpg
23b7b38870852a9f777eb863f5bd64c6
035233bec458802b456120cbc5d813998c4a5563
7126 F20101118_AAAHTV amarasekare_k_Page_005thm.jpg
8a162289bdaf73f33b0e770f314e1b2b
aacef89549a308f4eb44bd14ecc9e89d9def46b6
38392 F20101118_AAAHUJ amarasekare_k_Page_025.QC.jpg
e74a4783b3c0c56dbe93c874d8acbfbf
e4863532cab54c547270021ef5751c6919d61c3b
9304 F20101118_AAAHTW amarasekare_k_Page_007thm.jpg
88a7017ca4001081384381ac0b50509c
b84a27c68e856bc61e6366aa63d403903e556245
9245 F20101118_AAAHUK amarasekare_k_Page_026thm.jpg
11bc4587001d21790bafcc640f51caba
9e40429e207bd2d455df2ad3d71af2732f6c6764
38358 F20101118_AAAHUL amarasekare_k_Page_026.QC.jpg
d7ab870940910ea3e8237a4c44f6dcf9
d1d09c09dbc94c0a348073fd0d285dcfaca7bda9
15707 F20101118_AAAHTX amarasekare_k_Page_008.QC.jpg
fb3b4f2fd9466089e5a097f9e7f7e33f
99dba4cd24bdaad2056b113bc78512c9f2e63d2d
9367 F20101118_AAAHVA amarasekare_k_Page_042thm.jpg
88726eb58beb0510f8648723ff2bd217
5fc80005cc38956d85afee2aac74b7b59542fc21
25587 F20101118_AAAHUM amarasekare_k_Page_027.QC.jpg
2a13f943c024ceb7639c0182272ae8c0
df89548c441ad422ef84f6a0173e38fa8ef9f4dd
F20101118_AAAHTY amarasekare_k_Page_011thm.jpg
dc032be07704184822c0fa5f4dd3f8e9
051fcf8c30b04663016654463f281f0eedec902c
7891 F20101118_AAAHVB amarasekare_k_Page_044thm.jpg
7d318862c8c3d8101712c542eb1a51f9
151d6f9a6ba447e4d27d19ed357f1e2633c9eae5
9450 F20101118_AAAHUN amarasekare_k_Page_028thm.jpg
9e296609e685fc0b404f2688223972da
ad621f70bc0825ed9038430ecb77b31658886a0f
35917 F20101118_AAAHTZ amarasekare_k_Page_012.QC.jpg
9f39a262c09d0032bf7aeab4000fc62d
e805080d8adc45e999a50820c5155229e4f69dbe
8754 F20101118_AAAHVC amarasekare_k_Page_046thm.jpg
7b1dc7be49cb7d5e3669bb6fe53a5519
4f2215248bcdf3a6a68dfc8e3f46708cbcfa8331
9497 F20101118_AAAHUO amarasekare_k_Page_029thm.jpg
a752c37b1392626a3fa0f43656a280fe
f9dd2be1bda2bdb1f44e7fb41a33a7e1a27b918c
8878 F20101118_AAAHVD amarasekare_k_Page_047thm.jpg
b482fd9cd18a13f12fdd7c26f9e213e3
2c1df446b9020ef83ec301385e3adce65d670e8e
8976 F20101118_AAAHUP amarasekare_k_Page_030thm.jpg
b2ff34cbaaa30048c5f5c23cdffdf37a
68192635f20c385ccdb5132f859a2b2a25924f91
9585 F20101118_AAAHVE amarasekare_k_Page_048thm.jpg
36393b083922fd5e278b4ad38b3196b0
17a0b66485ca1f8fe575171a263855c388bf8b9a
9172 F20101118_AAAHUQ amarasekare_k_Page_031thm.jpg
f23ba107c6a3af15e0c0ab55b2399f57
aa4f8e2a2056a1ab92552e2f331f6aa85a18b956
38428 F20101118_AAAHVF amarasekare_k_Page_048.QC.jpg
587eb747da823cf58b7a4fddb6803008
99a83c88b1b280d43080e08ab0c77bbb66d83e2f
34657 F20101118_AAAHUR amarasekare_k_Page_033.QC.jpg
875086e299e3922373ea48aa300ccccf
f10d32f5770658082b95feb1add3279c7275b76e
9150 F20101118_AAAHVG amarasekare_k_Page_049thm.jpg
ff25ecb5d129def5dc97e5092ea66bfc
407268a4c5096f176d679c9a2dba7c04a0cb24b7
9043 F20101118_AAAHUS amarasekare_k_Page_034thm.jpg
b9631897f17c48865379ce3a81055df0
c3f701f2e0fe17b1d45862e7f2d4685fac50b202
36843 F20101118_AAAHVH amarasekare_k_Page_050.QC.jpg
2464627857c8f5e80600d164d6d9c02c
ddd49425e82267ef357770dfd9fa2423b01857b2
37889 F20101118_AAAHUT amarasekare_k_Page_035.QC.jpg
c8171b6cf6b768b158484527609b5ad2
bcb2e522a930ec6b2e599812744a04db4c7b79ce
7580 F20101118_AAAHVI amarasekare_k_Page_051thm.jpg
00c231fb567c7a7aff2a7efeb1cbfa3d
469484fec2efb22672608326fd25ebbe8912ad45
2839 F20101118_AAAHUU amarasekare_k_Page_038thm.jpg
1e56063720d1b07e4075e57abd30e77e
fe6ce1f9d8e66882ac52fee2ae0db66ae28971b3
31582 F20101118_AAAHVJ amarasekare_k_Page_051.QC.jpg
091b72c31548ba106257a2af4391710f
3a431ac4f99736cd5a9157bb688d0336e926e48c
2532 F20101118_AAAHUV amarasekare_k_Page_039thm.jpg
b2f698a3fba18a7cf8cabc9aed83a22b
76bd6efc9b56b15651def166cf4d4f3b3bf79335
16344 F20101118_AAAHVK amarasekare_k_Page_055.QC.jpg
d0f74c8fcf229be634fb7b3b0c0faff2
4d92f3242806111629ac4bc9f4e121f6fd4b96da
9048 F20101118_AAAHUW amarasekare_k_Page_040thm.jpg
b46a9a09e5dc72736ee096d01c7124b9
9ea2f794a7c86b100d79e21ab163f3545dccbd66
9354 F20101118_AAAHVL amarasekare_k_Page_057thm.jpg
5a8b50c8dfb0caf5b921773265b9d5f0
074070e13df61b464c56200efafe46062f065e63
36203 F20101118_AAAHUX amarasekare_k_Page_040.QC.jpg
7e64c2a5e49eaa105b7fa17e4ca99aef
ab4ddf4f29bba9c6dd9b8a207a8c9c6c6c690a76
8894 F20101118_AAAHWA amarasekare_k_Page_072thm.jpg
11045951a3cf84c50f801f64017fda42
8ced6afe7e7c5388c6f61f030f9d4b07334decba
37241 F20101118_AAAHVM amarasekare_k_Page_057.QC.jpg
2b7baebdc499aa7f560e66391f25cd04
0914c1550ed88e08443f276e4e95716a27409aa4
37587 F20101118_AAAHWB amarasekare_k_Page_073.QC.jpg
4c91fe600d13a09fa7920fb2052bcc66
20bcf985af3736b1835e0e66ef14b93edcdf9ce0
9124 F20101118_AAAHVN amarasekare_k_Page_058thm.jpg
6396fbc96ef6959ac4b5f66fe23863b0
4fc2070a90b93db7a6370f0c166246bdd5186641
9496 F20101118_AAAHUY amarasekare_k_Page_041thm.jpg
210848e8107f2123e8893ae9bf89feac
84bde67cac9c2d1f4897b6c1c53be2b349cedf93
2189 F20101118_AAAGTA amarasekare_k_Page_066.txt
7e63ed0f8ecae3665eeceea4eb19aeaa
80fadc8a6a6ffcc39d8579ec05a718411f33dc5b
9243 F20101118_AAAHWC amarasekare_k_Page_074thm.jpg
ca812fe07b1a3ccf9cec2bf04e0cbb61
2a0c7f0cc5c0cec2eecdfdbba87016064c2e1089
37781 F20101118_AAAHVO amarasekare_k_Page_059.QC.jpg
9c0096702f112e35d0d0747f282afe22
7b98b9562efa38b56c75cbcceb376bc0fc72a0d7
39647 F20101118_AAAHUZ amarasekare_k_Page_041.QC.jpg
36d87d14d56692088b7d82dc22249b68
598fa972c3652564742b6da31e297adc7ad6587a
36999 F20101118_AAAHWD amarasekare_k_Page_074.QC.jpg
2cdf79c1f71da7c9987e17cc06131183
66a535c81e8ff8d6f949ef9357d10d77a6025a22
36610 F20101118_AAAHVP amarasekare_k_Page_060.QC.jpg
34addbf67ca0c59f17f227e83951c289
11a5d59786de0822043d1dbeb25dc9ee04e02c65
F20101118_AAAGTB amarasekare_k_Page_095.tif
34e6ecd707060c93592a39aa64bd7a2d
1aecfa6946a9c9b46a26efe9ac054d67f6d227b0
8501 F20101118_AAAHWE amarasekare_k_Page_075thm.jpg
43817794ec1c3340b08dac1ac9ce2326
f0be5c50dd99d1f40b7ea2ce18e94d39fbd41fb0
31649 F20101118_AAAHVQ amarasekare_k_Page_062.QC.jpg
55df5dfad05eadb023fcfd5c1b76155b
52a10d7093cd31d9f5d5ea3188396accb1c72d2b
106425 F20101118_AAAGTC amarasekare_k_Page_104.jp2
8c5aecf91c1f4766e60f3437f1e69339
7820ac8eccd3cf4b79be1db9d308c55e90e9ab5f
32373 F20101118_AAAHWF amarasekare_k_Page_079.QC.jpg
9e9ab61f5499837d46cd28436ee6c1b8
398b12b0057f39db1d55534f9864a4f3d6b6dd1a
8946 F20101118_AAAHVR amarasekare_k_Page_063thm.jpg
c008cc353f695e6e6cccefcbcbd165a3
dc96a62cec88e79f7e22ac47d038cc8ab94e4b36
56771 F20101118_AAAGTD amarasekare_k_Page_014.pro
e00368fce2489e2b910b17b7d7dac675
c4842575542425a1a00e199bb42e6457b1e79687
37285 F20101118_AAAHWG amarasekare_k_Page_080.QC.jpg
e7705b6b50b5bf08a06045544fab0d34
f0dda07bef52f3080ced4f16e5b7dc60dd35a614
35219 F20101118_AAAHVS amarasekare_k_Page_063.QC.jpg
c036dc846627d64c7180d7cae815875b
73eb89f37611ff79c0cc2527f1ea3c07a3d1669e
36006 F20101118_AAAGTE amarasekare_k_Page_031.QC.jpg
e781567c7177e07b4eae50cdec84be6e
02a326bb00113dde3fb4d83ae94a543bf8d3f0f5
8858 F20101118_AAAHWH amarasekare_k_Page_081thm.jpg
1f9d9d959315754c99d4cbcdb484f0ef
beb75e3e4b2dd9be6bf6975b089c774198582e74
36703 F20101118_AAAHVT amarasekare_k_Page_064.QC.jpg
29b63ff098832c2b30b726e4ab89eb29
cee12d7e11742272ef087196cf725c67d0f58f71
117792 F20101118_AAAGTF amarasekare_k_Page_029.jpg
fbd59063ec6048790cd7c255659880cc
ac5812a26d0bbc959002d1a7a810275d1b8e8981
35703 F20101118_AAAHWI amarasekare_k_Page_081.QC.jpg
97a09543ec055f56c1ab66f9ab5bc751
2a7afee27d757aba116741e90fbf598f0d98076e
9180 F20101118_AAAHVU amarasekare_k_Page_066thm.jpg
5175489fcb4393fc31ade403535c07ed
bb308d1d198a1179633725c520bc9be11163dd19
132005 F20101118_AAAGTG amarasekare_k_Page_111.jpg
ab7a7a48950fdbbdee2bdf96f624f2b5
b7d5b5a0f74fd5621456d7d88773620caa59fff3
9169 F20101118_AAAHWJ amarasekare_k_Page_082thm.jpg
5d236f2ba20a056bfc57162561ce729c
1f8e291133d0ad60f9f30f5b6c1706211135a8ac
14503 F20101118_AAAHVV amarasekare_k_Page_068.QC.jpg
8583d843a9061bc55da8b1c95e7703bc
188e056493772de30f1667e3d81fbb785bc125c9
1217 F20101118_AAAGTH amarasekare_k_Page_004.txt
e56e484e4deeacf74996aa6a225104c9
db3abfb9dcb3ed6bad4e3dbe66defb6e6c001881
10831 F20101118_AAAHWK amarasekare_k_Page_083.QC.jpg
3a27f6bdbb144552fdb135ba6cce9e63
0a5525133b7d9ff0270456c984a410983d86ddc2
32133 F20101118_AAAGSU amarasekare_k_Page_054.jpg
4ca93bdb716578a7211e9a7bfc16869e
fd5c7c656a38c1f008ff43b0304737cbc0fce1da
2856 F20101118_AAAHVW amarasekare_k_Page_070thm.jpg
e2330a902e3503472a4d93f9212f234d
5d3e7d6ef7280b4125d8e16bc92c09137c3875e0
F20101118_AAAGTI amarasekare_k_Page_005.jp2
faca74f7bf47802ba9d0d15d9ab4f814
841e980cb1f745f12a3c26bcaa2843fc0eeafae5
5626 F20101118_AAAHWL amarasekare_k_Page_085thm.jpg
2fb15a50d8d22845821a5f5f963194fe
b723ec2766cfcfeaf1924849efca5c32704138cd
31809 F20101118_AAAGSV amarasekare_k_Page_083.jpg
a582041538287352d88947b74dfb5237
b7df3350ae62e722f7ec5823eaba5075f0636e29
11119 F20101118_AAAHVX amarasekare_k_Page_070.QC.jpg
e0c75a91b8bd2c79f9622d9ec6c4de3e
b1a15ff754584714b0990edcac41f038279837c0
F20101118_AAAGTJ amarasekare_k_Page_054thm.jpg
085b094a1fb4fc0c390084e2a7192147
9054d73890cd9be8d30f055e400ff9707cc6b86a
8008 F20101118_AAAHXA amarasekare_k_Page_099thm.jpg
c3c87a2fb9e0478dba9b58226c773c4b
7381cfcd9ca54f657f69abbdbb19d55b92855448
113226 F20101118_AAAGSW amarasekare_k_Page_049.jpg
851ab49d1ca6a9f827efb754240c6f75
ce2fd9af545028cebc2906adfbdae0aedcc75d70
3430 F20101118_AAAHVY amarasekare_k_Page_071thm.jpg
f349f8d5335386cb25682a36645532bd
15ca24fc1a3130ae364ed594b742ff91d89aded8
F20101118_AAAGTK amarasekare_k_Page_042.tif
bd42b6e7f433cd82d5e686e13cf4186a
574d0017bafc68a141f419b5c0704ed46643acd5
21138 F20101118_AAAHWM amarasekare_k_Page_085.QC.jpg
f7fdeee8363a572e81366552f7cba170
d573b94445e3d1aabb68b095eb77d515459d1364
32233 F20101118_AAAHXB amarasekare_k_Page_099.QC.jpg
67e3df5388628db71c8d4e08e11916e4
c0c67410e9aa64f7910719bd15df8e75fd1cc743
54049 F20101118_AAAGTL amarasekare_k_Page_015.pro
fa913ae9a2468b5b7213048a7c9a3dde
691ec34ce869f7ca8718b69d1b0cbdb24306754f
3272 F20101118_AAAHWN amarasekare_k_Page_086thm.jpg
3da88406b3e1fe47957cd348b800429c
c2516d2f46d5ac4c43e926938c872713d6278e60
2710 F20101118_AAAHXC amarasekare_k_Page_101thm.jpg
e1258d3460594d36059bea06f9ba6263
d28dbe02af67c1057885260bed4bf1a08b0d46d3
6770 F20101118_AAAGSX amarasekare_k_Page_067.pro
bc0222c253f9a27db7e14d12607b7b62
c414f425035569df72adffea9627d2a2ea93d8f6
12283 F20101118_AAAHVZ amarasekare_k_Page_071.QC.jpg
c7a63e017658d6c8f2e3f5e01245a758
9649d363dd7ed799f30c6f934c94e668bde6a6ae
55237 F20101118_AAAGUA amarasekare_k_Page_095.pro
7537b67a17c908360d0c0b22eaae7b3a
b9378814bd89b7bb1e9a99283f457f720255c423
9132 F20101118_AAAGTM amarasekare_k_Page_023thm.jpg
1a620ff6fa3983d0672171baada8e9ef
29759d3ca36d40bfbaa1af43794c3335bfc0ccd3
12541 F20101118_AAAHWO amarasekare_k_Page_086.QC.jpg
fb9851e46f30477305b9edc93b5527e0
0b34a6016cd3f521b0db777272086c4a5348fe30
3387 F20101118_AAAHXD amarasekare_k_Page_102thm.jpg
846f909ec162038d050a92871b6a5479
d4b47a1f75058b6dec24e6a639d90be02d2cf279
8420 F20101118_AAAGSY amarasekare_k_Page_079thm.jpg
57fb7dc257087c3f363be4d1a2321557
9e4a36de3ad2aff66a40043cad7737f7ccdc4f06
2176 F20101118_AAAGUB amarasekare_k_Page_096.txt
3e8b10953a6016901ca39abf0db7f25b
8b57341bb6662522bda97aeb9ea49bb548c9c7a6
51830 F20101118_AAAGTN amarasekare_k_Page_032.pro
365c83c0c9e0b7bba58e9e95e4821b26
84ddab7862e1073cfd37837a775d0b81b82fe4a6
9117 F20101118_AAAHWP amarasekare_k_Page_087thm.jpg
59db23291f0f567ee9ac4ce05beae58b
9cd68cd4bc51a4332dd5c023988ad527c19c95d7
8908 F20101118_AAAHXE amarasekare_k_Page_103thm.jpg
11543993a38604ea766c2f03a5a95ca4
1751fed950d31b2e970f08643405ff7b34259f77
26409 F20101118_AAAGSZ amarasekare_k_Page_037.QC.jpg
eb9f4aec15de23e6d34d4d2859e9f590
37878ffbf8374d4c8042990cbbed8fb18561f5aa
1458 F20101118_AAAGUC amarasekare_k_Page_037.txt
d26011ba29353a358321f0e1a0b10662
d17694bb2222307571d9ea6a66dcfd2aa9f69857
2132 F20101118_AAAGTO amarasekare_k_Page_094.txt
13c830f4d2779bfa80595b78cfc73822
157914c966a84738d2addc04a856462bf2bc08b6
37413 F20101118_AAAHWQ amarasekare_k_Page_087.QC.jpg
b9bad06b095697ee35e124d0a04ebb81
5d7eb388045a1758d2599be2b7e992d167a56587
8126 F20101118_AAAHXF amarasekare_k_Page_104thm.jpg
2e81d70dd68ae7097e0a9d72a6aee0a1
1aec685d27ed7c10f7a0dbf2d6229f519b24005e
8995 F20101118_AAAGUD amarasekare_k_Page_073thm.jpg
f01d98172c304cfc4561a4e0958834e6
77a95985b202fffd34ee3de140b22d1efedb51a6
F20101118_AAAGTP amarasekare_k_Page_085.tif
1ed98c68a3832980fc178165eb830ab9
7eef3586550cd2b4768c2f1d6254a08f01e47367
9640 F20101118_AAAHWR amarasekare_k_Page_089thm.jpg
0e5c6f2857cbb1f1b5f4176b8e462d12
8e95ea347b330dbe69ade15a772fc9626099dae1
32833 F20101118_AAAHXG amarasekare_k_Page_104.QC.jpg
46af243eff4ef8f1001620995258729a
2fbf9f5759e73e52c408f1a29baed3920b1fd8e5
2817 F20101118_AAAGUE amarasekare_k_Page_110.txt
c09dfdca1f4035bea5d899d7e3cf8008
638f5570f70b5beb69cf341585464f32c56c245e
118408 F20101118_AAAGTQ amarasekare_k_Page_020.jp2
b2efb897a27e5af024b4ad46a02c5c44
b5e91f3147bc3155ab6a5587d0666f71879d6a5b
38099 F20101118_AAAHWS amarasekare_k_Page_089.QC.jpg
7ac3fc11eef06c0a5132acfff6614aa3
0eb01f3c5366837d008d518c3aab87a108876e30
9582 F20101118_AAAHXH amarasekare_k_Page_106thm.jpg
1189ed998db8e5835d0ab64c25146d15
44eaca30117f9b9b92dd1ef21ea61d5421b3a4f9
F20101118_AAAGUF amarasekare_k_Page_100.tif
8cf26ca8da19e640926e3117a62ce898
915b59717ca6037c61e83a2b23187b72b84affd6
35854 F20101118_AAAGTR amarasekare_k_Page_032.QC.jpg
631a97770bcabc8700c02c5ed96dc0f2
30c9aea7aaf5ee290a0929e0c81a141c6e58a4dc
9192 F20101118_AAAHWT amarasekare_k_Page_091thm.jpg
de688e25c560f84840cb0bbf1fbf7b94
78d478f5b1573254f01be7bd87a5447ca6943618
121092 F20101118_AAAHAA amarasekare_k_Page_041.jpg
b59adfdf4cc0ad49e24fe0eb042e1721
f44d245bf078e40a92d939ea265f2deba46c10bd
35369 F20101118_AAAHXI amarasekare_k_Page_107.QC.jpg
c2654737c98529d1e1c3c84b24e8b0e0
aa20d9d44872938ff7e155832c4fd37997ef75b8
53779 F20101118_AAAGUG amarasekare_k_Page_036.pro
fb51f19ebd5996727e04fa8c7baf7887
ee5b2bb8a35acc36f8519973a76e8fe161078254
36919 F20101118_AAAGTS amarasekare_k_Page_042.QC.jpg
d76b5ce06c055ad488d8c16670d43538
c860740c7fb0dc67a94d331e2e09bc46331695f0
33750 F20101118_AAAHWU amarasekare_k_Page_092.QC.jpg
7384845330ad2308bb36da095fd95db9
22e1723e63f42cdfb2da20f34d78c0e2e4506cf5
112722 F20101118_AAAHAB amarasekare_k_Page_043.jpg
662aa4efef3ccc13d279e6609779bfb7
fa58add6b871b210b57feeecde35f0813b41b31c
9558 F20101118_AAAHXJ amarasekare_k_Page_108thm.jpg
2f4c5aad40e61267fa73bbcb58dee193
057ca20d93681a665bb4a1b32260dbd37be9fdc2
116610 F20101118_AAAGUH amarasekare_k_Page_035.jpg
5a489ac5a9a2059613b79d48ff7675e4
c62fb61dd2bd63b84b4e09b9f27f30c71207b514
38508 F20101118_AAAGTT amarasekare_k_Page_088.QC.jpg
d67111e68489c544e4616f2ec7686930
38d8aa6da12d3ff05fd47719425645639b2111f1
36358 F20101118_AAAHWV amarasekare_k_Page_094.QC.jpg
2dd7101e3487bbb4f1556f94c98d0d21
9d59d975c5294d1c092079561fa370e2f71f5764
95388 F20101118_AAAHAC amarasekare_k_Page_044.jpg
1200ae4aa7400bab62aba70a5d095105
19e5e48a98236e25f9504b2a25d206a408c38538
38454 F20101118_AAAHXK amarasekare_k_Page_108.QC.jpg
925183e21936d6651abd9b2e7ad27a24
04bd1df19b1bb5538228c10d1f70039aaf4d42c6
9385 F20101118_AAAGUI amarasekare_k_Page_065thm.jpg
9231078f776307d6eb0ca8bb4e510b8a
154df63a9138a5b8675fd9c660f9a4a946e9a20a
129266 F20101118_AAAGTU amarasekare_k_Page_005.jpg
b3585e32486ab4e46ec0e367a7c95bab
5d2c10932f4b96f127dfdee629667ab7575ead42
8988 F20101118_AAAHWW amarasekare_k_Page_095thm.jpg
4f05d7f832fbb31da5bd37cd9a985443
13bad060e85b22c33edf0821a978ae2a1dad82f3
100211 F20101118_AAAHAD amarasekare_k_Page_045.jpg
158867b8d8044101e41ec1aebc0dc105
a2664c524ce74e381fa8d41ca65ab34657628404
10010 F20101118_AAAHXL amarasekare_k_Page_110thm.jpg
7863c9ac5a80482ddfaba0485ab7bd66
21af596ceea647fe1c57aaae831151a471660291
F20101118_AAAGUJ amarasekare_k_Page_032.tif
eb22dbc3d31ca3ffdf24b353e2c80273
2e09b0c4644e3e636989b2e2446918ba5f894be7
36632 F20101118_AAAGTV amarasekare_k_Page_037.pro
7a535a78d186be3c22ed2700bbb1e34b
86af02fcf37d1ced718ca4d17531eb34c0a10973
38036 F20101118_AAAHWX amarasekare_k_Page_095.QC.jpg
db70576103b4ebe34ffa5899958c4831
7c92e01645846819945d654f73ef79c9682cf2db