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Application of the F1 Sterile Insect Technique (F1SIT) for Field Host Range Testing of Episimus utilis Zimmerman (Lepidoptera

Permanent Link: http://ufdc.ufl.edu/UFE0021849/00001

Material Information

Title: Application of the F1 Sterile Insect Technique (F1SIT) for Field Host Range Testing of Episimus utilis Zimmerman (Lepidoptera Tortricidae), a Candidate for Biological Control of Brazilian Peppertree
Physical Description: 1 online resource (71 p.)
Language: english
Creator: Moeri, Onour E
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: anacardiaceae, biocontrol, episimus, f1sit
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: Host range testing is used in weed biological control to demonstrate the safety of potential biological control agents. However, these laboratory tests may overestimate host range leading to ?false positives? where the insect accepts plant species that it would not normally accept in nature. As cage testing may inhibit the behavior of the potential biological control agent, open-field studies may provide a more realistic setting of environmental and ecological conditions that the biological control agent will encounter upon release in the proposed areas of introduction. Open-field studies, however, are prohibited in the area of introduction. Reproductively inactivated potential biological control agents produced as a result of the application of the F1 sterile insect technique (F1SIT) could be used to conduct field testing in a safe and temporary manner. A situation where this could be used is in the biological control of Brazilian peppertree, Schinus terebinthifolius Raddi (Anacardiaceae), an invasive, exotic species distributed widely throughout central and southern Florida. A leaf-rolling moth, Episimus utilis Zimmerman (Lepidoptera: Tortricidae) is a potential biological control agent of Brazilian peppertree. Traditional laboratory no-choice and choice tests performed with E. utilis produced ambiguous results where selected non-target species including fragrant sumac (Rhus aromatica L.), winged sumac (Rhus copallinum L.), poison sumac (Toxicodendron vernix (L.) Kuntze), pistachio (Pistacia vera L.) and cashew (Anacardium occidentale L.) were unexpectedly accepted as host species. Therefore, the use of F1SIT for field host range testing was investigated as a new approach for risk assessment of potential biological control agents. Male and Female virgin E. utilis adults were treated with increasing doses of radiation and either inbred or outcrossed to nontreated E. utilis adults. Five pairs of adults were placed in triangular waxed paper oviposition cages and allowed to mate and oviposit for two intervals of 5 days. The number of eggs laid (fecundity) and the number of eggs that hatched (fertility) were counted for each egg sheet per dose. As the dose of radiation increased, there were no significant changes in the fecundity of nontreated females mated with treated males, yet fewer eggs were laid by treated females. Fertility for both treated males and females decreased with increasing doses of radiation. The dose at which treated females were found to be 100% sterile was 200 Gy. There were no significant changes in the fecundity for F1 females and males resulting from treated parental males with increasing dose of radiation. Fertility for F1 females and males resulting from treated parental males declined as the dose of radiation increased. There was a moderate positive correlation for the F1 sex ratio of males to females with increasing dose of radiation. The dose at which F1 females and males were found to be 100% sterile was 225 Gy. Results from this study were similar to results found in other tortricid moths including the codling moth, Cydia pomonella (L.), and the false codling moth, Thaumatotibia leucotreta (Meyrick). As the dose of radiation increased, there was an increase in sterility, a decrease in fecundity for both treated female crosses, and a higher ratio of F1 males to females. This novel approach could be used to safely and temporarily test potential biological control agents in the field.
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 Onour E Moeri.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Cuda, James P.
Local: Co-adviser: Overholt, William A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021849:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021849/00001

Material Information

Title: Application of the F1 Sterile Insect Technique (F1SIT) for Field Host Range Testing of Episimus utilis Zimmerman (Lepidoptera Tortricidae), a Candidate for Biological Control of Brazilian Peppertree
Physical Description: 1 online resource (71 p.)
Language: english
Creator: Moeri, Onour E
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: anacardiaceae, biocontrol, episimus, f1sit
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: Host range testing is used in weed biological control to demonstrate the safety of potential biological control agents. However, these laboratory tests may overestimate host range leading to ?false positives? where the insect accepts plant species that it would not normally accept in nature. As cage testing may inhibit the behavior of the potential biological control agent, open-field studies may provide a more realistic setting of environmental and ecological conditions that the biological control agent will encounter upon release in the proposed areas of introduction. Open-field studies, however, are prohibited in the area of introduction. Reproductively inactivated potential biological control agents produced as a result of the application of the F1 sterile insect technique (F1SIT) could be used to conduct field testing in a safe and temporary manner. A situation where this could be used is in the biological control of Brazilian peppertree, Schinus terebinthifolius Raddi (Anacardiaceae), an invasive, exotic species distributed widely throughout central and southern Florida. A leaf-rolling moth, Episimus utilis Zimmerman (Lepidoptera: Tortricidae) is a potential biological control agent of Brazilian peppertree. Traditional laboratory no-choice and choice tests performed with E. utilis produced ambiguous results where selected non-target species including fragrant sumac (Rhus aromatica L.), winged sumac (Rhus copallinum L.), poison sumac (Toxicodendron vernix (L.) Kuntze), pistachio (Pistacia vera L.) and cashew (Anacardium occidentale L.) were unexpectedly accepted as host species. Therefore, the use of F1SIT for field host range testing was investigated as a new approach for risk assessment of potential biological control agents. Male and Female virgin E. utilis adults were treated with increasing doses of radiation and either inbred or outcrossed to nontreated E. utilis adults. Five pairs of adults were placed in triangular waxed paper oviposition cages and allowed to mate and oviposit for two intervals of 5 days. The number of eggs laid (fecundity) and the number of eggs that hatched (fertility) were counted for each egg sheet per dose. As the dose of radiation increased, there were no significant changes in the fecundity of nontreated females mated with treated males, yet fewer eggs were laid by treated females. Fertility for both treated males and females decreased with increasing doses of radiation. The dose at which treated females were found to be 100% sterile was 200 Gy. There were no significant changes in the fecundity for F1 females and males resulting from treated parental males with increasing dose of radiation. Fertility for F1 females and males resulting from treated parental males declined as the dose of radiation increased. There was a moderate positive correlation for the F1 sex ratio of males to females with increasing dose of radiation. The dose at which F1 females and males were found to be 100% sterile was 225 Gy. Results from this study were similar to results found in other tortricid moths including the codling moth, Cydia pomonella (L.), and the false codling moth, Thaumatotibia leucotreta (Meyrick). As the dose of radiation increased, there was an increase in sterility, a decrease in fecundity for both treated female crosses, and a higher ratio of F1 males to females. This novel approach could be used to safely and temporarily test potential biological control agents in the field.
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 Onour E Moeri.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Cuda, James P.
Local: Co-adviser: Overholt, William A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021849:00001


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1 APPLICATION OF THE F1 STERILE INSECT TECHNIQUE (F1SIT) FOR FIELD HOST RANGE TESTING OF Episimus utilis ZIMMERMAN (LEPIDOPTERA: TORTRICIDAE), A CANDIDATE FOR BIOLOGICAL CONTROL OF BRAZILIAN PEPPERTREE By ONOUR ELIZABETH MOERI A THESIS 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 2007

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2 2007 Onour Elizabeth Moeri

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3 To my parents

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4 ACKNOWLEDGMENTS I thank my major advisor James P. Cuda and co-adviser William A. Overholt for their support and guidance. I thank my other committ ee members, James L. Carpenter and Stephanie Bloem for sharing their vast know ledge of inherited sterility a nd providing an immeasurable amount of support throughout the project. I thank Judy Gillmore and the staff of the weed biological control lab, whom without their help with insect rear ing and plant cultivation, this project would not be possible. I thank Dr. Burrell Smittle, Carl Gillis, and Suzanne Fraser at the Florida Accelerator Services and Technology Gain esville, FL for their support and assistance with irradiation of the E. utilis moths. In addition, I thank my family and friends for always being there to listen and provide encouragement.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF FIGURES................................................................................................................ .........7 ABSTRACT....................................................................................................................... ..............8 CHAPTER 1 INTRODUCTION..................................................................................................................10 2 LITERATURE REVIEW.......................................................................................................14 The F1 Sterile Insect Technique (F1SIT)................................................................................14 Description.................................................................................................................... ..14 History........................................................................................................................ .....14 Field Application of F1SIT..............................................................................................16 Host Range Testing Protocols................................................................................................19 No-Choice Tests..............................................................................................................19 Choice Tests................................................................................................................... .20 Open Field Testing..........................................................................................................20 Schinus terebinthifolius Raddi................................................................................................22 Taxonomy....................................................................................................................... .22 Common Names..............................................................................................................23 Description.................................................................................................................... ..23 Distribution................................................................................................................... ...23 Environmenta l Impacts.......................................................................................................... .24 Ecosystem...................................................................................................................... ..24 Human and Animal Health..............................................................................................25 Beneficial Uses................................................................................................................25 Controlling Brazilian Peppertree............................................................................................26 Mechanical Control.........................................................................................................26 Physical Control..............................................................................................................26 Chemical Control.............................................................................................................27 Biological Control...........................................................................................................27 Episimus utilis Zimmerman....................................................................................................28 Taxonomy....................................................................................................................... .28 Biology........................................................................................................................ ....28 History of Introduction of E. utilis ..................................................................................29

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6 3 USE OF THE F1 STERILE INSECT TECHNIQUE (F1SIT) AS A TOOL FOR FIELD HOST RANGE TESTING OF Epismus utilis ZIMMERMAN (LEPIDOPTERA: TORTRICIDAE), A CANDIDATE BIOLOGICAL CONTROL AGENT OF BRAZILIAN PEPPERTREE..................................................................................................38 Introduction................................................................................................................... ..........38 Materials and Methods.......................................................................................................... .41 Colony Rearing................................................................................................................41 Radiation Biology Study.................................................................................................42 Statistical Analysis for Radiation Biology Study............................................................44 Inherited Sterility Methodology......................................................................................45 Statistical Analysis fo r Inherited Sterility.......................................................................46 Results........................................................................................................................ .............46 Radiation Biology Study.................................................................................................46 Inherited Sterility............................................................................................................ .46 Discussion..................................................................................................................... ..........47 4 CONCLUSIONS....................................................................................................................59 LIST OF REFERENCES............................................................................................................. ..62 BIOGRAPHICAL SKETCH.........................................................................................................71

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7 LIST OF FIGURES Figure page 2-1 Dense stands of Schinus terebinthifolius in Florida...........................................................31 2-2 Morphology of Schinus terebinthifolius ............................................................................32 2-3 Native distribution of Brazilian pe ppertree in South America by country........................33 2-4 Worldwide distribution of Brazilian peppertree................................................................34 2-5 Distribution of Brazilia n peppertree in Florida..................................................................35 2-6 A-F. Life cycle of E. utilis .................................................................................................36 2-7 Effect of E. utilis on Brazilian peppertree.........................................................................37 3-1 Results of laboratory no -choice tests performed with E. utilis ..........................................50 3-2 Potted BP plant covered by a clear acrylic cylinder..........................................................51 3-3 Materials used for irradiation of E. utilis moths................................................................52 3-4 Cesium-137 Gammacell 1000 irradiator (F.A.S.T. Gainesville, FL)...............................52 3-5 Waxed paper oviposition cham ber for mating and ovipositing by E. utilis moths............53 3-6 Fecundity (mean number of e ggs laid) per mated female of E. utilis adults for three crosses........................................................................................................................ ........54 3-7 Fertility (mean percentage of eggs that hatched) of E. utilis adults for three crosses treated with increasing dose of gamma radiation..............................................................55 3-8 Fecundity (mean number of eggs laid) of F1 crosses of E. utilis adults as a result of radiation administered to parental males...........................................................................56 3-9 Fertility (mean percentage of eggs that hatched) of F1 crosses of E. utilis adults as a result of radiation administ ered to parental males.............................................................57 3-10 Percentage of F1 E. utilis adult males as a result of radiation administered to E. utilis parental males................................................................................................................. ...58

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8 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science APPLICATION OF THE F1 STERILE INSECT TECHNIQUE (F1SIT) FOR FIELD HOST RANGE TESTING OF Episimus utilis ZIMMERMAN (LEPIDOPTERA: TORTRICIDAE), A CANDIDATE FOR BIOLOGICAL CONTROL OF BRAZILIAN PEPPERTREE By Onour Elizabeth Moeri December 2007 Chair: James P. Cuda Cochair: William A. Overholt Major: Entomology and Nematology Host range testing is used in weed biological control to demonstrate the safety of potential biological control agents. However, these labor atory tests may overestim ate host range leading to false positives where the insect accepts plan t species that it would not normally accept in nature. As cage testing may inhibit the behavior of the potential biological control agent, openfield studies may provide a more realistic setti ng of environmental and eco logical conditions that the biological control agent will encounter upon re lease in the proposed ar eas of introduction. Open-field studies, however, are prohibited in the area of intro duction. Reproductively inactivated potential biological control agents pr oduced as a result of th e application of the F1 sterile insect technique (F1SIT) could be used to conduct fiel d testing in a safe and temporary manner. A situation where this could be used is in the biological control of Brazilian peppertree, Schinus terebinthifolius Raddi (Anacardiaceae), an invasive, exotic species distributed widely throughout central and southern Fl orida. A leaf-rolling moth, Episimus utilis Zimmerman (Lepidoptera: Tortricidae) is a potential biolog ical control agent of Brazilian peppertree. Traditional laboratory no-choice and choice tests performed with E. utilis produced ambiguous results where selected non-target species including fragrant sumac ( Rhus aromatica L.), winged

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9 sumac ( Rhus copallinum L.), poison sumac ( Toxicodendron vernix (L.) Kuntze), pistachio ( Pistacia vera L.) and cashew ( Anacardium occidentale L.) were unexpected ly accepted as host species. Therefore, the use of F1SIT for field host range testi ng was investigated as a new approach for risk assessment of potential biolog ical control agents. Male and Female virgin E. utilis adults were treated with incr easing doses of radiation and e ither inbred or outcrossed to nontreated E. utilis adults. Five pairs of adults were pl aced in triangular waxed paper oviposition cages and allowed to mate and oviposit for two intervals of 5 days. The number of eggs laid (fecundity) and the number of eggs that hatched (fertility) were counted for each egg sheet per dose. As the dose of radiation increased, there were no significant change s in the fecundity of nontreated females mated with treated males, yet fewer eggs were laid by treated females. Fertility for both treated males and females decr eased with increasing dos es of radiation. The dose at which treated females were found to be 100% sterile was 200 Gy. There were no significant changes in the fecundity for F1 females and males resulting from treated parental males with increasing dose of radiation. Fertility for F1 females and males resulting from treated parental males declined as the dose of radiat ion increased. There was a moderate positive correlation for the F1 sex ratio of males to females with in creasing dose of radiation. The dose at which F1 females and males were found to be 100% st erile was 225 Gy. Results from this study were similar to results found in other to rtricid moths including the codling moth, Cydia pomonella (L.), and the false codling moth, Thaumatotibia leucotreta (Meyrick). As the dose of radiation increased, there was an increase in ster ility, a decrease in f ecundity for both treated female crosses, and a higher ratio of F1 males to females. This novel approach could be used to safely and temporarily test potential biological control agents in the field.

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10 CHAPTER 1 INTRODUCTION Host-specificity tests are used in weed biological control to determine whether or not a potential biological control candida te is safe to release in the field. Potential candidates are routinely subjected to caged labor atory tests that are either no-c hoice (subjected to non-target plants only) or choice (subjected to host plant and selected n on-target plants) (Marohasy 1998; Withers et al. 1999). The laboratory physiological host range is measured by larval development (i.e. survival, length of time to complete develo pment) and adult reproduction, which are used to predict the field ecological host range (Julien and White 1997). Some biologists believe that host-specificity tests often overe stimate host range, which leads to the rejection of acceptable candidates (Sands and Van Driesc he 2000). For example, in no-c hoice starvation tests (entire lifetime of an insect) the range of plant species on which larvae can complete their development is often broader than the range of species that females accept for oviposition (Schoonhoven et al. 1998). A cage environment where oviposition test s take place also may yield false-positive results because it creates a restri ctive environment where the pre-alighting cues of a female such as visual and olfactory cues may be inhibited cau sing an increase in the number of test plants accepted leading to a false pos itive result (Marohasy 1998). Because cage testing may inhibit normal be havior, open-field studies can provide a more realistic setting in which insects can display an array of behaviors (Clement and Cristofaro 1995). However, field testing can pose envir onmental risks in the geographical area of introduction and is therefore prohibited. Although some field testing can o ccur in the country of origin of the weed (Zwlfer and Harris 1971), a ll test plant species in the area of introduction may not be represented in the natural enemys native range. More importantly, it may not be

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11 possible to import all the required test plants into the countries of origin of the target weed because of quarantine restrictions. The use of reproductively inactivated or limited biological control agents may be a possible solution to field testing in proposed areas of introduction. According to LaChance (1985), there are three genetic methods of contro l that have been developed and field tested against Lepidoptera. These include the sterile insect tec hnique (SIT), the F1 sterile insect technique (F1SIT), and backcross ster ility. Both SIT and F1SIT involve the release of insects that have been genetically altered by radiation, provi ding either full or part ial sterility, and will introduce sterility into the wild population. Backcross sterilit y is different from SIT and F1SIT because there is no use of radiation and it introdu ces sterility factors into the population that can persist indefinitely because of production of multiple generations (LaChance 1985). Both backcross sterility and F1SIT may offer a better level of suppression because the suppressive effects may persist for one or more generations (LaChance 1985). Through the application of F1 sterility, however, candidate insect weed biological control agents could be safely released for field host range testing because they will produce only a single generation of insects, which are sterile. Advantages of this approach include exposure of the potential biological control candidate to the actual environm ental conditions it would experien ce post-release, prediction of true field host range in the area of introduction, and the ability to suspend releases of irradiated insects if non-target species are being attacked, without risk of permanent establishment (Carpenter et al. 2001a). This novel approach co uld possibly be applied to exotic lepidopterans that are potential weed biological control agents for field hos t range testing (Dunn 1978; Cullen 1990; Greany and Carpenter 2000). This would a llow the insect to be observed under actual

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12 conditions that it would be subjected to upon release and obs ervation of insect behavior including oviposition, ho st-finding, and larval feeding prefer ences (Carpenter et al. 2001a). An example of a situation where F1SIT could be used for field host range testing is in the biological control of Brazilian peppertree Schinus terebinthifolius Raddi (Anacardiaceae), a dioecious evergreen tree native to Brazil, Paraguay, and Argen tina that was introduced into Florida in 1898 as an ornamental (Austin 1978 ; Ewel et al. 1982). Currently, Brazilian peppertree is distributed widely throughout central and southern Florida, and is listed as a prohibited plant by the Florida Department of Environmental Protecti on (FLDEP 1993), as a noxious weed by the Florida Department of Agri culture and Consumer Services (FLDACS 1999) and as a Category 1 invasive exotic species by the Florida Exotic Pest Plant Council (FLEPPC 2007) because it is drastically altering native plan t communities. It also has exhibited invasive behavior in California (Randa ll 2000), Hawaii (Hight et al. 2002), and Texas (Gonzalez and Christoffersen 2006) as well as in subtropical regions of at leas t 20 different countries (Ewel et al. 1982). In 1994, several natural enemies of Brazilian peppertree were imported into a quarantine facility in Florida as candidate s for classical biological control (Cuda et al. 1999). One of these was a South American leaf-rolling moth, Episimus utilis Zimmerman (Lepidoptera: Tortricidae). Larvae of E. utilis feed by scraping the surface of the Br azilian peppertree leaflets. As they mature, the developing larvae are capable of co mpletely defoliating the plant (Martin et al. 2004). Laboratory host range testing of E. utilis produced ambiguous results; non-target plant species were unexpectedly accepted as developmen tal hosts in both no-choice and choice tests (J.P. Cuda unpubl. data). The behavior of the insects may have been inhibited by the caged laboratory environment, producing false positive results where the insects accept plants that

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13 they would not normally accept in a natural envi ronment (Withers et al. 1999). Therefore, we believe that the broad physio logical host range exhibited by E. utilis is not indicative of the behavior in a natural environmen t (ecological host range). For these cases where laboratory tests do not produce definitive results, F1SIT could be an additional t ool to complement laboratory host specificity testing. The F1 sterile insect technique is an approach that involves using radiation to partially sterilize male insects, resulting in the production of sterile progeny. Some of the effects of F1SIT include reduced egg hatch and a si ngle generation of offspring that are highly sterile and mostly male (North 1975). Compared to SIT, F1SIT uses a lower dose of radi ation resulting in increased quality and competitiveness of the moths (North 1975). Studies also have shown that using partially sterilizing doses of radiat ion may be more effective than using fully sterilizing doses of radiation to suppress pest populations (Toba et al. 1972; Proverbs et al. 1978). In order to use F1SIT for field host range testing of pot ential biological co ntrol agents, the dose of radiation used to provide sterility in the F1 generation must be determined along with the effects of radiation on the F1 generation. The performance of the irradiated insects also must be comparable to the non-irradiated insects. Th erefore oviposition rate, survival rates for F1 larvae, larval development, and host-finding behavior need to be examined in order for field host range testing to be complimentary to la boratory testing in terms of insect competence. The objective of the current study was to determine the dose of radiation that would ensure full sterility in the F1 generation and verify the effects of F1 sterility in E. utilis

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14 CHAPTER 2 LITERATURE REVIEW The F1 Sterile Insect Technique (F1SIT) Description The F1 Sterile Insect Technique (F1SIT) is an autocidal method of pest control whereby a minimum dose of radiation is applied to male ins ects causing the insects to be partially sterile. Upon mating with nontreated females, there is a reduction of progeny in the filial or F1 generation. Alternative terms for F1SIT include inherited sterilit y, inherited partial sterility, partial sterility, semi-sterili ty, delayed sterility, and F1 sterility (LaChance 1985; Carpenter et al. 2005). In the case of Lepidoptera, chromosomes are considered to be holokinetic (no primary site of microtubule attachment) due to a lack of centromeres which resu lt in sister chromatids separating by parallel disjunction during mitotic metaphase. Each chromosome also has a large, localized kinetochore plate (centromere) wher e the spindle microtubules attach during cell division. The plate covers most of the chromosome ensuring that a majority of radiation-induced breaks will not lead to a loss of chromosome fragments. Due to re duced fragment loss, there is a reduction in the amount of sterility in the parental generation. The F1 generation, however, inherits a greater degree of sterility as a re sult of rearrangement of chromosomes and production of genetically unbalanced gamete s (LaChance et al. 1970; Squire 1973; Carpenter et al. 2005). History The idea of sterilizing insect s was conceived in the early 20th century by three researchers working independentl y. These scientists (A.S. Sere brovskii, F.L. Vanderplank and E. F. Knipling) each played a role in developi ng the sterile insect technique (SIT) (Klassen and Curtis 2005). Through their efforts, SIT would b ecome an important tool in pest management strategies.

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15 The first major success using SIT in an inse ct eradication program occurred in the late 1950s against New World screwworm Cochliomyia hominivorax (Coquerel), a parasite of warmblooded animals that significantly affects lives tock health and producti on (Wyss 2000; Klassen and Curtis 2005). The goal of the program was to release sterile male flies to mate with wild females for the purpose of reducing screwworm popul ations. The first successful release was in 1951 on Sanibel Island, Florida. Sterile male flies were released from airplanes as part of a release-recapture experiment that resulted in the reduction of w ild populations of the New World screwworm on Sanibel Island, but not complete eradi cation. Later releases of the sterilized flies on the island of Curacao, however resulted in complete eradication of the flies (Klassen and Curtis 2005). In 1958, an eradication program began in Florida with airplane releases of up to 1,160 flies per km2 per week at certain hot spots (Kla ssen and Curtis 2005). The releases ended in 1959 with complete eradication of the flies (Klassen and Curtis 2005). The successful outcome of the pilot program resulted in a global campaign that led to the eradication of the New World screwworm in the USA, Mexico, Belize, Guatemala, Honduras, El Salvador, Nicaragua, Costa Rica, Panama north of the canal, some Cari bbean islands, and Libya (Vargas-Teran et al. 2005). Additionally, there are many other examples of successful programs using sterile insects to control pest populations in cluding the codling moth ( Cydia pomonella (L.)), fruit flies including the Caribbean fruit fly ( Anastrepha suspense (Loew)); Medite rranean fruit fly ( Ceratitis capitata (Wiedemann)); melon fly ( Bactrocera cucurbitae (Coquillett)); Mexican fruit fly ( Anastrepha ludens (Loew)); oriental fruit fly ( Bactrocera dorsalis Hendel); Queensland fruit fly ( Bactrocera tryoni (Froggatt)); West Indian fruit fly ( Anastrepha obliqua (Macquart)), the onion maggot ( Delia antiqua (Meigen)), pink bollworm ( Pectinophora gossypiella (Saunders)), and several species of tsetse flies ( Glossina spp.) (Klassen and Curtis 2005).

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16 Although SIT has been used to control lepidopt eran pests, the quality and competitiveness of the released insects is reduced due to the hi gh doses of radiation required to achieve complete sterility. Lepidoptera are highly radioresistant and therefore re quire high doses of radiation (LaChance et al. 1985). To solve this problem, pa rtial sterility was invest igated (Proverbs 1962). Instead of a dose of radiation th at would completely sterilize ma les, a lower dose of radiation could be applied, resulting in pa rtially sterile males that when outcrossed to fertile females produce a single generation of offspring. This ge neration is characterized by reduced egg hatch. In their study of radiation-induced gene tic anomalies in the silkworm moth, ( Bombyx mori (L.)) Astaurov and Frolova (1935), first reported inherited sterility in the mid-1930s in the Soviet Union (Carpenter et al. 2005). Soon after this first report of inherited st erility, OstriakovaVarshaver (1937) confirmed inherited sterility in the greater wax moth, Galleria melonella (L.) (Carpenter et al. 2005). Almost 30 years later, Proverbs (1962) demonstrated the effects of partial sterility in the codling moth in Canada. The significant findings of these early studies led to further research on partial st erility and its use in pest suppr ession (Astaurov and Frolova 1935; Ostriakova-Varshaver 1937; Proverbs 1962). Field Application of F1SIT As with the use of SIT, the F1SIT approach also is environmentally friendly because it, too, minimizes the use of insecticides to suppress pests. This is achieved by introducing sterility into the wild populations thro ugh the release of partially sterile males and fully sterile females. F1SIT is particularly useful for controlling many ec onomically important spec ies of Lepidoptera. Multiple studies involving inher ited sterility and pest lepidopte ran species have been conducted, including those on fall armyworm ( Spodoptera frugiperda (J.E. Smith)) (Carpenter et al. 1985,1997), corn earworm ( Helicoverpa zea (Boddie)) (Carpenter et al. 1986, 1987, 1989), cabbage looper ( Trichoplusia ni (Hubner)) (North and Holt 1969), Mediterranean flour moth

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17 ( Ephestia kuehniella (Zeller)) (Ayvaz and Tunbilek 2006), potato tuberworm ( Phthorimaea operculella (Zeller)) (Makee and Saour 1997), codling moth (Bloem et al. 1999a,b), false coding moth ( Thaumatotibia leucotreta (Meyrick)) (Bloem et al. 2003; Ho fmeyr et al. 2005), and cactus moth ( Cactoblastis cactorum (Berg)) (Carpenter et al. 2001a,b). Each of these studies produced similar results including an F1 generation that was more sterile than the parental generation yet was competitive with the native population. This s uggested greater potential for pest suppression compared with using a fully sterilizing dos e of radiation (North 1975; LaChance 1985). The codling moth is a serious pest of appl es and pears that developed resistance to the insecticides once used to c ontrol it. Autocidal control methods such as SIT and F1SIT have now been developed and incorporated into an area-wide integrated ma nagement plan to control this pest (Bloem et al. 2005). Prove rbs (1962) believed that control of the codling moth could be achieved by releasing sterile male moths. Fiel d tests were conducted in Canada, the United States, Europe, the former Soviet Union, and Switzerland, confirming the effectiveness of the SIT method (Proverbs 1982; LaChance 1985). Because the release of sterile males in the SIT program had been so effective, Knipling (1970) proposed that the release of partially sterile codling moths may complement the sterile releases thereby achieving a greater level of success. Studies by Proverbs (1971) and Proverbs et al. (1 973) compared the resu lts of releasing fully sterile codling moths with those of partially sterile moths in laboratory and field cage studies conducted in Canada. They found that partially st erile moths were more competitive in terms of sperm quality and suppression of reproduction. A lthough the results of partial sterility were promising, potential damage from F1 larvae on the fruit was consider ed to be too much of a risk (Proverbs et al. 1978). Bloem et al. (1999a,b) found that releasi ng partially sterile male moths resulted in a higher level of comp etitiveness than in males treated w ith higher doses of radiation.

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18 Despite concerns about economic damage by F1 larvae, it was conclude d that suppression would occur more quickly with F1 males mating with wild females in addition to continued releases of partially sterile male moths (Bloem et al. 1999a). Another example of the utility of the F1SIT approach in terms of field application is the suppression of pest introductions. For example, the cactus moth, Cactoblastis cactorum (Berg), which is native to South America, was released in Australia as a biolog ical control agent of several invasive species of Opuntia cacti (Zimmerman et al. 2000). The moth was so successful in controlling the invasive Opuntia species that it was later released in Africa, New Caledonia, Hawaii, Mauritius, the Caribbean Islands, the Cayman Islands, St Helena, Ascension Island, and Pakistan (Zimmerman et al. 2000). In 1989, the cactus moth arrived in Florida following the release of the insect in the Caribbean for biological control of several native Opuntia species (Habeck and Bennett 1990). The moth currently threatens native Opuntia species throughout the southern United States and Mexico (Zimmerman et al. 2000; Hight et al. 2005) It was suggested by Carpenter et al. (2001a,b) that F1SIT could be used to control the spread of the cactus moth. The effects of F1 sterility on fecundity, fertility, a nd competitiveness in the cactus moth demonstrated that 200 Gy would be the optimum dose of radiation to en sure survival and F1 sterility (Carpenter et al. 2001a,b) Field studies also have been conducted to determine mating behavior and overflooding ratios (Hi ght et al. 2003, 2005). Overall, F1SIT has been shown to be an effective method for controlling a variety of lepidopteran pests. Because this technique has been shown to predictably control reproducti on in Lepidoptera, a nove l application of F1SIT could be used to safely test potenti al weed biological control candidates in the field for risk assessment if results of traditional laboratory host range testing were ambiguous (Bloem and Carpenter 2001; Carpenter et al. 2001a,b; Tate et al. 2007).

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19 Host Range Testing Protocols One of the key steps in weed biological control programs is the evaluation of the host range of candidate biological c ontrol agents of invasive, exotic plants. Promising natural enemies (typically insects) are identified and fu rther studied in their native ranges to determine their potential for contro lling an invasive, exotic plant. Cand idate biological control agents are typically subjected to rigorous caged laborator y trials involving no-c hoice and choice tests (Marohasy 1998; Withers et al. 1999). The host range (or ho st specificity) of an insect is used to evaluate any risks to potential nontarget species in the proposed area of introduction (Littlefield and Buckingham 2004). Therefore, a wide range of more than 50 potential host plant species from those most closely related to more distan tly related families are tested, with emphasis on native, endangered, and economically importa nt species (Wapshere 1974; McEvoy 1996; Schaffner 2001). These tests invol ve exposing the potential host plant species to the larval and/or adult stages of the insect (Schaffner 2001). Results are used to determine whether the potential candidate is safe to release. No-Choice Tests Initially, a potential biological control agent (neonate and nave adult) is exposed to a series of test plant species and the target sp ecies separately (Maroha sy 1998; Sands and Van Driesche 1999). The purpose of the no-choice or starvation tests is to determine whether the insect is restricted to a single host plant species (Schaffner 2001) The results of the no-choice test identify the range of plant species that can support complete insect development and reproduction in the absence of the ta rget species. A starvation test is the most conservative type of host range test because it will invoke more of a response from the insect compared with choice tests because of the starved state of the insect (McEvoy 1996).

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20 One of the problems with testing taxonomically or chemically-related plants in no-choice tests is that many of these sp ecies will support some developm ent in a confined laboratory setting. A Canadian study reveal ed that only 4% of Canadian biological control agents which fed on non-target plants in no-choi ce laboratory tests actually fe d on these plants in the field (Harris 2004). In most cases, the range of pl ants determined by the no-choice test was 70% broader than the field host range (Harris 2004). Select ion in laboratory no-c hoice tests is based primarily on larval development, whereas in fi eld host-range tests, the focus is more on the behavior of the a dult (Harris 2004). Choice Tests When test plant species are accepted in a la boratory no-choice test, these species need to be further tested to rule out a ny risk of attack in the field (H arris 2004). Choice tests can be designed to include test plants eith er with or without the target pl ant and involve mobile stages of the potential biological control agent, usually the adult stage (Schaffner 2001). The main objective is to retest plant sp ecies on which the insect fed, ovi posited, or completed development in the no-choice tests (Schaffner 2001). In a ch oice test, it is important to demonstrate which plants the biological control agent will select when given multiple choices, including the host plant, as this is more representative of th e natural environment where the insect will be introduced (Cullen 1990; Littlefi eld and Buckingham 2004). Alt hough related species may have been attacked, if there is greater attack on the host plant, this may indicate speci fic behavior of the biological control agent (Harris 1964). Open Field Testing Results from the laboratory host specificit y tests are used to determine whether a candidate biological control agent is safe to release in the field. The main point of these tests is to identify the laboratory (or physiological) host range whic h is then used to estimate or

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21 predict the field (or ecological) host range of the candidate biol ogical control agent (Julien and White 1997). Some biologists be lieve that these tests overes timate the true host range, consequently leading to the rejection of otherwise acceptabl e biological control candidates (Dunn 1978; Marohasy 1998; Cullen 1990). One su ch example involves no-choice starvation tests (entire lifetime of an insect) where the rang e of plant species on which the immature stages of a candidate biological control agent completes its development is often broader than the range of plant species a female biological control agent will accept for oviposition (Schoonhoven et al. 1998; Schaffner 2001). Likewise, a cage environment for testing the females oviposition behavior also may yield false-pos itive results when a female accep ts a test plant that would not normally be selected in the field (Marohasy 1998). Therefore laboratory host specificity tests by their nature often produce am biguous results (Dunn 1978; Culle n 1990; Marohasy 1998). Because cage testing may inhibit normal behavi or, open-field studies can provide a more realistic setting where insects can display an a rray of behaviors (Cleme nt and Cristofaro 1995; Littlefield and Buckingham 2004). Fi eld tests can serve as another tool to supplement laboratory tests by focusing on host finding a nd oviposition behavior of the candidate biological control agent when exposed to an array of potential hos t plants. The goal of the field tests is to reexamine non-target hosts in a mo re natural setting that were a ttacked in laboratory tests. Among the first researchers to use open-field testing were A ndrs and Angalet (1963) in Italy. They examined the safety of the weevil, Microlarinus lypriformis (Wollaston) as a proposed biological control agent of puncturevine ( Tribulus terrestris L.). The importanc e of a free-choice environment without the restri ction of a laboratory cage was proposed by Dunn (1978). He suggested using partially opened fi eld cages to allow insects to l eave if they came into contact with an unacceptable plant (Dunn 1978). Other suggestions by Dunn (1978) and later by Cullen

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22 (1990) were to release sterile insects in the area of introducti on or only unmated females or males, to exterminate insects after use, or to rely on natural die off of the insects. However, these types of studies are prohibited in the area of introduction due to federal and state regulatory restrictions. Th erefore, field tests can be done only in the country of origin or native range of the insect. The main difficulty with field testing in the count ry of origin or native range of the insect is that it may not be possibl e to export critical test plant species from the proposed release area to the country of origin fo r testing. Other problem s associated with open field testing include availability of insects in the native range where they are subjected to parasitism or predation and discrimination of dama ge associated with other insect species in the area. Despite these limitations, at least 19 specie s of insects and one mite species have been released based on incorporation of open field te sts in the insects na tive region (Clement and Cristofaro 1995, Bredow et al. 2007, Gandolfo et al. 2007). The use of laboratory and field hostrange testing may not be required for all candidate biological cont rol agents, but for some that are in fact safe and actually pose very little ri sk, it could mean the difference between being accepted or rejected by regulatory agencies. Schinus terebinthifolius Raddi Taxonomy Brazilian peppertree is a member of the Fam ily Anacardiaceae. The family consists of 60 to 80 genera worldwide comprising around 600 sp ecies (Cronquist 1981). Most species are tropical and include trees, shr ubs, and woody vines. Some of the well-known genera include mango ( Mangifera ), sumac ( Rhus ), pistachio ( Pistacia ), cashew ( Anacardium ), and poison ivy ( Toxicodendron ) (Cronquist 1981). The genus Schinus includes 29 species and is native to parts of South America including Argentina, southern Brazil, Uruguay, Paraguay, Chile, Bolivia, and Peru (Barkley 1944, 1957).

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23 Common Names Brazilian peppertree has many different common names as a result of its spread around the world. In Hawaii, it is known as wilelaiki and nani-o-hilo Christmas berry is also a common name in Hawaii as well as in Guam. False pepper or faux poivrier are common names in the French Riviera whereas warui is used in Fiji. In nati ve Brazil and Argentina, common names include aroeira, aroeira negra, aroeira vermelha, aroeira de Minas, aroeira de praia, corneiba and chichita (Argentina). In Cuba, Brazi lian peppertree is known as copal whereas in Puerto Rico it is known as pimienta de Brasil Within the United States, names include Brazilian pepper, pink pepper and peppertree (Morton 1978, Cuda et. al 2006). Description Brazilian peppertree is a large evergreen shr ub or small tree that can grow up to 7.5 m in height and has compound leaves with 3-11 (usua lly 7-9) leaflets that produce a peppery or turpentine-like smell when crushed (Fig 2-1). The flowers are small, white, unisexual and found in short-branched clusters. The fruits appear in bunches as glossy green drupes ripening to a bright red color from October to December in the northern hemisphere. The bright red berries and shiny green leaves contribute to the popular ity of Brazilian peppertree as an ornamental during the holiday season (Fig 2-2) (Morton 1978; Ewel et al. 1982). Distribution Brazilian peppertree is native to Brazil, Para guay, and Argentina (Fig 2-3). The mid to late 1800s marked the beginning of the distribution of Brazilian peppertree throughout the world as an ornamental (Barkley 1944, 1957). Brazilian pe ppertree is considered an invasive weed in the subtropical regions of at least 20 different countries (Ewe l et. al 1982), including Bermuda, Bahamas, Australia, American Sa moa, Fiji, Island of Mauritius, Micronesia, New Caledonia, Reunion Island, South Africa, and Tahiti (Habeck et. al 1994; USDA NRCS 2007) (Fig 2-4).

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24 Brazilian peppertree has also been introduced in to other South American countries, parts of Central America, Mediterranean Europe, North Africa, and southern Asia (Morton 1978). Because of its attractive green leaves and re d berries, Brazilian peppertree is a favorite ornamental in Florida, Texas, Hawaii, Arizona, and California as well as parts of the Caribbean including the Bahamas, Commonwea lth of Puerto Rico and US Virgin Islands (Habeck et. al 1994; USDA NRCS 2007). However, Brazilian peppe rtree is now considered an invasive, exotic weed in Florida, Texas, California, and Hawa ii because it is altering native plant communities by displacing native species and changing comm unity structure (Hosaka and Thistle 1954; Yoshioka and Markin 1991; Randall 2000; Hight et al. 2003; FLEPPC 2007). Currently, Brazilian peppertree is distributed widely throughout central and s outhern Florida. According to Wunderlin and Hansen (2004), voucher specimens ha ve been received by statewide herbaria from 34 different Florida counties (Fig 2-5). Environmental Impacts Ecosystem Brazilian peppertree was not popul ar when first introduced into Florida as an ornamental in 1898, but eventually was commonly cultivat ed, and ultimately invaded natural areas (Alexander and Crook 1974; Austin 1978). Invasion occurs in dist urbed as well as undisturbed areas, including hammocks, pinelands, mangrove forests, canal banks, roadsides, and abandoned pastures where Brazilian peppertree produces de nse monospecific stands (Loope and Dunevitz 1981; Ewel et. al 1982; Bennett et al 1990). Seed dispersal of Brazilian peppertree occurs by wildlife; raccoons ( Procyon lotor L.) and opossums ( Didelphus virginianus Kerr) aid in local dispersal whereas frugivorous birds such as robins ( Turdus migratorius L.) are responsible for long-distance dispersal (Ewel et al 1982; Panetta and McKee 1997). Because of dispersal by wildlife along with its tolerance of extreme moisture conditions and ability to grow in shady

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25 environments, Brazilian peppertree ra pidly became a serious threat to the biodiversity of Florida (Ewel 1979). By the early 1990s, th e southern and central portions of the state were heavily infested, comprising an estimated > 280,000 ha (1 million acres) of Brazilian peppertree (Habeck 1995). Based on recent estimates from aerial surveys of Florida, approximately 300,000 ha of terrestrial ecosystems were infe sted as of 1997 (Ferriter 1997). Human and Animal Health Sap and volatiles produced by flowers of Braz ilian peppertree can cause allergic reactions in humans (Ewel et al. 1982). Allergenic properties of the plant are due in part to the fact that Brazilian peppertree is closely related to the more familiar poison ivy ( Toxicodendron radicans L. Kuntz), poisonwood ( Metopium toxiferum L. Krug), and poison oak ( Toxicodendron toxicarium Salib. Gillis) (Ewel et al. 1982). Reactions to Br azilian peppertree include dermatitis, eye inflammation, facial swelling, severe itchi ng, rash, respiratory ir ritation, sneezing, sinus congestion, and headaches (Morton 1969, 1978). Se verity of the symptoms depends on the individual. Animals such as horses (Equus caballus L.) and cattle ( Bos taurus L.) also are susceptible to allergenic propertie s of Brazilian peppertree. Reactions of the animals to Brazilian peppertree may include dermatitis, fatal colic, eye swelling, and enteritis. The fruit of Brazilian peppertree also has been found to produce a narco tic or toxic effect on birds (Morton 1978). Beneficial Uses In Brazil, Brazilian peppertree has various ec onomic uses. For instance, the wood is used for construction. The bark produces a resinous extr act, which is used to preserve fishing lines and nets. Brazilian peppertree also has some medicinal value used in homeopathy as a remedy for gout, muscular atony, pain associated with ar thritis, strain of tendons intestinal weakness, inertia of the reproductive organs, skin complaints, chills, tumors lymphatic swellings, diarrhea, and hemoptysis (Morton 1978). Brazilian peppertr ee serves as an important nectar source for

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26 commercial honey bees in both Florida and Hawa ii (Sanford 1988; Yoshioka and Markin 1991). According to Sanford (1978), Brazilian peppertree is one of Floridas best nectar producing plants resulting in honey that has a peppery taste and is popular locally. The fruit of Brazilian peppertree also is used as a spice and sold in gourmet stores in a dried form in addition to decorative uses in wreaths made in Hawaii (Mor ton 1978; Yoshioka and Markin 1991; Habeck et al. 1994). Controlling Brazilian Peppertree Mechanical Control Hand pulling seedlings and small plants is feas ible until the plant reaches several feet in height. It is extremely important to remove the root system so that resprouting will not occur. Larger plants require the use of bulldozers, fr ont-end loaders, root rake s and other specialized heavy equipment (Langeland 1990). Removal may be successful when used in combination with other control methods such as physical or chemi cal control. If mechanical methods are used alone, soil disturbance often can lead to plant re-growth (Langeland 1990). Physical Control The use of fire, soil removal, and flooding ar e physical tactics that may be used to stress plants. This stress can leave the plant in a weak ened condition possibly le ading to die-off. The use of fire has had mixed results and may prove to be a liability in some areas due to property damage and human injury (Langeland 1990; Randa ll 2000). Soil removal is costly and labor intensive. Because established Brazilian peppe rtree plants can withstand extended hydroperiods (Langeland 1990; Ewe 2004) flooding may interfere w ith water conservation efforts. Therefore in order to be effective, these techniques shoul d be used in combination with other methods of control.

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27 Chemical Control Currently, the most widely used method fo r managing Brazilian peppertree is chemical control. According to Ewel et al. (1982), several herbicides have been shown to be effective for controlling Brazilian peppertree. In their study, the effectiveness of five different herbicides was tested: a dicamba + 2,4-D combination, gra nular dicamba, an isopropylamine salt of glyphosphate, a triazine compound, a nd an ester formulation of triclopyr. The triazine compound proved to be the most effective and longest lasting herbicide. However, most of the non-target vegetation in the area of application also was killed. The most effective (and labor intensive) control with limite d non-target effects was achieved by using a low dose of triclopyr (basal application) (Ewel et al 1982). A number of herbicides are currently recommended to control Brazilian peppertree in Florida (imazapyr, triclopyr ester and amine, dicamba, hexazinone, and tebuthiuron). Thes e herbicides are applied specifi cally to the cut stump, basal bark, and foliage of the plant (Langeland 1990; Gioeli and Langeland 1997). Although herbicides are commonly used, they can remain in the soil and pose unwanted environmental effects. Cost also is an issue as are the unknow n effects of abiotic factors such as rain, wind, temperature, soil chemistry, and water chemistry wh ich can inhibit the efficacy of the herbicides (Langeland 1990). Biological Control A sustainable and environmentally friendly solution to control Brazilian peppertree is classical biological control. Th is process involves the introduction of natural enemies from the native range of the plant. Potential candidate s must undergo a rigorous screening process to determine their host specificity. The goal is to permanently establish host-specific biological control agents that will reduce the competitivenes s of the weed (Cuda et al. 1999; Scoles et al. 2005; Cuda et al. 2006). Results of exploratory surveys conducte d in Brazil identified several

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28 potential biological control agents of Brazili an peppertree. In the 1990s, three candidate biological control agents from Brazil were in troduced under permit into an approved Florida containment facility (FBCL, Gainesville, FL). The three Brazilian peppertree natural enemies included a thrips Pseudophilothrips ichini Hood (Thysanoptera: Phl aeothripidae), a sawfly Heteroperreyia hubrichi Malaise (Hymenoptera: Pergid ae), and a leafrolling moth Episimus utilis Zimmerman (Lepidoptera: Tortricidae) (Cuda et al. 1999). To date, host-range testing has been completed for the thrips as well as the sawf ly and is in the final stages for the leafroller. Episimus utilis Zimmerman Taxonomy The larvae of the genus Episimus are commonly known as leafro llers or leafbud feeders. There are 32 New World species of Episimus found predominantly in tr opical regions (Heppner 1994). Unfortunately, little is known about the bi ology of many of the species. There are nine species of Episimus in the United States (with all nine found also in Florida) ranging from northern Mexico to southern Canada. Many of th e species are similar in appearance with minor differences in maculation and geni talia (Zimmerman 1978; Heppner 1994). Biology Episimus utilis was first described by Zimmerman (1978) who examined specimens collected in Hawaii where it was introduced in the 1950s (Krauss 1963) and in neotropical Brazil, which is part of its native range (Heppner 1994, Martin et al. 2004). Upon investigating the biology of E. utilis in a containment laboratory in Gainesv ille, FL, the entire life cycle (Fig 26) was found to be approximately 42 days, with multiple generations produced throughout the year (Martin et al. 2004). The duration of the larval stage also was found to be 24 days and included five instars (Martin et al 2004). Four of the instars ar e pale green in color followed by a bright red 5th instar. Larvae range in size from 1.5 mm to 15 mm (Zimmerman 1978; Martin

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29 et. al 2004). One of the diagnostic ch aracteristics of th e larval stage of E. utilis is a dark lateral bar on the head capsule (Zimmerman 1978). Th e larvae feed by scrapi ng the surfaces of Brazilian peppertree leafle ts (Fig 2-7). Early instars web t ogether adjacent leaf lets, while later instars usually roll a single leaflet to form a sh elter in which to conti nue feeding (Yoshioka and Markin 1991). As the feeding process continue s through larval development, the plant can be completely defoliated, resulting in injury or death of young plants and preventing reproduction of older plants (Cuda et al. 1999, 2006) Simulated herbivory studies have shown that growth and reproduction of Brazilian peppertr ee are inhibited when the plan t is subjected to multiple defoliation events (Treadwell and Cuda 2007). Pupae are brown in color and average 8 mm in length (Martin et al. 2004). During this 12day stage, males and females can be separate d by the number of abdominal segments. The female pupae have three movable segments with f our fused segments in the terminal portion of the abdomen, whereas the male pupae have four m ovable segments and the apex with only three fused segments. The genital pore in female pupae al so serves as a diagnostic characteristic for separating the sexes as it is s trongly drawn headward (van der Geest 1991). Adults are small grayish brown moths with distin ctive markings on the forewings (Zimmerman 1978; Martin et al. 2004). The adults also exhibit sexual dimor phism; the pale area on the male forewing can be used to separate the male from the female (Zim merman 1978). The adults live an average of 7 days with females laying an average of 34.0 5.1 eggs. The in cubation period for the egg stage averaged 6 days (Martin et al. 2004). History of Introduction of E. utilis Measures to control Brazilian peppertree biologically in Hawaii were undertaken by the release of three different bi ological control agents in 1954 (Krauss 1963; Bennett et al. 1990; Habeck et al. 1994). However, only two of the th ree agents released actua lly established, one of

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30 these was the moth E. utlis. Despite becoming widely distribu ted on the islands of Kauai, Oahu, Molokai, Maui, and Hawaii, E. utilis was not found to be sufficiently abundant to destroy a significant amount of foliage (Zimmerman 1978; Yoshioka and Markin 1991; Julien and Griffiths 1998). One of the reasons why E. utilis was unable to attain high population densities was due to biotic interference fr om non-specific parasitoids releas ed in the early 1900s to control sugarcane pests, specifically leafrollers of the genus Hedylepta (= Omiodes ) Meyrick (Swezey 1907; Krauss 1963; Yoshioka and Markin 1991; Martin et. al 2004). However, E. utilis is still widely distributed on Brazilian pe ppertree throughout the islands.

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31 Figure 2-1. Dense stands of Schinus terebinthifolius in Florida (Photo by Vic Ramey, University of Florida/IFAS Center for Aquatic and I nvasive Plants. Used with Permission).

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32 Figure 2-2. Morphology of Schinus terebinthifolius (Photo by Ann Murray, University of Florida/IFAS Center for Aquatic and I nvasive Plants. Used with Permission).

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33 Figure 2-3. Native distribution of Brazilian peppertree in South Am erica by country (Cuda et al. 2006).

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34 Figure 2-4. Worldwide distri bution of Brazilian peppert ree (Cuda et al. 2006).

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35 Figure 2-5. Distribution of Br azilian peppertree in Florida (Wunderlin and Hansen 2004).

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36 Figure 2-6 A-F. Life cycle of E. utilis ; A) Egg. B) Young larva. C) Mature larva. D) Pupa. E) Adult male moth. F) Adult female moth. (Photo credit: Sean McCann).

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37 Figure 2-7. Effect of E. utilis on Brazilian peppertree, A) Larv ae feeding by scraping the surface of a BP leaflet, B) Larval damage of a BP plant (right) followed by complete defoliation (left) (Photo credit: Ve ronica Manrique and J. P. Cuda).

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38 CHAPTER 3 USE OF THE F1 STERILE INSECT TECHNIQUE (F1SIT) AS A TOOL FOR FIELD HOST RANGE TESTING OF Epismus utilis ZIMMERMAN (LEPIDOPTERA: TORTRICIDAE), A CANDIDATE BIOLOGICAL CONTROL AGENT OF BRAZILIAN PEPPERTREE Introduction Throughout history, humans have sought ways to better manage pests affecting our plants, animals, and health. In the ear ly 20th century, three scientis ts (A.S. Serebrovskii, F.L. Vanderplank, and E.F. Knipling) independently estab lished the basis of what would become known as the sterile insect tec hnique (SIT) (Klassen and Curtis 2005). The idea was to release irradiated and, hence, sterile male insects to ma te with wild females, which would lead to a reduction in pest populations and their damaging effects on crops and liv estock (Klassen and Curtis 2005). Although a high dose of radiation is needed to provi de complete sterility, the F1 sterile insect technique (F1SIT) uses a lower dose of radiatio n, providing partial sterility and a reduced number of progeny. Lepidoptera are very radioresistant and re quire a large dose of radiation to ensure sterility (L aChance 1985). With the use of F1SIT, however, a lower dose could be applied resulting in a more competitive insect (North 1975). Various studies have used this technique to demonstrate control of populati ons of pest Lepidoptera, including the codling moth, Cydia pomonella (L.) (Bloem et al. 1999 a,b), false codling moth Thaumatotibia leucotreta (Meyrick) (Bloem et al. 2003), and cactus moth Cactoblastis cactorum (Berg) (Carpenter et al. 2001a,b). Because application of F1SIT has been widely studied in pest management programs, this well known approach has potential for evalua ting the risks of releas ing exotic lepidopteran candidates for weed biological control (Dunn 1978; Cullen 1990; Greany and Carpenter 2000). Before a candidate weed biologi cal control agent can be releas ed into the environment, the safety of the organism must be demonstrated. Host range testing is a process of screening potential biological control agents to minimize the risk of damage to non-target plant species.

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39 The tests involve several different plant species cl osely and distantly related to the family of the targeted host plant (Wapshere 1974). Related pl ant species that are ec onomically important, endangered, or native are of hi gh priority and also tested (McEvoy 1996; Schaffner 2001). Two phases in host-range testing, no-choice and choi ce tests, are performed in the laboratory (Marohasy 1998; Withers et al. 1999). No-c hoice tests involve larval development and oviposition tests on a single non-target species. Choice tests, however involve exposing the insect to two or more plants and typically in clude the host plant (McEvoy 1996; Schaffner 2001). Although these tests are designed to provide a realistic estimate of field host range, caged laboratory tests often overestimate host range be cause of unnatural behavior exhibited by some natural enemies as a result of being in a caged e nvironment. This type of behavior may produce false positives, or acceptance of plants as hosts that woul d not normally be accepted by the potential biological control agent in nature (Withers et al. 1999). A more natural or realistic testing approach is the use of open field te sts. These tests can be done only in the native range of the target weed. However, there are certain limitations including the need to import test plant species th at may be subjected to regulatory restrictions, availability of the potential biological control agent, and po ssible mortality of the agent by specialized predators and parasitoids. Field testing in the ar ea of introduction could be done in a safe, temporary manner for potential lepidopteran biological control agents by using the F1 sterile insect technique. Advantages of this approach include the expos ure of the biol ogical control agent to the actual environmen tal conditions it would experience if approved for release, prediction of true field host range and ability to reverse releases of the biological control agent without permanent establishment if non-target dama ge is detected (Bax et al. 2001; Carpenter et al. 2001a).

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40 An example of a situation where F1SIT could be used is in cla ssical biological control of Brazilian peppertree ( Schinus terebinthifolius ). This invasive, exotic weed is native to Brazil, Paraguay, and Argentina and was introduced into Florida as an ornamental in 1898 (Austin 1978; Ewel et al. 1982). Brazilian peppertree is di stributed widely throughout central and southern Florida and listed by the Florida Exotic Pest Plant Council (FLEPPC) as a Category 1 invasive weed because it is drastically a ltering native plant communities. Th e invasive nature of Brazilian peppertree also has been documented in Hawaii (Hight et al. 2002), California (Randall 2000), and Texas (Gonzalez and Christoffersen 2006) as well as in the subtropical regions of at least 20 additional countries (Ewel et al. 1982). As a result of field surveys conducted in Brazil, three potential biological control candidates were imported into a Florida contai nment laboratory in 1994. One of these was a leafroller Episimus utilis Zimmerman (Lepidoptera: Tortricida e). The insect was released in 1954 in Hawaii, but despite becoming widely di stributed throughout the islands, it was not found to be sufficiently abundant to severely damage populations of the plant (Bennett et. al 1990; Yoshioka and Markin 1991; Habeck et. al 1994). Later, it was di scovered that biotic interference from generalist parasitoids and predators probab ly prevented the insect from reaching its full biotic potential (Krauss 1963; Ma rtin et. al 2004). Although E. utilis was ineffective in providing successful control of Br azilian peppertree in Hawaii, it could provide effective control in Florida because Florida may provide more favorable ecological condi tions and less biotic mortality from introduced and native parasitoids and predators (Martin et al. 2004). Larvae of E. utilis inflict damage by feeding on leaflets, which can eventually lead to the defoliation of the plant (Cuda et al. 1999). Recently, a simulate d herbivory study was conducted that showed Brazilian peppertree is vulnerable to sustai ned defoliation (Treadwell and Cuda 2007). The

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41 biology of E. utilis and methodology for its laboratory rearin g were investigated by (Martin et al. 2004). Laboratory no-choice tests with E. utilis produced ambiguous result s; non-target plant species including fragrant sumac ( Rhus aromatica L.), winged sumac ( Rhus copallinum L.), poison sumac ( Toxicodendron vernix (L.) Kuntze), pistachio ( Pistacia vera L.) and cashew ( Anacardium occidentale L.) were unexpectedly accepted as developmental hosts (Fig 3-1) (J.P. Cuda unpubl. data). We believe that the broad physiological host range exhibited by E. utilis is not indicative of the true behavior of this insect in a natural envi ronment (ecological host range). For these cases where the results of labor atory tests lead to false positives, F1SIT could be an additional tool to elucidate the actual or ecological host range of the candidate biological control agent. The objectives of the current study were to determine a dose of radiation that would sterilize the F1 generation of E. utilis and to verify the effects of radiation in E. utilis Materials and Methods Colony Rearing Pairs of adult E. utilis moths (24 48 hr old) were set up on individual Braz ilian peppertree plants planted in 3.8 L (1-gallon) pots (20 cm he ight x 22.5 cm diam.). Each plant was enclosed in a clear acrylic cylinder (45 cm height x 15 cm diam.) with six evenly spaced ventilation holes (6.5 cm diam.). The top of the cylinder was co vered with a sheer polye ster fabric (Jo-Ann Fabrics #449-1676 white casa organza) a nd all six circular ventilati on holes each were covered with a mesh, screen size of 150 x 150 (Green.tek Inc., Edgerton, WI). The sheer polyester fabric was fastened to the top of the cylinde r by a metal ring clasp (14.3-21.6 cm) and further sealed with a rubber band to prevent small la rvae from escaping (Fig 3-2). Two additional access holes in the cylinder (2.5 cm diam) were plugged with #5 rubber stoppers. Each cylinder also was provided with a Gatorade feeder (15 ml glass vial with a 5 cm piece of dental wick

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42 soaked in Gatorade) as a nectar source for the adults. The use of Gatorade was previously shown to increase lifespan of adu lts and fecundity (total number of eggs laid) in females (Martin et. al 2004). When approximately 90% larval damage of the plant wa s observed, bouquets of five stems of Brazilian peppertree leaves (1-5 days old) in plastic vials (40 ml) with water (field collected in Ft. Pierce, FL) were placed near the top of the plant in each cylinder as needed. Plants used for colony rearing were sprayed twice weekly with an organic insecticide consisting of 1 Tbsp (15 ml) each of isopropyl alc ohol (70%), insecticidal oil, and Ivory liquid soap mixed in 1 gallon (3.8 liters) of water to protect against softbodied pests. Average temperature and humidity of the rearing room were maintained at 25.8 4.0C and 40-70% RH as recorded by a Fisher Scientific Thermo-Hygro digital maximum-minimum temperature and relative humidity recording instrument. Temperat ure and relative humidity recorded within the cylinder were 24.9 3.8C and 60-80% RH, respectively. A photoperiod of 14:10 (L:D) was maintained by a programmable timer connected to sets of two 60 cm 20 watt fluorescent bulbs (one standard and one Gro-Lux) per shelf of colony plants. Colony rearing and experiments with E. utilis were conducted at the University of Florida, Department of Entomology and Nematology Containment Faci lity (Gainesville, FL). Radiation Biology Study The procedures used for the radiation biology study were based on methodologies developed previously for the codling moth Cydia pomonella (L.) (Bloem et al. 1999a,b), false codling moth Thaumatotibia leucotreta (Meyrick) (Bloem et al. 2003), and cactus moth Cactoblastis cactorum (Berg) (Carpenter et al. 2001a,b). The E. utilis moths were collected from the colony at the 5th instar (red larval stage) or the pupal stag e and placed individually into separate clear plastic 30 ml (1 oz) diet cups with a 3.5 cm piece of mois tened filter paper added to each cup to maintain humidity. The cups we re checked each morning at the same time and

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43 adults were removed upon emergen ce. Male and female virgin E. utilis adults (<24hr old) were collected and individually exposed to gamma radiation in plastic sn ap-cap vials (12 ml) within an aluminum-lined cardboard caniste r (8.8 cm height x 7.5 diam.) (Fig 3-3). Doses of 0, 50, 100, 150, 200, 250, and 300 Gy were administered by using a Cesium-137 Gammacell 1000 irradiator with a dose of 12-13 Gy/min (Fig 3-4) (F.A.S.T. Gainesville, FL). The dose rate was determined by a dosimetry study using Far West dosimetry film. Two canisters each containing 10 of the plastic vials were placed on top of each other within the irradiator. Dosimetry film was placed in three different positions within four di fferent vials (one from the top and bottom of each canister). Results indicated that to minimize the variance between th e levels of irradiation, the canister containing the plastic vi als should only be positioned at the bottom of the irradiator. Five treated (T) male or female moths we re placed inside a triangular waxed paper oviposition chamber (30 x 19 x 12cm) with an equa l number of either treated (T) or nontreated (N) adult moths of the opposite gender (Fig 35). The oviposition chamber was then placed inside a 1-gallon plastic s ealable freezer bag (Ziploc) to maintain relative humidity and suspended on a string line to maximize the use of the limited amount of space in the containment laboratory. Each oviposition chamber included a 2 cm piece of cotton dental wick soaked in Gatorade as a nectar source and a small leaf disc of Pistacia vera L. (2.4 cm x 2.4 cm). Due to inconsistent oviposition in preliminary experiment s with nontreated moths, small leaf discs of Brazilian peppertree were substituted with Pistacia vera L. A phytochemical study of the leaves and bark of Schinus terebinthifolius had previously found that its compounds show a greater similarity to compounds isolated from Pistacia species than to those isolated from other species of Schinus (Campello and Marsaioli 1975). It was later determined that the leaf material was not a factor in the preliminary results, yet the Pistacia vera L. was used throughout the rest of the

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44 experiments for consistency. The moths were allowed to mate and lay eggs for 2 intervals of 5 days to take into account the 7 day average lifespan for the adults (Martin et al. 2004). After the first 5-day period, they were tran sferred to a new oviposition chambe r. At the end of the 10-day period, the females were collected, preserved in et hyl alcohol (80%), and subsequently dissected to determine their mating status (presence of spermatophores or in flated bursa copulatrix) (Ferro and Akre 1975). The egg sheets were then in cubated for a period of 7 days at 24.6 4.5C, 5080% RH, and a photoperiod of 14:10 L:D which corre sponded to the developmental time of the egg stage (Martin et al. 2004). The total number of eggs laid (fecundity) and the number of eggs that hatched (fertility) we re then counted for each egg sheet per radiation dose. Five replicates of 3 different crosses (treatments) (N x T T x N and T x T ) were completed for each dose of radiation. Temperature and relative humidity in the experiment room were 24.6 4.5C and 50-80% RH, respectively, w ith a photoperiod of 14:10 (L:D). Temperature and relative humidity within the oviposition chambe r also were recorded as 24.1 3.8C and 60-80% RH, respectively. The newly emerged E. utilis moths were irradiated using a Cesium-137 Gammacell 1000 irradiator (Fig 3-4) at the Flor ida Accelerator Services and Technology, Florida Department of Agriculture and Cons umer Services, Division of Plant Industry (Gainesville, FL). Statistical Analysis fo r Radiation Biology Study In order to determine the effect of radiat ion dose on fecundity, lin ear regressions using fecundity as the response variab le (Y) and radiation dose as th e treatment variable (X) were performed. A separate model was f itted for each of the treatments. The effect of radiation dose on fertility was determined by performing simple linear regressions of radiation dose pr edicting fertility for each of the crosses. In some cases, a polynomial model was indicated by th e scatter plots of the data. Th e alpha level for all of the

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45 regressions was p=0.05. The sex ratio was recorded for F1 males and analyzed using a simple linear regression. All regression anal yses were performed using S-plus 7.0 for Windows (Insightful Corp.). Inherited Sterility Methodology Based on the findings from the radiation biology study, five doses of radiation were chosen to be additionally evaluated for the cross N x T (nontreated female x treated male) in order to achieve inherited sterility. These doses included 125, 150, 175, 200, 225 Gy and a control (0 Gy). The protocol was the same as previ ously described, with th e exception that the F1 egg sheets were each placed on a Brazi lian peppertree plant in a 3.8 L (1-gallon) pot enclosed by a clear acrylic cylinder (45 cm height x 15 cm diam.) in order to rear the F1 generation. Average temperature and relative humidity reco rded within a cy linder were 25.2 3.6C and 70-90% RH, respectively. When the larvae ha tched, they were allowed to develop on the plant. At the 5th instar (red larval stage) or the pupal stage, th e insects were collected and each individual was placed in a separate clear plastic diet cup 30 ml (1 oz) with a 3.5 cm piece of moistened filter paper to maintain humidity. Upon emergence, each F1 female or male was outcrossed with a nontreated adult moth of the opposite sex. These F1 crosses were done in single pairs (1 female x 1 male). The protocol for the single pair crosses was the same as previously described for the radiation biology study. Ten crosses of F1 females and males were attempted for each dose, but due to virgin females and/or limited emergence of adults, there was a rang e of 5-12 replications for each gender per dose. Temperature and rela tive humidity in the experiment room were 25.6 4.4 C and 40-70% RH, respectively, with a photope riod of 14:10 (L:D). The temperature and relative humidity recorded inside the oviposition chamber were sl ightly higher, averaging 26.1 4.9C and 50-60% RH, respectively.

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46 Statistical Analysis for Inherited Sterility To determine the effects of radiat ion on the reproductive biology of F1 offspring of irradiated males, linear regressi ons of radiation dose administer ed on fecundity and fertility of the offspring were performed, fitting polynomial f unctions where appropriate. The sex ratio was recorded for F1 males and analyzed using a simple regre ssion. Alpha level for each of the factors was p=0.05. The regression analyses al so were performed using S-plus 7.0 for Windows (Insightful Corp.). Results Radiation Biology Study The effects of the radiation treatments on the adults of E. utilis were dependent upon the dose of radiation and gender irradiated. In irradi ated males, no significant changes in fecundity of mated females were observed as radiation dose increased (N x T ; F = 3.673; df = 1, 31; P = 0.06456), whereas in irradiated females, significan tly fewer eggs were la id as dose increased (T x N ; F = 11.62; df = 1,32; P < 0.05; T x T ; F = 16.85; df = 1, 31; P < 0.05) (Fig. 3-6). Fertility for treated females also decr eased with increasing radiation dose (T x N ; F = 56.31; df = 2, 30; P < 0.05; T x T ; F= 53.74; df = 2, 30; P < 0.05) and the same effect was observed for treated males crossed with nontreated females (N x T ; F = 57.35; df = 1,31; P = < 0.05) (Fig. 3-7). Additionally, the dos e of radiation at which treate d females were found to be 100% sterile was 200 Gy, whereas males irradiated at 200 Gy still had a residual fertility of 18%. Mating was confirmed in all adult female moths used in the experiments as determined by the presence of spermatophores or inflated bursa copulatrix (Ferro and Akre 1975). Inherited Sterility With respect to the fecundity for F1 females, there was no sign ificant relationship between the dose of radiation administered to the treat ed male in the parent al cross and fecundity

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47 observed in the F1 generation (F1 x N ; F = 0.06699; df = 1, 42; P = 0.797; N x F1 ; F = 1.114; df = 2, 52; P = 0.336) (Fig. 3-8). Per cent egg hatch for treatments with the F1 males and females both declined with an increased dose of radiation (N x F1 ; F= 40.31; df = 2, 52; P < 0.05; F1 x N ; F = 58.28; df = 1, 43; P < 0.05) (Fig. 3-9). For both the F1 female and male treatments, 100% sterility was found at 225 Gy, wh ich was the minimum dose at which no viable offspring could survive. In addition, the ratio of F1 males to females was more or less correlated with an increase in radiation dose, although the p-value was bord erline (F= 7.517; df = 1, 4; P = 0.05181) (Fig. 3-10). Discussion The F1 sterile insect technique (F1SIT) has been used in severa l studies to control various pest Lepidoptera. The technique provides a safe, environmentall y friendly approach to pest management. Early studies by Proverbs (1962), w ho first documented partial sterility in the codling moth found that when males were partia lly sterilized and mate d to wild females the number of progeny was reduced and they were mo stly male and highly sterile. Subsequent studies by North (1975) and LaChance (1985) compared the use of partial sterility with complete sterility in Lepidoptera. They determined that a partially sterilizing dose of radiation would increase competitiveness, possibly cause a delay in development, and lower quality sperm in the F1 generation. Recent laboratory and field studies have confirmed these effects in the codling moth, (Bloem et al. 1999a,b) and false codling moth (B loem et al. 2003), both belonging to the Family Tortricidae. The results of this study were similar to the result s found in the previous studies. Higher doses of radiation resulted in an increase in sterility, a higher ratio of F1 males to females, and a declining trend in fecund ity for both treated female cros ses. Irradiated females of E. utilis were found to be 100% sterile at 200 Gy, which is similar to the female false codling moth but

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48 was more radioresistant than the female codling moth where complete sterility was observed at 100 Gy. When treated males of E. utilis were mated with nontreated females, the sterility of the F1 generation was likewise similar to that reported fo r the other tortricids. In particular, the dose at which E. utilis was found to be 100% sterile was 225 Gy whereas the dose for the codling moth was 250 Gy (Bloem et al. 1999a,b), and the ra nge of partial sterility for the false codling moth was 150-200 Gy (Bloem et al. 2003). Because our findings were similar to the other studies, we determined that a dose of radiation of 225 Gy will provide 100% sterility and ensure a reduced number of progeny which are more steril e than their parents and mostly male. The F1 sterile insect technique could therefore be appropriately appl ied in other areas of pest management including the testing procedures of potential biological control agents involved in control of invasive, exotic weeds. For example, Brazilian peppertree is a highly invasive, exotic weed in Florida and E. utilis historically is an established bi ological control agent of Brazilian peppertree in Hawaii where no non-target impacts have been documented. Laboratory no-choice and multiple choice host range tests performed with E. utilis showed that the physiological host range of this insect is broader than expected (J.P. Cuda unpublish. data). The cage environment in laboratory screening tests pr obably inhibited the behavior of the insects and produced false positive results, where the insects accept plants that they would not normally accept in nature (Withers et al. 1999). Therefore, field testing in the proposed area of introduction would provide the most accurate results. In this study, we found that a dose of 225 Gy can be applied to E. utilis adult male moths and upon mating with nontreated female moths, complete sterility in the F1 generation is assured. Based on fecundity results of nontreated female s mated with irradiat ed male parents, no significant relationship was found between treatment dose and fecundity of females, therefore

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49 suggesting that the number of eggs laid would be similar to non-irradiat ed moths. Fertility recorded in the irradiated male moth treatments (N x T ), however, would be greatly reduced. Normal oviposition behavior as well as larval damage and fe eding would occur under natural conditions except that the F1 generation would be unable to reproduce. Performance of irradiated E. utilis males should be similar to nontreated males based on results of previous studies examining the effects of radiation on other tortricid mo ths (Bloem et al. 1999a,b, 2003) The safety of the technique can also be furt her insured by the fact that most of the F1 progeny will be males, therefore limiting the number of matings. The objectives of this study were to identify a dose of radiation that when administered to E. utilis a candidate biological control agent for cont rol of Brazilian peppertree, would result in complete sterility in the F1 generation and confirm the effects of radiation in E. utilis Using the F1 sterile insect technique (F1SIT) in addition to laboratory host range testing can provide a temporary and reversible way to test potential biological control agents in the proposed area of release as proposed by Bax et al. 2001. Further studi es will be needed to address the performance of irradiated biological contro l agents including oviposition, la rval feeding preferences and survival, and host-finding behavior.

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50 Figure 3-1. Results of laboratory no-choice tests performed with E. utilis (ANOC Cashew, COCO European Smoke Tree, PICH Chin ese Pistachio, RHAR Fragrant Sumac, RHCO Winged Sumac, RHGL Smooth Sumac, SCTE Brazilian peppertree, TOVE -Poison Sumac).

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51 Figure 3-2. Potted BP plant covere d by a clear acrylic cylinder.

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52 Figure 3-3. Materials used for irradiation of E. utilis moths. Figure 3-4. Cesium -137 Gammacell 1000 irradiator (F.A.S.T. Gainesville, FL).

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53 Figure 3-5. Waxed paper oviposition ch amber for mating and ovipositing by E. utilis moths.

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54 Figure 3-6. Fecundity (mean number of eggs laid) per mated female of E. utilis adults for three crosses (N x T T x N and T x T ) treated with increas ing doses of gamma radiation. Lines shown are leas t -squares regression lines, T x T ; y = 81.7731 0.2125x; R2 = 0.352; T x N ; y = 71.6725 0.1511x; R2 = 0.266; N x T ; y = 68.6534 0.0946x; R2 = 0.106.

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55 0 10 20 30 40 50 60 70 80 90 100 050100150200250300350Dose (Gy)Percent Egg Hatch N x T T x N T x T Figure 3-7. Fertility (mean percenta ge of eggs that hatched) of E. utilis adults for three crosses (N x T T x N and T x T ) treated with increasing dose of gamma radiation. N x T ; y = 64.4286 0.1999x; R2 = 0.649 ; T x N ; y = 60.0056 0.6843x + 0.0017x2; R2 = 0.790; T x T ; y = 63.6209 0.7619x + 0.0020x2; R2 = 0.782.

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56 Figure 3-8. Fecundity (mean nu mber of eggs laid) of F1 crosses (F1 x N N x F1 ) of E. utilis adults as a result of radi ation administered to parent al males. Lines shown are least squares regression lines, F1 x N ; y = 148.0383 0.0536x; R2 = 0.002; N x F1 ; y = 99.3422 + 0.2139x 0.0017x2; R2 = 0.041.

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57 0 10 20 30 40 50 60 70 80 90 100 0125150175200225Dose (Gy)Percent Egg Hatch F1 x NT NT x F1 Figure 3-9. Fertility (mean percentage of eggs that hatched) of F1 crosses (F1 x N N x F1 ) of E. utilis adults as a result of radiation ad ministered to parental males. F1 x N ; y = 63.8547 0.3176x; R2 = 0.575 ; N x F1 ; y = 55.1634 0.6017x +0.0016x2; R2 = 0.608.

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58 Figure 3-10. Percentage of F1 E. utilis adult males as a result of radiation administered to parental males. Line shown is l east-squares regression line, y = 54.9846 +0.1159x, R2=0.653.

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59 CHAPTER 4 CONCLUSIONS Host range testing in weed biological cont rol has long been a heavily debated issue. Typical protocols involve the expos ure of one or more life stages of an insect to a variety of plants in no-choice and multiple choice conditions to determine if the potential biological control agent would damage or reproduce on non-target ho sts: plant species ther eby rendering it unsafe for release. These host range tests typically are conducted in cages in an artificial laboratory environment and may result in behavioral responses that would not be seen in nature. This type of testing procedure often can result in false positives where the insect may accept plants outside of their normal host range (Withers et al.1999). Because of fede ral restrictions, actual field testing can only be done in th e native range of the ta rget weed. While this type of testing would provide the most realistic approximation of the true host range for the potential biological control agent several obstacles ar e involved. Critical test plant sp ecies of interest to the USA may not be available in the native range, a nd if so, there may be resistance by foreign governments to their importation. Additionally, it may be difficult to find qualified people with the facilities necessary to conduc t the studies, and sending trained sc ientists and materials to the native range may be very costly. A possible solution to field testing in the area of introduction may lie in the use of insects that have been reproduct ively inactivated by the F1 sterile insect technique (F1SIT). Numerous studies involving important econom ic Lepidopteran pests have doc umented the effects of using radiation to achieve inherited st erility. Some of these effects include increased competitiveness compared with complete steril ity, a decline in the number of progeny, a predominantly male biased sex ratio, a nd a highly sterile F1 generation that may expe rience delayed development along with producing lower quality sper m (Proverbs 1962, North 1975, LaChance 1985).

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60 Results from studies by Bloem et al. (1999a,b, 2003) showed that the perf ormance of irradiated adult moths were comparable to non-irradiated moths. The purpose of this study was to determine th e dose of radiation that would ensure full sterility in the F1 generation and to verify effects of radiation in E. utilis a candidate biological control agent of Brazilian peppertree. The situ ation presents a prime example of an invasive, exotic weed that has a promising potential biological cont rol agent, yet host specificity tests in the laboratory proved to be ambigious. We found a broader host range than what was observed in Brazil, the insects native range or Hawaii, where the insect was released and attacks only Brazilian peppertree. Identifying the dose of radiation that produces a fully sterile F1 generation in E. utilis would allow the biological control agent to be field tested in Florida in a safe and nonpermanent manner (Bax et al. 2001). This additi onal tool would provide a realistic indication of host range because E. utilis could be released in the area of potential introduction. The sterile F1 generation could then be allowed to devel op normally on appropriate test plants. Results for E. utilis were similar to other inherited st erility studies on tortricid moths including those reported for the codling moth (Bloem et al. 1999a,b) and false codling moth (Bloem et al. 2003). The fecundity of irradiated E. utilis females in the parental generation decreased significantly with increased dose, wher eas fecundity for nontreated females outcrossed to irradiated males did not differ from the contro ls. However, the fertility of both irradiated females and males significantly declined with incr eased dose, especially for irradiated females due to a higher sensitivity to radiation. When incr easing doses of radiation were applied to parental males outcrossed to nontreated females, there was no significant effect on the fecundity of the F1 females or males when outcrossed to nontreat ed moths of the opposite sex. Fertility of the F1 females and males declined significantly with increased dose, yet F1 males were found to

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61 be more sterile than F1 females. A significantly greater number of F1 males were produced with higher doses of radiation applied to the parental male. For all three tortricids, effects of increased dose of radiation resulted in an overa ll decline in the fecundity and fertility of the parental and F1 generations. Additionally, with increased dose of radiation, a higher ratio of F1 males to females was found. The dose at which irradiated E. utilis females were 100% sterile was the same as for the false codling moth, yet E. utilis proved to be more resistant to radiation than irradiated codling moth females. In term s of the dose of radiati on required for inherited sterility, E. utilis proved to be more ra dioresistant than the false codling moth, but was comparable to the codling moth. The results of this study have shown that a dose of radiation can be applied to E. utilis adult males resulting in a reduced number of progeny that are ster ile and largely male. The use of this technique for field host range testing can be done in a safe manner without posing any environmental risks because the F1 generation will be sterile. Th is tool, used in addition to laboratory testing, would be us eful for clarifying any unnatura l behavior observed in the laboratory leading to overestim ation of host range. Furthe r study of the performance of irradiated insects compared to nontreated insects will need to be completed to ensure that hostfinding behavior, oviposition, and larval survival along with feeding preferences can be compared in laboratory tests with field tests us ing reproductively inactivat ed biological control agents. If approved by state and federal regulatory officials, future studies will involve the use of partially sterilizing doses of radi ation applied to males outcrossed to nontreated females released into field cages with appropriate test plants.

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62 LIST OF REFERENCES Alexander TJ, Crook AG. 1974. Recent vegetational changes in Southern Florida. In: Gleason PJ, editor. Environments of South Flor ida: Present and Past. Miami: The Miami Geological Society. p 61-72. Andrs LA, Angelet GW. 1963. Notes on the ecology and host specificity of Microlarinus lareynii and M. lypriformis (Coleoptera: Curculionidae) and the biological control of puncture vine, Tribulus terrestris Journal of Economic Entomology 56:333-340. Astaurov BI, Frolova SL. 1935. Artificia l mutations in the silkworm ( Bombyx mori L.). V. Sterility and spematogenic anaomalies in the progeny of irradiated moths concerning some questions of general biological and mu tagenic action of X-rays. Biologicheskii Zhurnal 4:861-894. Austin DF. 1978. Exotic plants and their effect s in Southeastern Florida. Environmental Conservation 5(1):25-34. Ayvaz A, Tunbilek A. 2006. Effects of gamma ra diation on life stages of the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralida e). Journal of Pest Science 79:215-222. Barkley F. 1944. Schinus L. Brittonia 5:160-198. Barkley F. 1957. A study of Schinus L. Lilloa Revista de Botanica Tomo 28:110. Bax N, Carlton JT, Mathews-Amos A, Haedrich RL Howarth FG, Purcell JE, Rieser A, Gray A. 2001. The Control of Biological Invasions in the World's Oceans. Conservation Biology 15:1234-1246. Bennett FD, Crestana L, Habeck DH, Berti-Fil ho E. 1990. Brazilian PeppertreeProspects for Biological Control. In: Delfosse ES, edito r; Proc. VII. International Symposium on Biological Control of Weeds 1989; Rome, Italy. Bloem KA, Bloem S, Carpenter JE. 2005. Impact of moth suppression/eradication programmes using the sterile insect technique or inhe rited sterility. In: Dyck VA, Hendrichs J, Robinson AS, editors. Sterile Insect Technique Principles and Practice in Area-Wide Integrated Pest Management. Dordrecht: Springer. Bloem S, Bloem KA, Carpenter JE, Calkins CO 1999a. Inherited Sterility in Codling Moth (Lepidoptera: Tortricidae): E ffect of Substerilizing Doses of Radiation on Field Competitiveness. Environmental Entomology 28(4):669-674. Bloem S, Bloem KA, Carpenter JE, Calkins CO 1999b. Inherited Sterility in Codling Moth (Lepidoptera: Tortricidae): E ffect of Substerilizing Doses of Radiation on Insect Fecundity, Fertility, and Control. Annals of the Entomological Society of America 92:222-229.

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71 BIOGRAPHICAL SKETCH Onour received a BS in marine science and biology with a minor in chemistry at the University of Miami in 2002. Immediately followi ng graduation, she received an internship at the Smithsonian Marine Station in Fort Pierce that involved working on the interaction of nutrients and salinity in the mangroves of the Indian River Lagoon. Following the internship, Onour held positions at the Indian River Rese arch and Education Ce nter working for Dr. William Overholt on the Biological Control of Weed s and at the USDA in Fort Pierce with Dr. Erin Rosskopf on Alternatives to Methyl Brom ide-Weed and Disease Control. She quickly developed an interest in the bi ological control of weeds and working with insects which lead to her pursuit of a masters degree in entomology and nematology at the University of Florida.