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Evaluation of the Green Lacewing Chrysoperla rufilabris Burmeister (Neuroptera

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Title:
Evaluation of the Green Lacewing Chrysoperla rufilabris Burmeister (Neuroptera Chrysopidae) as a Biological Control Agent of the Yellowmargined Leaf Beetle Microtheca ochroloma Stal (Coleoptera: Chrysomelidae)
Physical Description:
1 online resource (71 p.)
Language:
english
Creator:
Nino Beltran, Angie A
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
Cave, Ronald David
Committee Co-Chair:
Leppla, Norman C
Committee Members:
Webb, Susan E

Subjects

Subjects / Keywords:
biocontrol -- chrysoperla -- crucifers -- ipm -- lacewings -- microtheca
Entomology and Nematology -- Dissertations, Academic -- UF
Genre:
Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
The production of crucifers in organic farms in thesouthern United States has been highly affected by the yellowmargined leafbeetle, Microtheca ochroloma Stål(Coleoptera: Chrysomelidae). This insect was detected for first time in thecountry in 1945, and until now no host-specific natural enemy has been reportedin the area. Therefore, the goal of this study was to evaluate the effectivenessof the generalist predator Chrysoperlarufilabris Burmeister (Neuroptera: Chrysopidae) as a biological controlagent for the management of the yellowmargined leaf beetle in organic farms. Chrysoperlarufilabris larvae have been reported feeding on M. ochroloma eggs and larvae. Therefore, the first experiment ofthis thesis quantified the development time and predation rate of C. rufilabris larvae feeding on eggs andfirst instars of M. ochroloma atconstant temperatures of 10, 15, 20 and 25°C. The predator was able to developat temperatures between 15 and 25°C. Chrysoperlarufilabris spent, from egg hatch to adult emergence, 75.5 ± 1.7 and 54.0 ±1.6 days at 15°C, 34.7 ± 0.7 and 31.7 ± 0.9 days at 20°C, and 26.6 ± 0.6 and 21.4± 0.7 days at 25°C when fed eggs and first instars of M. ochroloma, respectively. During the larval stage, the predatorkilled a mean total of 633.9 ± 38.5 eggs and 217.7 ± 18.3 first instars at15°C, 592.1 ± 30.5 eggs and 224.8 ± 18.9 first instars at 20°C, and 496.1 ±28.7 eggs and 218.5 ± 17.6 first instars of M.ochroloma at 25°C.  Prey preference of C. rufilabris among M. ochroloma eggs and first instars and Myzus persicae Sulzer (Hemiptera: Aphididae) nymphs was evaluatedin the second experiment of this thesis. The predator showed a 5.2-foldpreference for aphids over M. ochrolomaeggs and larvae. When only eggs and larvae of M. ochroloma were offered in Petri dishes, the predator did notshow any preference. However, when the immature stages of M. ochroloma were offered to the predator on bok choy plants, C. rufilabris killed more first instarsthan eggs due to differences in the location of the prey. In the third experiment of thisthesis, the performance of C.rufilabris feeding on M. ochroloma larvaeunder field conditions on an organic farm was evaluated. All possiblecombinations of cage-uncaged bok choy plants and with-without predator wereevaluated. The release of the predator did not cause an important effect on M. ochroloma populations. Differences inthe number of prey on the last sampling period were more related to the use ofcages. The results of myresearch suggest that C. rufilabris mightnot be a promising biological control agent of M. ochroloma. Augmentative releases of C.rufilabris to control M. ochrolomain organic farms probably would not be cost effective for the growers. Efforts should be focused on attracting, enhancing,and conserving natural populations of C.rufilabris present in the organic farms.
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 Angie A Nino Beltran.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Cave, Ronald David.
Local:
Co-adviser: Leppla, Norman C.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0046041:00001

MISSING IMAGE

Material Information

Title:
Evaluation of the Green Lacewing Chrysoperla rufilabris Burmeister (Neuroptera Chrysopidae) as a Biological Control Agent of the Yellowmargined Leaf Beetle Microtheca ochroloma Stal (Coleoptera: Chrysomelidae)
Physical Description:
1 online resource (71 p.)
Language:
english
Creator:
Nino Beltran, Angie A
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
Cave, Ronald David
Committee Co-Chair:
Leppla, Norman C
Committee Members:
Webb, Susan E

Subjects

Subjects / Keywords:
biocontrol -- chrysoperla -- crucifers -- ipm -- lacewings -- microtheca
Entomology and Nematology -- Dissertations, Academic -- UF
Genre:
Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
The production of crucifers in organic farms in thesouthern United States has been highly affected by the yellowmargined leafbeetle, Microtheca ochroloma Stål(Coleoptera: Chrysomelidae). This insect was detected for first time in thecountry in 1945, and until now no host-specific natural enemy has been reportedin the area. Therefore, the goal of this study was to evaluate the effectivenessof the generalist predator Chrysoperlarufilabris Burmeister (Neuroptera: Chrysopidae) as a biological controlagent for the management of the yellowmargined leaf beetle in organic farms. Chrysoperlarufilabris larvae have been reported feeding on M. ochroloma eggs and larvae. Therefore, the first experiment ofthis thesis quantified the development time and predation rate of C. rufilabris larvae feeding on eggs andfirst instars of M. ochroloma atconstant temperatures of 10, 15, 20 and 25°C. The predator was able to developat temperatures between 15 and 25°C. Chrysoperlarufilabris spent, from egg hatch to adult emergence, 75.5 ± 1.7 and 54.0 ±1.6 days at 15°C, 34.7 ± 0.7 and 31.7 ± 0.9 days at 20°C, and 26.6 ± 0.6 and 21.4± 0.7 days at 25°C when fed eggs and first instars of M. ochroloma, respectively. During the larval stage, the predatorkilled a mean total of 633.9 ± 38.5 eggs and 217.7 ± 18.3 first instars at15°C, 592.1 ± 30.5 eggs and 224.8 ± 18.9 first instars at 20°C, and 496.1 ±28.7 eggs and 218.5 ± 17.6 first instars of M.ochroloma at 25°C.  Prey preference of C. rufilabris among M. ochroloma eggs and first instars and Myzus persicae Sulzer (Hemiptera: Aphididae) nymphs was evaluatedin the second experiment of this thesis. The predator showed a 5.2-foldpreference for aphids over M. ochrolomaeggs and larvae. When only eggs and larvae of M. ochroloma were offered in Petri dishes, the predator did notshow any preference. However, when the immature stages of M. ochroloma were offered to the predator on bok choy plants, C. rufilabris killed more first instarsthan eggs due to differences in the location of the prey. In the third experiment of thisthesis, the performance of C.rufilabris feeding on M. ochroloma larvaeunder field conditions on an organic farm was evaluated. All possiblecombinations of cage-uncaged bok choy plants and with-without predator wereevaluated. The release of the predator did not cause an important effect on M. ochroloma populations. Differences inthe number of prey on the last sampling period were more related to the use ofcages. The results of myresearch suggest that C. rufilabris mightnot be a promising biological control agent of M. ochroloma. Augmentative releases of C.rufilabris to control M. ochrolomain organic farms probably would not be cost effective for the growers. Efforts should be focused on attracting, enhancing,and conserving natural populations of C.rufilabris present in the organic farms.
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 Angie A Nino Beltran.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Cave, Ronald David.
Local:
Co-adviser: Leppla, Norman C.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0046041:00001


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EVALUATION OF THE GREEN LACEWING Chrysoperla rufilabris BURMEISTER (NEUROPTERA: CHRYSOPIDAE) AS A BIOLOGICAL CONTROL AGENT OF THE YELLOWMARGINED LEAF BEETLE Microtheca ochroloma STL (COLEOPTERA: CHRYSOMELIDAE) By ANGIE A LEJANDRA NIO BEL TR N A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUI REMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013 1

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2013 Angie Alejandra Nio Beltrn 2

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T o my dedicated parents Alejo and Ligia and my lovely family 3

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AC KNOWLEDGMENTS I would like to acknowledge my committee members Dr. Ronald Cave, Dr. Norm Leppla, and Dr. Susan Webb In particular, I am thankful to Dr. Cave for assisting me in all steps of this process H is continuous instruction, encouragement, and financial assistance helped me to successfully finish my studies and share my results in national and state meetings. I am also grateful to Dr. Leppla and Dr. W ebb for allowing me to use their lab oratory and office facilities during my stay in Gainesville, and for their thoughtful s uggestions and editorial comments that made substantial improvements to this study. I give thanks to the people at the Biological Con trol Research and Containment Laboratory (BCRCL) at the Indian River Research and Education Center in Ft. Pierce, e specially Christy Richardson, Isabella Daza, and Esteban Tapia for their help during the lab oratory and field experiments Thanks to the Departamento de Ciencia, Tecnologa e Innovacin of Colombia and Fulbright for sponsoring my masters education at the University of Florida and the USDA Organic Agricultural Research and Extension Initiative and the Florida Department of Agriculture for sup port of my research. Thanks to Valerie Quant and Diane Cordeau for providing space in their farms to conduct my research. I would also like to thank all my friends and colleagues in Gainesville for their unconditional friendship. I am thankful to Rodrigo Daz and Vernica Manrique for being my family in Ft. Pierce and for their continuous advice regarding my research that help ed me to accomplish my goals. Finally, I would like to give special thank s to God and my family for their endless love and unconditi onal support that upheld me during my studies. 4

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T ABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 ABSTRACT ..................................................................................................................... 9 CHAPTER 1 GENERAL INTRODUCTION .................................................................................. 11 2 LITERATURE REVIEW .......................................................................................... 14 Microtheca ochroloma Stl (Coleoptera: Chrysomelidae) ....................................... 14 Distribution, Biology, and Ecology .................................................................... 14 Control Methods ............................................................................................... 16 Chrysoperla rufilabris Burmeister (Neuroptera: Chrysopidae) ................................ 18 Distribution and Biology .................................................................................... 18 Chrysoperla rufilabris as a Biological Control Agent ......................................... 19 3 DEVELOPMENTAL TIME AND KILLING RATE OF CHRYSOPERLA RUFILABRIS (BURMEISTER) (NEUROPTERA: CHRYSOPIDAE) FED MICROTHECA OCHROLOMA ( STL ) (COLEOPTERA: CHRYSOMELIDAE) ...... 22 Introduction ............................................................................................................. 22 Materials and Methods ............................................................................................ 23 Plant Material ................................................................................................... 23 Stock Colonies ................................................................................................. 24 Experimental Design ........................................................................................ 24 Statistical Analyses .......................................................................................... 25 Results .................................................................................................................... 25 Developmental Time ........................................................................................ 25 Mortality ............................................................................................................ 27 Killing Rate ....................................................................................................... 27 Discussion .............................................................................................................. 29 4 PREY PREFERENCE OF CHRYSOPERLA RUFILABRIS (NEUROPTERA: CHRYSOPIDAE) BETWEEN IMMATURE STAGES OF MICROTHECA OCHROLOMA (COLEOPTERA: CHRYSOMELIDAE) AND MYZUS PERSICAE (HEMIPTERA: APHIDIDAE) ................................................................................... 38 Introduction ............................................................................................................. 38 Materials and Methods ............................................................................................ 39 5

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P lant Material ................................................................................................... 39 Sto ck Colonies ................................................................................................. 39 Experimental Design ........................................................................................ 40 Predation on M. ochroloma Eggs and Larvae on Host Plants .................... 40 Prey Preference among M. ocholoma Eggs and Larvae and M. persicae Nymphs in a Petri Dish ........................................................................... 40 Statistical Analyses .......................................................................................... 41 Results .................................................................................................................... 42 Predation on M. ochroloma Eggs and Larvae on Host Plants .......................... 42 Prey Preference among M. ocholoma E ggs and Larvae and M. persicae Nymphs in a Petri Dish .................................................................................. 42 Discussion .............................................................................................................. 43 5 PERFORMANCE OF CHRYSOPERLA RUFILABRIS (BURMEISTER) (NEU ROPTERA: CHRYSOPIDAE) FEEDING ON MICROTHECA OCHROLOMA (STL) (COLEOPTERA: CHRYSOMELIDAE) LARVAE UNDER FIELD CONDITIONS IN AN ORGANIC FARM ....................................................... 49 Introduction ............................................................................................................. 49 Materials and Methods ............................................................................................ 50 Plant Material ................................................................................................... 50 Stock Colonies ................................................................................................. 51 Experimental Design ........................................................................................ 51 Statistical Analyses .......................................................................................... 52 Results .................................................................................................................... 52 Discussion .............................................................................................................. 54 6 CONCLUSIONS ..................................................................................................... 61 LIST OF REFERENCES ............................................................................................... 64 BIOGRAPHICAL SKETCH ............................................................................................ 71 6

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L IST OF TABLES Table page 3-1 M ean ( SE) number of days for development of Chrysoperla rufilabris larvae feeding on Microtheca ochroloma eggs at three constant temperatures. Number within parentheses equals sample size. ................................................ 34 3-2 Mean ( SE) number of days for development of Chrysoperla rufilabris larvae fee ding on first instar Microtheca ochroloma at three constant temperatures. Number within parentheses equals sample size. ................................................ 34 3-3 Mean ( SE) number of Microtheca ochroloma eggs killed per day per Chrysoperla rufilabris larva at three constant temperatures. Number within parentheses equals sample size. ....................................................................... 34 3-4 Mean ( SE) number of Microtheca ochroloma first instars killed per day per Chrysoperla rufilabris larva at three constant temperatures. Number within parentheses equals sample size. ....................................................................... 35 3-5 Mean ( SE) total number of Microtheca ochroloma eggs killed per Chrysoperla r ufilabris larva at three constant temperatures. Number within parentheses equals sample size. ....................................................................... 35 3-6 Mean ( SE) total number of Microtheca ochroloma first instars killed per Chrysoperla rufil abris larva at three constant temperatures. Number within parentheses equals sample size. ....................................................................... 35 4-1 Mean number ( SE) of Microtheca ochroloma eggs and first instar s and Myzus persicae nymphs kil led by first instar Chrysoperla rufilabris ................... 47 5-1 Mean ( SE) number of Microtheca ochroloma larvae, leaf damage percentage, and dry weight of bok choy leaves in fieldcage study. Sample size for all means was eight. .............................................................................. 58 7

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L IST OF FIGURES Figure page 2-1 D evelopmental stages of Microtheca ochroloma. .. ............................................. 21 2-2 Developmental stages of Chrysoperla rufilabris ................................................ 21 3-1 Percentage of Chrysoperla rufilabris that survived to the next developmental stage at three constant te mperatures ................................................................ 36 3-2 Dead Chrysoperla rufilabris pharate adult after pupal eclosion. ......................... 37 4-1 Bok choy plant confined in an insect rearing sleeve. .......................................... 48 5-1 Experimental setup of bok choy plants in the field.. ............................................ 58 5-2 Number of Microtheca ochroloma eggs, larvae, and adults on caged and uncaged bok choy plants with and without larvae of Chrysoperla rufilabris .. ...... 59 5-3 Number of Chrysoperla rufilabris larvae on caged and uncaged bok choy plants. ................................................................................................................. 60 8

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A bstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requ ir ements for the Degree of Master of Science EVALUATION OF THE GREEN LACEWING BURMEISTER (NEUROPTERA: CHRYSOPIDAE) AS A BIOLOGICAL CONTROL AGENT OF THE YELLOWMARGINED LEAF BEETLE STL (COLEOPTERA: C HRYSOMELIDAE) By A ngie Alejandra Nio Beltrn A ugust 2013 Ch air: Ronald D.Cave Cochair: Norman Leppla Major: Entomology and Nematology T he production of crucifers in organic farms in the southern United States has been highly affected by the yellowmargined leaf beetle, Microtheca ochroloma Stl (Coleoptera: Chrysomelidae). This insect was detected for first time in the country in 1945, and until now no host specific natural enemy has been reported in the area. Therefore, the goal of this study was to evaluate the effectiveness of the generalist predator C hrysoperla rufilabris Burmeister (Neuroptera: Chrysopidae ) as a biological control agent for the management of the yellowmargined leaf beetle in organic farms Chrysoperla rufilabris larvae have been reported feeding on M. ochroloma eggs and larvae. Therefore, the first experiment of this thesis quantified the development time and predation rate of C. rufilabris larvae feeding on eggs and first instars of M. ochroloma at constant temperatures of 10, 15, 20 and 25C. The predator was able to develop at temperatures between 15 and 25C. Chrysoperla rufilabris sp ent, from egg hatch to adult emergence, 75.5 1.7 and 54.0 1.6 days at 15C, 34.7 0.7 and 31.7 0.9 days at 20C, and 26.6 0.6 and 21.4 0.7 days at 25C when fed eggs and first 9

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instar s of M. ochroloma, respectively. During the larval stage, the predator killed a mean total of 633.9 38.5 eggs and 217.7 18.3 first instars at 15C, 592.1 30.5 eggs and 224.8 18.9 first instars at 20C, and 496.1 28.7 eggs and 218.5 17.6 first instars of M. ochroloma at 25C. Prey preference of C. rufil abris among M. ochroloma eggs and first instars and Myzus persicae Sulzer (Hemiptera: Aphididae) nymphs was evaluated in the second experiment of this thesis. The predator showed a 5.2fold preference for aphids over M. ochroloma eggs and larvae. When only eggs and larvae of M. ochroloma were offered in Petri dishes, the predator did not show any preference. However, when the immature stages of M. ochroloma were off ered to the predator on bok choy plants, C. rufilabris killed more first instars than eggs due to differences in the location of the prey. In the third experiment of this thesis, the performance of C. rufilabris feeding on M. ochroloma larvae under field conditions on an organic farm was evaluated. All possible combinations of cageuncaged bok cho y plants and withwithout predator were evaluated. The release of the predator did not cause an important effect on M. ochroloma populations. Differences in the number of prey o n the last sampling period were more related to the use of cages. The results of my research suggest that C. rufilabris might not be a promising biological control agent of M. ochroloma. Augmentative r eleases of C. rufilabris to control M. ochroloma in organic farms probably would not be cost effective for the growers Efforts shou ld be focused on a ttracting, enhancing, and conserving natural populations of C. rufilabris present in the organic farms. 10

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CHAPTER 1 GENERAL INTRODUCTION The organic food industry has grown rapidly in the United States during the last two decades. Since 1 990, the demand for organic food has increased 20% or more per year, and around 73% of the fresh production is sold in traditional markets (Nguyen et al. 2008). In the 2007 Census of Agriculture, there w ere 172 organic farms in Florida, of which 57 produced vegetables, potatoes, and melons. Among the vegetables produced, crucifers constituted a large and important group which includes broccoli, cabbage, cauliflower, collards, kale, mustard, radish, turnips, and some Asian vegetables ( e.g. bok choy, Chinese broccoli) (Web b 2010, Lamberts et al. 2011). Crucifer crops are conventionally and organically cultivated for various purposes, including human food (cooked or fresh), oil, medicinal benefits (Fenwick et al. 1982), and animal fodder. In order for a produc t to qualify as organic, it must meet the standards of the National Organic Program (NOP) (Ferguson 2004, Nguyen et al. 2008). According to the NOP, arthropod pest management in organic crops must incorporate ecological approaches, including augmentation or introduction of natural enemies, development of suitable habitat for natural enemies, and nonsynthetic control methods (Ferguson 2004, Zehnder et al. 2007). Since the use of synthetic pesticides is not an alternative for organic growers, it is vital to develop information about more ecofriendly control strategies. Organic growers must deal with many insects that infest crucifers. The yellowmargined leaf beetle, Microtheca ochroloma Stl (Coleoptera: Chrysomelidae), has become a major pest of turnips, m ustard, and Chinese cabbage in organic farms (Webb 2010). This insect is a problem for organic growers because: 1) it is active during 11

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cool seasons (late fall, winter, and early spring) when crucifers are produced in Florida and the state becomes an import ant supplier for other parts of the country due to a reduced production of other states (Olson 2011); 2) it feeds on leaves which are the marketed part of the plant, thus reducing the quality of the product (Chamberlin and Tippins 1948, Ameen and Story 199 7a); 3) it is an invasive species, and no effective, host specific natural enemy is present in Florida; and 4) organic growers are not allowed to use foliar insecticides which are commonly used to control this pest (Bowers 2003). Biological control is used to manage many economically important insect pests. Montemayor (2010) and Montemayor and Cave (2011, 2012) evaluated the generalist predator Podisus maculiventris Say (Hemiptera: Pentatomidae) as a control agent against M. ochroloma. They found that P. m aculiventris fully develops when feeding on the immature stages of M. ochroloma at temperatures above 10C, and has good potential to diminish populations of this pest in the field. My laboratory and field studies evaluated another generalist predator, Chr ysoperla rufilabris (Burmeister), commonly known as the green lacewing. This predator is often used in augmentative programs (Ridgway and Jones 1968, New 1975), is commercially available, and has been used successfully against other foliagefeeding beetles including the Colorado potato beetle ( Leptinotarsa decemlineata Say). Green lacewing larvae feed on a diversity of prey, principally small, soft bodied arthropods (Tauber et al. 2000). However, Obrycki et al. (1989) indicated that even if a prey is cons umed by the predator, this does not mean it is suitable prey for survival and development of the predator. For a prey type to be optimal, it must: 1) coincide in time 12

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and space with the predator, 2) be easily recognized and captured by the predator, and 3) enable adequate predator reproduction and development (Canard et al. 1984). Chrysoperla rufilabris larvae have been observed on crucifers infested by M. ochroloma (R. D. Cave, personal communication), and laboratory bioassays confirmed that they feed on l arvae and eggs of the pest (Montemayor and Cave 2009). But no information is available about the suitability of yellowmargined leaf beetle eggs and larvae as nutritional resources for the predators development. My overall research goal was to understand f urther the effectiveness of C. rufilabris as an applied biological control agent for the management of the yellowmargined leaf beetle in organic farms. My specific objectives were to: 1. Quantify the development time and predation rate of C. rufilabris larvae feeding on eggs and larvae of M. ochroloma at four different temperatures. 2. Evaluate the prey selection preference of C. rufilabris when exposed to immature stages (eggs and larvae) of M. ochroloma and the green peach aphid, Myzus persicae Sulzer. 3. Evaluat e the performance of C. rufilabris feeding on M. ochroloma larvae under field conditions on an organic farm. 13

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CHAPTER 2 LITERATURE REVIEW Microtheca ochroloma Stl (Coleoptera: Chrysomelidae) Distribution, B iology, and E cology Microtheca ochroloma, comm only known as the yellowmargined leaf beetle (Woodruff 1974), is a nonindigenous pest that feeds on crucifers (Chamberlin and Tippins 1948, Oliver and Chapin 1983). It was reported for the first time in the United States in 1945 (Chamberlin and Tippins 19 48) and has become established in Texas, Louisiana, Mississippi, Alabama, Georgia, North Carolina, California, and Florida (Chamberlin and Tippins 1948, Balsbaugh 1978, Oliver and Chapin 1983, Staines 1999, Guillebeau 2001, Gilbert et al. 2011 ). It also ha s been reported as a serious pest in Brazil, Argentina (where it is native), and Uruguay (Chamberlin and Tippins 1948). The adult stage of this beetle is characterized by its small size (about 5 mm long) and its black or dark brown color with a conspicuous pale yellow or white border and four rows of punctures on the elytra (Chamberlin and Tippins 1948, Woodruff 1974) (Fig. 2 1 d ) The female lays elongate, bright orange eggs, singly or in small clutches, on the soil or under fallen leaves (Woodruff 1974, B owers 2003) (Fig. 2 1 a ) The larvae vary from gray to yellow brown, are covered with fine hairs, and have a dark, sclerotized head capsule (Woodruff 1974, Fasulo 2005) (Fig. 2 1 b ) The yellowmargined leaf beetle goes through four instars (Ameen and Story 1997a). After the last instar, the insect ceases to feed and spins a pupal cocoon of a fibrous material that is excreted through the anal opening (Oliver and Chapin 1983). The pupal cocoon is spun on the undersides of dry leaves (Woodruff 1974, Fasulo 2005) (Fig. 2 1 c) The pharate adult remains in the cocoon until its cuticle becomes sclerotized and fully pigmented (Oliver and Chapin 14

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1983). Males are smaller than females and their posterior abdominal segment is decurved. For more precise sex recognition, t he presence of two knoblike structures on the last abdominal segment can be observed in females during the pupal stage (Oliver and Chapin 1983, Ameen 1996). Copulation occurs 56 days after emergence (Oliver and Chapin 1983, Fasulo 2005). Ameen and Story (2007) determined that the life cycle from egg to adult lasts about 27 days at 20C, with an average of 7.8 days for eggs, 10 days for larvae, three days for prepupae, and 5.6 days for pupae. Microtheca ochroloma is an important pest of crucifers, including cabbage ( Brassica oleracea L. var. capitata), napa cabbage ( B. pekinensis Lour.), collard ( B. oleracea var. acephala ), mustard ( B. juncea Cosson), radish ( Raphanus sativus L.), turnips ( B. rapa L.), mizuna, mibuna ( B. rapa L. subsp. Japonica, Japanese leafy vegetables), and watercress ( Nasturtium officinale L.) (Chamberlin and Tippins 1948, Woodruff 1974, Ameen and Story 1997b, Bowers 2003, Balusu and Fadamiro 2011). Although the yellowmargined leaf beetle feeds on a wide variety of crucifers, it shows a preference for turnips over other crucifers (Chamberlin and Tippins 1948, Ameen and Story 1997b, Balusu and Fadamiro 2011). Plants of the family Brassicaceae contain secondary metabolites called glucosinolates (Kjr 1981, Mithen 2001). Glucosinolates ar e hydrolyzed by the enzyme myrosinase to produce different compounds that may serve as a defense against bacteria, fungi, and insects, or as attractants for crucifer specialist pests (Sutherland 1977, Fenwick et al. 1982). Hopkins et al. (2008) reviewed the role of glucosinolates in multitrophic interactions. Many factors affect the quantity and quality of glucosinolates among and within plants, including variety, climate, cultivation conditions, and plant tissue (Fenwick et al. 1982). As a result, the products formed through hydrolysis of 15

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glucosinolates differ among plants, which is the case of differences in the volatile profiles of turnips compared to cabbage and collards (Balusu and Fadamiro 2011). According to Balusu and Fadamiro (2011), M. ochroloma pr eference for turnips is mediated by plant volatiles; preliminary studies suggest isothiocyanate as the attractant. Both larvae and adults of M. ochroloma consume foliage (Chamberlin and Tippins 1948, Ameen and Story 1997b). Feeding injury consists of holes chewed in the leaf; larvae produce large holes in foliage because they feed collectively (Chamberlin and Tippins 1948, Woodruff 1974). Microtheca ochroloma is considered a cool season pest (Ameen 1996). In Florida, this beetle is active from October to May (Bowers 2003, Balusu 2011). Aestival behavior has been reported for the yellowmargined leaf beetle during hot weather seasons (Chamberlin and Tippins 1948, Oliver and Chapin 1983, Bowers 2003). During the fall, M. ochroloma adults migrate from wild mus tard plants, considered their summer aestivation sites, to cruciferous crops (Balusu 2011). According to Bowers (2003), M. ochroloma resumes feeding and oviposition when favorable environmental and resource conditions are present, which indicates that it undergoes quiescence rather than a true diapause. Laboratory experiments showed that at 30C food consumption and survivorship of immature M. ochroloma decrease, which may be due to aestivation (Manrique et al. 2012). Control M ethods The application of sy nthetic insecticides has been the main control option for this pest (Menezes Jr. et al. 2005). However, the use of synthetic foliar insecticides is not allowed in organic production (Bowers 2003, Ferguson 2004). Various biopesticides and botanical insectic ides approved by the Organic Material Review Institute (OMRI) have 16

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been evaluated against M. ochroloma. According to Balusu and Fadamiro (2012), Entrust WP and PyGanic applied in the field were the most effective formulations against larvae and adults, c ausing 100% mortality within 24 h. In general, the entomopathogenic formulations showed slow activity and the maximum mortality obtained was 50%. Plant extracts, such as p defumo ( Nicotiana tabacum L., Solanaceae), ramo de cinamomo ( Melia azedarach L., M eliaceae), and DalNeem (a commercial product extracted from Azadirachta indica A. Juss, [Meliaceae]), have been shown to cause high mortality of yellowmargined leaf beetle larvae and adults (Dequech et al. 2008). Cultural control strategies for pest manag ement in crops involve implementation of modified standard agricultural practices to prevent pest problems. Two cultural methods, intercropping and the use of straw mulch, have been evaluated to reduce the impact of M. ochroloma on crucifers. Bowers (2003) tested an intercropping system using mizuna ( B. rapa var. Kyona) and oak leaf lettuce ( Lactuca sativa L. var. Berenice), but it did not prevent beetle colonization of host plants. Similarly, Manrique et al. (2010) evaluated the use of straw mulch to reduce the abundance of M. ochroloma in turnips. However, an opposite effect was observed, with a higher population density of the pest in plots with straw mulch. There are no reports of host specific natural enemies of M. ochroloma. However, in Florida the i ndigenous generalist predators P. maculiventris (Say) (Hemiptera: Pentatomidae), C. rufilabris (Burmeister) (Neuroptera: Chysopidae), and Hippodamia convergens (Say) (Coleoptera: Coccinellidae) have been observed preying on larvae, adults and pupae of the yellowmargined leaf beetle (Montemayor and Cave 2009). Stiretrus decastigmus Herrich Schaeffer (Hemiptera: Pentatomidae) and Toxomerus 17

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duplicatus Wiedemann (Diptera: Syrphidae) have been reported preying on adults and larvae of M. ochroloma in Brazil (Ponc io et al. 2010, Sturza et al. 2011). Podisus maculiventris is able to complete its development by feeding on eggs and larvae of M. ochroloma (Montemayor and Cave 2011). Development al time, consumption rate, and effectiveness of this predator as a biological control agent of M. ochroloma were evaluated in laboratory and fieldcage experiments. The results showed that P. maculiventris might play an important role in the management of this pest in organic farms (Montemayor and Cave 2011). Chrysoperla rufilabr is Burmeister ( Neuroptera: Chrysopidae ) Distribution and B iology Commonly known as the green lacewing, C. rufilabris is a cosmopolitan, generalist predator that is commonly found in manaltered ecosystems (New 1975, Tauber and Tauber 1983). It is abundant in the southeastern United States and Mexico where it is adapted to humid conditions and considered a lateseason (late July and August) species (Dinkins et al. 1970, Tauber and Tauber 1983). Chrysoperla rufilabris lays eggs that are oval and pos ted on a slender, pliable stalk glued to the substrate (Canard et al. 1984) (Fig. 2 2 a ). The larva is campodeiform with setae along the body, and the head is strongly chitinized with curved mandibles and maxillae that protrude forwards, forming a channel for passage of food (Fig. 2 2 b ) (Canard et al. 1984). The green lacewing larva goes through three instars. Two molts occur during the active period and the last molt occurs inside an ovoid cocoon made by numerous layers of silk which protect the pupa (Fig. 2 2 c) T he green lacewing pharate adult undergoes a final molt within minutes or hours after emerging from the cocoon (Duelli 1984). After emergence, the adult takes flight, irrespective of the presence of 18

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food. Apparently, this first flight is required to induce oviposition, therefore few females lay eggs in the habitat in which they emerge (Canard et al. 1984, Duelli 1984, Ventura et al. 2007). The adults are light green with large and broadly oval wings that have a rich and regular venation (Canard et al. 1984) The antennae are long, filiform, and multisegmented. The head has no ocelli, but has golden and prominent compound eyes. Adults are not predaceous; they feed on honeydew or nectar and receive essential amino acids from mutualistic yeasts (Burke and Marti n 1956). Time of development of C. rufilabris from hatching to adult emergence ranged from 16 to 27 days at temperatures between 24C to 30C when Aphis gossypii Glover was its prey (Burke and Martin 1956). According to Butler and Ritchie (1970), the clos ely related species Chrysoperla carnea (Stephens) is not able to reach adulthood at a constant temperature of 15C and the duration of the immature stages takes 40 days, whereas at 25C it takes approximately 24 days to develop from egg to adult. Chrysoperla rufilabris as a B iological C ontrol A gent Green lacewing larvae feed on a diversity of prey, principally small, soft bodied arthropods (Tauber et al. 2000). This predator is considered an excellent aphid killer (Hydorn and Whitcomb 1979, Canard et al. 1984, Dean and Schuster 1995, Chen and Liu 2001, Knutson and Tedders 2002); however, it has been evaluated as a biological control agent against other economically important pests, including many species of whiteflies (Legaspi et al. 1994, Dean and Schust er 1995), moths (Lingren et al. 1968, Hydorn and Whitcomb 1979), leafhoppers (Daanel and Yokota 1997), and mites (Canard et al. 1984). Montemayor and Cave (2009) reported C. rufilabris feeding on eggs and larvae of M. ochroloma. However, the efficacy of t his prey for green lacewing development is 19

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unknown. Two species of green lacewing, C. rufilabris and C. carnea, have been documented feeding on other beetle eggs and young larvae (Nordlund 1991, HoughGoldstein et al. 1993, Sablon et al. 2013). Chrysoperl a rufilabris larvae preyed on and killed L. decemlineata eggs and larvae, and they reduced the beetle population by 99.7% and 97.9% when 5 and 10 larvae/cage were released, respectively (Nordlund 1991). The predator also reduced the pests population on av erage by 84% in field experiments (Nordlund 1991). Laboratory experiments showed that all stages of C carnea consumed mainly eggs and first and second instars of L. decemlineata (Sablon et al. 2013). The green lacewing third instar killed four times more prey than the first two instars, and the prey handling time increased as prey size increased. No studies have been done to determine if C. carnea is able to complete its life cycle feeding only on L. decemlineata. Hydorn and Whitcomb (1979) found that us ing Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) to rear C. rufilabris significantly reduced the predators survival to maturity and fecundity, and the length of the larval stage was prolonged. 20

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Figure 2 1 De velopmental stages of Microtheca ochroloma: (a) eggs, (b) larvae, (c) pupae, and (d) adults. Photos b y Angie Nio. Figure 2 2 De velopmental stages of Chrysoperla rufilabris : (a) eggs, (b) larvae, (c) pupae, and (d) adults. Photos b y Angie Nio. 21

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CHAPTER 3 DEVELOPMENTAL TIME AND KILLING RATE OF CHRYSOPERLA RUFILABRIS (BURMEISTER) (NEUROPTERA: CHRYSOPIDAE) FED MICROTHECA OCHROLOMA ( STL ) (COLEOPTERA: CHRYSOMELIDAE) Introduction The organic food industry demands the development of pest control strategies that can be adopted by far mers. Since synthetic pesticides are not an alternative for organic growers, it is necessary to originate methodologies that incorporate ecological approaches for the management of pest problems (Ferguson 2004, Zehnder et al. 2007). The production of cruci fers in organic farms has been highly affected by the presence of the yellowmargined leaf beetle, M. ochroloma Stl (Webb 2010). This invasive species, native to Argentina, was first detected in the United States in 1945 (Chamberlin and Tippins 1948) and i s now established in several states. Microtheca ochroloma feeds on leaves of plants of the family Brassicaceae, including turnips, mustard, and Chinese cabbage among others (Chamberlin and Tippins 1948, Woodruff 1974, Ameen and Story 1997b, Bowers 2003, B alusu and Fadamiro 2011). The insect is active in late fall, winter, and early spring (Ameen 1996, Bowers 2003), coinciding with the time of crucifer production in Florida. Restrictions on the use of synthetic foliar insecticides and the lack of effective host specific natural enemies in Florida leave organic growers with few alternatives to reduce the economic impact of this pest in their crops. In search for a more ecofriendly alternative, Montemayor and Cave (2009) examined crucifer plants in organic farms to find native natural enemies that could be used against M. ochroloma. A variety of generalist predators were observed preying on the beetle, including P. maculiventris Say (Hemiptera: Pentatomidae), H. convergens 22

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Say (Coleoptera: Coccinellidae), and C. rufilabris Burmeister (Neuroptera: Chrysopidae). My study evaluates the potential of C. rufilabris as a control agent against M. ochroloma. Chrysoperla rufilabris commonly known as the green lacewing, because it has been used in pest control program s involving augmentative releases (Ridgway and Jones 1968, New 1975), is mass produced by commercial insectaries, and has been successfully used against other foliagefeeding pests (Nordlund 1991). The green lacewing larva, which is the predacious stage, f eeds on a diversity of prey, principally small, soft bodied arthropods (Tauber et al. 2000). Laboratory bioassays confirmed that C. rufilabris feeds on larvae and eggs of M. ochroloma (Montemayor and Cave 2009). However, Obrycki et al. (1989) point out that even if a prey is consumed, it is not evidence of its suitability for the survival and development of the predator. An optimal prey type must coexist in time and space with the predator, be easily recognized, captured, and accepted, and enable adequate predator reproduction and development (Canard et al. 1984). No information is available about the suitability of yellowmargined leaf beetle as a nutritional resource for the development of C. rufilabris Therefore, the objective of this study was to quantif y the developmental time and killing rate of C. rufilabris larvae when eggs and first instar s of M. ochroloma were offered at four constant temperatures. Materials and Methods Plant M aterial Turnip and bok choy ( Brassica rapa L ) were used as host plants t o rear M. ochroloma. Seeds were planted in 72cell trays containing sterilized soil mix (Fafard super fine germinating mix). Twoweek old seedlings were transplanted into 3.8L plastic pots containing a mixture of soil and fertilizer (Osmocote Classic 14N 14P, 14K). 23

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Stock C olonies Adults and larvae of M. ochroloma were collected in the field and transported to the Biological Control Research and Containment Laboratory at the Indian River Research and Educational Center in Ft. Pierce. The insects were confined in Bug Dorms (60 60 60 cm) with two host plants which were changed twice weekly. To obtain eggs and first instars, 20 pairs of males and females were collected from the colony and placed in a plastic box (18 13.5 9 cm) with a screen mesh clo th in the lid for ventilation. Microtheca ochroloma females oviposited the eggs on the edges of white paper towels or Kimwipes (Kimberly Clark) which were collected every two days. Turnip or bok choy leaves were offered as food. Adults of C. rufilabris we re purchased from RinconVitova Insectaries, Inc. (Ventura, CA). The company packed the adults in a cylindrical container with available food and a piece of moistened sponge and placed in a cooler. The number of adults alive was 4 times the quantity expect ed. After arrival, 30 pairs were placed in plastic containers and fed an artificial diet. The eggs collected from the colony were placed singly in plastic vials and stored in an environmentally controlled chamber at 25C, 75% RH, and 12L:12D photoperiod un til emergence. Experimental D esign An individual larva of C. rufilabris was housed in a plastic Petri dish ( 5.5 cm diameter ) with a hole (2.5cm diameter ) in the top covered by screen mesh cloth Each Petri dish contained a piece of moistened, white filter paper placed at the bottom to maintain appropriate humidity. A piece of bok choy leaf (3 cm2) was also provided as a food source or substrate for the prey and changed daily. 24

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Eggs or first instars of M. ochroloma were offered ad libitum to each instar of C. rufilabris Natural mortality of the prey was recorded by comparison with the control treatment, which consisted of a fixed number of prey in a Petri dish without the predator. In all treatments, dead prey were replaced daily. Prey mortality in the treatments with a predator was corrected by the mortality in the controls (Abbott 1925). Treatments at each of the t h ree test temperatures had 20 replicates. Killing rate was recorded as the number of prey killed per instar per day, total number of prey ki lled per instar, and total number of prey killed per larva. Developmental time for the predator was measured in number of days to complete development from egg hatch to adulthood. Mortality of the predators was also recorded. Statistical A nalys e s Data on k illing rate and development time was analyzed using analysis of variance (ANOVA). Treatment means were compared using the Tukey Kramer HSD test. Mortality was analyzed using a C hi square test. The level of association for mortality and temperature was based on the Pearson coefficient. JMP 8 (SAS Institute Inc. 2008) with a significance level of 5% was used for all statistical analyses. Results Chrysoperla rufilabris larvae were able to complete their development at 15, 20, and 25C, but not at 10C. Firs t instars at 10C lived on average 39 6.8 d. Therefore, no results for killing rate and developmental time were obtained at this low temperature. Developmental T ime The number of days spent by C. rufilabris in each stadium and stage when fed M. ochroloma eggs decreased as temperature increased (Table 31). The time spent to develop from egg hatch to adult emergence decreased from 75 d at 15C to about 27 d 25

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at 25C. Mean developmental times of all instars and the pupa at 15C were significantly longer than at the other two temperatures (F=199.99; df=2,48; P<0.0001 for first instar, F=191.43; df=2,48; P<0.0001 for second instar, F=64.29; df=2,42; P<0.001for third instar, F=165.13; df=2,27; P<0.0001 for pupae, F=371.35; df=2,27; P<0.0001 for total development time). No significant difference was detected for third instar developmental time at 20 and 25C. For the first and second instars and the pupa, the predator took about 1.31.4 times longer to develop at 20C than at 25C. The number of days from egg hat ch to adult emerge of C. rufilabris larvae fed M. ochroloma first instars also decreased as temperature increased, but C. rufilabris larvae held at 15C developed more rapidly on first instars than on eggs of M. ochroloma (Table 32).The number of days spe nt in each instar and the pupal stage varied significantly among temperatures (F=146.20; df=2,57; P<0.0001 for first instar, F=136.05; df=2,49; P<0.0001for second instar, F=13.96; df=2,41, P<0.0001 for third instar, F=237.74; df=2,23; P<0.0001 for pupae, F=183.79, df=2,23, P<0.0001 for total development). There was no significant difference in developmental time of third instars between 20C and 25C. For the other stages, the predator took 70% longer to develop from egg hatch to adult stage at 15C than a t 20C, which was 48% longer than at 25C. A significant difference was detected for time of development between larvae fed with eggs and larvae fed with first instar M. ochroloma at all three temperatures (F=133.12; df=1,3; P=0.0014 at 15C, F=7.8; df=1,2 0; P=0.011 at 20C, F=27.66; df=1,27; P<0.0001). Mean developmental time of C. rufilabris fed eggs at 15C was 40% longer than the mean developmental time of the predators fed larvae. At 20 and 25C, the mean developmental time of the predators fed eggs was 9% and 24% longer, respectively, than it was for predators fed larvae. 26

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Mortality The number of individuals of C. rufilabris that completed development feeding on eggs and first instars of M. ochroloma was reduced at the lowest temperature (Fig. 31). Th e Pearson coefficient of association showed a relationship between mortality and temperature ( X2=19.6; df=2; P<0.0001for C. rufilabris fed on eggs; X2=12.35; df=2; P=0.0021 for C. rufilabris fed on larvae). The highest mortality occurred during the pupal stage for all temperatures regardless the type of prey offered. When C. rufilabris was fed M. ochroloma eggs, only 10% of the initial cohort reached adulthood at 15C, whereas at 20 and 25C, 65% and 75% survived, respectively (Fig. 31a). For C. rufilabris fed M. ochroloma first instar, the highest mortality occurred at 15C, at which only 15% of individuals pupated whereas 45% and 70% reached pupal stage at 20 and 25C, respectively (Fig 31b). For those individuals that emerged from the pupa, only two at 15C, none at 20C, and two at 25C became adults; all other individuals died as pharate adults (Fig. 32). Killing Rate The number of M. ochroloma eggs killed per day by C. rufilabris larvae increased as the temperature and instar increased (Table 33) One way ANOVA detected significant differences among temperatures for the mean numbers of prey killed per day per instar (F= 21.79; df= 2,49; P<0.0001 for first instar; F=12.99; df=2,46; P<0.0001 for second instar; F=15.62; df=2,41; P<0.0001 for third instar). The mean daily killing rates of the second and third instar at 20 and 25C were not significantly different, but they were significantly higher than the mean daily killing rate of both stages at 15C. First instar predators killed on average about 34 prey eggs per day at 20 and 25C, but less than three per day at only 15C. Second instar predators killed on average about 1011 27

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prey eggs at the two highest temperatures, but no more than six per day at 15C. Third instar predators killed on average a bout 66 prey eggs per day at 20 and 25C but less than 32 per day at only 15C. The number of M. ochroloma first intars killed per day by C. rufilabris larvae increased as the temperature and instar increased (Table 34). Significant differences for mean daily killing rate of first instar prey per predator instar were found among temperatures (F= 18.59; df=2, 57; P<0.0001 for first instar, F=13.34; df= 2,49; P<0.0001 for second instar, F=24.97; df=2,41; P<0.0001 for third instar). The mean daily killing rat e of first instar prey by second instar predators did not differ between 20 and 25C. However, the mean number of prey killed per day by predator larvae held at those two temperatures was significantly greater than at 15C. The number of prey killed per day increased with temperature; the predator killed about two times more prey per day at 25C than at 15C. The total number of M. ochroloma eggs killed per C. rufilabris larvae at 15C, 20C and 25C decreased as the temperature increased but increased fro m first to third instar (Table 35). The mean total number of prey eggs killed per instar and per larva was significantly different among temperatures (F=20.96; df=2,49; P<0.0001 for first instar, F=8.18; df=2,46; P<0.0001 for second instar, F=4.99; df=2,4 6; P=0.0113 for third instar, F=4.86; df=2,42; P=0.0127 for larval stage). The number of prey eggs killed at 20C did not differ significantly from the number killed at 25C. However, the total killing rate per instar and per larva was significantly higher at 15C compared to 25C. First, second, and third instar predators killed, on average, 35, 65, and 574 prey eggs, respectively, at 15C, whereas they killed, on average, about 20, 47, and 471 prey eggs, 28

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respectively, at 20 and 25C. Chrysoperla rufilabri s killed, on average, about 90 more prey eggs at 15C than at 20 and 25C during its larval stages. The mean total number of prey killed per third instar predator and per larva for all three instars was not significantly different among temperatures (Table 3 6). Significant differences were detected for first and second instar predators among temperatures (F=4.58; df=2,55 P=0.0144 for first instar, F=3.31; df=2,48; P=0.0450 for second instar). The mean total number of prey killed for both early predator ins tars was not significantly different between 15 and 20C and 20 and 25C. However, the total number of prey killed at 15C was about 1.3 times higher than at 25C for first and second instar predators. The total number of prey killed per C. rufilabris larv a at all three temperatures ranged from 218 to 225. Discussion One of the first steps in a biological control program is evaluation of the suitability of the target pest as prey for the natural enemy to be released. It is important to know the number of pr ey that the predator is able to consume or kill and the effect of the prey on the development and survival of the predator. Developmental time of C. rufilabris was significantly affected by temperature. The number of days spent in each stadium and the tota l time from egg hatch to adult emergence decreased at higher temperatures (Tables 3 1 and 32). For the most part, an insects body temperature varies with environmental temperature, when it increases the metabolism of the insect speeds up, and, consequent ly, the rate of development also increases, causing a reduction in the length of each stage (Jervis and Kidd 1996). Developmental time can also be affected by the quality of the larval food (Canard et al. 1984). Chrysoperla rufilabris spent more time developing from egg hatch to adult 2 9

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when it fed on eggs than it did when it fed on first instars of M. ochroloma. Montemayor and Cave (2011) found that the developmental time of P. maculiventris was significantly shorter when the predator fed on fourthinstar M. ochroloma than when it fed on eggs. Chrysoperla rufilabris development was significantly prolonged when it was fed with T. castaneum pupae and prepupae than when fed eggs and larvae of the potato tuber moth, Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae) (Hydorn and Whitcomb 1979). Legaspi et al. (1994) found that developmental time of C. rufilabris larvae feeding on the sweetpotato whitefly, Bemisa tabaci (Gennadius) (Hemiptera: Aleyrodidae), was longer than for those larvae feeding on lepid opteran prey. Temperature also influences the survival of the predator. Chrysoperla rufilabris was not able to complete development at 10C when feeding on eggs of M. ochroloma. eggs of different species of Chrysopidae range from 6.1 to 10C with an average of 8.9 1.4C, so the fact that C. rufilabris larvae died at 10C indicates that the lower temperature threshold for larvae might be close to that value. My results showed that the survival of the predator is reduced at lower temperatures. The quality of prey also plays an important role in the predators survival. Some prey might be nutritionally inadequate, resulting in high mortality or abnormal development of the predator ( Canard et al. 1984). At 25C, 75% of the predators fed eggs of M. ochroloma reached the pupal stage and all of them reached adulthood. In the case of the predators fed with first instar M. ochroloma, 70% pupated, of which only 13% became adults. When C. ru filabris larvae were fed the aphids M. persicae and A. gossypii 100% of individuals survived to adulthood, whereas all predators fed Lipaphis erysimi Kaltenbach (Hemiptera: Aphididae) died before adult emergence (Chen and Liu 30

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2001). Chrysoperla rufilabris larvae provided with eggs and larvae of B. tabaci never pupated (Legaspi et al. 1994), and the survival of the predator was significantly less for individuals reared on T. castaneum than for those given any other prey (Hydorn and Whitcomb 1979). The quali ty of a prey can be highly influenced by their diet. Specialized herbivores develop many adaptations that allow them to resist, metabolize, or assimilate secondary plant substances (Francis et al. 2001). These adaptations vary from chemical modification of toxins into a nontoxic compounds to sequestration of toxins as defense substance against natural enemies. Plants of the family Brassicaceae are known to synthes ize glucosinolates as secondary metabolites (Kjr 1981, Fenwick et al. 1982, Fahey et al. 2001) The degradation of glucosinolates by an enzyme present in specialized plant cells leads to the production of different compounds that act as infochemicals in plant insect interactions and can also have toxic effects against insects or fungi (Hopkins et al. 2009). It is unknown if M. ochroloma is able to sequester glucosinolat es from its host plant and use them as defens ive compound. H owever, it might explain the higher mortality and inability of the predator to develop when fed M. ochroloma first instars Francis et al. (2001) reported that Adalia bipunctata L. (Coleoptera: Coccinellidae) was unabl e to reach the adult stage and higher mortality was observed when fed Brevicoryne brassicae L. (Hemiptera: Aphididae) compared to individuals fed a nonspeciali st aphid reared on two mustard species. Mortality was related to the presence of isothiocyanates, a product of glucosinolate degradation. Chrysoperla rufilabris readily consumed eggs and first instars of M. ochroloma. The number of prey killed per day incr eased with temperature; the predator consumed twice as many prey at 25C as it did at 15C (Tables 33 and 34). A similar pattern was 31

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reported for the generalist predator P. maculiventris (Montemayor and Cave 2011).The rate at which food passes through th e gut is affected by temperature (Jervis and Kidd 1996). At high temperatures, food is processed faster and more prey are needed to fill the gut. An empty gut directly affects the predators hunger, forcing it to consume more food, which translates to kill ing more prey. When the total number of prey killed was compared among temperatures, the predator killed more eggs per instar and larval stage at 15C than at 20 and 25C, which can be attributed to the longer time required by the predator to complete dev elopment at the lowest temperature (Tables 35 and 3 6). Montemayor and Cave (2011) observed similar results for the total number of prey killed by P. maculiventris ; the predator killed a more M. ochroloma larvae at 15C than at 20C. Podisus maculiventris killed 50% more eggs of M. ochroloma during its developmental period at 25C than did C. rufilabris One possible explanation for this difference between the two predators is the dissimilarity in the life history phases of these two predators. Despite hav ing similar total developmental times (27 d for C. rufilabris 23 d for P. maculiventris ), P. maculiventris goes through five instars and molts directly to an adult without going through a pupal stage, whereas C. rufilabris only goes through three instars and then molts to a pupal stage in which it ceases feeding. Therefore, I conclude that P. maculiventris has greater potential as a biological control agent for M. ochroloma eggs and larvae than does C. rufilabris Similar results were found for first and second instars of C. rufilabris fed first instar M. ochroloma; however, for the last instar and the total killing rate for larvae, the average total number of prey killed did not differ among temperatures (Table 36). The inability of C. rufilabris to com plete development when fed first instar M. ochroloma suggests that the prey might be nutritionally inadequate. A diet of M. 32

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ochroloma eggs allowed the predator to reach adulthood, which, along with the high number of eggs that the predator killed, suggests that the predator might have potential to control the egg population of M. ochroloma in crucifer crops. However, it is necessary to determine if the predator will search for and consume eggs under natural conditions. It is also recommended to evaluate the effect on the biology and survival of the predator when it is fed mixed diets, including eggs and larvae of M. ochroloma and other soft bodied arthropods that it might encounter on crucifers. The best seasons to release this predator in Florida for the co ntrol of M. ochroloma are late fall and early summer when temperatures are warmer. Thus, performance of the predator should be evaluated at fluctuating temperatures and under field conditions. 33

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Table 31. Mean ( SE) number of days for development of Ch rysoperla rufilabris larvae feeding on Microtheca ochroloma eggs at three constant temperatures. Number within parentheses equals sample size. Chrysoperla rufilabris stage Temperature (C) 1st 2nd 3rd Pupa Total 15 15.5 0.5 (14 ) a 10.5 0.3 (14) a 15.9 0.7 (11) a 36.0 1.3 (2) a 75.5 1.7 (2) a 20 5.7 0.4 (19) b 5.2 0.2 (18) b 7.6 0.6 (16) b 16.8 0.5 (13) b 34.7 0.7 (13) b 25 4.3 0.4 (19) c 3.9 0.2 (18) c 6.1 0.5 (18) b 11.6 0.5 (15) c 26.6 0.6 (15) c Means followed by the same lowercase letter within a column are not significantly different ( P > 0.05). Table 32. Mean ( SE) number of days for development of Chrysoperla rufilabris larvae feeding on first instar Microtheca ochroloma at three constant temperatures. Number within parentheses equals sample size. Chrysoperla rufilabris stage Temperature (C) 1st 2nd 3rd Pupae Total 15 10.2 0.3 (20) a 7.6 0.2 (15) a 10.2 0.7 (14) a 29.0 0.8 (3) a 54 .0 1.6 (3) a 20 5.6 0.3 (20) b 4.4 0.2 (17) b 6.8 0.7 (13) b 14.3 0.4 (9) b 31.7 0.9 (9) b 25 3.3 0.3 (20) c 3.0 0.2 (20) c 5.4 0.6 (17) b 10.2 0.4 (14) c 21.4 0.7 (14) c Means followed by the same lowercase letter within a column are not significantly different ( P > 0.05). Table 33. Mean ( SE) number of Microtheca ochroloma eggs killed per day per Chrysoperla rufilabris larva at three constant temperatures. Number within parentheses equals sample size. Chrysoperla rufilabris instar Temperature (C) 1st 2nd 3rd 15 2.3 0.2 (14) a 6.3 0.9 (12) a 32.3 5.4 (10) a 20 3.3 0.2 (19) b 9.7 0.7 (18) b 66.9 4.1 (16) b 25 4.2 0.2 (19) c 12.1 0.7 (19) b 65.4 3.9 (18) b Me ans followed by the same lowercase letter within a column are not significantly different ( P > 0.05). 34

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Table 34 Mean ( SE) number of Microtheca ochroloma first instars killed per day per Chrysoperla rufilabris larva at three constant temperatures. Num ber within parentheses equals sample size. Chrysoperla rufilabris instar Temperature (C) 1st 2nd 3rd 15 1.3 0.2 (20) a 3.6 0.4 (15) a 18.8 2.4 (14) a 20 2.0 0.2 (20) b 5.7 0.4 (17) b 29.4 2.5 (13) b 25 2.8 0.2 ( 20) c 6.4 0.4 (20) b 41.9 2.2 (17) c Means followed by the same lowercase letter within a column are not significantly different ( P > 0.05). Table 35 Mean ( SE) total number of Microtheca ochroloma eggs killed per Chrysoperla rufilabris larva at three constant temperatures. Number within parentheses equals sample size. Chrysoperla rufilabris instar Temperature (C) 1st 2nd 3rd Total 15 35.5 2.1 (14) a 64.6 3.9 (12) a 573.8 39.8 (11) a 633.9 38.5 (10) a 20 20.5 1.8 (19) b 49.8 3.9 (18) b 520.7 33.0 (16) ab 592.1 30.5 (16) ab 25 18.8 1.8 (19) b 44.8 3.1 (19) b 422.2 31.1 (18) b 496.1 28.7 (18) b Means followed by the same lowercase letter within a column are not significantly different ( P > 0.05). Table 36. Mean ( SE) total number of Microtheca ochroloma first instar s killed per Chrysoperla rufilabris larva at three constant temperatures. Number within parentheses equals sample size. Chrysoperla rufilabris instar Temperature (C) 1st 2nd 3rd Total 15 12.6 0.8 (18) a 27.5 2.2 (15) a 178.8 16.7 (14) a 217.7 18.3 (14) a 20 11.5 0.7 (20) ab 23.7 2.1 (16) ab 195.8 17.3 (13) a 224.8 18.9 (13) a 25 9.4 0.7 (20) b 20.1 1.9 (20) b 186.3 16.1 (15) a 218.5 17.6 (15) a Means followed by the same lowercase letter within a column are not significantly different ( P > 0.05). 35

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Figure 31 Percentage of Chrysoperla rufilabris that survived to the next developmental stage at three constant temperatures when fed M. ochroloma eggs (a) and first instars (b). Initial cohort consisted of 20 first instar predators. 36

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Figure 32. Dead Chrysoperla rufilabris pharate adult after pupal eclosion. Photo b y A ngie Nio. 37

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CHAPTER 4 PREY PREFERENCE OF CHRYSOPERLA RUFILABRIS (N EUROPTERA: CHRYSOPIDAE) BETWEEN IMMATURE STAGES OF MICROTHECA OCHROLOMA (COLEOPTERA: CHRYSOMELIDAE) AND MYZUS PERSICAE (HEMIPTERA: APHIDIDAE) Introduction The production of crucifers o n organic farms in the southeastern US is threatened by the presence of the yellowmargined leaf beetle, Microtheca ochroloma Stl (Coleoptera: Chrysomelidae) This insect, indigenous to Argentina, was first detected in U nited States in 1945 (Chamberlin and Tippins 1948) and is now established in several states, including Texas, Louisiana, Mississippi, Alabama, Georgia, North Carolina, California, and Florida. Microtheca ochroloma consumes the foliage of plants of the family Brassicaceae (Chamberlin and Tippins 1948, Woodruff 1974, Ameen and Story 1997b, Bowers 2003, Balusu and Fadamiro 2011) and is active during late fall, winter, and early spring (Ameen 1996, Bowers 2003), which coincides with the time of crucifer production in Florida. The use of synthetic insecticides is the only effective method to control this pest (Menezes Jr. et al. 2005). The use of synthetic insecticides is not allowed in organic farming, however, growers require the development of methodologies that incorporate ecological approaches for the management of pest problems (Ferguson 2004, Zehnder et al. 2007). Biological control might have an important role as a potential alternative to control pests in organic farms, but no host specific natural enemies of this pest have been reported in Florida. Therefore, it is necessary to evaluate the potential of nat ive natural enemies that can be used against M. ochroloma. The green lacewing, Chrysoperla rufilabris Burmeister (Neuroptera: Chrysopidae) was observed preying on the M. ochroloma on crucifers in organic farms in Florida (Montemayor and Cave 2009). The green lacewing larva, which is the 38

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predacious stage, feeds on small, soft bodied arthropods (Tauber et al. 2000). Laboratory bioassays confirmed that C. rufilabris feeds on eggs and larvae of M. ochroloma and that it is able to complete its development fee ding on eggs of this pest (see Chapter 3). However, even if a prey enables adequate survival and development of the predator, it must also coexist in time and space with the natural enemy (Canard et al. 1984). U n der natural conditions, M. ochroloma adults lay eggs at the base of the plant, on the soil, or under fallen leaves, and it is not known if the predator will look for and prey on them in these sites. It is also important to assess the effect that the presence of other insect pests might have on the performance and consumption rate of the predator. Chrysoperla rufilabris feeds on a wide variety of insects (Hydorn and Whitcomb 1979, Canard et al. 1984, Nordlund and Morrison 1990, Nordlund 1991, Legaspi et al. 1994, Tauber et al. 2000) but is usually considered to be an important predator of aphids. No information is available about the effect that the presence of aphids might have on the killing rate of immature stages of M. ochroloma. Therefore, the objective of this study was to evaluate the prey selec tion preference of C. rufilabris when exposed to immature stages (eggs and larvae) of M. ochroloma and the green peach aphid, Myzus persicae Sulzer (Hemiptera: Aphididae) Materials and Methods Plant Material Turnip and bok choy ( Brassica rapa L ) were gr own as described in Chapter 3. Bok choy plants used in the experiments had six true leaves. Stock C olonies Adults and larvae of M. ochroloma were collected in the field and used to establish the laboratory colony as described in Chapter 3. 39

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Bok choy plants infested with all stages of M. persicae were confined in Bug Dorms (60 60 60 cm). Dead plants were replaced with new, clean plants when necessary. Adults of C. rufilabris were purchased from RinconVitova Insectaries, Inc. (Ventura, CA) and a colony was established in the laboratory as described in Chapter 3. Experimental Design Predation on M. ochroloma E ggs and L arvae on H ost P lants A bok choy plant was confined within a white insect rearing sleeve ( 60 cm wide 70 cm long ) (Fig. 4 1). On the plant, a first instar C. rufilabris was offered one of the following prey items: (1) M. ochroloma eggs ; (2) M. ochroloma eggs and first instars ; or (3) M. ochroloma first instars. Each treatment had 10 replicates. Treatments with eggs or larvae only had 18 individuals per plant and treatments in which eggs and larvae were offered together had nine individuals of each type per plant. Eggs of M. ochroloma were placed at the base of the plant and on the soil around the base of the plant, whereas M. ochroloma l arvae were placed on the leaf blades. The predator larvae were placed on the leaves of the plants. Chrysoperla rufilabris larvae were starved at least 12 hours prior to experimentation, with only water provided via a moistened cotton ball. All cages were k ept in a rearing room at constant 25oC, with 75% RH and 12L: 12D photoperiod. The number of prey killed was counted at the end of 48 hours. Prey P reference among M. ocholoma Eggs and Larvae and M. persicae N ymphs in a Petri Dish An individual first insta r of C. rufilabris was housed in a Petri dish (diameter: 5.5 cm) with a hole in the top sealed with a screen mesh cloth Predator larvae were starved at least 12 hours before experimentation; water was provided through a 40

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moistened cotton ball. A piece of moistened, white filter paper ( 5.5 cm diameter ) was placed at the bottom of each Petri dish to maintain appropriate humidity. A 3cm2 section of bok choy leaf was added as a food source or substrate for the prey Four combinations of prey were offered to f irst instar C. rufilabris : (1) M. ochroloma eggs and first instars; (2) M. ochroloma eggs and M. persicae nymphs; (3) M. ochroloma first instars and M. persicae nymphs; and (4) all three types of prey together. Another three treatments were included in whi ch only one of the three types of prey was offered to the predator. Natural mortality of the prey was recorded for each combination by holding a fixed number of prey in a Petri dish without the predator. There were seven and three replicates for each treat ment with predators and control treatments without predators, respectively. The number of prey offered varied according to the type of prey. For treatments where a single prey type was provided, 18 individuals were offered. For treatments where two or three prey types were provided, 9 and 6 individuals of each type were offered, respectively. The Petri dishes were kept in environmentally controlled chambers at constant 25oC, with 75% RH and 12L: 12D photoperiod. The number of prey killed was counted at th e end of 24 hours. Statistical Analyse s For each experiment, the mean number of prey killed was compared with a oneway ANOVA. Prey preference data was analyzed using G test for goodness of fit (Sokal and Rohlf 1995). In treatments in which three types of prey were offered, means were separated with a modified Tukey test for proportional data (Elliott and Reisch 2006) 41

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Result s Predation on M. ochroloma Eggs and Larvae on Host Plants Chrysoperla rufilabris first instars were able to find and prey on both eg gs and first instars of M. ochroloma on bok choy plants. Given a choice, the predator attacked more M. ochroloma first instars eggs (G= 17.47; X2(0.05, 1) = 3.84; P<0.001). Chrysoperla rufilabris preyed on a higher proportion of larvae (3.70.6) than eggs (0.70.6) of M. ochroloma within 48 hours. All eggs killed were located at the base of the plant; none of the eggs placed on the soil, close to the base of the plant, were killed or damaged. Oneway ANOVA did not detect significant differences (F=0.087; df =1, 16; P=0.77 for eggs, F=0.03, df=1, 16; P=0.86 for larvae) among the treatments with a single prey type offered and the treatment with both prey types offered for the number of prey killed. Prey P reference among M. ocholoma E ggs and L arvae and M. persi cae N ymphs in a Petri D ish First instars of C. rufilabris preyed on eggs and first instars of M. ochroloma and nymphs of M. persicae when offered singly or in combination (Table 41). For the treatments in which only two prey types were offered, nymphs of M. persicae were always preferred over eggs or larvae of M. ochroloma (G= 20.58; X2(0.05, 1) = 3.84; P<0.001). When beetle eggs and larvae were offered as prey without aphids, the predator did not show a preference (G= 0.043; X2(0.05, 1) = 3.84; P=0.83). The killing rate of M. persicae nymphs by C. rufilabris was higher than that of eggs and larvae of M. ochroloma. In the paired prey treatments, first instar C. rufilabris killed 5.5 times more aphid nymphs than beetle eggs or larvae. 42

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When the predator was given three options of prey, a significant preference was detected (G= 19.50; X2(0.05, 1) = 3.84; P<0.001). The Tukey test showed that M. persicae nymphs were preferred over eggs and larvae of M. ochroloma ( X2= 30.84; df= 2, 125; P<0.0001; Tukeys q= 6.89 and 6.48 for nymphs vs. eggs and nymphs vs. larvae respectively; q0.05= 3.31). First instar C. rufilabris killed 4.5 and 4.0 times more M. persicae nymphs than eggs and larvae of M. ochroloma, respectively (Table 41). The predator killed twice as many eggs and larvae of M. ochroloma when provided separately than it did when these two prey were provided together. The killing rate of eggs and larvae decreased about 73% when each prey was provided along with M. persicae nymphs. Discussion Plant morpholog y has an important effect on the foraging behavior of Chrysopidae (Clark and Messina 1998). Patterns of searching behavior and predator consumption rates change considerably when predator and prey interact on a whole plant compared to less complex environm ents ( e.g Petri dishes) (Reynolds and Cuddington 2012b, a). When C. rufilabris first instars were fed eggs and larvae of M. ochroloma placed on a homogeneous arena ( e.g. a bok choy leaf piece in a Petri dish) (see Chapter 3), the predator killed 89% and 40% more eggs and larvae, respectively, in 24 hours than the number of eggs and larvae killed when placed on a whole bok choy plant. This is similar to the observations of Reynolds and Cuddington (2012a) who reported that C. carnea consumed more pea aphids Acyrthosiphon pisum Harris (Hemiptera: Aphididae), when housed in a Petri dish than on whole plants. Such differences might be explained by the fact that eggs were placed at the base of the plant or on the soil, natural oviposition sites, where apparentl y they are less accessible or 43

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conspicuous to the predator. In the case of first instars of M. ochroloma, the predator has to explore a larger area, so a more time is required to locate the prey, which, together with the time needed to kill and consume it, reduces the time available to find and kill other prey. In my study, when both prey were offered simultaneously on a plant, the predator showed a preference for larvae, killing a higher number of first instars than eggs. The reason for this preference may be attributed to the intraplant distribution of the predator in the field. Chrysopid larvae are usually found on the leaves of the plant; only 5% of chrysopids are collected from the soil surface compared to 95% of hemerobiids (Szentkirlyi 1986). Accordi ng to Canard et al. (1984), an optimal prey type must coexist in space with the predator. In the field, C. rufilabris might not provide good control of eggs of M. ochroloma, which are laid mainly on the soil and fallen leaves or at the base of the plant. Prey preferences of C. rufilabris are affected by the type of prey offered and the age of the predator. Chrysoperla rufilabris has been reported feeding on a wide variety of soft bodied arthropods (Hydorn and Whitcomb 1979, Canard et al. 1984, Nordlund and Morrison 1990, Nordlund 1991, Legaspi et al. 1994, Tauber et al. 2000). However, this predator has been used mainly in biological control of aphids. My results showed that when immature stages of M. ochroloma were offered as prey along with aphids in the confined space of a Petri dish, C. rufilabris first instar consumed unequal proportions of each prey, preferring aphids in all cases. Nordlund and Morrison (1990) evaluated the preference of second and third instars of C. rufilabris when provided with eggs and larvae of the tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), and nymphs and adults of the cotton aphid, A. gossypii The predator preferred tobacco budworm larvae over cotton aphids and aphids were preferred over H. virescens eggs 44

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When second and third instars of C. carnea were offered Pieris brassicae L. (Lepidoptera: Pieridae) eggs and larvae and Brevicoryne brassicae L. (Hemiptera: Aphididae) nymphs, C. carnea second instars preferred aphids over lepidopteran eggs and larvae, w hereas the third instar preferred P. brassicae larvae over aphids and aphids over P. brassicae eggs (Huang and Enkegaard 2010). For a biological control program to be successful when generalist predators are used, it is important that the target pest be am ong the preferred prey (Nordlund and Morrison 1990). First instars of C. rufilabris did not choose any of the immature stages of M. ochroloma over aphids, however, it is not known if the prey preference of second and third instars of C. rufilabris is the s ame as that of first instars. In my study, the number of eggs and larvae of M. ochroloma killed by first instar C. rufilabris was reduced about 73% in the presence of M. persicae nymphs. Similar results were reported by Huang and Enkegaard (2010) who fou nd that predation on eggs of P. brassicae was completely absent or reduced by 80% in the presence of B. brassicae when offered to secondand thirdinstar C. carnea respectively. Secondinstar C. carnea consumed fewer P. brassicae larvae in the presence o f aphids. Despite the preference of first instar C. rufilabris for M. persicae nymphs over immature stages of M. ochroloma in the laboratory, it is important to evaluate the predation on these two types of prey in the field. A low population of aphids on bok choy plants in the field might not have a great impact on the killing rate of immature stages of M. ochroloma by the predator. Additionally, the presence of aphids in the crop can be beneficial for biological control by lacewings. In many cases, a low or moderate populations of other prey allows the predator to survive when numbers of the target pest are low (Ables et al. 1978). Studies have also showed that many species of the family 45

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Chrysopidae are attracted by aphids sex pheromones (Boo et al. 2003, Zhu et al. 2005, Koczor et al. 2010). According to Kunkel and Cottrell (2007), C. rufilabris laid more eggs on pecan seedlings infested with aphids than on uninfested plants. 46

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Table 41. Mean number ( SE) of Microtheca ochroloma eggs and first instar s a nd Myzus persicae nymphs killed by first instar Chrysoperla rufilabris Treatments Prey killed N L E N L N E E L N L E Nymphs (N) 3.8 0.4 a 4.7 0.5 a 4.7 0.6 a 7.6 0.8 Larvae (L) 1.0 0.4 b 0.9 0.5 b 1.6 0.4 a 3.4 0.4 Eggs (E) 0.8 0.4 b 0.9 0.6 b 1.7 0.4 a 3.4 0.4 Means followed by the same lowercase letter within a column are not significantly different ( P > 0.05). Sample size equals 7 for all treatments 47

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Figure 41. Bok choy plant confined in an insect rearing sleeve. A bamboo stick was used to support the sleeve and rubber bands around the edge of the pot prevented insect escape. Photo b y Angie Nio. 48

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CHAPTER 5 PERFORMANCE OF CHRYSOPERLA RUFILABRIS (BURMEISTER) (NEUROPTERA: CHRYSOPIDAE) FEEDING ON MICR OTHECA OCHROLOMA (STL) (COLEOPTERA: CHRYSOMELIDAE) LARVAE UNDER FIELD CONDITIONS IN AN ORGANIC FARM Introduction The invasive species Microtheca ochroloma, commonly known as the yellowmargined leaf beetle, is considered one of the most detrimental pests i n crucifer crops. This beetle feeds on economically important vegetables belonging to the Brassicaceae family, including cabbage ( Brassica oleracea L. var. capitata), napa cabbage ( B. pekinensis Lour.), collard ( B. oleracea var acephala), mustard ( B. junc ea Cosson), radish ( Raphanus sativus L.), mizuna, mibuna ( B. rapa subsp. japonica, Japanese leafy vegetables), watercress ( Nasturtium officinale L.), and turnips ( B. rapa L.), the latter being the most preferred (Chamberlin and Tippins 1948, Woodruff 1974, Ameen and Story 1997b, Bowers 2003, Balusu and Fadamiro 2011). The first M. ochroloma specimen reported in the United States was intercepted in Louisiana in a shipment of grapes coming from Argentina (Chamberlin and Tippins 1948). Now, M. ochroloma is es tablished in several southern states, including Florida, where it is active during the coolest months of the year, October through April, the primary production season for crucifers in the state (Bowers 2003). Efforts to develop strategies that reduce the economic impact of this pest and maintain populations at tolerable levels have not been fruitful. Restriction by the National Organic Program on the use of synthetic insecticides (Ferguson 2004, Nguyen et al. 2008), and the lack of specialist natural enemi es in Florida, leave organic growers without alternatives to control M. ochroloma (Bowers 2003). A promising strategy for the management of this pest is the manipulation of native generalist predator populations. 49

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One of the predators found by Montemayor a nd Cave (2009) preying on M. ochroloma in the field is the green lacewing, C hrysoperla rufilabris Burmeister (Neuroptera: Chrysopidae) Chrysoperla rufilabris naturally occurs in many agroecosystems in North America (McEwen et al. 2007). This generalist predator consumes small, soft bodied arthropods, including aphids, scales, whiteflies, eggs and larvae of moths and butterflies, thrips, and mites among others. This predator and the closely related species C. carnea have been used successfully to control t he Colorado potato beetle, also a chrysomelid, under laboratory conditions (Nordlund 1991, Sablon et al. 2013). In fieldcage experiments, both species also have controlled other pests effectively (Ridgway and Jones 1968, New 1975, Legaspi et al. 1994). L aboratory experiments confirmed that C. rufilabris feeds on eggs and larvae of M. ochroloma, and according to temperature conditions in Florida, the predators development when feeding on this pest might be favored during late fall and early summer which overlaps with the active season of the pest (see Chapter 3). However, the potential of C. rufilabris as a biological control agent of field populations of M. ochroloma is unknown. Therefore, the goal of this study was to evaluate the performance of C. rufil abris feeding on M. ochroloma larvae under field conditions on an organic farm. Materials and Methods Plant M aterial Bok choy seeds ( Brassica rapa L.), variety Joi choi, were seeded in 50cell trays. Four week old seedlings were transplanted individually i nto 3.8L plastic pots containing a mixture of soil and fertilizer (Osmocote classic 14N, 14P, 14K). Four weeks after 50

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transplanting, the plants were taken to the field and their pots were buried in a single row with 30 cm between adjacent plants, in a field bed at the White Rabbit Acres Organic Farm at Vero Beach, FL. Drip irrigation was provided to each plant through an inverted plastic bottle with holes in the lid. Stock C olonies Adults and larvae of M. ochroloma were collected in the field, and a colony was established at the Biological Control Research and Containment Laboratory at the Indian River Research and Educational Center in Ft. Pierce, FL (as described in Chapter 3). Adults of C. rufilabris were purchased from RinconVitova Insectaries, I nc. (Ventura, CA) and a colony was established in the laboratory (as described in Chapter 3). Experimental Design Four treatments were evaluated: (1) plants confined in cages with predators; (2) plants confined in cages without predators; (3) uncaged plant s with predators; and (4) uncaged plants without predators. For treatments 1 and 2, plants were housed inside a white insect rearing sleeve (60 cm wide 70 cm high) sealed around the rim of the buried pot and secured with elastic bands to prevent the esca pe of insects. Bamboo sticks were used to tie the cage closed at the top and provide space around the plant (Fig. 5 1a). Ten first instars of M. ochroloma were placed on each plant using a fine paint brush. In the treatments with predators, five C. rufilab ris first instars were released per plant. A second release of prey and predators was done two weeks after the first release. Each treatment had eight replicates. The experiment was conducted during 3 29 March 2013. Treatments were randomized within the bed (Fig. 5 1b). All plants were monitored every four days for four weeks, and the numbers of M. ochroloma eggs, 51

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larvae, and adults and C. rufilabris larvae per plant were recorded. Excess adults and eggs of M. ochroloma were removed to maintain similar densities of individuals on all plants. At the end of four weeks, the aboveground parts of each plant were taken to the laboratory and carefully examined. The numbers of prey and predators were counted. Each plant was gently washed and four damaged leaves w ere randomly chosen per plant to measure the leaf area removed as a result of M. ochroloma larval feeding. Each leaf was placed under a glass sheet and photographed into a digital format using an EOS Rebel T4i camera. Once all images were in digital format the level of defoliation was measured using the image analysis software Scion Image Beta as described by O'Neal et al. (2002). Then, each plant was placed in a paper bag, dried at 45C for two weeks, and the dry weight of the plant was measured on an Ohaus Scout Pro. Statistical Analyse s Number of prey found on the plants was transformed using square root. Arcsine transformation was used to convert percentage of defoliation. Untransformed data are reported in tables and figures. Means were compared using analysis of variance (ANOVA). Treatment means were separated using Tukey Kramer HSD test. JMP 8 (SAS Institute Inc. 2008) with a significance level of 5% was used for all statistical analyses. Results During the data collection period, the mean maximum t emperature was 22.9 4.3C and the mean minimum temperature was 8.7 5.2C. Six of the eight replicates for the caged treatment without predators were severely infested with aphids, which reduced plant growth and masked the effect of M. ochroloma larvae on the plants. Therefore, this treatment was excluded from the statistical analysis. 52

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During the first two weeks of sampling, the number of M. ochroloma larvae decreased with time and few eggs and adults were found in the treatments without cages (Fig. 5 2 ). After the second release of prey and predators, the numbers of M. ochroloma larvae and eggs tended to increase with time for the uncaged treatments but not the caged plants with predators. In the caged treatments, the number of individuals at each sampl ing period varied considerably due to the difficult access to some parts of the plant and the area around it, which did not allow thorough inspection of the plant. Therefore, mean comparisons among treatments were done only for the number of insects at the last sampling period. Oneway ANOVA detected significant differences in the number of larvae of M. ochroloma that survived to the last sampling on bok choy plants (F= 3.80; df= 2, 21; P=0.0391) (Table 51). Caged plants with predators had about three times fewer M. ochroloma larvae than uncaged plants with predators. In the treatment with caged plants with predators, the total number of M. ochroloma larvae placed on the plants was reduced by about 75%. The number of beetle larvae on uncaged plants did not differ between treatments. The number of C. rufilabris larvae found on the plants decreased with time for both treatments in which the predator was released (Fig. 53). Only 20% and 10% of the predators released in the treatments with and without cages, respectively, were found in the last sampling. In the treatments without cages, the C. rufilabris larvae apparently were able to move between plants. Predators were found on the cages next to the plants where they were released. Only two predators reached adulthood, both on caged plants infested with aphids. The percentage of foliar area removed by M. ochroloma was lower for caged plants with predators (Table 51). Significant differences among treatments were 53

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detected for the percentage of foliage area loss (F=8.10; df=2, 21; P=0.0025). Microtheca ochroloma consumed three times more foliage from uncaged plants without predators than from caged plants with predators. Foliage area loss did not vary between uncaged plants with and without predators. Foliage area loss on uncaged plants with predators did not differ significantly from that of caged plants with predators. Caged plants with predators had a significantly lower dry weight than uncaged plants with predators (F= 3.46; df= 2, 21; P=0.05). Dry weight of uncaged plants with predators was 1.6 times more than that of caged plants with predators (Table 51). No significant differences were detected between the treatments without cages. Dry weight for uncaged plants without predators did not differ significant ly from the dry weight of caged plants with predators. Four of eight caged plants with predators were infested with aphids and these plants had the lowest dry weight values for that treatment. Ants and lady beetles were observed preying on aphids on uncaged plants, and these predators apparently helped keep the aphid population at low numbers. Discussion When the number of larvae of M. ochroloma was compared among treatments, the lowest number of the pest were found on caged plants with predators, while the highest number was observed on uncaged plants with predators, which suggests that the isolation of prey and predators through cages played an important role. On caged plants, the predator killed on average 75% of the larvae, whereas on plants without cages, the proportion of prey killed was about 22%. The differences in predation rates might be attributed to new oviposition by female M. ochroloma which had free access to the plants without cages and which were present at every sampling period (Fig. 5 2). Although adults and eggs of M. ochroloma were removed from the plants, larvae from 54

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undetected eggs or larvae moving from nearby plants may have replaced the killed prey, or these could have allowed the prey populations on plants to reach high numbers so the predators were no longer effective at controlling them. According to Grasswitz and Burts (1995), releases of C. rufilabris were insufficient to keep densities of the green apple aphid, Aphis pomi De Geer, below damaging levels when plant growth was vigorous and aphid populations were high. Another possibility is that the predators interplant movement on caged plants was limited, whereas on uncaged plants the C. rufilabris larvae were able to move to other plants. On two occasions, I found predator s walking on the exterior of cages next to uncaged plants on which they were released. When the number of M. ochroloma larvae was compared between the treatments without cages, no significant difference was detected. If predators had any impact on M. ochr oloma populations, no evidence of additional mortality caused by the predator was apparent. Unfortunately, the two caged treatments could not be compared because the caged plants without predators were excessively infested with aphids. At one point, the p opulation of aphids was so high that the plant leaves were covered with honeydew and M. ochroloma larvae were not able to feed on the plant tissue or move among the leaves. The number of predators recorded on uncaged plants was very low (Fig. 53)., Only a bout 20% of the predators were found at the next sampling period after each release, and the numbers decreased as time passed, which explains the similarity in the number of prey present in the two uncaged treatments. A decrease in the predator numbers may have been due to a number of factors. One well known aspect is the cannibalistic behavior displayed by C. rufilabris The sibling species C. carnea showed strong cannibalistic tendencies as a response to food deprivation; this behavior allows 55

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neonate larv ae to survive when food is scarce (Duelli 1981). Another factor might be death by starvation. When the prey population is low, the predator might not be able to easily find sufficient prey. Many times I noticed that M. ochroloma larvae dislodged from the plant when disturbed, which helped them avoid predation. Knutson and Tedders (2002) demonstrated that starvation of first instar C. rufilabris is an important source of mortality. Environmental conditions also play a very important role in the population dy namics of prey and predators. The mean minimum temperature during the experiment was 8.7C, which is below the lower developmental threshold reported for C. downesi and C. harrissi larvae (McEwen et al. 2007). Temperatures below the mean minimum temperatur e were registered on 20 days during the sampling period, which may have decreased survivorship of M. ochroloma larvae. Defoliation was lowest in plants with predators confined in cages. In that treatment, the defoliation was a result of feeding by M. ochro loma larvae only, and as the predator killed prey the damage did not increase significantly. On the other hand, in the treatments without cages, defoliation occurred by feeding of M. ochroloma larvae and adults. The predators might have been able to prevent damage caused by larvae by killing them, but they were not able to control adults because C. rufilabris larvae only feed on soft bodied arthropods (Canard et al. 1984). Dry weight of bok choy plants was lower for caged plants with predators than for unc aged plants with predators. In this case, the cage might have restricted plant growth by limiting space and sunlight to the plant. The cage also limited access to other natural enemies that control aphids. Ants and ladybeetles were found preying on aphids on uncaged plants, whereas on caged plants the only predators present that could control aphids were C. rufilabris As a result, four plants confined in cages were highly infested 56

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with aphids. The dry weights of these plants were the lowest among the repli cates for that treatment. Aphids on crucifers cause yellowing and curling of leaves, and plants may be stunted or die (Webb 2010). Further studies are needed to determine the effectiveness of C. rufilabris in organic farms. These studies should include the dispersal behavior of the adults. Species of Chrysoperla display two dispersal strategies: preoviposition flight and nocturnal movement during the reproductive period (McEwen et al. 2007). According to Duelli (1984), C. carnea undergoes obligatory flight after emergence before reacting to stimuli like food or oviposition sites that extend its dispersal capacity in large areas. Moreover, once the reproductive period starts, C. carnea flies and disperses during most nights of its life unless temperatures are too low. This dispersal behavior has been considered an important constraint to augmentative biological control by lacewings (Knutson and Tedders 2002). Releases of C. rufilabris to control M. ochroloma in organic farms probably would not be cost effectiv e for the growers. Application systems can be an important cause of mortality of the predator (Daanel and Yokota 1997) and both larvae and adults disperse. Production and shipment of this predator by suppliers is expensive, so augmentative releases would not be economically viable if constant releases are needed to reduce a pest population. A preferred strategy might be to attract and enhance oviposition by C. rufilabris adults. The use of synthetic baits and artificial food sprays has been evaluated to manipulate lacewing populations ( Hagen et al. 1976, Boo et al. 2003, Kunkel and Cottrell 2007, Koczor et al. 2010). Attracting, enhancing and conserving C. rufilabris should be investigated for management of M. ochroloma in organic crucifer production. 57

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Tab le 5 1. Mean ( SE) number of Microtheca ochroloma larvae, leaf damage percentage, and dry weight of bok choy leaves in fieldcage study. Sample size for all means was eight. Treatments Number of M. ochroloma larvae Leaf damage percentage Dry weight Pred ator cage d 5.0 2.4 a 1.6 0.2 a 18.3 3.6 a Predator uncaged 15.7 4.8 b 3.1 0.8 ab 30.1 2.7 b No predator uncaged 11.4 3.1 ab 5.0 0.7 b 22.5 3.2 ab Means followed by the same lowercase letter within a column are not sig nificantly different ( P > 0.05). Figure 51. Experimental setup of bok choy plants in the field (a) and distribution of the treatments within the bed (b). Photos b y Angie Nio. Caged plant Unc aged 58

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Figure 52. Number of Microtheca ochroloma eggs, larvae, and adults on caged and uncaged bok choy plants with and without larvae of Chrysoperla rufilabris A second release of prey and predators was done two weeks after the initial release. Second Second Second 59

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Figure 53. Number of Chrysoperla rufilabris larvae on caged and un ca ge d bok choy plants. Second 60

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CHAPTER 6 CONCLUSIONS The main goal of my thesis was the evaluation o f a potential biological control agent C hrysoperla rufilabris Burmeister (Neuroptera: Chrysopidae) for management of the yellowmargined leaf beetle, M icrot heca ochroloma Stl (Coleoptera: Chrysomelidae) o n organic farms. I concentrated on three main aspects: (1) the suitability of immature stages (eggs and first instars) of M. ochroloma for the development and survivorship of the predator and its prey killi ng rate at three temperatures; (2) prey preference among eggs and larvae of M. ochroloma and aphid nymphs ; and (3) performance of the predator in the field feeding on larvae of M. ochroloma. C hrysoperla rufilabris is able to develop at temperatures between 15C and 25C, but a high mortality was registered at 15C. The number of days spent by C. rufilabris from egg hatch to adult emergence decreased from 75 d and 54 d at 15C to about 27 d and 22d at 25C when fed eggs and first instars of M. ochroloma, re spectively. The m ean developmental time of C. rufilabris fed eggs at 15C was 40%, at 20C was 9% an d at 25C was 24% longer than the mean developmental time of the predators fed larvae. The number of M. ocholoma eggs and first instars killed daily per instar of C. rufilabris increased with temperature. The predator killed twice as many prey daily a t 25C as it did at 15C However, the total number of eggs of M. ocholoma killed per C. rufilabris larvae was higher at 15C compared to 20 and 25C because the predator took more time to develop. When the predator was fed with first instar M. ochroloma, the total number of larvae killed was similar regardless of temperature. The survival of 61

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the predator was reduced at lower temperatures; only 10 and 15% of the initial cohort reached pupal stage at 15C, whereas 75% and 70% did at 25C when fed eggs and larvae of M. ochroloma, respectively. Pupae of C. rufilabris reared on a diet consisted only on M. ochroloma eggs, emerged as adults; whereas only a lower proportion of pupae fed first instar M. ochroloma reached adulthood. Chrysoperla rufilabris did not exhibit any preference when eggs and first instars of M. ochroloma were offered simultaneous ly in P etri dishes. However, when the predator was given a choi ce between immature stages (eggs and first instars) of M. ochroloma and nymphs of M. persicae, it preferred aphids in all cases. O n a whole plant infested only with M. ochroloma eggs and first instars, C. rufilabris killed more first instars than eggs whi ch might be related to the location of the prey on the plant The predator spends more time searching for prey on the leaves of the plant which is where M. ochroloma larvae are usually found, whereas M. ochroloma eggs are usually oviposited at the base of the plant or on the surrounding soil. In the fieldcage experiment, differences in the number of M. ochroloma among treatments were more related to the effect caused by the cages. If predators had any impact on M. ochroloma populations, no evidence of additional mortality cau sed by the predator was evident. The number of predators found on the plants after initial release decreased with time. Only about 20% of the predators were founded at the next sampling period after release. Based on my results, releas es of C. rufilabris to control M. ochroloma in organic farms probably would not be cost effective for the growers. The poor survival of the predator when fed M. ochroloma first instars, and the fact that none of the immature 62

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stages of M. ochroloma were pre ferred by the predator, suggests that this predator might not p rovide satisfactory control of this pest. Production and shipment of C. rufilabris by suppliers is expensive, so augmentative releases would not be economically viable if constant releases are needed to reduce a pest population. A preferred strategy might be to attract and enhance oviposition by C. rufilabris adults in the field. 63

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LIST OF REFERENCES Abbott, W. S. 1925. A method of computing the effectiveness of an insecticide. J. E con. E ntom ol. 18: 265267. Ables, J. R., S. L. Jones, and D. W. McCommas. 1978. Response of selected predator species to different densities of Aphis gossypii and Heliothis virescens eggs. Environ. Entomol. 7: 402404. Ameen, A. O. 1996. The biology and ecology of t he yellowmargined leaf beetle, Microtheca ochroloma Stl ,( Coleoptera: Chrysomelidae) on crucifers. PhD dissertation, Louisiana State University, Baton Rouge, LA. Ameen, A. O., and R. N. Story. 1997a. Biology of the yellowmargined leaf beetle ( Coleoptera: C hrysome lidae) on crucifers. J. Entomol Sci 32: 478486. Ameen, A. O., and R. N. Story. 1997b. Feeding preferences of larval and adult Microtheca ochroloma (Coleoptera: Chrysomelidae) for crucifer foliage. J Agr Entomol 14: 363368. Balsbaugh, E. U., J r. 1978. A second species of Microtheca Stl (Coleoptera: Chrysomelidae) found in North America. Coleopts. Bull. 32: 219222. Balusu, R. 2011. Chemical ecology and management of yellowmargined leafbeetle Microtheca ochroloma Stal (Coleoptera: Chrysomelidae). PhD dissertation, Auburn University, Auburn, AL. Balusu, R. R., and H. Y. Fadamiro. 2011. Host finding and acceptance preference of the yellowmargined leaf beetle, Microtheca ochroloma (Coleoptera: Chrysomelidae), on cruciferous crops. Environ. Entomol. 40: 1471 1477. Balusu, R. R., and H. Y. Fadamiro. 2012. Evaluation of organically acceptable insecticides as stand alone treatments and in rotation for managing yellowmargined leaf beetle, Microtheca ochroloma (Coleoptera: Chrysomelidae), in organic cruci fer production. Pest Manag Sci 68: 573 579. Boo, K. S., S. S. Kang, J. H. Park, J. A. Pickett, and L. J. Wadhams. 2003. Field trapping of Chrysopa cognata (Neuroptera: Chrysopidae) with aphid sex pheromone components in Korea. J Asia Pacific Entomol 6 : 29 36. Bowers, K. 2003. Effects of within field location of host plants and intercropping on the distribution of Microtheca ochroloma (Stl) in mizuna. M.Sc. thesis, University of Florida, Gainesville, FL. Burke, H. R., and D. F. Martin. 1956. The biolog y of three chrysopid predators of the cotton aphid. J. Econ. Entomol. 49: 698 700. 64

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Fenwick, G. R., R. K. Heaney, W. J. Mullin, and C. H. VanEtten. 1982. Glucosinolates and their breakdown products in food and food plants. CRC. Cr Rev. Food. Sci. 18: 123201. Ferguson, J. J. 2004. General guidelines for organic crop production, University of Florida, Gainesville, FL. http://ufdc.ufl.edu/IR00000276/00001 Francis, F., G. Lognay, J. P. Wathelet, and E. Haubruge. 2001. Effects of a llelochemicals from f irst (Brassicaceae) and second ( Myzus persicae and Brevicoryne brassicae ) trophic l evels on Adalia bipunctata. J. Chem Ecol 27: 243256. Grasswitz, T. R., and E. C. Burts. 1995. Effe ct of native natural enemies and augmentative releases of Chrysoperla rufilabris (Burmeister) and Aphidoletes aphidimyza (Rondani) on the population dynamics of the green apple aphid, Aphis pomi De Geer. International J Pest Manag 41: 176183. Guillebeau P. 2001. Crop profile for leafy greens in Georgia. USDA crop profiles. http://www.ipmcenters.org/cropprofiles/docs/GAleafgreen.pdf Hagen, K. S., P. Greany, E. F. Sawall, and R. L. Tassan. 1976. Tryptophan in artificial honeydews as a source of an attractant for adult Chrysopa carnea Environ. Entomol 5: 458468. Thermal requirements for development of aphidophagous Coccinellidae (Coleoptera), C hrysopidae, Hemerobiidae (Neuroptera), and Syrphidae (Diptera): some general trends. Oecologia. 76: 455 460. Hopkins, R. J., N. M. van Dam, and J. J. A. van Loon. 2008. Role of glucosinolates in insect plant relationships and multitrophic interactions. Ann u. Rev. Entomol. 54: 5783. HoughGoldstein, J. A., G. E. Heimpel, H. E. Bechmann, and C. E. Mason. 1993. Arthropod natural enemies of the Colorado potato beetle. Crop Prot. 12: 324334. Huang, N. X., and A. Enkegaard. 2010. Predation capacity and prey pref erence of Chrysoperla carnea on Pieris brassicae Biocontrol 55: 379385. Hydorn, S. B., and W. H. Whitcomb. 1979. Effects of larval diet on Chrysopa rufilabris Fla. Entomol. 62: 293298. Jervis, M., and N. Kidd. 1996. Insect natural enemies : practical approaches to their study and evaluation, Chapman & Hall, London, UK. Kjr, A. 1981. Glucosinolates and related compounds. Food Chemistry 6: 223234. Knutson, A. E., and L. Tedders. 2002. Augmentation of green lacewing, Chrysoperla rufilabris in cotton in T exas. Southwest. Entomol. 27: 231239. 66

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Koczor, S., F. Szentkirlyi, M. A. Birkett, J. A. Pickett, E. Voigt, and M. Tth. 2010. Attraction of Chrysoperla carnea complex and Chrysopa spp. lacewings (Neuroptera: Chrysopidae) to aphid sex pheromone components and a synthetic blend of floral compounds in Hungary. Pest Manag Sci 66: 13741379. Kunkel, B. A., and T. E. Cottrell. 2007. Oviposition response of green lacewings (Neuroptera: Chrysopidae) to aphids (Hemiptera: Aphididae) and potential attractants on pecan. Environ. Entomol. 36: 577 583. Lamberts, M. L., E. J. McAvoy, D. D. Sui, A. J. Whidden, and C. A. Snodgrass. 2011. Production of major Asian vegetables in Florida, Electronic Data Information Source (EDIS) HS740. University of Florida, Gainsville, F L. http://edis.ifas.ufl.edu/cv139. Legaspi, J. C., R. I. Carruthers, and D. A. Nordlund. 1994. Life history of Chrysoperla rufilabris (Neuroptera: Chrysopidae) provided sweetpotato whitefly Bemisia tabaci (Homoptera: Aleyrodidae) and other food. Biol Cont rol 4: 178 184. Lingren, P. D., R. L. Ridgway, and S. L. Jones. 1968. Consumption by several common arthropod predators of eggs and larvae of two Heliothis species that attack cotton. Ann. Entomol. Soc. Am. 61: 613618. Manrique, V., C. O. Montemayor, R. D Cave, E. A. Skvarch, and B. W. Smith. 2010. Effect of straw mulch on populations of Microtheca ochroloma (Coleoptera: Chrysomelidae) and ground predators in turnip Brassica rapa in Florida. Fla Entomol 93: 407411. Manrique, V., R. Diaz, C. Montemayor D. Serrano, and R. D. Cave. 2012. Temperaturedependent development and cold tolerance of Microtheca ochroloma (Coleoptera: Chrysomelidae), a pest of cruciferous crops in the southeastern United States. Ann. Entomol. Soc. Am. 105: 859864. McEwen, P. K., T. R. New, and A. E. Whittington. 2007. Lacewings in the crop environment. Cambrid ge University Press, Cambridge, MA Menezes Jr., A. O., A. Y. Mikami, A. K. Ide, and M. U. Ventura. 2005. Feeding preferences of Microtheca punctigera (Achard) (Coleoptera: Chrysomelidae) for some Brassicaceae plants in multiplechoice assays. Scientia Agricola 62: 7275. Mithen, R. 2001. Glucosinolates biochemi stry, genetics and biological activity. Plant Growth Regul 34: 91103. Montemayor, C. O. 2010. Evaluation of a pr edator and a fungus as biological control agents of the yellowmargined leaf beetle, Microtheca ochroloma Stl (Coleoptera: Chrysomelidae). MSc thesis, University of Florida, Gainesville, FL. 67

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Montemayor, C. O., and R. D. Cave. 2009. Prospects for biological control of the yellow margined leaf beetle, Microtheca ochroloma Stl. P. Fl. St. Hortic. Soc. 122: 250 252. Montemayor, C. O., and R. D. Cave. 2011. Development time and predation rate of Podisus maculiventris (Hemiptera: Pentatomidae) feeding on Microth eca ochroloma (Coleoptera: Chrysomelidae). Environ. Entomol. 40: 948954. New, T. R. 1975. The biology of Chrysopidae and Hemerobiidae (Neuroptera), with reference to their usage as biocontrol agents: a review. T. Roy. Ent. Soc. London. 127: 115140. Nguyen T. V., A. Wysocki, and D. Treadwel. 2008. Economics of the Organic Food Industry in Florida, Electronic Data Information Source (EDIS) FE732. Univers ity of Florida, Gainesville, FL http://edis.ifas.ufl.edu/fe732. Nordlund, D. A. 1991. Predation of Color ado potato beetle (Coleoptera: Chrysomelidae) eggs and larvae by Chrysoperla rufilabris (Neuroptera: Chrysopidae) larvae in the laboratory and field cages. J. Entomol. Sci. 26: 443449. Nordlund, D. A., and R. K. Morrison. 1990. Handling time, prey preference, and functional response for Chrysoperla rufilabris in the laboratory. Entomol. Exp. Appl. 57: 237242. O'Neal, M. E., D. A. Landis, and R. Isaacs. 2002. An inexpensive, accurate method for measuring leaf area and def oliation through digital image anal ysis J. Econ. Entomol. 95: 11901194. Obrycki, J. J., M. N. Hamid, A. S. Sajap, and L. C. Lewis. 1989. Suitability of corn insect pests for development and survival of Chrysoperla carnea and Chrysopa oculata (Neuroptera: Chrysopidae). Environ. Entomol. 18: 11281130. Oliver, A. D., and J. B. Chapin. 1983. Biology and distribution of the yellowmargined leaf beetle, Microtheca ochroloma Stal, with notes on Microtheca picea (Guerin) (Coleoptera: Chysomelidae) in Louisiana. J. Georgia. Entomol. Soc. 18: 22923 4. Olson, S. M. 2011. Introduction. Vegetable production guide for Florida, Electronic Data Information Source (EDIS) HS710. University of Florida, Gainesville, FL. http://edis.ifas.ufl.edu/cv100. Poncio, S., S. T. B. Dequech, V. S. Sturza, R. A. D. Lissne r, L. F. Perlin, P. K. Rosalino, and L. D. Ribeiro. 2010. First report of Stiretrus decastigmus in Brazil preying Microtheca ochroloma. Cienc. Rural 40: 12031205. Reynolds, P. G., and K. Cuddington. 2012a. Effects of plant gross morphology on predator consumption rates. Environ. Entomol. 41: 508515. 68

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BIOGRAPHICAL SKETCH A ngie Nio was born in Bogot, Colombia. She gr aduated with a ba chelors degree in applied biology from the Universidad Militar Nueva Granada in Colombia in 2007. For her bachelor s thesis, she w orked on biological control of mites in flower crops. From 2010 to 2011, she worked as a research assistant with t he Biological Control Research g roup and the Biodiversity and Ecology of Wild Bees Research g roup at the Universidad Militar Nueva Granada. Sh e began her Master of Science degree program in the Entomology and Nematology Department in August 2011. She received a F ulbright C olciencias scholarship from the Departamento Administrativo de Ciencia, Tecnologa e Innovacin and Fulbright to support her during her study program. She is a member of the Sociedad Colombiana de Entomologa, the Entomological Society of America, and the Florida Entomological Society. She presented talks about her research at the annual meetings of the Entomological Society of America in 2012 and at the Florida Academy of Sciences and the Sociedad Colombiana de Entomologa in 2013. 71