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Evaluation of Rhyzobius lophanthae (Blaisdell) and Cryptolaemus montrouzieri Mulsant (coleoptera

Permanent Link: http://ufdc.ufl.edu/UFE0024433/00001

Material Information

Title: Evaluation of Rhyzobius lophanthae (Blaisdell) and Cryptolaemus montrouzieri Mulsant (coleoptera Coccinellidae) as Predators of Aulacaspis yasumatsui Takagi (Hemiptera: Diaspididae)
Physical Description: 1 online resource (79 p.)
Language: english
Creator: Thorson, Greta
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aulacaspis, cryptolaemus, cycas, rhyzobius
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The objective of this research was to gain a better understanding of Rhyzobius lophanthae (Blaisdell) and Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) as augmentative biological control agents of Aulacaspis yasumatsui Takagi (Hemiptera: Diaspididae), a severe pest on Cycas revoluta Thunberg. This study quantitatively evaluated the consumption of A. yasumatsui by both predators at 18 and 24degree C. The larval developmental periods, pupation periods, adult longevities, and fecundities of the two predators were compared. The effect of releases of R. lophanthae on A. yasumatsui populations and the time frame in which control can be seen on an infested plant were examined through a greenhouse and field study. Both R. lophanthae and C. montrouzieri were able to complete larval development when feeding on male and female A. yasumatsui whereas only R. lophanthae completed larval development when feeding only on female scales at both temperatures. Larval survivorship was significantly greater in R. lophanthae than in C. montrouzieri. Larval development time and pupation period were significantly greater for C. montrouzieri when feeding on both male and female scales. Female R. lophanthae are able to produce eggs when feeding on A. yasumatsui, whereas C. montrouzieri are unable to produce eggs at 18oC or 24oC. This study revealed the rates of consumption by R. lophanthae were higher than that of C. montrouzieri as well as the ineffectiveness of both beetles in targeting female A. yasumatsui, implicating their inability to control the increase of scale populations. In the greenhouse study, treatments with higher numbers of beetles consumed the greatest mean number of scales and a greater proportion of the total scale population. Healthy scale infestations were reduced by 10% following the introduction of R. lophanthae in treatments with 4 and 6 beetles whereas treatments with 2 beetles were reduced by 35%. Initial beetle feeding damage was observed on infested plants during the first 8 d during a field study where plants were treated with 0, 100, 200, or 300 beetles. With the absence of beetles and low numbers of larvae, there were no significant differences in damage to scales among treatments at later time points. The level of scale infestation increased over the course of the study following release of R. lophanthae indicating the ineffectiveness of the predator.
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 Greta Thorson.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Cave, Ronald D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024433:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024433/00001

Material Information

Title: Evaluation of Rhyzobius lophanthae (Blaisdell) and Cryptolaemus montrouzieri Mulsant (coleoptera Coccinellidae) as Predators of Aulacaspis yasumatsui Takagi (Hemiptera: Diaspididae)
Physical Description: 1 online resource (79 p.)
Language: english
Creator: Thorson, Greta
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aulacaspis, cryptolaemus, cycas, rhyzobius
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The objective of this research was to gain a better understanding of Rhyzobius lophanthae (Blaisdell) and Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) as augmentative biological control agents of Aulacaspis yasumatsui Takagi (Hemiptera: Diaspididae), a severe pest on Cycas revoluta Thunberg. This study quantitatively evaluated the consumption of A. yasumatsui by both predators at 18 and 24degree C. The larval developmental periods, pupation periods, adult longevities, and fecundities of the two predators were compared. The effect of releases of R. lophanthae on A. yasumatsui populations and the time frame in which control can be seen on an infested plant were examined through a greenhouse and field study. Both R. lophanthae and C. montrouzieri were able to complete larval development when feeding on male and female A. yasumatsui whereas only R. lophanthae completed larval development when feeding only on female scales at both temperatures. Larval survivorship was significantly greater in R. lophanthae than in C. montrouzieri. Larval development time and pupation period were significantly greater for C. montrouzieri when feeding on both male and female scales. Female R. lophanthae are able to produce eggs when feeding on A. yasumatsui, whereas C. montrouzieri are unable to produce eggs at 18oC or 24oC. This study revealed the rates of consumption by R. lophanthae were higher than that of C. montrouzieri as well as the ineffectiveness of both beetles in targeting female A. yasumatsui, implicating their inability to control the increase of scale populations. In the greenhouse study, treatments with higher numbers of beetles consumed the greatest mean number of scales and a greater proportion of the total scale population. Healthy scale infestations were reduced by 10% following the introduction of R. lophanthae in treatments with 4 and 6 beetles whereas treatments with 2 beetles were reduced by 35%. Initial beetle feeding damage was observed on infested plants during the first 8 d during a field study where plants were treated with 0, 100, 200, or 300 beetles. With the absence of beetles and low numbers of larvae, there were no significant differences in damage to scales among treatments at later time points. The level of scale infestation increased over the course of the study following release of R. lophanthae indicating the ineffectiveness of the predator.
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 Greta Thorson.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Cave, Ronald D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024433:00001


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EVAL UATION OF Rhyzobius lophanthae (BLAISDELL) AND Cryptolaemus montrouzieri MULSANT (COLEOPTERA: COCCIN ELLIDAE) AS PREDATORS OF Aulacaspis yasumatsui TAKAGI (HEMIPTERA: DIASPIDIDAE) By GRETA THORSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009 1

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2009 Greta Thorson 2

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To m y family for their constant support and encouragement, as well as past and present colleagues and mentors who helped inspire me along the way 3

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ACKNOWL EDGMENTS I thank my family for their enthusiasm in helping me collect insects and willingness to store countless specimens in thei r freezers over the years. Id especi ally like to thank my major professor and committee members for lending thei r experience and encouragement. Id like to also thank my past mentors who inspired me to pursue entomology as a profession. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 LIST OF ABBREVIATIONS........................................................................................................10 ABSTRACT...................................................................................................................................11 CHAPTER 1 REVIEW OF LITERATURE.................................................................................................13 Introduction................................................................................................................... ..........13 Aulacaspis yasumatsui Takagi........................................................................................14 Rhyzobius lophanthae (Blaisdell)....................................................................................18 Cryptolaemus montrouzieri Mulsant...............................................................................19 Objectives...............................................................................................................................20 2 LIFE HISTORY OF RHYZOBIUS LOPHANTHAE AND CRYPTOLAEMUS MONTROUZIERI FEEDING ON AULACASPIS YASUMATSUI ..........................................26 Introduction................................................................................................................... ..........26 Materials and Methods...........................................................................................................27 Insects..............................................................................................................................27 Experimental Design.......................................................................................................28 Results.....................................................................................................................................29 Egg Duration...................................................................................................................29 Female Scales Only As Prey for Larvae..........................................................................29 Male and Female Scales as Prey For Larvae...................................................................31 Pupation Period...............................................................................................................32 Adult longevity................................................................................................................ 32 Fecundity...................................................................................................................... ...33 Discussion...............................................................................................................................33 3 RHYZOBIUS LOPHANTHAE AND CRYPTOLAEMUS MONTROUZIERI CONSUMPTION RATE OF AULACASPIS YASUMATSUI .................................................46 Materials and Methods...........................................................................................................47 Insects..............................................................................................................................47 Experimental Design.......................................................................................................48 Results.....................................................................................................................................49 Larvae..............................................................................................................................49 5

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Adults ..............................................................................................................................50 Discussion...............................................................................................................................51 4 FIELD RELEASE STUDY OF RHYZOBIUS LOPHANTHAE .............................................58 Introduction................................................................................................................... ..........58 Materials and Methods...........................................................................................................59 Greenhouse Study............................................................................................................59 Insects.......................................................................................................................59 Experimental design.................................................................................................60 Field Study.................................................................................................................... ...60 Insects and plants.....................................................................................................60 Experimental design.................................................................................................61 Results.....................................................................................................................................62 Greenhouse Study............................................................................................................62 Field Study.................................................................................................................... ...62 Discussion...............................................................................................................................64 LIST OF REFERENCES...............................................................................................................75 BIOGRAPHICAL SKETCH.........................................................................................................79 6

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LIST OF TABLES Table page 3-1 Daily and total consumption of 2nd and 3rd instar female Aulacaspis yasumatsui by Rhyzobius lophanthae and Cryptolaemus montrouzieri larvae and adults at 18oC...........54 3-2 Daily and total consumption of 2nd and 3rd instar female Aulacaspis yasumatsui by Rhyzobius lophanthae and Cryptolaemus montrouzieri larvae and adults at 24oC...........55 3-3 Daily and total consumption of 2nd and 3rd instar male and female Aulacaspis yasumatsui by Rhyzobius lophanthae and Cryptolaemus montrouzieri larvae at 18oC.....56 3-4 Daily and total consumption of 2nd and 3rd instar male and female Aulacaspis yasumatsui by Rhyzobius lophanthae and Cryptolaemus montrouzieri larvae at 24oC.....57 4-1 Number of Rhyzobius lophanthae observed on cycad plants at field sites after their release................................................................................................................................73 7

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LIST OF FI GURES Figure page 1-1 Adult female Aulacaspis yasumatsui .................................................................................21 1-2 Adult male Aulacaspis yasumatsui ....................................................................................22 1-3 Adult female Aulacaspis yasumatsui and Pseudaulacaspis cockerellii ............................23 1-4 Dorsal and ventral view of adult and 3rd instar Rhyzobius lophanthae..............................24 1-5 Dorsal and ventral view of adult and 3rd instar Cryptolaemus montrouzieri .....................25 2-1 20 x 9 mm Petri dish....................................................................................................... ...35 2-2 Egg duration time of Rhyzobius lophanthae feeding on female Aulacaspis yasumatsui and Cryptolaemus montrouzieri feeding on Maconellicoccus hirsutus ..........36 2-3 Age-specific larval survivorship of Rhyzobius lophanthae and Cryptolaemus montrouzieri feeding on female Aulacaspis yasumatsui ...................................................37 2-4 Larval development time of Rhyzobius lophanthae and Cryptolaemus montrouzieri feeding on female Aulacaspis yasumatsui at 18oC............................................................38 2-5 Larval development time of Rhyzobius lophanthae feeding on female Aulacaspis yasumatsui at 24oC.............................................................................................................39 2-6 Age-specific larval survivorship of Rhyzobius lophanthae and Cryptolaemus montrouzieri feeding on male and female Aulacaspis yasumatsui ....................................40 2-7 Larval development time of Rhyzobius lophanthae and Cryptolaemus montrouzieri feeding on male and female Aul a caspis yasumatsui at 18oC.............................................41 2-8 Larval development time of Rhyzobius lophanthae and Cryptolaemus montrouzieri feeding on male and female Aulacaspis yasumatsui at 24oC.............................................42 2-9 Pupation period of Rhyzobius lophanthae and Cryptolaemus montrouzieri .....................43 2-10 Age-specific survivorship of adult female Rhyzobius lophanthae feeding on female Aulacaspis yasumatsui.......................................................................................................44 2-11 Mean daily egg production per female Rhyzobius lophanthae feeding on female Aulacaspis yasumatsui.......................................................................................................45 3-1 Feeding damage to Aulacaspis yasumatsui by Rhyzobius lophanthae larva.....................53 4-1 Field release Site 1.............................................................................................................66 8

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4-2 Field release Site 2.............................................................................................................67 4-3 Field release Site 3.............................................................................................................68 4-4 Adult R. lophanthae were manually shaken onto a C. revoluta plant infested with A. yasumatsui. .........................................................................................................................69 4-5 Average number of 2nd and 3rd instar female A. yasumatsui consumed per R. lophanthae adult in treatments of 0, 2, 4, 6, and 8 beetles per leaf...................................70 4-6 Proportion of total A. yasumatsui population consumed by R. lophanthae in treatments of 0, 2, 4, 6 and 8 b eetles per leaf in a greenhouse..........................................71 4-7 The mean number of healthy A. yasumatsui per leaflet undamaged by R. lophanthae over time in a greenhouse..................................................................................................72 4-8 The percentage of leaf area infested by A. yasumatsui over time in treatments of 0, 100, 200 or 300 R. lophanthae over all field sites.............................................................74 9

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LIST OF ABBRE VIATIONS CAS Aulacaspis yasumatsui known commonly as the cycad aulacaspis scale, Asian cycad scale, cycad scale, sago palm scale, snow scale, and Thai scale. RH Relative humidity 10

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Masters of Science EVALUATION OF Rhyzobius lophanthae (BLAISDELL) AND Cryptolaemus montrouzieri MULSANT (COLEOPTERA: COCCIN ELLIDAE) AS PREDATORS OF Aulacaspis yasumatsui TAKAGI (HEMIPTERA: DIASPIDIDAE) By Greta Thorson May 2009 Chair: Ronald Cave Major: Entomology and Nematology The objective of this research was to gain a better understanding of Rhyzobius lophanthae (Blaisdell) and Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) as augmentative biological control agents of Aulacaspis yasumatsui Takagi (Hemiptera: Diaspididae), a severe pest on Cycas revoluta Thunberg. This study quantitatively evaluated the consumption of A. yasumatsui by both predators at 18 and 24 C. The larval developmental periods, pupation periods, adult longevities, and f ecundities of the two predators were compared. The effect of releases of R. lophanthae on A. yasumatsui populations and the time frame in which control can be seen on an infested plant were examined through a greenhouse and field study. Both R. lophanthae and C. montrouzieri were able to complete larval development when feeding on male and female A. yasumatsui whereas only R. lophanthae completed larval development when feeding only on female scales at both temperatures. Larval survivorship was significantly greater in R. lophanthae than in C. montrouzieri. Larval development time and pupation period were significantly greater for C. montrouzieri when feeding on both male and female scales. Female R. lophanthae are able to produce eggs when feeding on A. yasumatsui, whereas C. montrouzieri are unable to produce eggs at 18oC or 24oC. This study revealed the rates of consumption by R. lophanthae were higher than that of C. montrouzieri as well as the 11

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12 ineffectiveness of both beetles in targeting female A. yasumatsui implicating their inability to control the increase of scale populations In the greenhouse study, treatmen ts with higher numbers of b eetles consumed the greatest mean number of scales and a greater proportion of the total scale population. Healthy scale infestations were reduced by 10% following the introduction of R. lophanthae in treatments with 4 and 6 beetles whereas treatments with 2 bee tles were reduced by 35%. Initial beetle feeding damage was observed on infested plants during the first 8 d during a field study where plants were treated with 0, 100, 200, or 300 beetles. With the absence of beetles and low numbers of larvae, there were no significant differences in damage to scales among treatments at later time points. The level of scale infestation increased ov er the course of the study following release of R. lophanthae indicating the ineffectiveness of the predator.

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CHAP TER 1 REVIEW OF LITERATURE Introduction Cycas revoluta Thunberg, commonly referred to as ki ng sago, is a popular plant in the landscape environment of Florida, being widely distributed throughout th e state in botanical gardens and residences. Since 1996, C. revoluta in Florida has been attacked by the cycad aulacaspis scale, Aulacaspis yasumatsui Takagi. This scale not only pr esents a substantial threat to landscape cycad plants in Florida, but also worldwide. The scale is native to a region stretching from the Andaman Islands to Viet nam, Thailand, southern China, and likely Cambodia, Laos, Malaysia, and Myanmar (How ard et al. 1999; Muni appan 2005). The first known outbreak outside of its native range occurred at the Bogor Bo tanical Garden in Java in the 1980s (Haynes 2005). Since its introduction in the US, A. yasumatsui has spread to Alabama, California, Georgia, Hawaii, Louisiana, South Carolina, and Texas (Broome 2000). Its destruction is also seen in the West I ndies, Guam, Hong Kong, Singapore, Taiwan, New Zealand, Costa Rica, and Africa (Weissling et al. 1999; Hodges et al. 2004; Moore et al. 2005; Germain and Hodges 2007). Threats of further spread are a major concern to Australia and India which currently do not have any recorded outbreaks (Muniappan and Viraktamath 2006). The scale destroys plants and gives them an unsightly snow-covered appearance. Heavy infestations form a dense multilayered covering of nearly 465 scales per square centimeter (Weissling et al. 1999) and result in the death of the cycad (Heu 2003). The scale is successful in Florida due to the warm temperatures which allo w development from egg to adult in less than one month; females produce 100 eggs on average. Init ial infestations in Miami were treated with systemic insecticides from October 1996 to January 1998, although re-infestations were common (Howard et al. 1999). 13

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Aulacaspis yasumatsui Takagi Aulacaspis y asumatsui was originally described fro m Bangkok, Thailand in 1972 (Takagi 1977). Belonging to the family Dias pididae, these scales have a waxy outer coating that forms a protective barrier for both adult and eggs (Weissling et al 1999). Females (Figure 1-1) are 1.21.6mm in length and pyriform in shape with an irregular pear-shaped covering, whereas males (Figure 1-2) are 0.5-0.6 mm in lengt h, with tricarinate coverings. Males are much more abundant on infested plants than fe males (Howard et al. 1999). In 1996, A. yasumatsui was found infesting ornamental cy cad plants in Miami, Florida. The pest quickly spread throughout southern Fl orida, aided by the ornamental industrys transport of cycad plants (McLaughlin 1998). Howa rd et al. (1999) observed that within 16 days from initial infestation during April and June, a m ean infestation of 69.6 scales per leaflet, with 84.8% being in first instar and the remainder in second instar, could be seen on the abaxial surface of C. revoluta leaflets. Approximately 18.1% of scales were in the third instar after 28 days and 89.1% after 41 days, 68.2% of whic h had produced eggs. Number of days for development was significantly less between August a nd September, but resulted in similar scale densities. Studies have shown that Cycas species native to China often had significantly higher infestations of A. yasumatsui than other host plants (Howard et al. 1999). This suggests that cycads in China were likely the original host plants of scales because A. yasumatsui readily uses Cycas species as hosts, while infestations on cycad species in other genera are considerably less frequent. Aulacaspis yasumatsui has been found infesting cycads of the families Cycadaceae, Zamiaceae, and Stangeriaceae (Howard et al. 1999). Aulacaspis yasumatsui causes injury by piercing plant ti ssue with its styl et mouthparts and sucking sap out of the leaves. As a result, necrosis of the leaves is evident by the yellow-brown 14

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coloration beginning at the leaflet tips. Initial in festations are seen on the underside of leaves and, as the population increases, sc ales move to the topside of l eaflets. A heavy buildup of scales is often observed by a snowy appearance detectable up to 2 m away. Control of A. yasumatsui is difficult due to several factors. First, it was discovered that the scale is capable of infesting the primary and second ary roots of plants as deep as 60 cm into the ground, where they are not easily observed, a nd can therefore be transported unknowingly (Howard et al. 1999). Additionally, scales can escape freezing temperatures by hiding in overlapping plant material from previous years growth in th e central cone of C. revoluta plants which cannot be reached with normal chemical treatments (Broome 2002). Immediate control was further hindered by the strong resemblance of Pseudaulacaspis cockerelli (Comstock) (Figure 1-3) and Pinnaspis strachani (Cooley) to A. yasumatsui which can be differentiated by its orange-colored body and eggs and swollen pr ostigma. Initial contro l measures targeting P. cockerelli were ineffective against this pest which was later correctly identified by Dr. Avas B. Hamon, Department of Plant Industry, Florid a Department of Agriculture and Consumer Services, Gainesville (Howard et al. 1996). Chemical control of A. yasumatsui has evolved quickly in the last ten years in an effort to control this invasive scale fr om moving to other parts of the U.S. The earliest treatment recommended drenching plants with malathion (Walters et al. 1997; Weissling et al. 1999). Malathion is a fast acting, broad spectrum in secticide that can be toxic if misused, causing phytotoxicity in new growth and death of bene ficial organisms (Hodges et al. 2003; Emshousen and Mannion 2004b). Horticultural oil and fish o il were also among the first chemicals used to control the spread of A. yasumatsui. Organocide (95% fish oil) consiste ntly reduced scale populations when 15

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infestations were m ild. Treatment with horticultural oil has also been eff ective, but full coverage of infested plants is difficult primarily due to plant architecture wherein cycad leaves curl down and inward. Oil sprays were unable to be applied in uniform thickness and result in a build up of past oil treatments after several applications (H odges et al. 2003). Weekly or biweekly sprayings for several months is required to control scales on leaflets and stems (Walters et al. 1997; Meyerdirk 2002; Hodges et al. 2003). Treatment with Organocide and Ortho Horticultural Mineral Oil in studies by Caldwell (2003) resulted in less then 50% control on the undersides of leaves where A. yasumatsui are most dense. Mixed treatments with oil and the contact insecticide Sevin (carbaryl), resulted in grea ter scale mortality than oil trea tments alone, but increase the potential to negatively affect biological contro l agents (Hodges et al 2003). Efforts to control scales on all parts of infested plants in cluding the roots have included application of a soil drench containing up to 0.1 L of imidacloprid per 18.9 L of water; however, the label rate proved to be not as effective in controlling heavy scale in festations (Howard and Weissling 1999; Hodges et al 2003; Emshousen and Mannion 2004b). Cygon, with the active ingredient dimethoate, has been applied to plants in studies with overwintering nymphs from mid-May, June, and July by drenching the entire plant (Caldwell 2003). Root-infesting A. yasumatsui were treated by soil drenching with Cygon 2E at a rate of 0.06 L per 3.8 L of water. Cygon 2E caused mortal ity in 85% of crawlers on the underside of leaves. Root drench treatments resulted in 95% mortality of scales 31 d after treatment. This percentage was significantly higher than natural mortality of 16% in the control. Application of this chemical has proven to be highly detrimental to natural enemies. Despite its effectiveness, Cygon 2E is not labeled for use on cycads and is t hus illegal to use on infested plants. The EPA 16

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published in the Federal Register a cancelati on notice of dim ethoate products, including Cygon, for residential use (U.S. EPA 2002). A study by Emshousen and Mannion (2004a) rev ealed that Distance, with the active ingredient pyriproxyfen, an insect growth regulator, provided excellent control of A. yasumatsui Distance acts to inhibit metamorpho sis, therefore making it less likely to affect biological control agents and humans. This insecticides effectiv eness in disrupting the development of scales requires a whole life cycle in orde r to provide control. Treatments with Distance have resulted in 100% mortality of eggs and 99% of adult fe males after 8 weeks. Due to threats of A. yasumatsui moving to other cycad species in Florida, pyriproxyfen was te sted on many of these plants. Treatments were able to control at least 75% of adult female A. yasumatsui and eggs on lightly infested plants. Distance did not control scale well on densely infested plants, but showed no signs of phytotoxicity. It is difficult to eliminate A. yasumatsui because scales can be transported unknowingly on the roots of plants and unsettled crawlers can become airborne up to a half mile (Broome 2000). Due to scale outbreaks and the inability to control them in Florida, there are severe restrictions on the export of C. revoluta outside of Florida. Mo rtality of 70-100% of the ornamental cycads in Hong Kong has been attributed to this same pest, and may be indicative of the level of destruction possibl e in Florida (Hodgson and Martin 2001). This suggests that there is a need for better understanding the influence of biological control agen ts on scale control and the development of methods for effective release of these agents Two natural enemies, the parasitoid Coccobius fulvus (Compere and Annecke) and the predator Cybocephalus nipponicus Endrody-Younga, were released in Florida in 1998 to control scales (Howard et al. 1999; Hodges et al. 2003; Muniappan 2005) Re-releases showed that 17

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control is possible during som e parts of the year (Hodges et al. 2003). Further studies of C. fulvus by Wiese and Mannion (2005) indicate that the wasp was successfully parasitizing scales, although the beetles have failed to significantly control the scale. Sixteen lady beetles (Coccinellidae) have been identified on cycads infested by A. yasumatsui in southern Florida (Cave 2006). Rhyzobius lophanthae (Blaisdell), an Australian lady beetle, has been observed feeding on A. yasumatsui, but isolated populations have been found only in downtown Tampa and Tallahassee (Cave 2006). Rhyzobius lophanthae has been an effective biological control agent in Hawaii, Italy, and Guam, but its limited distribution in Florida could be hampering its effectiveness as a biological control agent in terms of dispersal across all of southern Florida. Another Australian lady beetle, Cryptolaemus montrouzieri Mulsant, has been frequently encountered throughout southern Florida on C. revoluta plants infested with A. yasumatsui Research into the effectiveness of both species of lady beetles as predators of A. yasumatsui would be useful in determining their significance as biological control agents. Rhyzobius lophanthae (Blaisdell) Rhyzobius lophanthae (= Lindorus lophanthae Blaisdell) has been identified as an important natural enemy of ma ny armored scale species (Yus 1973; Rosen 1990). The beetle was introduced from New South Wales into Calif ornia by Albert Koebele between 1889 and 1892, and successfully controlled black scale, Saissetia oleae (Bern) (Greath ead 1973). In 1894, R. lophanthae was introduced into Hawaii to control Aspidiotus destructor Signoret (Honda 1995; McLaughlin 1998). Subsequently, R. lophanthae has been recommended for release in many insect pest management programs. Rhyzobius lophanthae has proven to be an effective biological control agent worldwide, specifically controlling Carulaspis juniperi (Bouch) in Italy, Chrysomphalus dictyospermi (Morgan) in Morocco, Parlatoria blanchardi (Targ ioni) in Israel, 18

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Aulacaspis tegalens is (Zhut) in East Africa, and Aspidiotus nerii Bouch in Greece. However, R. lophanthae failed to control Aonidiella aurantii (Maskell) in Califor nia (Honda 1995). Although R. lophanthae currently lives in ar eas of Florida where A. yasumatsui is a problem, the beetle has been unable to regulate pest populations through natural interactions, leading to concerns over the ability of R. lophanthae to disperse to isolated plants in the urban and suburban landscapes. Rhyzobius lophanthae (Figure 1-4) is a small predat ory beetle, with adult females approximately 2.5 mm in length and 1.8 mm in wi dth. Adult males are 2.4 mm in length and 1.7 mm in width. The head in both sexes is reddishbrown in color and cove red with setae. The antennae are nine-segmented and the elytra are black-brown (Smir noff 1950; Stathus 2002). There are currently no data quantifying the capacity of R. lophanthae to prey upon A. yasumatsui Consumption on other prey and fecund ity was measured by Stathus (2000) in Greece. There is concern about the adequate number of R. lophanthae needed to control an infestation of A. yasumatsui on a plant. Two companies, Gard ening Zone and IPM of Alaska, both suggest this predator should be augmentativel y released onto cycads to facilitate control of A. yasumatsui These companies recommend releasing 20-40 beetles for each infested plant, but are unclear about the plant size treated. Cryptolaemus montrouzieri Mulsant Cryptolaemus montrouzieri (Figure 1-5) was imported from Australia into California in 1872. It is used frequently in the biological c ontrol of mealybugs and soft scales (Cooper 1985; Heidari and Copland 1992) Adults range betwee n 3.4 and 4.5 mm in length and between 2.4 and 3.1 mm in width. The head, prothorax and elytral ap ices are reddish in co lor; the remainder of the body is black. Its dorsal surface is densely pu nctate, apart from the humeral callus which is almost devoid of punctures. Apar t from being found in California, C. montrouzieri is also 19

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distributed throughout central a nd southern Florida (Gordon 1985). In feeding studies com paring C. montrouzieri to another predatory beetle, Adalia bipunctata (Linnaeus) it was discovered that both beetles are not selective feeders and that, overall, C. montrouzieri eats six times fewer scales and has a smaller gut capacity (Magro et al. 2002). Fecundity life tables were developed based on studies of C. montrouzieri feeding on the pink hibiscus mealybug, Maconellicoccus hirsutus (Green), in Trinidad (Persad et al. 2002). Because C. montrouzieri is frequently encountered on cycads infested with A. yasumatsui (Cave 2006), a comparative feeding study would indicate the effectiveness of C. montrouzieri as a biological control agent of the pest. Objectives The aim of this study was to determine how the life history and feeding behavior of R. lophanthae and C. montrouzieri compare when A. yasumatsui on C. revoluta is provided as prey. The research also studied the effectiveness of R. lophanthae in field release trials. Therefore, data were collected to answer these specific questions: Are the consumption rates of R. lophanthae and C. montrouzieri larvae and adults feeding on A. yasumatsui significantly different at two different temperatures? Are the larval development times, pupati on periods, adult longe vities, and female fecundities of R. lophanthae and C. montrouzieri preying on A. yasumatsui significantly different? How many R. lophanthae adults should be released on a plant to provide significant control of A. yasumatsui and how long do the beetle s persist on the plant? 20

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Figure 1-1. Adult fem ale Aulacaspis yasumatsui (Hodges et al. 2003). 21

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Figure 1-2. Adult m ale Aulacaspis yasumatsui 22

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Figure 1-3. Adult fem ale Aulacaspis yasumatsui and Pseudaulacaspis cockerellii (Hodges et al. 2003). 23

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Figure 1-4. Dorsal and vent ral view of adult and 3rd instar Rhyzobius lophanthae (Cave 2006). 24

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25 Figure 1-5. Dorsal and vent ral view of adult and 3rd instar Cryptolaemus montrouzieri

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CHAP TER 2 LIFE HISTORY OF RHYZOBIUS LOPHANTHAE AND CRYPTOLAEMUS MONTROUZIERI FEEDING ON AULACASPIS YASUMATSUI Introduction Members of the family Coccinellidae, commonly called lady beetles, are the most widely used predator in biological control programs and are successful in controlling aphids, mealybugs, scales, whiteflies, psyllids, and mites (Obr ycki and Kring 1998). Since the 1890s, nearly 40 coccinellid species have been introduced into th e U.S. for control of pest insects (Frank and McCoy 2007). Rhyzobius lophanthae, a species of lady beetle native to New South Wales, was first introduced into California fo r control of black scale, Saissetia oleae (Olivier) (Greathead 1973). This lady beetle has been effective in the biological control of Aspidiotus destructor Signoret in Hawaii, Carulaspis juniperi (Bouch) in Italy, Chrysomphalus dictyospermi (Morgan) in Morocco, P arlatoria blanchardi (Targioni) in Israel, Aulacaspis tegalensis (Zhut) in East Africa, and Aspidiotus nerii Bouch in Greece (Honda and Luck 1995; McLaughlin 1998). The short developmental period, lack of parasitoids, ab sence of diapause, and consumption of pest populations by both larvae and adults makes R. lophanthae successful in biological control programs (Stathas 2000). Cryptolaemus montrouzieri, known commonly as the mealybug destroyer, is a generalist feeder native to Australia and ha s been used primarily to control Planococcus citri (Risso), a pest in citrus. Introductions have also been made to control Coccus viridis (Green), Pulvinaria psidii (Maskell), Dysmicoccus boninsis (Kuwana), Dysmicoccus brevipes (Cockerell), Ferrisia virgata (Cockerell), Maconellicoccus hirsutus (Green), Nipaecoccus nipae (Maskell), Planococcus citri (Risso), Pseudococcus comstocki (Kuwana), Pseudococcus longispinus (Targioni-Tozzetti), Pseudococcus maritimus (Ehrhorn), Pseudococcus viburni (Signoret), Saccharicoccus sacchari 26

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(Cockerell), Dactylopius tomentosus (Lam arck), and Eriococcus araucariae (Muskell) (Frank and McCoy 2007). The lady beetle is distribut ed throughout central and southern Florida (Gordon 1985). Considerable loss of cycads in the urban lands cape which has been attributed to the cycad aulacaspis scale, Aulacaspis yasumatsui, has resulted in decreased production of cycads by the ornamental industry in Florida (Hodges et al. 200 3). The scale was introduced accidentally into the U.S. from Thailand (McLaughlin 1998), with th e initial detection occurring in Miami, FL in 1998. Aulacaspis yasumatsui causes injury by piercing plant ti ssue to remove plant sap. As a result, necrosis of the leaves is evident by th e yellow-brown coloration beginning at the leaflet tips and leads to eventual death of plants. Initial infestations are seen on the unde rside of leaves and, as the population increases, scales move to the topside of leaflets. Aulacaspis yasumatsui has been found infesting plant species in the families Cycadaceae, Zamiaceae, and Stangeriaceae (Howard et al. 1999). Sixteen lady beetle species have been identified on cycads infested by A. yasumatsui in southern Florida (Cave 2006). Of these, R. lophanthae and C. montrouzieri were selected for this study based on their success in other biological control programs. There is no knowledge of the development time, longevity, and fecundity of R. lophanthae and C. montrouzieri feeding on A. yasumatsui on Cycas revoluta plants. Therefore, this study was conducted to obtain this information in order to compare the efficacy of bot h lady beetles as biological control agents of A. yasumatsui in Florida. Materials and Methods Insects Aulacaspis yasumatsui was reared on C. revoluta plants in 3.7 L pots and maintained in a greenhouse with 30% RH. Plants were fertilized and watered regularly according to growers 27

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recomm endations to maintain the health of plants. Uninfested C. revoluta were exposed to infested plants with active crawlers by interlocking leaflets fo r 1 week, allowing crawlers to settle on the clean foliage. Mode rately infested plants with 2nd and 3rd instar A. yasumatsui were obtained in approximately 1 month following exposure at 30oC. Adult R. lophanthae were obtained from Rincon-Vitova Inse ctaries (Ventura, California) a nd kept in 20 20 20 cm Bug Dorms (BioQuip, Inc., Rancho Dominguez, CA) with water-saturated cotton balls and infested C. revoluta plants at 25oC, 60% RH and 14:10 (L:D) photoperiod. Eggs of R. lophanthae used in this study were produced from females feeding on A. yasumatsui. Adult C. montrouzieri were unable to produce eggs while feeding on A. yasumatsui (personal observation), therefore, eggs were obtained from the Florida Department of Agriculture and Consumer Services, Division of Plant Industry in Gainesville, FL. Eggs of C. montrouzieri were produced from females feeding on M. hirsutus Experimental Design Eggs and pupae of R. lophanthae and C. montrouzieri were placed individually in 20 9 mm Petri dishes with screen lids (Figure 2-1) Following larval and adult emergence, the predators were supplied daily with fresh C. revoluta leaflets infested with 10-20 2nd and 3rd instars of male and female or just female A. yasumatsui. Ten larvae and ten adults of each sex of both predator species were kept in environmental chambers set at 18oC and 24oC with 60% relative humidity and 14:10 (L:D) photoperiod. Pr edators were checked daily for molting and death. Adult longevity and age specific surv ivorship were measured in this study. Ten mating pairs of R. lophanthae and C. montrouzieri were isolated for 48 h in 20 9 mm Petri dishes with screen lids and lined with moist filter paper. Pairs were selected based on observation of copulation. Dishes were supplied daily with C. revoluta leaflets infested with ~20 2nd and 3rd instars of male and female A. yasumatsui Adult females were then placed 28

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individu ally into Petri dishes for the remainder of their life and provided new C. revoluta leaflets with scales ad libitum Presence of predator eggs was checked by visually inspecting leaflets and manually removing the cover of female scales The number of eggs produced and number of females alive were recorded daily to determine fecundity. Each study (set of 10 females) was repeated five times for both temperatures. Means of development time, pupation period, longevity, an d fecundity were statistically compared between predator species and temperatures using an analysis of vari ance (ANOVA) (Proc GLM, SAS Institute, 2001) and a t-test to separate mean s. Data for all 5 studies were combined during the analysis. Means are reported with their stan dard error. Population growth parameters of R. lophanthae feeding on female A. yasumatsui at both temperatures were calculated by computation of the net reproductive rate (Ro= l x m x), the intrinsic rate of increase (rm= ( ln Ro) / T), and mean generation time (T= l x m x x / l x m x). Results Egg Duration Duration time for R. lophanthae eggs from females reared on A. yasumatsui was significantly greater (t=8.9693, df =97, P<0.001) than that for C. montrouzieri eggs reared on Maconellicoccus hirsutus at 18oC (Figure 2-2). There was no significant difference (t=0.4471, df=97, P>0.5) between species at 24oC. Female Scales Only As Prey for Larvae Survivorship of larvae feeding only on female scales varied between the two predators and the two temperatures (Figure 2-3). There was a sharp decrease in survivorship at 18oC within the first 20 days for R. lophanthae larvae and likewise was observed until day 18 for C. montrouzieri. At 24oC, larvae of both species had a sharp decrease in survivorship within the first 7 days. Cohort survivorship was 50% on day 10 for R. lophanthae and on day 12 for C. 29

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montrouzieri at 18oC, whereas 50% survivorsh ip of the cohort at 24oC occurred on day 4 for R. lophanthae and on day 7 for C. montrouzieri. At 18oC, 2nd instar R. lophanthae developed in the fewest number of days (6.8 0.4), whereas the longest developmen t time was observed during the 4th instar (17.1 1.4 d) (Figure 2-4). Development time in the 4th instar was almost twice as l ong as all other instars, with 1st and 3rd instars developing in 10.3 0.6 d and 9.7 0. 6 d, respectively. Development time was not significantly different between 1st and 3rd instars. Total larval development time of R. lophanthae averaged 43.9 2.9 d at 18oC. The minimum development time was 38 d and the maximum was 64 d. At this temperature, larvae of C. montrouzieri did not reach the 4th instar; larvae in the 3rd instar died in 1.0 3.3 d. Larval de velopment was longest during the 1st instar (5.8 3.7 d) (Figure 2-4). At 24oC, 3rd instar R. lophanthae developed in the fewest number of days (3.8 0.3), whereas the longest developmen t time was observed during the 4th instar (5.9 0.3 d). Development time was identical (5.2 0.3 d) for both 1st and 2nd instars (Figure 2-5). Total larval development time of R. lophanthae at 24oC averaged 20.1 1.3 d, with a minimum of 17 d and a maximum of 23 d. Larvae of C. montrouzieri did not reach 2 nd instar at 24oC. First instars died on average in 5.2 2.6 d (n=36). Comparisons of instar development time between species can only be made for 1st and 2nd instars at 18oC since no C. montrouzieri larvae survived the 3rd instar at 18oC and none survived the 1st instar at 24oC (Figures 2-4, 2-5). During the 1st (t= 5.3795, df= 50, P<0.0001) and 2nd (t= 8.6734, df= 23, P<0.0001) instars at 18oC, R. lophanthae took significantly longer to develop than C. montrouzieri 30

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Male and Female Scales as Prey For Larvae Survivorship of R. lophanthae and C. montrouzieri larvae feeding on m ale and female A. yasumatsui decreased considerably with in the first 25 days at 18oC and 24oC (Figure 2-6). Cohort survivorship was 50% on day 14 of development of R. lophanthae and on day 18 of C. montrouzieri at 18oC. Cohort survivorship at 24oC was 50% on day 12 of R. lophanthae and on day 14 of C. montrouzieri At 18oC, 2nd instar R. lophanthae developed in the fewest number of days (7.2 1.3), whereas the longest developmen t time was observed during the 4th instar (16.7 4.2 d) (Figure 2-7). Development time in the 3rd and 4th instars was almost twice as long as development of 1st and 2rd instars. Total larval development time for R. lophanthae averaged 45.5 20.1 d at 18oC, whereas C. montrouzieri total development time averaged 85.1 15.0 d. Development time for C. montrouzieri was shortest during the 2nd instar (10.1 2.9 d), whereas the longest development time was observed during the 4th instar (41.2 7.3 d) at 18oC (Figure 2-7). Development time was more than 3 times longer during the 4th instar than during the 1st and 2nd instars, whereas development time was twice as long for 4th instar compared to 3rd instar. Mean larval development times were significan tly different between species (t= 5.1049, df= 21, P<0.001). Minimum and maximum development times for R. lophanthae were 26 d and 61 d, respectively. Minimum and ma ximum development times for C. montrouzieri were 32 d and 101 d, respectively. Later instars of R. lophanthae had progressively longer development times at 24oC (Figure 2-8), with total larval development time averaging 22.1 6.5 d. Larvae of C. montrouzieri had the shortest developmen t time (8.6 2.9 d) during the 2nd instar, whereas the longest development time occurred in both 1st and 4th instars (13.6 and 13 .4 d, respectively). Total larval development time averaged 47.8 16.4 d in C. montrouzieri There was a significant 31

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difference between the two predators m ean larval development times (t= 4.4872, df= 23, P=0.0002). Minimum development time for R. lophanthae was 17 d and the maximum was 41 d Larvae of C. montrouzieri developed in a minimum of 22 d and a maximum of 67 d. Variation occurred in the development time fo r each instar between the two predators and the two temperatures (Figures 27, 2-8). Development time for al l instars at both temperatures was always greater for C. montrouzieri compared to R. lophanthae. At 18oC, instar development times were only significantly diffe rent between species for the 1st (t=2.2910, df=19, P=0.0336) and 4th (t=6.0517, df=8, P=0.0003) instars. There was a significant difference observed between species during the 1st (t=7.4398, df=23, P<0.0001), 2nd (t=4.0072, df=17, P=0.0009), 3rd (t=2.8646, df=15, P=0.0118) and 4th (t=2.8077, df=12, P=0.0158) instars at 24oC. Pupation Period The pupation periods for R. lophanthae and C. montrouzieri differed significantly at both temperatures (Figure 2-9) Pupation was longer for C. montrouzieri than for R. lophanthae at 18oC (t=7.8284, df=9, P<0.001) and at 24oC (t=8.2968, df=9, P<0.0001). Adult longevity Adult mortality of R. lophanthae feeding only on female scales reached 50% on day 99 at 18oC and on day 94 at 24oC (Figure 2-10). Longevity data for C. montrouzieri on a diet of female scales only were not obtained be cause the larvae failed to complete development. Longevity data for C. montrouzieri and R. lophanthae on a diet of male and female scales were not obtained. Female R. lophanthae longevity averaged 95.5 2.9 d (n=6) at 18oC, whereas longevity at 24oC averaged 104.3 24.3 d (n=9). Minimu m and maximum longevity at 18oC were 92 d and 100 d, respectively. Minimum and maximum longevity at 24oC were 67 d and 126 d, respectively. 32

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Male R. lophanthae longevity averaged 116.1 19.7 (n=14) at18oC, whereas longevity at 24oC averaged 102.6 29.0 (n=12). Minimu m and maximum longevity at 18oC were 74 d and 129 d, respectively. Minimum and maximum longevity at 24oC were 60 d and 128 d, respectively. Fecundity Female R. lophanthae began laying eggs on average 15.7 4.4 d after adult emergence at 18oC and 26.1 8.2 d at 24oC (Figure 2-11). Seven females kept at 18oC produced a total of 84 eggs and nine beetles kept at 24oC produced 208 eggs within 100 days. Fifty eggs were produced by one female maintained at 24oC over its lifetime. Eggs were laid individually on the top surface of female A. yasumatsui armor or laid both singly or in groups of 2 to 3 eggs beneath the armor of female scales that had been preyed upon. Eggs were yellow in color and eventually became transparent prior to hatching. Peak daily egg production at both temperatures was at about 55 5 d (Figure 2-11). During the 100 day oviposition period, females kept at 18oC produced on average 0.2 0.2 eggs per female per day. Females kept at 24oC over a 126 day period produced on average 0.2 0.3 eggs per female per day. The maximum number of eggs produced by a single female in one day at 18oC was 6 on day 54. The maximum number of eggs produced by a single female in one day at 24oC was 12 on day 51. Female R. lophanthae feeding on female scales at 18oC had a net reproductive rate (Ro) of 24.6, mean generation time (T) of 96.1, and intrinsic rate of increase (rm) of 0.01986. At 24oC, the net reproductive rate (Ro) was 69.2, the mean generation time (T) was 75.3, and the intrinsic rate of increase (rm) was 0.04085. Discussion Rhyzobius lophanthae is a better predator of A. yasumatsui than C. montrouzieri. At 18oC, egg duration of R. lophanthae is longer than that of C. montrouzieri, whereas there is no 33

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difference at 24oC. Longer development time of the egg and 1st instars may be disadvantageous when eggs are exposed to fluctuations in temperature, humidity, precipitation and predators for longer periods of time, resulting in higher ra tes of mortality. Larval survivorship was significantly greater in R. lophanthae than in C. montrouzieri. Only R. lophanthae was able to complete development when offered only female A. yasumatsui. When presented with a mixed diet of male and female A. yasumatsui, both predators completed larval development and had higher leve ls of survivorship, in dicating that time was spent feeding on male scales. Similar result s were recorded by Stathas (2000) when R. lophanthae preyed on A. nerii Immature male A. yasumatsui were consumed by both predators on greater than 50% of days in the adult longevity study. Larval development time and pupation period were significantly greater for C. montrouzieri compared to R. lophanthae when feeding on both male and female scales, which may s upport the production of mo re generations of R. lophanthae. Female R. lophanthae are able to produce eggs when feeding on A. yasumatsui, whereas C. montrouzieri are unable to produce eggs at 18oC or 24oC. This indicates that C. montrouzieri relies on other food sources to produce eggs and th erefore would not be able to sustain healthy populations in areas where plants are solely infested with A. yasumatsui (Frank and McCoy 2007). Adult longevity and egg production by R. lophanthae at 18oC and 24oC were similar, indicating that this pred ator is capable of surviving and re producing within that temperature range. Similar peaks in production of eggs at both temperatures indicate that fluctuations in temperature would not effect on the time frame in which the greatest numbers of eggs are produced. Further comparisons are need ed to determine if egg production by R. lophanthae coincides with increased infestations of A. yasumatsui between June and September in Florida. 34

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Figure 2-1. 20 x 9 mm Petri dish 35

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Figure 2-2. Egg duration time of Rhyzobius lophanthae feeding on female Aulacaspis yasumatsui and Cryptolaemus montrouzieri feeding on Maconellicoccus hirsutus Numbers inside bars indicate sample size. 36

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Figure 2-3. Age-specific larval survivorship of Rhyzobius lophanthae and Cryptolaemus montrouzieri feeding on female Aulacaspis yasumatsui only. 37

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Figure 2-4. Larval development time of Rhyzobius lophanthae and Cryptolaemus montrouzieri feeding on female Aulacaspis yasumatsui only at 18oC. Number in each bar represents sample size. 38

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0 1 2 3 4 5 6 7 8 1234 InstarDays 20 20 20 20 Figure 2-5. Larval developm ent time of Rhyzobius lophanthae feeding on female Aulacaspis yasumatsui only at 24oC. Number in each bar represents sample size. 39

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Figure 2-6. Age-specific larval survivorship of Rhyzobius lophanthae and Cryptolaemus montrouzieri feeding on male and female Aulacaspis yasumatsui 40

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Figure 2-7. Larval development time of Rhyzobius lophanthae and Cryptolaemus montrouzieri feeding on male and female Aul a caspis yasumatsui at 18oC. Number in each bar represents sample size. 41

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Figure 2-8. Larval development time of Rhyzobius lophanthae and Cryptolaemus montrouzieri feeding on male and female Aulacaspis yasumatsui at 24oC. Number in each bar represents sample size. 42

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Figure 2-9. Pupation period of Rhyzobius lophanthae and Cryptolaemus montrouzieri Number in each bar represents sample size. 43

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Figure 2-10. Age-specific survivorship of adult female Rhyzobius lophanthae feeding on female Aulacaspis yasumatsui only. 44

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45 Figure 2-11. Mean daily egg production per female Rhyzobius lophanthae feeding on female Aulacaspis yasumatsui only.

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CHAP TER 3 RHYZOBIUS LOPHANTHAE AND CRYPTOLAEMUS MONTROUZIERI CONSUMPTION RATE OF AULACASPIS YASUMATSUI Introduction Predaceous coccinellids have been used more often than any other predatory organisms for biological control (Obrycki 1998). The su ccess of some scale-feeding coccinellids was hypothesized by Clausen (1940) to di rectly reflect the physical characteristics of the diaspid cover, the scales developmenta l stage, and the nu tritional value provided (Honda and Luck 1995). Rhyzobius lophanthae has been an effective biological control agent against diaspidid scales in Hawaii, Italy, and Guam (Heu et al. 2003; Moore et al. 2005) through inundative releases (Stathas 2002). Cave ( 2006) reported isolated populations of the beetle on cycads in downtown Tampa and Tallahassee, FL. However, its limited distribution in the state could be hampering its effectiveness as a biologi cal control agent desp ite the ability of R. lophanthae to produce as many as 8 generations per year feeding on the scale insect Chrysomphalus dictyospermi (Morgan) in Morroco (Smirnoff 1950). Limited distribution of coccinellid species m ay also reflect the suita bility of the environmen t to sustain populations. Cryptolaemus montrouzieri has proven effective in controlling mealybug and scale infestations dating back to the 1800s, having be en shipped worldwide for use in biological control programs (Bartlett 1974). This species is most often us ed to control grape, citrus and greenhouse mealybugs. Cryptolaemus montrouzieri, unlike R. lophanthae, has been frequently encountered throughout southern Florida on Cycas revoluta plants infested with Aulacaspis yasumatsui Aulacaspis yasumatsui originally described from Bangkok, Thailand (Takagi 1977), was first reported in 1996 infesting cyca d plants in Miami, Florida. Du e to the popularity of cycads in 46

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the urban landscape, the pest quickly spread throughout southern Florida (McLaughlin 1998). Initial contr ol of A. yasumatsui was attempted through the use of the insecticide, malathion and the introduction of two natura l enemies, the parasitoid Coccobius fulvus (Compere and Annecke) and the predator Cybocephalus nipponicus Endrody-Younga (Howard et al. 1999; Hodges et al. 2003; Muniappan 2005). Because of the widespread damage caused by A. yasumatsui the ineffectiveness of initial control measures, and potential to spread to uninfested parts of the world, biological control agents have been sought. More information is needed to determine the level of control that could be provided by R. lophanthae and C. montrouzieri Therefore, this study was conducted to analyze the rate of consumption of scales by R. lophanthae and C. montrouzieri throughout their development at tw o constant temperatures. Materials and Methods Insects Aulacaspis yasumatsui was reared on C. revoluta plants kept in 3.7 L pots and maintained in a greenhouse with 30% RH. Plants were fertilized and watered regularly according to growers recommendations to main tain plant health. Uninfested C. revoluta plants were exposed to infested plants with active crawlers by inte rlocking leaves for 1 week, allowing crawlers to settle on the uninfested foliage. Mode rately infested plants with 3rd instar A. yasumatsui were obtained in approximately 1 month at 30oC (Howard et al. 1999). Adult R. lophanthae were obtained from Rincon-Vitova Inse ctaries (Ventura, California) a nd kept in 20 20 20 cm Bug Dorms (BioQuip, Inc., Rancho Dominguez, CA) with water-saturated cotton balls and infested C. revoluta plants at 25oC, 60% RH and 14:10 (L :D) photoperiod. Adult C. montrouzieri were unable to produce eggs while feeding on A. yasumatsui (personal observation), therefore eggs were obtained from the Florida Department of Agriculture and Consumer Services, Division of 47

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Plant Industry in Gainesville, FL. Eggs of C. montrouzieri were produced from females feeding on M. hirsutus Experimental Design Ten 1st instars and 10 adults of each sex of R. lophanthae and C. montrouzieri were placed individually in 20 9 mm Petri dishes with screen lids and provided daily with C. revoluta leaflets infested with 10-20 2nd and 3rd instar female A. yasumatsui. First instar and male scales were manually removed using a small pain t brush. Beetles were kept in environmental chambers set at 18oC and 24oC, with 60% RH and 14:10 (L:D) photoperiod. Adult R. lophanthae used in the test were the F1 progeny of adult beetles purchased from Rincon-Vitova, reared on a diet of A. yasumatsui whereas C. montrouzieri were F1 progeny of beetles feeding on M. hirsutus. Adults were not starved prior to being studied. Bo th larvae and adults were scored daily until pupation or death, respectively, for number of scales consumed. Consumed scales were indicated by an absence of the female scales body due to complete consumption or damaged from partial feeding that resulted in a de ad scale with a hole in its armor (Figure 3-1). The study was repeated five times for both temperatures. In a second experiment, ten 1st instars of R. lophanthae and C. montrouzieri were evaluated using the same protocol stated a bove. Dishes were provided daily with 10-20 2nd and 3rd instar female and 20-40 3rd instar male A. yasumatsui on C. revoluta leaflets First instars and male 2nd instars were manually removed using a sm all paint brush. Larvae of both predator species were scored daily until pupation or death, respectively, for number of female scales consumed and visible damage to male scales The study was repeated five times for both temperatures. Mean consumption rates were compared be tween predator species and temperatures using an analysis of variance (ANOVA) (Proc GLM, SAS Institute, 2001) and a t-test to separate 48

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m eans. Means are reported with their standard error. Data for all 5 studies were combined during the analysis. Results Larvae Daily consumption varied cons iderably between larvae of R. lophanthae and C. montrouzieri feeding on female A. yasumatsui at 18oC and 24oC (Tables 3-1, 3-2). Daily and total consumption increased with development in to subsequent instars for both species at 18oC. Daily and total consumption of scales was significantly greater for R. lophanthae during the 1st (t=3.0165, df=429, P=0.0027), 2nd (t=4.4164, df=161, P<0.001) and 3rd (t=5.8763, df=201, P<0.001) instars at 18oC compared to C. montrouzieri (Table 3-1). Daily consumption was 2 times greater for 1st instar R. lophanthae, 7 times greater for 2nd instar, and 5 times greater for third instar compared to daily cons umption by the respective instars of C. montrouzieri at 18oC. Larvae of C. montrouzieri failed to complete the 4th instar while feeding on female scales. Total consumption of scales wa s significantly greater by R. lophanthae than C. montrouzieri during the 1st (t=150.4072, df=50, P<0.001), 2nd (t=9.6746, df=24, P<0.001) and 3rd instars (t=17.7223, df=21, P<0.001) at 18oC. At 24oC, daily consumption by 1st instar R. lophanthae was significantly greater (t=3.2040, df=323, P=0.0015) than by C. montrouzieri Larvae of C. montrouzieri failed to complete the 2nd instar. Total consumption by 1st instars was also greater in R. lophanthae (t=3.2979, df=323, P=0.0011) at this temperature; daily and tota l consumption were about 2 times greater for R. lophanthae compared to C. montrouzieri Consumption by cohorts of R. lophanthae and C. montrouzieri larvae feeding on male and female A. yasumatsui varied significantly at 18oC and 24oC (Tables 3-3, 3-4). Daily consumption was greater by R. lophanthae during the 3rd (t=7.7791, df=223, P<0.001) and 4th 49

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(t=3.7159, df=395, P=0.002) instars at 18oC compared to C. montrouzieri (Table 3-3). Daily consumption by R. lophanthae was 27 times greater for 3rd instars and 3 times greater for 4th instars compared to 3rd and 4th instars of C. montrouzieri. Total consumption by each instar at 18oC again revealed higher le vels of consumption of A. yasumatsui by R. lophanthae during the 2nd (t=2.1202, df=16, P=0.0500), 3rd (t=26.3366, df=15, P<0.001), and 4th (t=4.8122, df=7, P=0.0019) instars compared to C. montrouzieri The average total consumption of scales during the entire larval development reflected a similar trend (t=18.4397, df=65, P<0.001). Total consumption by R. lophanthae was 9 times greater for 3rd instars, 2 times greater for 4th instars, and 3 times greater for the entire larval period compared to C. montrouzieri. At 24oC, cohorts of R. lophanthae feeding on male and female A. yasumatsui consumed significantly greater numbers of scales daily during the 3rd (t=1.9731, df=187, P<0.001) and 4th (t=3.3381, df=166, P=0.0010) instars than C. montrouzieri (Table 3-4). Daily consumption was 2 times greater in 4th instar R. lophanthae compared to 4th instar C. montrouzieri at 24oC. Total consumption of scales was greater by C. montrouzieri during the 1st (t=8.6049, df=23, P<0.001) and 2nd (t=7.9293, df=18, P<0.001) instars co mpared to total consumption by R. lophanthae at 24oC; there was no significant difference between sp ecies for the other instars nor total larval period (Table 3-4). Adults Adult female R. lophanthae consumed greater numbers of fe male scales daily than male beetles at 18oC (t=3.1391, df=424, P=0.0018) (Table 3-1). The total average consumption was also significantly greater for female R. lophanthae (t=8.2328, df=18, P<0.001). At 24oC, adult female R. lophanthae consumed significantly greater numbers of female A. yasumatsui daily (t=6.9095, df=1695, P<0.001) than males. Average total consumption by female R. lophanthae compared to males was significan tly higher (t=9.1913, df=19, P<0.001). 50

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Discussion When offered a diet of only female A. yasumatsui R. lophanthae larvae were able to complete development at 18oC and 24oC, unlike C. montrouzieri. This suggests that female scales may not provide proper nutrition for deve lopment by the latter species. The lesser consumption of female scales by C. montrouzieri may also reflect mechanical difficulty of larvae to penetrate the armor of female A. yasumatsui Structural differences in the mandibles of generalist coccinellids versus that of species specialized for feeding on armored scales may affect the rate of consumption and developmen t of predators. In a study by Honda and Luck (1995), the mandibular structures of adult R. lophanthae and Chilocorus cacti (L.) were compared when feeding on hard and soft scales. Differences in the structures of each species mandibles were better adapted to removing scale covers of some species over others and therefore had an effect on a b eetles selection of a host. Female R. lophanthae consumed approximately two-thirds more scales than males, as was similarly observed by Stathas (2000). Re search on longevity of beetles under these conditions did not show significant variation in life spa n, therefore this difference in consumption may indicate the need for more resources to begin oviposition. Higher numbers of A. yasumatsui were consumed at 24oC than at 18oC which may be due to a shorter development time at the higher temperature and the need for mo re nutrition in a shorter period of time to complete development. Both species were able to complete devel opment when presented a diet of male and female A. yasumatsui In this study, C. montrouzieri larvae were observe d feeding almost exclusively on male A. yasumatsui and selectively avoiding female scales. Male scales, being more numerous and having thinner coverings, may require less energy to consume. Individuals 51

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of R. lophanthae also consum ed significantly fewer fema le scales when offered both male and female A. yasumatsui. This study revealed the rates of consumption by R. lophanthae were higher than that of C. montrouzieri as well as the ineffectiveness of both beet les in targeting female A. yasumatsui implicating their inability to cont rol the increase of scale populations Further investigation into biological control agents that can more prec isely control fluctuati ons in populations of A. yasumatsui by targeting reproductively viable females is greatly needed. 52

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Figure 3-1. Feeding damage to Aulacaspis yasumatsui by Rhyzobius lophanthae larva. 53

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Table 3-1. D aily and total consumption of 2nd and 3rd instar female Aulacaspis yasumatsui by Rhyzobius lophanthae and Cryptolaemus montrouzieri larvae and adults at 18oC. N is the number of days on which the average daily consumption rate is based; n is the number of individuals on which the averag e total consumption rate is based Means and standard errors are compared across co lumns to compare the same instar in both species. Means with the sa me letter are not significantly different (p>0.05). 18oC Rhyzobius lophanthae Cryptolaemus montrouzieri Daily Consumption N MeanSE N MeanSE 1st 232 0.3.5 a 199 0.1.3 b 2nd 138 1.2.1 a 25 0.2.4 b 3rd 182 2.7.7 a 21 0.5.5 b 4th 120 2.4.3 Adult Males 295 2.3.3 Adult Females 131 2.7.4 Rhyzobius lophanthae Cryptolaemus montrouzieri Total Consumption n MeanSE n MeanSE 1st 20 2.7.4 a 32 0.5.8 b 2nd 20 8.1.9 a 6 1.8.5 b 3rd 20 25.6.9 a 3 5.0.4 b 4th 20 39.0.4 Larvae 20 75.4.6 Adult Males 14 121.9.5 Adult Females 6 175.8.2 54

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Table 3-2. D aily and total consumption of 2nd and 3rd instar female Aulacaspis yasumatsui by Rhyzobius lophanthae and Cryptolaemus montrouzieri larvae and adults at 24oC. N is the number of days on which the average daily consumption rate is based; n is the number of individuals on which the averag e total consumption rate is based. Means and standard errors are compared across co lumns to compare the same instar in both species. Means with the sa me letter are not significantly different (p>0.05). 24oC Rhyzobius lophanthae Cryptolaemus montrouzieri Daily Consumption N MeanSE N MeanSE 1st 112 0.2.4 a 213 0.1.3 b 2nd 109 2.0.0 3rd 76 4.8.6 4th 118 4.9.4 Adult Males 876 2.6.5 Adult Females 821 3.1.6 Rhyzobius lophanthae Cryptolaemus montrouzieri Total Consumption n MeanSE n MeanSE 1st 21 1.1.3 a 36 0.5.7 a 2nd 21 10.0.0 3rd 21 17.9.6 4th 21 29.4.9 Larvae 21 58.4.8 Adult Males 12 194.0.6 Adult Females 9 281.0.8 55

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Table 3-3. D aily and total consumption of 2nd and 3rd instar male and female Aulacaspis yasumatsui by Rhyzobius lophanthae and Cryptolaemus montrouzieri larvae at 18oC. N is the number of days on which the averag e daily consumption ra te is based; n is the number of individuals on which the av erage total consumption rate is based. Means and standard errors are compared ac ross columns to compare the same instar in both species. Means with the same lett er are not significantly different (p>0.05). 18oC Rhyzobius lophanthae Cryptolaemus montrouzieri Daily Consumption N MeanSE N MeanSE 1st 40 0.1.3 a 216 <0.1.2 a 2nd 31 0.1.3 a 140 <0.1.2 a 3rd 12 0.8.2 a 213 <0.1.2 b 4th 58 0.7.2 a 339 0.2.8 b Rhyzobius lophanthae Cryptolaemus montrouzieri Total Consumption n MeanSE n MeanSE 1st 6 0.3.2 a 17 0.2.1 a 2nd 5 0.8.5 a 13 0.5.1 b 3rd 5 7.5.8 a 12 0.8.3 b 4th 4 15.4.7 a 5 5.5.3 b Larvae 4 24.0.1 a 5 7.3.9 b 56

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Table 3-4. D aily and total consumption of 2nd and 3rd instar male and female Aulacaspis yasumatsui by Rhyzobius lophanthae and Cryptolaemus montrouzieri larvae at 24oC. N is the number of days on which the averag e daily consumption ra te is based; n is the number of individuals on which the av erage total consumption rate is based. Means and standard errors are compared ac ross columns to compare the same instar in both species. Means with the same lett er are not significantly different (p>0.05). 24oC Rhyzobius lophanthae Cryptolaemus montrouzieri Daily Consumption N MeanSE N MeanSE 1st 38 0.3.6 a 192 0.2.5 a 2nd 36 0.7.0 a 109 0.7.3 a 3rd 45 1.5.4 a 144 1.0.6 b 4th 55 1.7.1 a 113 0.8.3 b Rhyzobius lophanthae Cryptolaemus montrouzieri Total Consumption n MeanSE n MeanSE 1st 9 0.3.2 a 16 2.9.9 b 2nd 6 0.8.5 a 14 3.9.9 b 3rd 6 7.5.8 a 9 16.06.8 a 4th 6 15.4.7 a 6 13.5.2 a Larvae 6 24.9.1 a 6 36.45.8 b 57

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CHAP TER 4 FIELD RELEASE STUDY OF RHYZOBIUS LOPHANTHAE Introduction Rhyzobius lophanthae is an important natural enemy of many armored scale species and has been introduced into Hawaii, Italy, and Guam for control of scales (Yus 1973; Rosen 1990; Heu et al. 2003; Moore et al. 2005). This species is an effective biological control agent due to biological factors such as its pr ey specificity, high fecundity and adult longevity as well as ecological factors including its lack of diapau se, high mobility, lack of parasitism and rapid population development (Stathas et al. 2002). Beetles are able to feed on armored scales by straddling the scale while maintaining contact wi th the substrate and in serting their mandibles under the scale cover while pressing away from the substrate. Studies with R. lophanthae demonstrated the predators ability to handle sm aller scales, especially the immature stages (Honda 1999). Aulacaspis yasumatsui Takagi is an invasive armored scale native to a region stretching from the Andaman Islands to Vietnam, Thailand, southern China, and likely Cambodia, Laos, Malaysia, and Myanmar (Howard et al. 1999; Muniappan 2005). Si nce its accidental introduction into the US in 1996, A. yasumatsui has spread to more th an 7 southern states (Broome 2000). This species has also been detrimen tal to plants in the West Indies, Guam, Hong Kong, Singapore, Taiwan, New Zealand, Costa Rica, and Africa (Weissling et al. 1999; Hodges et al. 2004; Moore et al. 2005; Germain and Hodges 2007). The scale gives plants an unsightly snow-c overed appearance that when unmanaged can form dense multilayered coverings of nearly 3,000 s cales per square inch (Weissling et al. 1999), eventually leading to the death of plants (Heu et al. 2003). Manage ment of the scale infestations is hindered by its abil ity to infest the primary and secondary r oots of plants as deep as 60 cm into 58

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the ground (Howard et al. 1999). Addition ally, scales can escape freezing temperatures by hiding in overlapping plant material from previous years growth in the central cone of Cycas revoluta plants, which cannot be reached with nor mal chemical treatments (Broome 2002). While R. lophanthae is commercially available as a biological control agent information regarding the adequate number of R. lophanthae needed to control an infestation of A. yasumatsui per plant is unknown. This study was undertak en to assess the number of beetles needed to control an infestation of A. yasumatsui on a given leaf area over a period of time in a greenhouse and urban environment. The amount of consumption by R. lophanthae on plants will provide a standard for treating infested plants. Materials and Methods Greenhouse Study Insects Aulacaspis yasumatsui was reared on C. revoluta plants kept in 3.7 L pots and maintained in a greenhouse with 30% RH. Plants were fertilized and watered regularly according to growers recommendations to maintain the hea lth of plants throughout the study. Clean plants were exposed to infested plants with active cr awlers by interlocking leaves for 1 week, allowing crawlers to settle on the clean foliage. Moderately infested plants with 3rd instar A. yasumatsui were obtained in approximately 1 month at 30oC (Howard et al. 1999). Adult R. lophanthae were obtained from Rincon-Vitova Inse ctaries (Ventura, California) a nd kept in 20 20 20 cm Bug Dorms (BioQuip, Inc. Rancho Dominguez, CA) with water-saturated cotton balls and infested C. revoluta plants at 25oC, 60% RH and 14:10 (L:D) photoperiod. 59

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Experimental design Cycas revoluta plants infested with 10-50 2nd and 3rd instar male and 5-30 2nd and 3rd instar female A. yasumatsui per leaflet were chosen. The initial infestation per leaf was determined by sampling 6 leaflets at random and counting the number of 2nd and 3rd instar female A. yasumatsui The number of leaflets per leaf was also counted. Individual leav es were enclosed by a cylindrical mesh bag that was secured with rubber bands at the leaf base a nd at the tip just past the last leaflet. Mesh bags a llowed conditions on the leaf to remain similar to those of the greenhouse at 29oC with 21% RH recorded with a HOBO Data Logger. Bags were large enough to allow movement along upper and lower sides of leaves. Velc ro closure along the underside of the leaf allowed for access to ba gs without removing them from the leaf. Beetles were starved for 48 h prior to study. Leaves were randomly assigned treatments of 0, 2, 4, 6, or 8 adult R. lophanthae with a sex ratio of 1:1. Bee tles were manually shaken into each bag. Six leaflets were sampled at random every 3 days from each leaf. The number of consumed scales and live, undamaged scales in the sample and number of live beetles in the cage were recorded. Dead R. lophanthae were replaced with new beetles to maintain treatment numbers for 2 weeks. Each treatment was replicated 4-5 times. The average number of female A. yasumatsui consumed versus the number of scales alive and undamaged by R. lophanthae was analyzed using a t-test. Field Study Insects and plants Adult R. lophanthae were obtained from Rincon-Vitova Insectaries (Ventura, California) and kept in vials with suga r water-saturated cotton balls and leaflets of infested C. revoluta plants at 25oC, 60% RH and 14:10 (L:D) photoperiod. Commercially produced C. revoluta plants in 9 L containers and mature 1m tall in ground plan ts used in this study were naturally infested 60

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prior to being selected. A ll plants were m aintained at each si te for 6 months prior to this study without the use of chemical cont rol. Plants chosen had 10-50% of their leaf area covered in scales, were healthy and had > 5 leaves at the be ginning of the study. Experimental design Selected plants were found at one of three site s with 12 containerized plants at Site 1, 4 in ground plants at Site 2 and 12 in ground plants at Site 3. Site 1 (F igure 4-1) and Site 2 (Figure 42) were located less than one mile from one anot her in a suburban area. Both sites represented the typical landscape for homeowners Plants at both sites were separated by at least 2 m from one another. Site 3 (Figure 4-3) was situated in a high foot-traffic area in downtown Tampa that uses cycads in a business landscape. Plants were sa mpled prior to releasing predators. At Site 1 and Site 2 parasitism by Coccobius fulvus (Compere and Annecke) was observed with a maximum of 6 parasitized scales per sample of 6 leaflets and a maximum of 21 parasitized scales at Site 3. The beetle Cybocephalus nipponicus Endrdy-Younga on a single day was also observed on all plants at Site 2 with a maximum of 36 beetles and minimum of 3 beetles on one plant. Infested plants at each site were randomly assigned 0, 100, 200 or 300 adult R. lophanthae with a 50:50 sex ratio. Just afte r sunset at 6pm, beetles were tr ansferred from plastic vials onto plants where the leaves met the trunk by manually shaking the vial (Fig ure 4-4). Plants were sampled every 4 d from September until November during a peak in A. yasumatsui infestation Each plant was scored in the field for level of s cale infestation as percenta ge of leaf area covered (low 10-40%, medium 41-70%, an d heavy 71-100%), number of R. lophanthae larvae and adults observed, presence of other scale predators and b eetle predation damage to scales. Consumption rates were determined by sampling 6 leaflets randomly from each plant every 8 d and by counting the number of live scales present on ea ch leaflet, occurrence of feeding damage by beetles and parasitoid emergence holes observed using a stereomicroscope. 61

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Results Greenhouse Study The greatest m ean number of scales consumed per beetle occurred in the treatment of 8 beetles per leaf (Figure 4-5) with a decreasing number of scales consumed as the number beetles in each treatment decreased. High ra tes of beetle mortality were observed in all treatments. In order to determine the consumption of beetles pr ior to death, assuming they did not consume the maximum number of scales possible yet they lived long enough to consume some scale, all dead beetles were counted as half of one individua l when calculating the average number of scales consumed. Similar trends in consumption were observed in all treatments demonstrating an increase in consumption over time. Treatments with higher numbers of beetles c onsumed a greater propo rtion of the total scale population (Figure 4-6). The proportion of scales consumed by treatments of 8 beetles was significantly greater then those with 2 beetle s over the entire study a nd greater then treatments with 4 beetles in the first 9 d. Six beetles cons umed significantly more scales then 2 beetles during the whole period except 9 d after release. Based on regression lines for each treatment, 50% mortality of scales on one leaf is possible in 12, 16, 79 and 525 d, respectively, for treatments of 8, 6, 4 and 2 beetles. Healthy scale infestations were reduc ed by 10% following the introduction of R. lophanthae in treatments with 4 and 6 beetles whereas treatments with 2 beetles were reduced by 35% (Figure 4-7). Field Study Adult R. lophanthae were observed on plants during the fi rst 12 d at Site 1, first 4 d at Site 2, and first 8 d at Site 3 following their release (Figure 4-4). Af ter 4 d, plants with 100 beetles released at Site 1 had an average of 5 beetles per plant, with a minimum of 0 and 62

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m aximum of 14. Plants with 200 beetles released had 11 beetles on averag e per plant, with a minimum of 2 and maximum of 21. Plants with 300 beetles released had on average 17 beetles per plant, with a minimum of 14 and maximum of 19. After 8 d, all plants at Site 1 had 6 beetles per plant. At Site 2, 5 beetles were observed 4 d after the release of 100 beetles and 6 beetles were observed on plants af ter 300 beetles were released. Plan ts at Site 3 with 100 beetles released had an average of 1 bee tle per plant 4 d after release. Plants treate d with 200 beetles had an average of 3 beetles per plant, with a mini mum of 0 and maximum of 6, while plants treated with 300 beetles had an average of 2 beetles per plant, with a minimum of 0 and maximum of 3. After 8 days at Site 3, 1 beetle was observed on each of two plants treated with 200 and 300 beetles. No beetles were found on s ubsequent days at Sites 2 or 3. Larvae of R. lophanthae were observed 20-28 d following rel ease of adults at Site 1 and 12-24 d at Site 3 (Table 4-1). No larvae were obse rved at Site 2. Five larvae were seen on day 20 at Site 1 with a decline in the number of larvae observed on following days, with 2 larvae at 24 d and 1 larva at 28 d. At Site 3, 1 larva was obs erved on day 12 and again on day 24. There was no correlation between the number of beetles re leased and the number of larvae observed. Initial beetle feeding damage was observed on infested plants during the first 8 d. With the absence of beetles and low numbers of larv ae, there were no signifi cant differences in damage to scales among treatments at later time points. The level of scale infestation increased over the course of the study following release of R. lophanthae (Figure 4-8). At Site 1, all control plants had a heavy infestation 20 d af ter the initial release. All plants at this site had a medium to heavy infestation by day 36, regardless of treatment Plants at Site 2 maintained very low to medium infestations during the entire study. At Site 3, control plants were heavily infested after 36 d. All other plants at Site 3 had medium to heavy infestations after 24 d. Levels of A. 63

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yasumatsui infestation on 4 plants w ere so high that 44 d after release, the plants were removed due to poor health and to decrease the further spread of scales. Several species of predatory beetles and evidence of parasitism were observed on the plants. Adults of C. nipponicus were found at all three sites, w ith the largest population at Site 2. As many as 36 beetles were observed on one plant at Site 2. The coccinellids Chilocorus stigma (Walker) and Exochomus childreni Mulsant were both observed at Sites 2 and 3, while Curinus coeruleus Mulsant adults were present only at Site 3. As many as 4 C. stigma, 7 E. childreni and 1 C. coeruleus were observed on a single plant at one time. The presence of C. fulvus at Sites 1 and 2 was also observed. Discussion In the greenhouse study, 8 beetle s consumed more scales than treatments with 2, 4 or 6 beetles. These results may suggest that with a la rger number of beetles, individuals were better able to search leaf areas fo r undamaged scales to feed on. Th e average number of scales consumed by 8 beetles was significantly greater th en that of 2 beetles. There was no significant difference between all other treatments. Analysis of the proportion of to tal scales consumed indicated that increased nu mbers of beetles are able to control populations of A. yasumatsui in the shortest amount of time. These results follow with the previous resu lts indicating that the application of more beetles to a plant may result in the control of scales bellow an economic injury level in a shorter period of time. Beetle mortality rates were high, with as many as 5 beetles in a treatment found dead. The high humid ity in the greenhouse and restricted movement of beetles to any other part of the plant ma y have contributed to their mortality. Scale populations initially decreas ed after the release of R. lophanthae in all treatments and then held relatively constant for all treatments except those with 2 beetles. These results were unexpected. The restricted search area should have increased the likelihood of adults finding healthy scales 64

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and should h ave resulted in higher levels of control than those observed. Because beetles were starved prior to their release the data may reflect initial heavy feeding damage. In the field study, the high mobility of R. lophanthae after release may have contributed to its dispersal away from treatment plants despite the availability of scales on the plants. Interference competition as a result of large numbers of beetles in each treatment may also have influenced this result. The presence of large numbers of C. nipponicus at Site 2 may have been effective in maintaining scale infestations at lo w levels. All other beetle species were observed on plants in which scale populati ons were increasing, therefore popul ations of those species were apparently not effective in controlling A. yasumatsui Both Site 2 and Site 3 were located along roadways which may have made the sites less idea l for populations of beetles to establish with the movement of air as vehicles passed by. Given that the infestation of A. yasumatsui was higher on all plants at the end of the field study and the high mortality of beetles and low leve l of control of scales in the greenhouse, I would not recommend using R. lophanthae as a biological control agent following the same parameters of this experiment. Similar releases have taken place at field sites in Guam, however, in those releases beetles were ke pt in mesh sleeves on plants for several weeks before bags were removed (Moore et al. 2005). This may have allo wed beetles to spend more time laying eggs on the plant before dispersing. 65

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Figure 4-1. Field release Site 1. Bl ue represents water, brown is a residence, black is a paved surface, green area is grass, a nd dark circles represent individual plants used and their treatment (replicate-number of beetles released). 66

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Figure 4-2. Field release Site 2. Black objects are paved surfaces, green represents grass, and dark green circles indicate individual pl ants used and their treatment (replicatenumber of beetles released). 67

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Figure 4-3. Field release Site 3. Black indicates a paved surfa ce, brown indicates a building, green represents grass, and dark green ci rcles represent individual plants used and their treatment (replicate-num ber of beetles released). 68

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Figure 4-4. Adult R. lophanthae were manually shaken onto a C. revoluta plant infested with A. yasumatsui. 69

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0 1 2 3 4 5 3691215 Days after releaseMean number of scales consumed per beetle 0 2 4 6 8 Figure 4-5. Average number of 2nd and 3rd instar female A. yasumatsui consumed per R. lophanthae adult in treatments of 0, 2, 4, 6, and 8 beetles per leaf. Data account for mortality of adult beetles in a greenhouse. 70

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0 0.1 0.2 0.3 0.4 3691215 Days after releaseProportion of scale population consumed 0 2 4 6 8 Figure 4-6. Proportion of total A. yasumatsui population consumed by R. lophanthae in treatments of 0, 2, 4, 6 and 8 beet les per leaf in a greenhouse. 71

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0 2 4 6 8 10 12 14 3691215 Days after releaseMean number of undamaged scales per leaflet 0 2 4 6 8 Figure 4-7. The mean number of healthy A. yasumatsui per leaflet undamaged by R. lophanthae over time in a greenhouse. 72

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Table 4-1. N umber of Rhyzobius lophanthae observed on cycad plants at field sites after their release. Days after release Site Treatment 4 8 12 16 1 0 0 0 0 0 0 0 0 0 0 0 0 0 100 0 0 0 0 1 0 0 0 14 6 1 0 200 11 0 0 0 21 5 0 0 2 0 0 0 300 14 1 0 0 19 2 0 0 19 4 0 0 2 0 0 0 0 0 100 5 0 0 0 200 0 0 0 0 300 6 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 100 0 0 0 0 1 0 0 0 1 1 0 0 200 0 0 0 0 0 0 0 0 6 1 0 0 300 3 0 0 0 1 0 0 0 0 0 0 0 73

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0 10 20 30 40 50 60 70 80 90 100 4812162024283236404448525660 Days after releasePercentage of leaf area covered 0 100 200 300 Figure 4-8. The percentage of leaf area infested by A. yasumatsui over time in treatments of 0, 100, 200 or 300 R. lophanthae over all sites in the field. 74

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LIST OF REFERE NCES BARTLETT, B. R. 1973. Introduction into Califo rnia of cold-tolerant biotypes of the mealybug predator Cryptolaemus montrouzieri and laboratory procedures for testing natural enemies for cold-hardi ness. Environmental Entomology 3: 553556. BROOME, T. 2000. The Asian cycad scale. The Cycad Newsletter 24(4): 6-7. BROOME, T. What is a Cycad? An Introduction. 2002. The Cycad Newsletter. Retrieved May 12, 2008 from http://cycadjungle.8m.com/cycad jungle/what%20is%20a%20cycad.htm l CALDWELL, D. L. 2003. The cycad aulacaspis scale, Aulacaspis yasumatsui : Management approaches and pesticide tr ial updates. Proceedings of the Florida State Horticultural Society 116: 347-350. CAVE, R. D. 2006. Biological control agen ts of the cycad aulacaspis scale, Aulacaspis yasumatsui Proceedings of the Florida Stat e Horticultural Society 119: 422-424. CLAUSEN, C. P. 1940. Entomophagous insects. McGraw-Hill. London. COOPER, S. 1995. Cryptolaemus montrouzieri : a predator for mealybug. British Cactus and Succulent Journal 3: 38-39. EMSHOUSEN, C., AND MANNION, C. 2004a. Taming Asian cycad scale ( Aulacaspis yasumatsui ). The Cycad Newsletter 27(1): 8. EMSHOUSEN, C., AND MANNION, C. 2004b. Ma nagement of cycad aulacaspis scale, Aulacaspis yasumatsui Takagi. Proceedings of the Florida State Horticultural Society 117: 305-307. FRANK, J. H., AND MCCOY, E. D. 2007. The risk of classical biological control in Florida. Biological Control 41: 151-174. Rhyzobius lophanthae. 2006. Gardening Zone. Retrieved August 16, 2006 from http://gardeningzone.com/product_inf o_Rhizobius_(Lindorus)_lopanthae_50_per _bottle.htm l GERMAIN, J. F., AND HODGES, G. S. 2007. First report of Aulacaspis yasumatsui (Hemiptera: Diaspididae) in Africa(Ivory Coast), a nd update on Distribution. Florida Entomologist 90: 755-756. GORDON, R. D.1985. North American Cocci nellidae. Journal of the New York Entomological Society 93: 105-107, 641-646. 75

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GREATHEAD, D. J. 1973. A re view of introductions of Lindorus lophanthae (Bla isdell) (Coleoptera: Coccinellidae) against hard scales (Diaspididae). Technical Bulletin of the Commonwealth Institute of Biological Control 16: 29-33. HATTINGH, V., AND SAMWAYS, M. J. 1994. Physiological and behavioral characteristics of Chilocorus spp.(Coleoptera: Coccinellidae) in the laboratory relative to effectiveness in the field as biocontrol agents. Journal of Economic Entomology 87: 31-38. HAYNES, J. 2005. Cycad Aulacaspis Scale: A Global Perspective. The Cycad Newsletter 28(5): 3-6. HEIDARI, M., AND COPLAND, M. J. W. 1992. Host finding by Cryptolaemus montrouzieri (Col.: Coccinellidae) a pred ator of mealybugs (Hom.: Pseudococcidae). Entomophaga 37: 621-625. HEU, R.A., CHUN, M., AND NAGAMINE, W. T. 2003. Sago palm scale, Aulacaspis yasumatsui Takagi (Homoptera: Diaspididae). State Departme nt of Agriculture New Pest Advisory No.99-01. HODGES G. S., HOWARD, F. W., AND BUSS, E. A. 2003. Update on management methods for cycad aulacaspis scale. Univ ersity of Florida Extension publication. ENY-680. HODGSON, C., AND MARTIN, J. H. 2001. Thr ee noteworthy scale in sects (Hemiptera: Coccoidea) from Hong Kong and Singapore, including Cribropulvinaria tailungensis, new genus and species (Coccidae) and the status of the cycadfeeding Aulacaspis yasumatsui (Diaspididae). Raffles Bulletin of Zoology 49: 227-250. HONDA, J. Y., AND LUCK, R. F. 1995. S cale morphological effects on feeding behavior and biological control potential of Rhyzobius lophanthae (Coleoptera: Coccinellidae). Ecology and Population Biology 88: 441-450. HOWARD, F. W., HAMON, A., MCLAUGHL IN, M., WEISSLING, T. AND YANG, S. 1999. Aulacaspis yasumatsui (Hemiptera: Sternorrhyncha: Diaspidae), a scale insect pest of cycads recently introduced into Florida. Florida Entomologist 82: 14-27. HUE, R. A., CHUN, M. E. AND NAGAMINE, W. T. 2003. Sago palm scale Aulacaspis yasumatsui Takagi (Homoptera: Diaspididae). Hawaii Department of Agriculture NPA #99-01. MAGRO, A., HEMPTINNE, J. L., CODRE ANU, P., GROSJEAN, S. AND DIXON, A. F. G. 2002. Does the satiation hypothesis acc ount for the differences in efficacy of coccidophagous and aphidophagous ladybird beet les in biological control? A test with Adalia bipunctata and Cryptolaemus montrouzieri BioControl 47: 537-543. 76

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MCLAUGHLIN, M. What's this white stuff on my cycad? 2008. Fairchild Tropical Garden. Retrieved September 19, 2006 from http://www.fairchildgarden.org/index.cfm ? section=fairchildn ews&page=articles& newsid=9 MOORE, A., IRIARTA, I. AND QUINTU GUA, R. 2005a. Asian cycad scale Aulacaspis yasumatsui Takagi (Homoptera: Diaspididae). Cooperative Extension Service, Agricultural and Natural Resources Division, Univers ity of Guam, Pest Sheet 2005-01. MOORE, A., MARLER, T., MILLER, R. H., AND MUNIAPPAN, R. 2005b. Biological Control of Cycad Aulacaspis Scale on Guam. The Cycad Newsletter 28(5): 6-8. MUNIAPPAN, R. 2005. Foreign exploration for natural enem ies of the cycad scale, Aulacaspis yasumatsui (Homoptera: Diaspididae) University of Florida Extension publication #EENY-096. MUNIAPPAN, R., AND VI RAKTAMATH, C. A. 2006. The Asian cycad scale Aulacaspis yasumatsui a threat to native cycads in India. Current Science 91: 868-870. OBRYCKI, J. J., AND KRING, T. J. 1998. Predaceous Coccinellidae in biological control. Annual Review of Entomology 43: 295-321. ONSET COMPUTER CORPORATION (1996-2007) Bourne, MA, USA PERSAD, A., AND KHAN, A. 2002. Comparison of the life table parameters for Maconellicoccus hirsutus Anagyrus kamali Cryptolaemus montrouzieri and Scymnus coccivora BioControl 47: 137-149. ROSEN, D. 1990. Armored Scale Insects: th eir biology, natural enemies and control. Elsevier,Amsterdam, 4B: 688. SAS INSTITUTE (2002-2003) Ve rsion 9.1. Cary, NC, USA. SMIRNOFF, W. 1950. Sur la biologie au Maroc de Rhyzobius (Lindorus) lophanthae Blaisdell (Coleoptera: Coccinellidae). Revue de Pathologie Vgtale et d Entomologie Agricole de France 29: 190. STATHAS, G. J. 2000a. Rhyzobius lophanthae prey consumption and fecundity. Phytoparasitica 28(3): 1-9. STATHAS, G. J. 2000b. The effect of temper ature on the development of the predator Rhyzobius lophanthae and its phenology in Greece. BioControl 45: 439-451. STATHAS, G. J. 2001. Studies on morphology and biology of immature stages of the predator Rhyzobius lophanthae (Blaisdell) (Col.: Coccinellidae). Journal of Pest Science 74: 113-116 77

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STATHUS, G. J., ELIOPOUL OS, P. A., KO NTODIMAS, D. C., AND SIAMOS, D. T. 2002. Adult morphology and life cycle under constant temperatures of the predator Rhyzobius lophanthae Blaisdell (Coleoptera: Co ccinellidae). Journal of Pest Science 75: 105-109. TAKAGI S. 1977. A new species of Aulacaspis associated with a cycad in Thailand (Homoptera: Cocoidea). Insecta Ma tsumurana New Series 11: 63-72. U.S. ENVIRONMENTAL PR OTECTION AGENCY. 2002. Dime thoate; Receipt of Requests for Amendments and Cancellati ons. Federal Register Environmental Documents 67: 1345-1348. WALTERS, T., SHROYER, E., AND ANDERS ON, L. 1997. Scale and south Florida Cycas. The Cycad Newsletter 20(1). WEISSLING, T. J., HOWARD, F. W. A ND HAMON, A. B. 1999. Cycad aulacaspis scale, Aulacaspis yasumatsui Takagi (Insecta: Hom optera: Sternorryhyncha: Diaspididae). University of Florid a Extension publication #EENY-096. WIESE, C., AMALIN, D., COE, R., AND MA NNION, C. 2005. Effects of the parasitic wasp, Coccobius fulvus on cycad aulacaspis scale, Aulacaspis yasumatsui at Montgomery Botanical Center, Miami, Flor ida. Proceedings of the Florida State Horticultural Society 118: 319-321. YUS, R. 1973. On the presence in the Iberian Peninsula of Rhyzobius lophanthae Blaisdell (Coleoptera: Cocci nellidae). Graellsia 29: 111. 78

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79 BIOGRAPHICAL SKETCH Greta Thorson received her Bachelor of Scien ce degree from the University of Delaware in 2006, majoring in entomology with a minor in landscape horticulture. From 2002 to 2004, she worked as a biological science technician at the USDA Beneficial Inse cts Research Laboratory in Newark, DE under the direction of Mr. Roge r Fuester. While working for the USDA she assisted with research on the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky) and gypsy moth parasitoids. During the summer s of 2003 and 2004 she worked at the USDA Bee Research Lab in Beltsville, MD for Mr. I. Ba rton Smith as a biological science technician tending to honey bee colonies and assisting with research. She worked in the Termite Research and Genetics lab of Dr. Susan Whitney King at the University of Delaware from May 2005 until July 2006, focusing on colony behavior and variation between Reticulitermes flavipies (Kollar) and Reticulitermes virginicus (Banks). Most recently she worked as a graduate research assistant with the University of Florida from 2006-2008.