INVASIVE SPECIES AND PLANT BIOSECURITY EDUCATION WITH A FOCUS ON REARING STINK BUGS (HEMIPTERA: PENTATOMIDAE) OF CONCERN TO FLORIDA

By

MORGAN PINKERTON

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2019

© 2019 Morgan Pinkerton

To my mom and dad, for teaching me strength and providing unconditional love and support throughout my life. To the rest of my family and friends. Thank you for the motivation and help throughout my graduate school experience.

ACKNOWLEDGMENTS

First, I would like to thank Dr. Amanda Hodges and Dr. Norman Leppla for their guidance and support throughout my master’s project. The youth outreach work was supported by two years of USDA-APHIS-PPQ Farm Bill funding (FY2016 Cooperative

Agreement Number 16-8212-2065 and FY2017 Award Number AP17PPQFO000C240).

Piezodorus guildinii research was funded in part by Bayer Crop Sciences, as of 2018 known as BASF, and the Biosecurity Research and Extension Laboratory.

I would like to thank Jennifer Carr, Harry Edwards, Trevor Forsberg, Alexander

Fernandez, and Ryan Dick for their assistance in rearing the colony of P. guildinii and aid in data collection for experiments. Additionally, I thank Sage Thompson, Brianna

Whitman, and Cleveland Ivey for support rearing and with data collection for the B. hilaris colony and experiments. I would like to thank Florida Department of Agriculture and Consumer Services-Division of Plant Industry for allocation of space in their quarantine facility to rear Bagrada hilaris and perform experiments. I would especially like to thank Phillip Lake and David Davidson for assistance with quarantine procedures and questions. I would also like to thank Dr. John Palumbo for collecting and sending live B. hilaris specimens to establish the colony.

I wish to thank all the middle and high school teachers that allowed for us to give guest lectures on invasive species and plant biosecurity to their students. I also thank the schools that hosted us as well as the parents that gave us permission to survey their children. I thank the students that participated in the outreach events and surveying. I thank Nicole Casuso for her development and leadership during the first year of the youth outreach program. I would like to thank Sage Thompson for her assistance with outreach events and Ariane McCorquodale for the development of the website for the

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outreach program. I thank Craig Frey for his continuation of the youth outreach program to more schools and students in the future.

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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 9

LIST OF FIGURES ...... 10

ABSTRACT ...... 11

CHAPTER

1 LITERATURE REVIEW ...... 13

Importance of Invasive Species and Plant Biosecurity Education ...... 13 Invasive Species and Plant Biosecurity ...... 13 Impacts of Invasive Species on Florida ...... 13 Public Outreach and Education ...... 15 Laboratory Reared Insects Emphasizing Stink Bugs ...... 16 Challenges of Rearing Insects in the Laboratory ...... 16 History of Stink Bugs as Pests ...... 20 The Redbanded Stink Bug, Piezodorus guildinii ...... 21 Geographic Distribution of Piezodorus guildinii ...... 21 Hosts and Damage ...... 22 Identification ...... 23 Life History ...... 24 Rearing Methods ...... 24 Control Methods ...... 26 Bagrada hilaris ...... 27 Geographic Distribution of Bagrada hilaris ...... 27 Hosts and Damage ...... 28 Identification ...... 29 Life History ...... 31 Rearing Methods ...... 32 Control Methods ...... 33

2 ENGAGING FLORIDA’S YOUTH TO INCREASE THEIR KNOWLEDGE OF INVASIVE SPECIES AND PLANT BIOSECURITY ...... 36

Introduction ...... 36 Expected Outcomes ...... 38 Purpose ...... 38 Objectives ...... 39 Materials and Methods...... 39 Audience ...... 39 Outreach Events ...... 39

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Surveys ...... 43 Results ...... 44 Student’s Comfort Level ...... 44 Student’s Knowledge Level ...... 45 Conclusion ...... 46

3 LABORATORY REARING OF THE REDBANDED STINK BUG, Piezodorus guildinii (WESTWOOD) ...... 51

Introduction ...... 51 Materials and Methods...... 53 Colony Source ...... 53 Cages ...... 54 Conditions ...... 54 Diet ...... 55 Rearing Procedure ...... 55 Experiment 1: Preference of Oviposition Substrate ...... 56 No-choice ...... 56 Choice ...... 57 Experiment 2: Identification of Size Disparities in Successive Generations of Laboratory Reared Piezodorus guildinii ...... 57 Results and Discussion...... 58 Experiment 1: Preference of Oviposition Substrate ...... 58 No-choice ...... 58 Choice ...... 59 Experiment 2: Identification of Size Disparities in Successive Generations of Laboratory Reared Piezodorus guildinii ...... 59 Conclusions ...... 60

4 LABORATORY REARING OF THE BAGRADA BUG, Bagrada hilaris (BURMEISTER) ...... 65

Introduction ...... 65 Materials and Methods...... 66 Colony Source ...... 66 Cages ...... 67 Conditions ...... 68 Diet ...... 68 Rearing Procedure ...... 68 Experiment 1: Effect of Photoperiod on Egg Hatching ...... 70 Experiment 2: Preference of Oviposition Substrate ...... 71 No-choice ...... 71 Choice ...... 72 Results and Discussion...... 72 Experiment 1: Effect of Photoperiod on Egg Hatching ...... 72 Experiment 2: Preference of Oviposition Substrate ...... 73 No-choice ...... 73

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Choice ...... 73 Conclusions ...... 74

5 IMPLICATIONS AND FUTURE DIRECTIONS OF RESEARCH ...... 79

LIST OF REFERENCES ...... 81

BIOGRAPHICAL SKETCH ...... 89

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LIST OF TABLES

Table page

3-1 Mean (μ) and standard deviations (σ) of body size of adult P. guildinii males over four generations...... 64

3-2 Mean (μ) and standard deviations (σ) of body size of adult P. guildinii females over four generations...... 64

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LIST OF FIGURES

Figure page

2-1 Survey questions for the ‘Invasive Species that Affect Plants’ topic...... 48

2-2 Survey questions for the ‘Plant Biosecurity-Local and Global Perspectives’ topic...... 49

2-3 Mean percentage of students in each class that received instruction on ‘Invasive Species That Affect Plants’ that improved, did not change, or decreased their score from the pretest to the posttest for the concept questions 2–7...... 50

2-4 Mean percentage of students in each class that received instruction on ‘Plant Biosecurity-Local and Global Perspectives’ that improved, did not change, or decreased their score from the pretest to the posttest for the concept questions 2–7...... 50

3-1 Adult cage utilized in rearing of P. guildinii...... 63

3-2 Immature cage utilized in rearing of P. guildinii...... 63

3-3 No-choice and choice test evaluating the total number of eggs laid by P. guildinii on three artificial substrates; dental wicks, cotton balls, and kim wipes...... 64

4-1 Adult cage utilized in rearing of B. hilaris...... 76

4-2 Immature cage utilized in rearing of B. hilaris...... 76

4-3 Percent of hatching of B. hilaris eggs in dark (0L:24D) and light (14L:10D) conditions...... 77

4-4 Average number of days it took for B. hilaris eggs to hatch in dark (0L:24D) and light (14L:10D) conditions...... 77

4-5 No-choice and choice test evaluating the total number of eggs laid by B. hilaris on three artificial substrates; dental wicks, cotton balls, and kim wipes. .. 78

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

INVASIVE SPECIES AND PLANT BIOSECURITY EDUCATION WITH A FOCUS ON REARING STINK BUGS (HEMIPTERA: PENTATOMIDAE) OF CONCERN TO FLORIDA

By

Morgan Pinkerton

May 2019

Chair: Amanda Hodges Major: Entomology and Nematology

Globally, invasive species cause many problems including significant economic losses, decreased biodiversity in natural ecosystems, and health hazards. In the United

States, it is estimated that invasive species cause annual losses of $120 billion. Florida is a high-risk state for the introduction of non-native species due to many deep-sea ports, international airports, tourist attractions, and a diversity of flora and fauna. Plant biosecurity is the strategic approach surrounding the prevention and mitigation of invasive species. Two invasive stink bugs (Pentatomidae) of concern to Florida are the redbanded stink bug, Piezodorus guildinii (Westwood) and Bagrada hilaris (Burmeister).

Piezodorus guildinii is now established in the southeastern United States including

Florida and causes substantial losses in legumes like soybean (Glycine max). Bagrada hilaris is an invasive species that impacts crucifers like cabbage (Brassica oleracea var. capitata) and was recently introduced into the United States but has not yet established in Florida. Current research aims to better understand the biology of these species, discover more effective management strategies and prevent spread to new areas.

Effective rearing of insect colonies is crucial to these investigations and provides

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valuable information on the pest’s biology. Despite the importance of invasive species and plant biosecurity education, the general public, and particularly, Florida’s youth remain largely uninformed about these topics. A youth outreach program was developed to educated Florida’s youth and the effectiveness of this two-year program was evaluated. Experiments surrounding insect rearing of P. guildinii and B. hilaris provided valuable case studies that were integrated into outreach programs.

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CHAPTER 1 LITERATURE REVIEW

Importance of Invasive Species and Plant Biosecurity Education

Invasive Species and Plant Biosecurity

An invasive alien species is a non-native organism that causes damage in a particular area (Meyerson et al. 2013). In the United States, it is estimated that invasive alien species cause losses of over $120 billion annually (Pimentel et al. 2005). A non- native species has the potential to become invasive when it is introduced into a new environment where it can establish and cause problems such as substantial economic losses, decreased biodiversity, introduction of human and animal health hazards, or disruption of native ecosystems. A variety of pathways for introduction of non-native species exist and increased globalization has resulted in goods and people moving around the world at a higher rate than ever before (Meyerson et al. 2013). Invasive species can be introduced intentionally, such as an on ornamental plant or unintentionally as an insect hitch hiking on a vehicle (Winberry and Jones 1973; Sargent et al. 2011). In order to prevent the spread of invasive species, biosecurity is utilized as a strategic and integrated approach to protect human, animal and plant health (Magarey et al. 2009). Specifically, plant biosecurity focuses on protecting all plants, including agricultural crops, ornamentals, and the natural environment. Biosecurity strategies can be implemented in assessment of risk, early detection, eradication efforts, and control of potentially invasive plant pests and pathogens.

Impacts of Invasive Species on Florida

Florida is a particularly high-risk state for the introduction of invasive species for many reasons including its geography. Florida also has 11 commercial seaports and

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eight main destination airports that receive a large portion of the United States’ imports before shipment to the rest of the country (Szyniszewska 2013). The many tourist attractions result in millions of potential travelers and source points for non-native species introductions every year. Moreover, Florida’s has a wide diversity of high value agricultural commodities including oranges, grapefruit, fresh market tomatoes, cabbage, cucumbers, and cattle. With the invasive species introduction rate being high in Florida, the $120 billion annual revenue of the agricultural industries in the state is put at risk

(FDACS 2016a).

In recent years, many problematic invasive species of arthropods and pathogens have impacted Florida. For example, the Mediterranean fruit fly, Ceratitis capitata

(Wiedemann), was detected in Florida multiple times during the last century and required costly eradication efforts and continued monitoring throughout the state (Steck

2002). Another example of a successful detection and eradication occurred with

Oxycarenus hyalinipennis (Costa), which was detected in the Florida Keys in 2010 and promptly eliminated (Sharma 2014). Similarly, the giant African snail, Lissachatina fulica

(Bowdich), was detected in South Florida in 2011 and has since been under an eradication program (FDACS 2018a). Significant public outreach has largely increased the success of this program with L. fulica.

Unfortunately, it is not possible to eradicate all invasive species that have established in Florida. Some economically important species that were introduced and have subsequently establish include the spotted-wing drosophila, Drosophila suzukii

(Matsumura) (Diptera: Drosophilidae) (Iglesias et al. 2009); Aulacaspis yasumatsui

Takagi (Hemiptera: Diaspididae) (Mannion et al. 2006); redbay ambrosia beetle,

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Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae) (FDACS 2018b); Candidatus

Phytoplasma palmae (Acholeplasmatales: Incertae sedis) (Elliott 2009); Huanglongbing

(Greening), Candidatus Liberibacter asiaticus Jagoueix et al. (Rhizobiales:

Phyllobacteriaceae) (Hodges and Spreen 2015); and Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) (Thomas 2005). Other pests of concern to

Florida that have not yet established, but are high risk to the state, include the old world bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae); Bagrada hilaris

(Burmiester) (Hemiptera: Pentatomidae), Asian longhorned beetle, Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae) ; light brown apple moth,

Ephiphyas postvittana (Walker) (Lepidoptera: Tortricidae); Phytophthora ramorum

Werres, de Cock & Man in’t Veld (Peronosporales: Peronosporaceae); and Oriental fruit fly, Bactrocera dorsalis Hendel (Diptera: Tephritidae). Many of these invasive species are subject to surveillance in Florida through statewide biosecurity activities of the state and federal government.

Public Outreach and Education

Outreach and education aid in translating scientific information into language that the public can easily understand and is obtained from many scientific fields (Larson et al. 2011). In terms of invasive species detection and management, outreach and education are imperative to achieving sustainable goals outlined in biosecurity plans.

These plans are important in resolving misconceptions about invasive species, creating dialog between scientists and the community, and delivering biosecurity information directly to the public.

Public outreach and education can play a vital role in the early detection of invasive species. The Invasive Plant Atlas of New England (IPANE) is a well-known

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example of citizen science for the detection of non-native plant species in New England

(Bois et al. 2011). Educating the public in invasive species identification can enable more comprehensive early. A study based on the IPANE program in Texas utilized a satellite network of public volunteers to report early detection information and compile an invasive species database for the state (Gallo and Waitt 2011). Results of this program also suggest that, with proper training, volunteer citizens can widen the area of search for early detection of invasive species at a local level.

Despite the great impact of invasive species currently in Florida and the potential for future introductions of new species, very little education on biosecurity has been delivered to the general public. Public outreach and education is crucial for volunteer- based networks to succeed and may aid in preventing establishment of additional invasive species in Florida (Pimentel et al. 2005; Bois et al. 2011; Burrack et al. 2012;

Andow et al. 2016; Stubbs et al. 2017). Of the existing outreach and education, the primary focus has been on adults and has excluded youths, a large and capable portion of the population. Future plant biosecurity efforts can benefit from involving Florida’s youth in invasive species detection and management.

Laboratory Reared Insects Emphasizing Stink Bugs

Challenges of Rearing Insects in the Laboratory

Rearing insects in the laboratory is often essential for many kinds of research.

Laboratory colonies offer readily available specimens for experiments, which can be used for a variety of studies such as pesticide and biological control agent interactions.

Insects needed for experimentation may only be available seasonally or may not occur in the natural environment within a geographic region. Specifically, quarantine rearing is often necessary for the development of risk assessments for potentially invasive

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insects. Additionally, basic life history knowledge resulting from rearing is imperative to the development of control methods for many pests. However, laboratory colonies have a limited genetic pool and over time, this can lead to inbreeding, genetic drift, or artificial selection of certain phenotypic traits. Understanding the limitations of laboratory rearing also is important for interpreting bioassay results (Sorensen et al. 2012).

Insects can adapt in response to the environment and these adaptations can be seen within single individuals or populations. These adaptations can be controlled genetically or based on environmental changes (Chevin et al. 2010). While this is beneficial for the rearing process as the insects respond to their conditions, reared insects may be different than those found in the wild. Inbreeding can lead to selection of certain traits coined as laboratory adaptation. Inbreeding depressions are well documented in rearing of a wide variety of insects. For example, rearing multiple generations of the sweet potato weevil, Cylas formicarius elegantulus (Fabricus)

(Coleoptera: Curculionidae), causes changes in developmental periods, fertility, body size, and starvation tolerance (Kuriwada et al. 2010). In the wild, traits that arise in a population are often selected for based on higher survival of individuals that possess that particular trait. However, survival in a laboratory setting is different due to the artificial conditions that prevent predation, severe weather events and other factors that would normally limit population growth and survival (Huettel 1976). Furthermore, colonies receive ample food and do not spend time searching for hosts as the insects normally would in the wild. The food source and microclimate is much more controlled in colonies, so these insects may not accurately represent populations found in a natural

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and uncontrolled environment, as insects can adapt or modify their behavior to adjust for the laboratory conditions (Chambers 1977).

Currently, there is not a single, or holistic measure for quality assurance in reared insects; however, some methods have been suggested. For example, it is important to evaluate the quality of insects that are used in sterile insect technique (SIT), such as the

Mediterranean fruit fly, Ceratitis capitata (Wiedemann). Quality assurance for SIT is important in ensuring that sterile males can perform adequately in the wild, as the sterilization process is direct manipulation of fitness. The quality assessment techniques of C. capitata were recently evaluated through genomic analysis which is a widespread technique currently utilized in many scientific fields and not just limited to insect quality assurance (Calla et al. 2014). Another study on light brown apple moth, Epiphyas postvittana (Walker), males reared for SIT suggested that flight of insects placed in a wind tunnel can be used as a measure of quality for sterile males (Brown et al. 2016).

Overall, many of the techniques for quality assurance of SIT would be applicable to insect rearing in general (Leppla 2009).

In addition to its application in rearing for SIT, quality assurance is needed in rearing insects for research such as for pesticide trials or development of biological control methods (Leppla 2013). For example, pest management methods are needed for the western tarnished plant bug, Lygus hesperus (Knight), an important pest of crops including alfalfa, Medicago sativa, and beans, Phaseolus vulgaris. Parameters have been identified to assess the fitness and quality of rearing L. herperus on different artificial diets (Portilla et al. 2011). The study utilized reproduction rate, stage weight, biomass accumulation, and fecundity to measure quality of different diets. As this study

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suggested, measuring multiple parameters may be necessary to properly evaluate the quality of mass rearing methods. Moreover, small size, slow development time, and mortality are all indicative of unfavorable environmental conditions (Zerbino et al. 2014).

Other studies have indicated that body size is an effective measurement of diet quality as it correlates to fecundity (Denno et al. 2000).

As previously noted, quality assurance for laboratory-reared insects is necessary because colonies can develop lower stress tolerance that results from the lack of external pressures, such as food insecurity and predation that would normally affect wild insects (Sorensen et al. 2012). In Drosophila melanogaster (Meigen) (Diptera:

Drosophilidae), stress resistance disparities were evaluated by comparing wild caught and laboratory reared insect’s ability to respond to drought or starvation. For starvation resistance, the 50% mortality rate decreased from 50.1 h to 35.9 h over the course of four years of maintaining the colony. Decline was also noted in smaller increments over several generations. Desiccation resistance also showed a similar decline over the course of rearing several generations of D. melanogaster (Hoffmann et al. 2001). This indicated that laboratory colonies may have different levels of abilities to overcome stressors. If the insects were being used to evaluate pesticides, reared-insects may have been more susceptible to the stressor which in this case, is the pesticide.

Accordingly, studies investigating control strategies for laboratory-reared insects may show higher effectiveness than in field insects due to artificially reduced stress tolerance. These genetic and behavioral deviations from wild caught populations can limit the applications of laboratory research. Understanding the limitations or variations

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in laboratory reared-insects will assist in the development of tests that are more applicable to field populations.

History of Stink Bugs as Pests

Stink bugs (Pentatomidae) represent a large family of polyphagous pests feeding on over 200 economically important species of plants (McPherson and McPherson

2000). Compared to many other plant feeding insects, stink bugs and other hemipterans, have the advantage of bypassing a plant’s natural defenses due to specific feeding strategies. Pentatomids utilize piercing sucking mouthparts to siphon out nutrients from various plant structures including seeds, leaves, stems, and fruits. A wider variety of plant herbivory defenses are often more effective on external feeders

(i.e. caterpillars or beetles in the mastication process), while internal feeding of stink bugs is a mechanism that avoids host defenses (Ali and Agrawal 2012).

Insects that feed internally often can avoid insecticides that are applied on the surface of plants. Although, insecticide on the surface of the plant would be less effective on pentatomids that feed internally, some contact and systemic insecticides are effective on stink bugs and similar pests (Panizzi 2000). Historically, early sprays of organophosphates used for lepidopteran pests controlled pentatomids indirectly

(McPherson and McPherson 2000). Due to the recent drive to reduce the use of broad- spectrum organophosphates and increase alternative insecticides, such as Bacillus thuringiensis (Bt), for controlling lepidopteran pests, stink bugs have emerged as secondary pests in many cropping systems. Consequently, research on control methods for stink bugs has increased.

The need to control pentatomids is a direct result of the potential damage, if left uncontrolled, these pests can cause in a wide variety of crops. The type of damage

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caused by pentatomids can be divided into five categories: localized wilting and necrosis, abscission of fruiting forms, morphological deformation of fruits and seeds, modified vegetative growth, and tissue malformation (Wiman et al. 2015). Pentatomid damage can cause lowered yields and reduced marketability of crops, and is particularly problematic at high population levels (McPherson and McPherson 2000). With current control mechanisms, the cost of controlling pentatomids in just soybean is estimated at over $60 million dollars annually. In addition to direct crop damage, feeding damage makes the plants more susceptible to secondary pathogens, such as fungi or bacteria, thus furthering yield losses both pre- and post-harvest (Wiman et al. 2015).

The Redbanded Stink Bug, Piezodorus guildinii

Geographic Distribution of Piezodorus guildinii

The redbanded stink bug, Piezodorus guildinii [Westwood] (Hemiptera:

Pentatomidae) is thought to originate from the Neotropics, but is now known to occur throughout South America, Costa Rica, Liberia, the Caribbean, and parts of the United

States (Panizzi 2005). In Brazil and Argentina, P. guildinii is a major economically important pest of leguminous crops, such as soybean, Glycine max (L.) Merr. In the

1970’s, P. guildinii established in North America, but it was not until 2002 that it was recorded as a pest species (Husseneder et al. 2016). Piezodorus guildinii is currently distributed throughout the southeastern United States and has expanded to crops other than soybean (Akin et al. 2011). As of 2017, P. guildinii has been recorded in Alabama,

Florida, Georgia, Louisiana, Mississippi, New Mexico, South Carolina, and Texas

(Pinkerton and Hodges 2017).

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Hosts and Damage

Piezodorus guildinii is polyphagous, but has a more restricted host range than many other stink bugs and is limited to legumes in the plant family Fabaceae (Smaniotto and Panizzi 2015). Soybean is the most cited host of P. guildinii due to its economic significance as one of the world’s leading crops rich in protein and oil (Silva et al. 2013).

In addition, P. guildinii has caused economic damage to beans, Phaseolus vulgaris L., peas, Pisum sativum L.; lentils, Lens culinaris Medik.; and other forage legumes.

Piezodorus guildinii is also known to occur on forage and cover crops like alfalfa,

Medicago sativa L., with high frequency throughout the year. For example, the host range of P. guildinii also includes Florida species of Crotalaria which suggests that wild plants can serve as hosts in addition to crops (Panizzi and Slansky Jr 1985).

Populations are sustained at adequate levels during the off seasons of economic hosts due to the variety of weedy legumes like Crotalaria spp. Piezodorus guildinii has also been recorded on plants other than legumes, but it is likely that these were just incidental occurrences and not reproductive hosts (Zerbino et al. 2014). Other potential hosts would need to be evaluated before determining that P. guildinii is reproductively active on these hosts.

The injury to plants by P. guildinii resembles most plant feeding pentatomids. The pest inserts its stylet into host tissues and using digestive enzymes, siphons out nutrients (Akin et al. 2011). Typically, pentatomid damage can be identified by small lesions on plant tissue that appear with light colored spots as nutrients are extracted.

For example, in soybean, P. guildinii feeds on the tissue of the bean that gives the pod a shriveled appearance (Depieri and Panizzi 2011). Inside the pod, the seeds are also shriveled and can be necrotic over time, leading to delayed plant maturity and reduced

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yields (Akin et al. 2011). Pentatomid damage can expose the plant to secondary pathogens, such as bacteria and fungi, that increase the decomposition rate and results in unmarketable produce in addition to yield losses (Koch et al. 2017). Piezodorus guildinii shows a significantly higher level of seed tissue damage and thus greater economic losses compared to other neotropical stink bug species, including Nezara viridula (Linnaeus) and Euschistus heros (Fabricius) (Depieri and Panizzi 2011).

Identification

Adult P. guildinii are relatively small (8-11 mm length, 4-6 mm width) compared to many pest stink bugs (Zerbino et al. 2014). Adults are light to dark green with a colored band on the pronotum ranging from cream colored to deep purple. A key morphological character is a fixed spine on the ventral side of the abdomen (King and Saunders 1984).

Thyanta spp. are often confused for P. guildinii based on the green body and red to brown pronotal stripe. However, Thyanta spp. are more shield shaped and have a finished green color while P. guildinii appears glossy. Thyanta spp. also lack the ventral abdominal spine seen in Piezodorus. Two other species that are commonly confused with P. guildinii are Nezara viridula and Chinavia hilaris (Say). These two species are also green in color but are significantly larger than P. guildinii and lack the abdominal spine and the colored pronotal band.

Nymphs of P. guildinii are oval shaped which is distinct from other common pentatomids in Florida. Earlier instars, typically 1st-3rd, have a black pronotum and head, and a red abdomen with black markings. Later instars, 4th and 5th, are green with black markings as well as several red and black stripes on the abdomen. When compared to

Thyanta nymphs, P. guildinii nymphs have fewer, thicker stripes on the dorsal abdomen

(Akin et al. 2011).

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Life History

Adult females lay eggs in double rowed, linear masses with an average of 15.8 eggs per clutch (Panizzi and Smith 1977). The number of eggs per clutch is lower than for other common pentatomid pests such as N. viridula and C. hilaris. Under ideal conditions, the average female lays a total of about 196 eggs (Zerbino et al. 2013).

Eggs are preferentially laid directly on host plants and develop in about 7.5 days

(Panizzi and Smith 1977). Piezodorus guildinii undergoes five nymphal instars that take about 3-13 days to develop before eclosion as adults. The total development time from egg to adult can be as short as 37-39 days (Panizzi and Smith 1977). Nymphs display high survivability in warm and humid conditions ranging from 50%-90% RH and 23-27°C

(Gomez et al. 2013). Adults appear to live longer in colder temperatures, which is suggestive of a diapause behavior common to other pentatomids. Adults become reproductively active in early spring and multiple generations occur annually. In

Louisiana, studies show that P. guildinii can have between four and eight generations per year with overlapping generations occurring around July (Akin et al. 2011).

Rearing Methods

The rearing of pest insect species, including P. guildinii, is important to provide specimens for research. Due to the economic damage caused by P. guildinii, colony insects are needed to develop control methods (Silva and Panizzi 2008). Many factors affect the development and reproductive success of laboratory reared insects including photoperiod, temperature, relative humidity, oviposition substrates, and diet (Cohen

2018).

Previous studies have investigated the optimal photoperiod and temperature for nymph development and adult reproduction in P. guildinii (Zerbino et al. 2013). The 2nd

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instar appears to be the most sensitive to temperature and mortality is high at temperatures below 20°C. This low survival rate at lower temperatures is even more significant when day length is shortened, suggesting that nymphs are unlikely to survive winter conditions. Laboratory colonies of P. guildinii are typically maintained at 23-27°C as this temperature range provides the least mortality (Gomez et al. 2013). In addition to the effect of temperature on all life stages, photoperiod appears to affect the time of development for 4th and 5th instars, and the reproductivity of adults (Zerbino et al. 2013).

Nymphs develop faster during a long day photoperiod (14L:10D) and is ideal for maintaining a successful colony. Humidity levels also affect the successful rearing of stink bugs. The distribution of P. guildinii in locations with high RH, such as Florida, suggests that the optimal rearing RH should also be high. A study in successful colonies were maintained at a relative humidity of 60 ± 10% in Paraguay (Gomez et al. 2013). In

Uruguay, P. guildinii was reared for experiments at a higher relative humidity of 80 ±

10% and also reported successful colonies (Zerbino et al. 2013).

Artificial oviposition substrates are commonly used for laboratory rearing of stink bugs and the preferred type varies for different species (Silva and Panizzi 2007). Host selection of P. guildinii has previously been evaluated using no-choice and choice tests; however, these methods could also be applicable to the evaluation of oviposition substrates (Molina and Trumper 2012). The optimal oviposition theory suggests that adults will select substrates based on optimizing offspring survival. Cotton balls are commonly used for rearing several species of stink bugs and one study showed that they were suitable for oviposition by P. guildinii (Silva and Panizzi 2007). However, a more recent study indicated that cotton balls were a poor oviposition substrate for P.

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guildinii despite its value in rearing of other pentatomids (Silva and Panizzi 2008). This study also evaluated cotton, synthetic wool string, and cheesecloth with synthetic wool string being the most effective artificial oviposition substrate (Silva and Panizzi 2008).

Other substrates, such as hanging tissue paper and string, utilized for rearing P. guildinii have not been evaluated for quality (Gomez et al. 2013).

Survival of P. guildinii is low when reared on green beans (Phaseolus vulgaris), a commonly used diet for other plant feeding stink bugs like N. viridula and Euschistus servus (Say) (Zerbino et al. 2014). Survival of P. guildinii is higher on fresh soybean pods compared to green beans, and it increases when soybean is mixed with raw peanuts. To date, a commercially available artificial diet for stink bugs is not available.

Attempts at developing artificial diets for P. guildinii have included a mixture of soybean, peanut, and green beans (Gomez et al. 2013). Current artificial diets for stink bugs increase nymph mortality (Fortes et al. 2006), or at the least, delays development time in comparison to a diet of fresh produce (Jensen and Gibbens 2000).

Control Methods

Monitoring is an important aspect of integrated pest management (IPM) as it is the first step in evaluating damage and identifying key pest problems in the field (Binns and Nyrop 1992). For stink bugs, monitoring usually includes weekly visual surveys to detect eggs, nymphs, adults, and damage (Akin et al. 2011). Additionally, sweep netting early in the morning or late in the evening is most effective to locate insects in the field.

In Louisiana and Arkansas, six P. guildinii per 25 sweeps is considered the economic threshold at which management should be initiated in soybean. Currently, a trap with a commercially available lure specific to P. guildinii does not exist. However, P. guildinii

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has been detected in traps baited with pheromone lures for E. heros in the Neotropics

(Borges et al. 1998).

Insecticides used for stink bugs, such as N. viridula and C. hilaris, have not been as effective against P. guildinii. Normally, in soybean and similar crops, stink bugs are controlled using insecticides containing pyrethroids (Akin et al. 2011). Specifically, pyrethroids with the active ingredient bifenthrin are the most effective against P. guildinii. Acephate, cypermethrin, and methamidophos also appear to be effective active ingredients for P. guildinii though dosage may depend on the given population (Baur et al. 2010). Chemical practices are often more effective when combined with cultural control methods. For P. guildinii, cultural control can include planting earlier maturing seeds, since stink bugs are often late season pests, or using insect resistant varieties of plants (Silva et al. 2013).

Bagrada hilaris

Geographic Distribution of Bagrada hilaris

The bagrada bug, Bagrada hilaris [Burmeister] (Hemiptera: Pentatomidae) formerly known as Bagrada cruciferarum, is an invasive stink bug recently introduced to the United States. Bagrada hilaris is also commonly referred to as the painted stink bug, the African stink bug, the mustard bug, the mustard painted bug, the colorful bug, and the caper bug though an approved common name does not exist (Palumbo et al. 2016).

Bagrada hilaris is native to Africa but is also widespread throughout the old world in parts of Asia and Europe. Bagrada hilaris causes major damage to crops in India,

Pakistan and many parts of Africa, with occasional outbreak population levels (Reed et al. 2013). Since being introduced to California in 2008, B. hilaris has spread throughout

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the southwestern United States establishing in Arizona, Nevada, Utah, New Mexico, and most recently Texas (Hoenisch et al. 2014) (Reed et al. 2013).

It is also important to note that in the United States, B. hilaris is one of three pests specifically controlled under the Federally Recognized State Managed

Phytosanitary Program (USDA 2015). For this reason, B. hilaris is only present in

Florida under maximum security quarantine conditions with strict protocols to ensure that it not be released into the environment. Despite being best known for citrus production, Florida also produces other high value commodities including cabbage

(Brassica oleracea). Based on data from 2010, Florida produced around 13% of cabbage in the United States and 8% of the country’s fresh market cabbage. In 2010

(Elwakil and Mossler 2010) and again in 2012 (FDACS 2013), cabbage was considered the 10th highest valued crop for Florida’s agricultural economy. If B. hilaris were to establish in Florida, this industry would suffer huge losses like those seen in the southwestern states of the United States

Hosts and Damage

Bagrada hilaris has a wide host range that covers economically important crops, weeds, and ornamental species. The primary hosts of importance are in the family

Brassicaceae and B. hilaris shows a strong preference for these plants (Palumbo et al.

2016). In areas where B. hilaris has established in the United States, economic damage occurs in broccoflower, broccoli, cabbage, cauliflower, Chinese cabbage, kale, collards, arugula, radish, turnip, mustards, and other Brassica spp. In Arizona and California, cole crop production was valued at over $1 billion dollars in 2011 (Huang et al. 2014a).

The industry has seen considerable losses since B. hilaris has established. In Arizona

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specifically, B. hilaris causes around 10% yield losses annually, but losses can be as high as 70% in outbreak occurrences (Palumbo et al. 2016).

Bagrada hilaris can survive on weedy hosts and migrate into crop fields at devastating population levels as soon as seedlings emerge early in the season

(Hoenisch et al. 2014). In addition to weedy hosts, B. hilaris can survive on less then suitable hosts when preferred crops are unavailable (Palumbo et al. 2016). Feeding damage appears particularly critical in younger or small plants including seedlings and

B. hilaris displays a preference for this stage (Hoenisch et al. 2014). Like other pentatomids, the piercing-sucking mouthparts of B. hilaris puncture the cuticle of the plant and siphon out nutrients. As a result, smaller chlorotic lesions are created on the leaves, stems, and other parts of the plant leading to wilting or desiccation (Halbert and

Eger 2010). In cotyledons, feeding by B. hilaris can kill the meristem and subsequent plant growth can be largely disfigured and even lack reproductive heads altogether

(Huang et al. 2014a). Furthermore, damage can weaken the plants and causing higher susceptibility to secondary pests and pathogens.

Identification

Eggs are laid singly or in small groupings containing 2-15 eggs although groupings as high as 75 eggs (Palumbo et al. 2016) has been recorded (Taylor et al.

2014). Eggs are oval and average 0.87–1.0 mm long by 0.55–0.75 mm wide.

Throughout development, eggs change from white or cream to pink or reddish orange near hatching (Palumbo et al. 2016). Nymphs undergo five instars all of which have been well described in the literature (Taylor et al. 2015). 1st instars have a red abdomen and dark brown to red head and thorax. Subsequent instars are primarily red with black to dark brown marking around the head and thorax. Most instars also have visible black

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to dark brown longitudinally oval spots on the dorsal abdomen. The 2nd instar is about 1 mm in length while the last instar reaches 5 mm in length before eclosion as an adult.

Adult B. hilaris are primarily black with orange to red, and white markings. Orange, red and white coloration are more prominent in hot, dry climates and fewer markings are seen in populations in cold or wet climates. On average, males are smaller around 5.9 mm in length and 3 mm in width while females are larger around 7 mm by 4 mm respectively (Palumbo et al. 2016).

Home gardeners, the general public, and some growers have occasionally mistaken nymphs for lady beetles (Coleoptera: Coccinellidae) (Reed et al. 2013). Based on this misidentification, growers do not manage B. hilaris as they believe the insects to be beneficial predators. The nymphs may initially give a beetle like appearance based on the red and black coloration and the rounded shape; however, coccinellid beetles have distinct hardened wing covers (elytra) while B. hilaris nymphs are soft-bodied.

Adults of B. hilaris can also be mistaken for other native insects. Although much larger in size than B. hilaris, the harlequin bug, Murgantia histrionica [Hahn], is sometimes mistaken for B. hilaris due to its orange to red and black colorations and feeding preference for cole crops (Huang et al. 2014b). Murgantia histrionica is significantly bigger than B. hilaris, and similar in size to Nezara viridula [Linnaeus] and other common stink bug species (Reed et al. 2013). Other commonly confused insects are in the genus Mormidea because this genus is similar in size to B. hilaris. However,

Mormidea spp. are black with white or cream coloration and lack any red or orange markings as seen in B. hilaris (Hoenisch et al. 2014). In addition, the host range of

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Mormidea spp. is different than B. hilaris as these insects primarily feed on grasses and are not considered a pest in crucifers.

Life History

Bagrada hilaris displays a unique oviposition strategy in which eggs are laid singly or in small groupings directly into the soil (Taylor et al. 2014). The eggs take about 2-6 days but can take as long as 20 days depending on temperature (Palumbo et al. 2016).

After hatching, nymphs undergo five instars prior to adulthood and often aggregate together on a host plant throughout all life stages. Nymph development can occur in as short as 17 days or as long as 81 days (Palumbo et al. 2016). The 1st instar is a non-feeding stage and is the shortest, averaging 2-3 days (Taylor et al. 2015). All other instars take 2-5 days with the 5th instar being the longest (Palumbo et al. 2016). In the states where B. hilaris is present in the United States, nymphs are typically seen around late February to June and again in November to early January (Taylor et al.

2015).

Upon molting into the adult stage, males and females mate 1-2 days after eclosion and each female produces on average 100-200 eggs in her lifetime. The average adult survive 8 to 26 days with the females living longer than the males

(Palumbo et al. 2016). In the southwestern United States, B. hilaris adults appear in higher population levels during the early spring and late fall which coincides with the presence of hosts including London rocket (Sisymbrium irio L.) and broccoli (B. oleracea) (Taylor et al. 2015).

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Rearing Methods

The rearing of B. hilaris was documented briefly by Taylor et al. 2015. This source reported having success with rearing B. hilaris, but procedural information is limited and of the documentation that does exist, comments on the stability of the colony over time are unavailable. However, rearing protocols for B. hilaris colonies without frequent infusions of field-collected insects have not been reported. This is particularly significant for a quarantine colony as quarantine procedures necessitate the isolation of the colony relying solely on colony females for new insects. To date, rearing of B. hilaris in a quarantine setting has not been cited in the literature.

With the limited information on B. hilaris rearing, mixed information occurs regarding an appropriate diet. One source indicates that Indian mustard (Brassica juncea (L.) Czern.) seeds or seedlings were sufficient for a rearing diet (Halbert and

Eger 2010). Another source indicate that cabbage (B. oleracea var. capitata L.) was successful for rearing (Verma et al. 1993). An additional paper indicated that colony insects were maintained on broccoli (B. oleracea var. italica L.) and sweet alyssum

(Lobularia maritima L.) (Huang et al. 2014b). Another, more recent paper claims that the fruiting structures of mesa pepperwort (Lepidium alyssoides A. Gray) were sufficient for rearing (Taylor et al. 2015).

The rearing temperature also appears variable throughout the literature but ranges from 25.5–40 °C (Taylor et al. 2015). Development time was significantly shorter in temperatures between 28-40°C (Palumbo et al. 2016). Early literature indicated that development time is fastest at 35°C while temperatures above 45° result in mortality

(Atwal 1959).

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In Taylor et al. (2015), experiments occurred in a laboratory within an established population area in Arizona and this was one of the few mentions of cage design in the literature. The colony was maintained in a large terrarium (61 cm by 32 cm by 41 cm) with paper towels as a substrate, plant cuttings for food, and wet cotton balls as a water source. However, it is important to note that this rearing design is not feasible for a quarantine facility since the cages contain higher densities of insects and may be less secure.

Rearing typically utilized sand as an oviposition site for B. hilaris as this species naturally lays its eggs under the soil (Taylor et al. 2014), but this method is not suitable for quarantine conditions and the purpose of security. In the Taylor et al. (2015) study, strips of cheesecloth were utilized as an artificial oviposition substrate and eggs were transferred to moistened filter paper using a paintbrush daily. Another study suggested that dry cotton wool is the preferred artificial substrate for oviposition, even when compared to cheesecloth, but these studies may be antiquated (Atwal 1959). To date, the suitability of artificial oviposition substrates for B. hilaris have not been evaluated.

Overall, more information on rearing is needed and protocols specific to quarantine conditions would benefit researchers in areas where B. hilaris is not established.

Control Methods

No reliable monitoring tools such as traps exist for B. hilaris. Current research is exploring an effective trapping method for B. hilaris though a commercially available trap has not yet been developed (Joseph 2014). Unlike other pentatomids, B. hilaris remains close to the ground and will not fly into traps. Susceptible crops are monitored for B. hilaris by visual inspection from mid-morning to late afternoon when bugs are most active (Palumbo et al. 2016). It is advised that seedlings are carefully inspected

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since B. hilaris prefers young plants. If insects are not visibly present, scouting for damage is also beneficial to monitoring for B. hilaris (Reed et al. 2013).

Recent research has focused on developing better control methods for B. hilaris.

The most common control method is chemical application, as with most stink bugs. In the past, Paris green, organochlorines, and cyclodienes were used to treat for B. hilaris in its native range (Palumbo et al. 2016). Current studies have demonstrated adequate control using pyrethroids (Palumbo et al. 2016). Globally, organophosphates, carbamates, and pyrethroids are utilized for control, but many of these chemicals are banned due to toxicity in the United States. Instead, growers in the United States typically use malathion, carbaryl, dimethoate, and chlorpyrifos to control B. hilaris with variable success rates. Since B. hilaris prefers younger cotyledons, early treatment is important for control (Reed et al. 2013). Bagrada hilaris is particularly problematic for organic producers as the efficacy of organic pesticides on stink bugs is very low.

Non-chemical practices alone appear to be less effective for control of B. hilaris.

When combined with chemical control methods, cultural control can aid in keeping population levels of B. hilaris lower (Palumbo et al. 2016). Some recommendations include removing weedy areas surrounding crops and destroying post-harvest crop residues. It is also suggested that transplants instead of direct seeding may prevent early feeding. Knowledge of biological control methods is limited, and more investigation is required in this area. Currently, no native predators of B. hilaris exist in the United

States although minor generalist predators are present (Palumbo et al. 2016). For the future, there is interest in biological control using species of parasitoid wasps that target

B. hilaris in its native range (Reed et al. 2013). However, classical biological control for

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nonnative species is a complex process that may take years to reach approval for release.

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CHAPTER 2 ENGAGING FLORIDA’S YOUTH TO INCREASE THEIR KNOWLEDGE OF INVASIVE SPECIES AND PLANT BIOSECURITY

Introduction

Strategic plant biosecurity policies and procedures have been developed and implemented to prevent the introduction of non-native invasive species that can be harmful to plants grown for human food and animal feed, maintained in established landscapes, and existing in the natural environment (Hodges and Stocks 2011). A non- native species is considered invasive if its establishment or spread is injurious to plants, animals or humans, or it is shown to be potentially injurious by risk analysis (FAO, IPPC

2007). A species may be introduced into a new environment intentionally, such as the introduction of a commercial ornamental plant, or unintentionally as an insect hitchhiker arriving on an agricultural commodity (Winberry and Jones 1973, Sargent et al. 2011).

Invasive species have become a global problem due to an increase in the potential pathways for introduction resulting from people and products, especially agricultural commodities, moving around the world at an accelerating (Meyerson et al. 2013). It is estimated that invasive species cause losses of over $120 billion annually in the United

States (Pimentel et al. 2005). Florida is a particularly high-risk state for the introduction of invasive species because of its temperate, subtropical and tropical habitats, and diversity of high-value agricultural commodities, including citrus, other fruit, vegetables, ornamental plants, and field crops (FDACS 2016b). Florida has eight main destination airports and 11 commercial seaports that receive passengers and commodities that

 Reprinted with permission from the Journal of Integrated Pest Management published by Oxford University Press.

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often continue to move throughout of the United States (Szyniszewska 2013). It also has many tourist attractions that bring millions of people to the state each year.

In recent years, many problematic invasive species of arthropods and pathogens have impacted Florida. For example, the Mediterranean fruit fly, Ceratitis capitata

(Wiedemann) (Diptera: Tephritidae), was detected multiple times during the last century and required costly eradication in addition to continued monitoring throughout the state

(Steck 2002). Another example of a successful detection and eradication occurred with

Oxycarenus hyalinipennis (Costa) (Hemiptera: Oxycarenidae), which was detected in the Florida Keys in 2010 and promptly eliminated (Sharma 2014). Similarly, the giant

African snail, Lissachatina fulica (Bowdich) (Stylommatophora: Achatinidae), was detected in 2011 and is currently being eradicated with public assistance in Miami-Dade

County (FDACS 2018a). Once widely distributed, however, it is not possible to eradicate all invasive species that have established in Florida, such as spotted-wing drosophila,

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) (Iglesias et al. 2009);

Aulacaspis yasumatsui Takagi (Hemiptera: Diaspididae) (Mannion et al. 2006); redbay ambrosia beetle, Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae) (FDACS

2018b); Candidatus Phytoplasma palmae (Acholeplasmatales: Incertae sedis) (Elliott

2009); Huanglongbing (Greening), Candidatus Liberibacter asiaticus Jagoueix et al.

(Rhizobiales: Phyllobacteriaceae) (Hodges and Spreen 2015); and Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) (Thomas 2005). In an attempt at early detection, surveillance is being conducted for some other pests of concern but not yet established in Florida, such as the Old World bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae); Bagrada hilaris (Burmiester) (Hemiptera:

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Pentatomidae); Asian longhorned beetle, Anoplophora glabripennis (Motschulsky)

(Coleoptera: Cerambycidae); light brown apple moth, Ephiphyas postvittana (Walker)

(Lepidoptera: Tortricidae); Phytophthora ramorum Werres, de Cock & Man in’t Veld

(Peronosporales: Peronosporaceae); and Oriental fruit fly, Bactrocera dorsalis Hendel

(Diptera: Tephritidae).

The general public, particularly youth, remain largely uninformed about plant biosecurity and invasive species. This lack of knowledge must be addressed because invasive species will continue to cause substantial economic losses, decrease biodiversity, introduce human and animal health hazards, and disrupt natural ecosystems (Hodges and Stocks 2010). To inform the public about these risks, a youth outreach project was developed to deliver information on plant biosecurity and invasive species to middle and high school students throughout Florida. The students received instruction on how to define and identify plant pests; distinguish between native, non- native and invasive pests; and the importance of early detection and rapid eradication of invasive species. This knowledge is crucial for volunteer-based networks to succeed in preventing the establishment of additional invasive species in Florida, as they have in the past (Pimentel et al. 2005, Bois et al. 2011, Burrack et al. 2012, Andow et al. 2016,

Stubbs et al. 2017).

Expected Outcomes

Purpose

The purpose of the outreach events was to raise awareness in Florida’s youth of risks posed by invasive species, deliver the concepts of plant biosecurity, and promote early detection of non-native species.

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Objectives

1. Evaluate current knowledge of Florida’s youth about invasive species and

plant biosecurity.

2. Measure the effectiveness of the outreach events targeting a youth

audience.

Materials and Methods

Audience

The target audience was 11- to 18-yr-old middle and high school students throughout Florida. Schools were selected based on the presence of biology, agriculture, or natural science classes, and if interested, teachers were contacted via e- mail to schedule outreach events. Thirteen classes were selected in the 2016–2017 school year at five different high schools in Alachua, Marion, and Palm Beach counties.

In the 2017–2018 school year, 25 classes were selected at an additional five high schools in Alachua, Brevard, Orange, and Palm Beach counties. Thus, over 2 yr, 730 students were surveyed on their knowledge of two topics with 359 participants in the

‘Plant Biosecurity-Local and Global Perspectives’ topic and 371 participants in the

‘Invasive Species that Affect Plants’ topic. Surveys with missing answers were discarded from the analysis. Classes usually had 15–25 students, but several larger auditorium-style events combined multiple classes to include 50–60 students.

Outreach Events

Each outreach event consisted of a 40-min presentation on one of the two topics selected by the teacher, 10 min of hands-on activity, and an additional 5–10 min for answering student questions. Teachers selected one of two topics to be presented to

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their students: ‘Plant Biosecurity-Local and Global Perspectives’ (Hodges and Stocks

2011) or ‘Invasive Species that Affect Plants’ (Hodges and Stocks 2010). The incorporated educational PowerPoint presentations were modified for the target audience from existing Protect U.S. scripted lectures (www.protectingusnow.org).

Presentations were based on survey results of a preliminary study completed in 2016 at two high schools with a total of 161 students. The presentations were modified to adequately explain the core concepts tested in the survey. Each presentation was co- delivered by two Doctor of Plant Medicine (www.dpm.ifas.ufl.edu) students, one being a coordinator that was different for each academic year. To ensure consistency between outreach events, presenters were trained prior to delivering a presentation by first attending one of the presentations, and then delivering both presentations for evaluation by a faculty member. Presentations were designed to be as interactive and engaging as possible, with periodic critical thinking questions. For example, questions such as ‘How do you think invasive species could affect what you eat?’ or ‘What are some of the reasons you can think of that caused the price of rice to increase in 2008?’ were used to engage students throughout the presentations.

Hands-on activities in the classrooms involved live agricultural insect pests, preserved insect displays, and pest-infested plants. Live insects were provided by the

Biosecurity Research and Extension Laboratory colonies reared for both outreach and research. Preserved insect displays contained curated insects from throughout Florida, including agricultural and ecological pests, non-native and invasive species, and beneficial or common insects. The students were provided with greenhouse-raised plants naturally infested with aphids (Hemiptera: Aphididae), whiteflies (Hemiptera:

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Aleyrodidae), and thrips (Thysanoptera). Dissecting microscopes were brought to every classroom so that students could study the organisms in detail. Insects rather than plant pathogens were provided because they are larger and easier to view. Each student was also given a magnifying hand lens, a pen, and a notepad to record observations throughout the outreach event.

At the end of the ‘Invasive Species that Affect Plants’ presentation, students received instruction on the term invasive species, and the issues and risks associated with establishment of non-native species. The presentation described a variety of invasive species including plant pests and pathogens, and plants. Several examples of invasive species that have established in the United States were covered, such as the invasive plant, Kudzu, Pueraria lobata (Lour.) (Fabales: Fabaceae); Huanglongbing

(Greening), Ca. L. asiaticus, a bacterial plant pathogen and its vector the Asian citrus psyllid, Diaphania citri Kuwayama (Hemiptera: Psyllidae); and soybean rust,

Phakopsora pachyrhizi Syd (Uredinales: Phakopsoraceae) (Hodges and Stocks 2010).

This topic also included prominent case studies on current pests of concern to Florida including B. hilaris; the brown marmorated stink bug, Halymorpha halys (Stål)

(Hemiptera: Pentatomidae); Laurel wilt, Raffaelea lauricola (Harrington, Fraedrich &

Aghayeva) (Ophiostomatales: Ophiostomataceae) and its vector the redbay abrosia beetle, X. glabratus; and the emerald ash borer, Agrilus planipennis Fairmaire

(Coleoptera: Buprestidae). Lastly, using messages from programs underway at agencies like the United States Department of Agriculture (USDA) and the United

States Department of Homeland Security-Customs and Border Protection (USDHS-

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CBP), it was reinforced that everyone, including the public, is responsible for protecting plants from invasive species.

In the ‘Plant Biosecurity-Local and Global Perspectives’ presentation, the terms biosecurity, bioterrorism, and agroterrorism were covered. By the end of the presentation, students should have been able to broadly define these terms. Plant biosecurity was described as protecting plant health, the food supply, and the overall environment using an integrated and strategic approach (Hodges and Stocks 2011).

Students gained an understanding of how plant biosecurity affects their lives, the consequences of globalization, and how this raises concerns about food security globally. This presentation also included information on bioterrorism, the use of a living organism as a weapon. Agroterrorism was more specifically defined as the release of a pest or pathogen with the intention of disrupting or destroying the food supply. For example, anthrax, Bacillus anthracis Cohn (Bacillales: Bacillaceae), was used by

Germany as a weapon of agroterrorism during World War II to kill livestock of the Allied forces (Croddy and Wirtz 2005). Another historic example of agroterrorism was release of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera:

Chrysomelidae) in World War II by Germany in an attempt to target the food supply of their enemies (Garrett 1996). The USDA Select Agents and Toxins List includes biological agents and toxins that could pose a severe threat to human and animal health, plant health, or to animal and plant products (FSAP 2018). Brief case studies of several current select agents were provided, including bacterial wilt, Ralstonia solanacearum (Smith) (Burkholderiales: Burkholderiaceae) race 3, biovar 2, and

Rathayibacter toxicus (Riley and Ophel 1992) (Actinomycetales: Microbacteriaceae).

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Also included was the previous select agent and toxin, Huanglongbing (Greening), Ca.

L. asiaticus, which was removed from the list after establishing in Florida and causing severe economic losses to citrus. Huanglongbing serves as an example to reinforce how invasive species can greatly impact agriculture (Hummel and Ferrin 2011).

Surveys

Students under the age of 18 were required to return a signed document from a parent or legal guardian giving consent for the student to be surveyed. After the consent forms were collected at the beginning of a class, each student received a packet containing a pre- and post-survey. The pre-survey was completed and collected before the presentation, and the post-survey was taken at the end of the class. Surveys were designed similarly with seven questions for both presentation topics (Figs. 2-1 and 2-2).

Question 1 asked each student to rate their level of understanding of the topic as none, minimal, general, or extensive. Based on preliminary studies in 2016, questions 2–7 were modified to ensure that the students could understand the questions and answers.

The questions were based directly on presentation content and had correct answers.

Answers given by the students were used to evaluate their knowledge of the topic before and after the outreach event. The surveys were numbered and given a specific class code, and each student had a survey identification number so that pre- and post- surveys could be paired for analysis. Survey results were analyzed using R 3.5.0 (R

Core Team 2013) with question 1 being evaluated separately from questions 2–7. All surveying was approved by the University of Florida, Institutional Review Board

(Protocol ID: IRB201602341).

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Results

Student’s Comfort Level

For the ‘Invasive Species that Affect Plants’ topic (question 1), students (n = 371) were asked to rate their level of comfort in their ability to define the term ‘invasive species’ as none, minimum, moderate or complete understanding of the definition.

Before the presentation, only 7.0% of students had a complete understanding of invasive species, 43.1% had a moderate understanding, 32.9% had a minimal understanding, and 17.0% had no understanding. Based on this pre-evaluation, over half of students in the surveyed classes had minimal or no understanding of invasive species despite the importance of this topic to the public. Following the presentation, complete understanding increased to 32.6% and the percentage of students with a moderate understanding increased to 55.8%. Furthermore, the students with a minimal understanding decreased to 9.2% and no understanding of the concept of invasive species dropped to 2.4%. Based on a paired t-test comparing the overall pre- and post- survey results for question 1, the results indicated that the outreach event significantly increased understanding of the topic of invasive species so that most students felt moderate to completely comfortable with the subject (P value 2.2e-16).

For the ‘Plant Biosecurity-Local and Global Perspectives’ presentation (question

1), students (n = 359) described their comfort in understanding the terms ‘biosecurity’ and ‘agroterrorism’. The comfort level was described as knowing neither terms, only one term, having a basic understanding of the two terms, or a strong understanding of both terms. In the pre-survey, only 0.8% stated that they had a strong understanding of both terms, 20.3% of students had a vague understanding, 24.5% claimed to only understand one of the two terms without specifying which term they understood, and

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another 54.3% of students indicated that they did not understand both terms. Thus, more than three-quarters of students did not understand the terms ‘biosecurity’ nor

‘agroterrorism’ or only understood one of the terms before the outreach event. After the presentation, 2.8% still did not understand both terms, 7.8% understood one term,

49.3% understood both terms at a basic level and 40.1% understood both terms completely. Analysis of question 1 results with a paired t-test indicated that students significantly increased their believed understanding of the two terms following the presentation (P value 2.2e-16). Increased comfort in these topics is a first step toward involving students in volunteer-based networks for invasive species and plant biosecurity, and encouraging their interest in related fields in the future.

Student’s Knowledge Level

Based on the information delivered, the student’s knowledge level was tested with multiple-choice questions (questions 2–7) on the pretests and posttests. Results were paired to measure improvement of individual students before and after the presentations. Students were grouped by class and results represented the percentage of students that improved their scores (Figs. 2-3 and 2-4).

For all 16 classes that received the ‘Invasive Species that Affect Plants’ presentation, the performance of most students improved (over 50%) with only three classes showing improvement in less than 75% of students (Fig. 2-3). The greatest improvement was in classes 1 and 2 where 100% of students improved their scores. A paired t-test indicated a significant change (P value 2.2e-16) in student scores following the presentation with an average increase of 1.95 questions answered correctly. Based on these findings, the program is highly effective in teaching students about invasive species.

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Responses of students in classes that received the ‘Plant Biosecurity-Local and

Global Perspectives’ presentation were more variable than for the first topic. At least

50% of students in 18 out of 22 classes showed improvement although this percentage was higher in several classes (Fig. 2-4). For this topic, the greatest improvement was in class 22 where 90.6% of students improved their scores. Students increased their scores in the posttest by an average of 1.05 questions answered correctly. A paired t- test indicated that students improved their scores significantly after receiving this presentation (P value 2.2e-16).

Overall, the students were more comfortable in their understanding of invasive species than plant biosecurity. The level of comfort in the topics was translated to learning and understanding of the material as indicated by the improvement of scores in the post-survey for both topics. However, the instructors were more interested in plant biosecurity, because this topic generated more discussion by the students due to its novelty in the classroom.

Conclusion

Based on survey results, the base level of knowledge about invasive species and plant biosecurity is low for most students. The outreach presentations and associated hands-on activities significantly increased the level of knowledge about invasive species and plant biosecurity for the majority of the students. Students understood most of the basic concepts in the presentations on ‘Plant Biosecurity-Local and Global

Perspectives’ and ‘Invasive Species that Affect Plants’ by the end of outreach events.

Although these concepts are universal, the outreach events could be adapted to a variety of classroom settings and incorporate plant pests and pathogens of local concern. The students and general public must be informed about these topics to

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decrease the impacts of invasive species on agriculture, communities and the environment.

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Presentation Topic: Overview – Invasive Species that Affect Plants 1. Rate your level of comfort with providing a response to the following statement: “Define an invasive species.” a. Wait, what is an invasive species? b. I am only slightly comfortable, but want to know more. c. I can jot down a few good bullet points. d. Very confident. I could write an essay on invasive species! 2. Kudzu vine was first introduced to the United States at the Philadelphia Centennial Exposition of 1876 as a. An ornamental plant b. Soil erosion control c. Forage for cattle d. Clothing fiber 3. Which 4 states are responsible for the majority of commercial citrus production citrus in the United States? a. Florida, California, Georgia, and South Carolina b. Florida, Georgia, Louisiana, and Texas c. Florida, California, Texas, and Arizona d. Florida, California, Virginia, and New Mexico 4. Huanglongbing (HLB) also known as Citrus Greening is a devastating bacterial disease vectored by the Asian Citrus Psyllid affecting citrus in the United States. What are some symptoms of this disease in the fruit? a. Soft sunken spots, extra sweet pulp b. Lopsided fruit, green rind, bitter taste c. Yellow and black cankers on the rind d. None of the above 5. Invasive species only threaten agricultural commodities. They are not problematic in landscapes or natural areas. a. True b. False 6. The U.S. Department of Agriculture (USDA) has a special agency and branch dedicated to protecting our agriculture and natural resources called “APHIS-PPQ”. What does this acronym stand for? a. Animal and Plant Health Investigation Service, Plant Production and Quality b. Animal and Plant Health Interrogation Services, Plant Protection and Quarantine c. Animal and Plant Health Inspection Service, Plant Protection and Quarantine d. Animal and Plant Health Inspector Station, Plant Protection and Quality 7. Who is responsible for protecting our agriculture and natural areas from invasive species? a. Regulatory Agencies and Private Industry Companies b. University researchers, extension agents, and conservationists c. Homeowners and the general public d. All of the above

Figure 2-1. Survey questions for the ‘Invasive Species that Affect Plants’ topic.

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Presentation Topic: Plant Biosecurity – Local and Global Perspectives 1. Rate your level of comfort with understanding the terms “biosecurity” and “agroterrorism”. a. I have not heard of either term before. b. I know one of these terms but not the other. c. I have a vague understanding of both terms. d. I can easily provide descriptions and examples related to these terms. 2. How are invasive species introduced to the United States? a. International commercial trade b. Traveler baggage c. Private citizens d. a and c e. All of the above 3. The United States Department of Agriculture (USDA) works very closely with the U.S. FDA to help keep our food safe for consumption. What does the acronym “FDA” stand for? a. Food and Drug Association b. Food Delivery Administration c. Food and Drug Administration d. Food and Drink Association 4. Anthrax is a fatal disease in livestock (and humans) because symptoms are seen too late for treatment. What type of organism causes this disease? a. Bacteria b. Fungus c. Virus d. Insect 5. In 2002, what act required the USDA “to establish and regulate a list of biological agents that have the potential to pose a severe threat to animal health and safety, plant health and safety, or to the safety of animal or plant products (Select Agents and Toxins List)”? a. The Food Quality Protection Act b. The Agricultural Bioterrorism Protection Act c. The Protection Against Biological Threats Act d. The Federal Insecticide, Fungicide, and Rodenticide Act 6. Citrus Greening, Candidatus Liberibacter asiaticus, is no longer listed as a Select Agent in the U.S. a. True b. False 7. Ricin is a poison that can be derived from the waste material left over from processing which plant? a. Coffee beans b. Canola c. Quinoa d. Castor beans

Figure 2-2. Survey questions for the ‘Plant Biosecurity-Local and Global Perspectives’ topic.

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Figure 2-3 100% 80% 60% 40% 20% 0% Percentage of Students 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Class Number Improved score No change Decreased score

Figure 2-3. Mean percentage of students in each class that received instruction on ‘Invasive Species That Affect Plants’ that improved, did not change, or decreased their score from the pretest to the posttest for the concept questions 2–7.

Figure 2-4

100%

80%

60%

40%

20%

Percentage of Students 0% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Class Number Improved score No change Decreased score

Figure 2-4. Mean percentage of students in each class that received instruction on ‘Plant Biosecurity-Local and Global Perspectives’ that improved, did not change, or decreased their score from the pretest to the posttest for the concept questions 2–7.

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CHAPTER 3 LABORATORY REARING OF THE REDBANDED STINK BUG, Piezodorus guildinii (WESTWOOD)

Introduction

Piezodorus guildinii (Westwood), the redbanded stink bug, is an invasive stink bug thought to have arrived in the United States in the 1900’s from the Neotropics. It was not until the early 2000’s that P. guildinii was cited as an economic pest of soybean

(Husseneder et al. 2016). As of 2017, this pest is found throughout the southeastern

United States in Florida, Georgia, Louisiana, Mississippi, New Mexico, South Carolina and Texas (Pinkerton and Hodges 2017). Piezodorus guildinii is a polyphagous pest of leguminous plants including soybean, Glycine max; alfalfa, Medicago sativa; peas,

Pisum sativum; lentils, Lens culinaris; and other forage legumes. This pest can also survive on wild hosts such as Crotalaria spp. suggesting that population levels can be sustained on weedy plants when economically important crops are unavailable (Zerbino et al. 2014).

Piezodorus guildinii, like other stink bugs, uses piercing sucking mouthparts to siphon nutrients out of host tissues. This causes chlorotic spots on leaves, stems, and fruit which can result in lowered yield or marketability of crops, necrotic lesions, plant or fruit deformation, and susceptibility to secondary pathogens such as fungi (McPherson and McPherson 2000). For example, in soybean, most of the damage is to the bean pod. Piezodorus guildinii feeding causes shriveled pods and the seeds inside can be deformed and necrotic (Depieri and Panizzi 2011).

Laboratory rearing of P. guildinii is difficult and not as well documented as other stink bug species such as Nezara viridula. However, developing rearing techniques for

P. guildinii is equally as important to fostering efficient research on this pest. One study

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investigated photoperiods and temperature for nymphal development and found that nymphs are unlikely to survive in cold temperatures (Zerbino et al. 2013). Furthermore, another study suggests that a humidity level of RH 60 ±10% was ideal for rearing P. guildinii (Gomez et al. 2013), while a different group found an RH of 80 ±10% shows higher survivability (Zerbino et al. 2014). For diet, green beans, which are commonly used for other stink bugs, show a low success rate specifically for P. guildinii (Gomez et al. 2013). There has been additional research on artificial oviposition substrates and previous studies have evaluated tissue paper, wool string, and cotton balls with mixed success rates (Gomez et al. 2013, Silva and Panizzi 2007, Silva and Panizzi 2008).

As a result of the severe damage to agronomic crops, P. guildinii has become a pest of interest for research to better understand its biology and develop methods of control. A sustainable rearing technique would be beneficial to this research.

Investigations related to insect pests have generally led to many discoveries and accomplishments in agriculture including development of management strategies to enhance food productions, and laboratory colonies have been important for several innovations. For example, sterile insect technique requires rearing sterile males for release to help control pests (Sorensen et al. 2012). Another example would be to maintain a colony in order to study readily available specimens. Regardless, quality assurance is imperative to obtaining accurate results for experiments. In a laboratory colony, inbreeding and unintended artificial selection of certain traits can change biology and population dynamics thus effecting research. Some important indicators of inbreeding within a colony include weight changes and decreased fecundity (Portilla et al. 2011). In stink bugs, colony specimens appear to be smaller than wild caught insects

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which may be indicative of other noteworthy reductions in fitness. For this reason, it is valuable to document this size disparity and its significance throughout successive generations of stink bugs. This study aims to develop a rearing method for P. guildinii and identify a trend in body size over four generations of stink bugs reared in the laboratory.

Materials and Methods

Colony Source

Insects were collected from three different sites on multiple occasions when establishing the colony. Insects were collected during December of 2016 on weedy patches at the UF/IFAS Gulf Coast Research and Education Center in Wimauma, FL.

Approximately 500 adults were collected from weedy Crotalaria spp. and hairy indigo

(Indigofera hirsuta). A second collection site was utilized during April and May of 2017 at the UF/IFAS Plant Science Research and Education Unit in Citra, FL where an additional 600 insects were collected from alfalfa (Medicago sativa) and red clover

(Trifolium pratense). Piezodorus guildinii was collected from wild populations in June and July of 2018 at a third site in the UF/IFAS Suwannee Valley Agricultural Extension

Center in Live Oak, FL. Approximately 400 insects were found on sorghum (Sorghum bicolor) over a two-week period at this site. Wild caught insects were held in a separate area, reared following the laboratory protocol, and eggs were collected from females and used to establish the colony.

In April of 2017, a plot of known hosts of P. guildinii was planted at the UF research station in Citra, FL to ensure future collection sites. The plants seeded at this site included soybean (Glycine max), hairy indigo, and alfalfa. This plot was replanted in

September of 2018 to continue to attract new specimens to add to the colony. Wild

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caught specimens remained separated in the laboratory from the colony to prevent introduction of pathogens, pesticides or other contaminants that could interfere with the health of the colony.

Cages

Two different sized cages were used depending on the stage of the insect. Adult cages were prepared from plastic Beanie Babies display cases purchased from Pioneer

Plastics (Pioneer Plastics, Inc., Eagan, MN; www.pioneerplastic.com) (Fig. 3-1). The display case measured 9.4 x 9.4 x 21 cm and had a removable lid for easy access to the insects. Three 5.1 cm diameter holes were drilled into the plastic. One hole was drilled on the top of the lid and two holes were drilled in the sides of the cage on opposite walls. Nylon netting (off-white, 150x150 mesh) was then cut to be slightly larger than the hole on the container lid. A hot glue gun was used to attach the mesh netting to the outside of the lid, being careful to seal any openings. Once this initial seal dried, the other side of the lid was glued along the edge of the hole to prevent insects from escaping or becoming caught between the mesh and the plastic.

Eggs and nymphs were contained in a cage made from rectangular Tupperware

(Tupperware Corporation, Orlando, FL; www.tupperwear.com) containers measuring

10.2 x 5.1 x 5.1 cm (Fig. 3-2). Holes were cut through the softer material of the lid with around 1.3 cm between the hole and the edge of the lid to ensure that the container can still seal properly. The same process was used to secure mesh netting over the holes as was used with the adult cages.

Conditions

The laboratory temperature was maintained between 24-27°C and a humidifier was used to keep to the relative humidity between 60-80%. The colony was housed on

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wooden rearing shelves with attached lights on each shelf that were on a timer set to a

14L:10D cycle.

Diet

Insects were fed on a schedule a mixed diet consisting of sweet peas and raw peanuts purchased regularly from a local supermarket. The peas were washed thoroughly to remove dirt or any pesticide residue and dried prior to insect feeding.

Leguminous plants including alfalfa, crotalaria, and hairy indigo were grown in a greenhouse to supply fresh plant cuttings.

Rearing Procedure

All adult cages were labeled with the date the cage was created (i.e. when the insects emerged as adults) and the generation. A maximum of 16 newly emerged, and unmated adults were placed in a single adult cage. Females and males were randomly assigned to cages with an average of 8:8 males to females. Fresh adult cages were prepared once per week and then adults were directly transferred from dirty cages using a paintbrush or gently by hand to the clean cage with fresh food. On feeding days, eggs were collected from the cages, by removal of the substrate they were laid on. Adult cages were lined with a paper towel folded to fit the bottom of the container. Three cotton balls were added to every adult cage as an oviposition substrate. Each adult cage had three peas pods and three peanuts on the bottom of the cage, and two peas pods attached to the lid of the cage with an insect pin. In addition, every cage had cuttings of a fresh leguminous plant held within a test tube filled with water. The test tube was leaned against the side of the cage to prevent it from spilling over and plant cuttings were replaced as needed. Other food sources were replaced consistently twice

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per week on Mondays and Thursdays. Adult cages were completely cleaned and changed once per week (Monday).

All immature cages were labeled with the generation number and the date the eggs were collected. A maximum of 30 eggs were placed into each immature container by movement of the oviposition substrate they were laid on to disturb the eggs as little as possible. Once the eggs hatch, nymphs in the immature cages are fed on the same schedule as the adult cages. Cages were lined with a paper towel cut to fit the bottom of the cage. Two cotton balls were added to the cage for crawling substrate and to absorb excess moisture. Each cage was given two peas pods and two peanuts in the bottom of the cage and an additional pea pod was pinned to the top of the cage using an insect pin. Nymphs were handled with a fine tipped paint brush to lower mortality caused by handling.

Experiment 1: Preference of Oviposition Substrate

No-choice

Three substrates (cotton balls, kim tech wipes [www.kcprofessional.com], and dental wicks) were evaluated using a no-choice test. Kim wipes and dental wicks were tested as both are utilized in laboratory colonies for other pentatomids in the Biosecurity

Research and Extension laboratory at the University of Florida. Three replicates of each substrate were set up prior to the introduction of insects with the only contents in the cage being the tested substrate and food. Cages were fed using the standard rearing procedures for the colony except for the oviposition substrate type within the cage. A large cage of second generation 5th instars were separated from the colony and upon emergence as adults were sorted into temporary males and female adult cages. Twenty insects were placed in each cage with a ratio of 10 males to 10 females. Twice per

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week, when the food was changed, all substrates and dead insects were removed from the cages and clean substrate was re-added. Substrates were examined at this time to minimize disturbances of the insects. Deaths were recorded and divided by sex. The number of eggs was recorded from all substrates within each cage. Egg number was used rather than number of clutches in order to account for the variability in clutch size.

Eggs were collected and recorded on each substrate until all female individuals in the cage died. Data was analyzed in R using an ANOVA and further evaluated with a Tukey

HSD procedure for significant differences between replicates and substrate type.

Choice

Three replicates of 10 males and 10 females were set up with a choice between all three substrates. Insects were able to choose between equal proportions of each substrate. Insects were reared, and data was collected in the same manner as the no- choice test with oviposition being recorded as number of eggs per substrate. Data was analyzed in R using an ANOVA and further evaluated with a Tukey HSD procedure to test the effect of both replicate and substrate type on the number of eggs laid.

Experiment 2: Identification of Size Disparities in Successive Generations of Laboratory Reared Piezodorus guildinii

For this experiment, wild caught insects were collected from Citra, FL in April

2017 and 10 cages were set up following the rearing procedures used for the colony.

Insects were reared accordingly until death. Dead insects were removed from the cages twice per week when the food was changed, stored in labeled containers, and put into a deep freezer for further measurement. Eggs were collected from the wild caught containers, labeled as generation one and reared following laboratory procedures.

Insects were continually reared until generation three. Dead insects from each

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generation were defrosted and measured with a Neiko 01407A Electronic Digital

Caliper. The caliper has a measurement range of 0-150 mm, a resolution of 0.01mm, and an accuracy of 0.02mm. Results were analyzed using an ANOVA and a Tukey test in R to test the effect of gender and generation on size.

Results and Discussion

Experiment 1: Preference of Oviposition Substrate

No-choice

Given no-choice of one of the three substrates, there does not appear to be a significant effect (F=2.195; df=2; p=0.114) on the mean number of eggs laid on each substrate by adult P. guildinii and the substrate type (Fig. 3-3). Additionally, there was a significant difference between the total number of eggs laid on each substrate by replicates (F=7.439; df=2; p<0.001) further supporting that the substrates have a variable effect on the number of eggs laid by female P. guildinii. Consequently, the results support that all three substrates perform equally as an artificial oviposition substrate given no alternatives. Given that the average female can lay as many as 200 eggs under ideal conditions (Zerbino et al. 2013), the total number of eggs laid on dental wicks, cotton balls and kim wipes was substantially lower than the potential eggs the females could have laid (1070, 1770, 1596 eggs respectively). Although the literature displays mixed results on the adequacy of cotton balls for oviposition by P. guildinii (Silva and Panizzi 2007, Silva and Panizzi 2008), experiment results supports those studies that indicated cotton balls are not a suitable substrate for P. guildinii.

Since the egg laying is lower than it could potentially be, all three substrates are likely not suitable for oviposition by P. guildinii.

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Choice

Unlike the no-choice test, there was not a significant difference in the total number of eggs laid between each replicate of the choice test indicating that the selection of substrate was less variable when insects were given the choice between all three substrates (Fig. 3-3). The results of the choice test indicated a significant effect of the substrate type on the mean number of eggs laid (F=5.868; df= 2; p=0.003). The highest total number of eggs were laid on the cotton balls, 408 eggs, across all three replicates which was significantly different than the total number of eggs, 99, laid on the dental wicks (p=0.002). However, a non-significant difference between the number of eggs laid on the cotton balls and the kim wipes (p=0.126) was seen. Previous studies have suggested mixed results for cotton balls as an artificial oviposition substrate for P. guildinii (Silva and Panizzi 2007, Silva and Panizzi 2008). Choice experiment results suggest cotton balls had the highest number of eggs of the three substrates tested, but as seen with the no-choice test, egg laying appears to be lower than the average potential of female P. guildinii.

Experiment 2: Identification of Size Disparities in Successive Generations of Laboratory Reared Piezodorus guildinii

A significant effect of gender occurred on both length (F=87.63; df=1; p<0.001) and width (F=29.71; df=1; p<0.001) of adult P. guildinii. Results confirm the sexual dimorphism previously recorded in this species (Tab. 3-1, 3-2). The effect of generation on insect size was separately analyzed for males and females based on these results. A significant effect of generation occurred on body width (F=37.51; df=3; p<0.001) and length (F=87.63; df=3; p<0.001) in females. Similarly, there was a significant effect of

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generation on body width (F=64.81; df=3; p<0.001) and length (F=33.98; df=3; p<0.001) in males as well.

Additional analysis with a Tukey HSD procedure showed significant differences in body size for males (Table 3-1) and females (Table 3-2) based on generation. For both sexes, the general trend was that body size decreased over subsequent generations reared in the laboratory. For females, a significant difference occurred in length from the wild caught population to the 1st generation (p=0.003), and between the

2nd generation and the 3rd generation (p=0.016) indicating a gradual decline in body size. However, in the males, the length was only significantly different between the wild caught population and the 1st generation (p<0.001). Overall, body length appears to change more drastically over subsequent generations of females compared to males based on experimental results, suggesting that females are more susceptible to selective pressures caused by rearing conditions than males.

A trend similar to body length was seen in body width of the females as a significant difference occur from the wild caught to the 1st generation (p<0.001) and from the 1st generation to the 2nd generation (p=0.024). The significant difference of the widths was the same in the males with a significant difference from the wild caught to the 1st generation (p<0.001) and from the 1st generation to the 2nd generation

(p<0.001). Results suggest that body width may be more stable than length in both sexes after the 2nd generation of P. guildinii reared in the laboratory.

Conclusions

The optimal oviposition theory suggests that adult females will select an oviposition site to optimize offspring survival (Molina and Trumper 2012). In the

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laboratory, artificial substrates are often utilized to better monitor insect colony growth and development and induce oviposition under highly controlled conditions (Leppla

2008). Nonetheless, preference and success of artificial substrates for oviposition differ for different types of insects or even closely related species. Cotton balls are effectively utilized in the rearing of several stink bug species, but the literature shows variable success with cotton balls as an artificial oviposition site in the rearing of P. guildinii

(Silva and Panizzi 2008,Silva and Panizzi 2007). Other literature has cited using tissue paper or string as an artificial oviposition substrate for P. guildinii, but prior to this research dental wicks have not been evaluated (Gomez et al. 2013, Silva and Panizzi

2008, Silva and Panizzi 2007). Experiment results suggest that cotton balls, dental wicks, and kim wipes perform equally as oviposition substrates for P. guildinii, but adults lay fewer eggs than they potentially could with all three substrates. These findings imply that none of the three evaluated substrates are ideal for oviposition by P. guildinii and this could have implications on colony stability, particularly level of oviposition, over time. To date, the most effective oviposition substrate for P. guildinii was live plant host material, and of known artificial substrate, the most effective is wool string for this species as tested in previous publications (Silva and Panizzi 2008). Future studies will evaluate additional artificial substrates and compare them to the number of eggs laid on wool string though not tested in this experiment. Additionally, although insects species are capable of adapting to rearing conditions for oviposition (Shimoji and Miyatake

2002), adaptation to artificial oviposition substrates may occur over multiple generations at which point colony stability may already be compromised. For this reason, a suitable

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artificial oviposition substrate applicable to wild caught and colony P. guildinii remains important.

Understanding the limitations of insect rearing is important for research as it can aid in the proper interpretation of bioassay results for field application (Sorensen et al.

2012). Insects can adapt to their surroundings at an individual or population level and these adaptations can be driven by genetics or environmental conditions (Chevin et al.

2010). In laboratory rearing, adaptations can be beneficial for fostering higher colony survival and reproductions, but adaptations may also lead to distinct differences in colony insects compared to wild populations. Laboratory conditions often simulate lower population selective pressures such as predation or unfavorable climatic conditions

(Huettel 1976). Results of experiments on body size over subsequent generations of P. guildinii could indicate that current rearing techniques may be imperfect. The smaller body size may also be indicative of a sub-optimal rearing condition such as an insufficient diet. Although insect size is smaller, after the 2nd generation, body size appears to stabilize and not significantly change over succeeding generations, which may indicate that current rearing conditions favor smaller bodied insects. More research is needed to determine if the smaller body size is genetically determined or linked to deficiencies in the rearing conditions and procedure.

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Figure 3-1. Adult cage utilized in rearing of P. guildinii. Cage was made from plastic containers (Pioneer Plastics, Inc., Eagan, MN; www.pioneerplastic.com). Holes were cut and covered with mesh for aeration on the lid and sides of cage.

Figure 3-2. Immature cage utilized in rearing of P. guildinii. Cage was made from plastic Tupperware containers (Tupperware Corporation, Orlando, FL; www.tupperwear.com) and had a clear blue lid. Holes were cut and covered with mesh for aeration on the lid.

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Oviposition of P. guildinii on Artificial Substrates 2000 1800 1600 1400 1200 1000 800

600 A Number ofeggs 400 A 200 B 0 Dental wicks Cotton ball Kim wipe Dental wicks Cotton ball Kim wipe No-choice substrates Choice substrates

Figure 3-3. No-choice and choice test evaluating the total number of eggs laid by P. guildinii on three artificial substrates; dental wicks, cotton balls, and kim wipes.

Table 3-1. Mean (μ) and standard deviations (σ) of body size of adult P. guildinii males over four generations.

Generation μlength (mm) σlength (mm) μwidth (mm) σwidth (mm) 0 9.371 A ± 0.459 5.459 a ± 0.272 1 8.534 B ± 0.533 4.930 b ± 0.288 2 8.351 B ± 0.441 4.631 c ± 0.270 3 8.146 B ± 0.558 4.587 c ± 0.228

Table 3-2. Mean (μ) and standard deviations (σ) of body size of adult P. guildinii females over four generations.

Generation μlength (mm) σlength (mm) μwidth (mm) σwidth (mm) 0 9.884 A ± 0.672 5.668 a ± 0.263 1 9.291 B ± 0.559 5.108 b ± 0.332 2 8.957 BC ± 0.650 4.872 c ± 0.380 3 8.684 C ± 0.742 4.756 c ± 0.409

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CHAPTER 4 LABORATORY REARING OF THE BAGRADA BUG, Bagrada hilaris (BURMEISTER)

Introduction

The bagrada bug, Bagrada hilaris, is an invasive stink bug from Africa that has recently been introduced to California in 2008. Bagrada hilaris is now spread and established in California, Arizona, Nevada, Utah, New Mexico, and west Texas

(Hoenisch et al. 2014). Currently, B. hilaris is one of three pests under the Federally

Recognized State Managed Phytosanitary Program (USDA 2015). In its native range and introduced areas, B. hilaris causes major damage to cole crops (Brassica spp.) such as broccoli, cauliflower, cabbage and kale (Palumbo et al. 2016). Other hosts include collards, radish, arugula, turnip and mustards (Huang et al. 2014a). In the southwestern United States, it is estimated that B. hilaris can cause around 10% yield losses in cole crops but as high as 70% in severe cases like outbreak pest population levels (Palumbo et al. 2016).

This pest is extremely problematic due to some unique behaviors not seen in other plant feeding stink bugs. Bagrada hilaris can employ an overwintering strategy surviving on weedy hosts such as wild mustards before moving into crops just as seedlings emerge early in the season. The young plant stage is the most susceptible to damage by B. hilaris as plant growth can terminate. Overall, this pest can cause irreversible damage producing desiccated or unmarketable plants, and in extreme cases, preventing reproductive heads from forming altogether in crops like broccoli

(Halbert and Eger 2010). Additionally, B. hilaris lays eggs individually beneath the soil, making control strategies difficult since contact insecticides or natural enemies may not be able to penetrate the soil (Taylor et al. 2014).

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Rearing strategies for B. hilaris are limited and knowledge on quarantine rearing has not been documented (Taylor et al. 2015). According to the literature, B. hilaris has been successfully reared on Indian mustard, Brassica juncea (L.) Czern. (Halbert and

Eger 2010), mesa pepperwort, Lepidium alyssoides A. Gray (Taylor et al. 2014), and cabbage, B. oleracea (Verma et al. 1993). The ideal rearing temperature range appears to be 25.5-34 °C (Taylor et al. 2015). Still, the range of humidity levels for this pest have not been documented.

In Florida, B. hilaris has not yet established and due to its regulatory status, is only present in Florida under maximum security containment protocols. However, with cabbage being the 10th highest valued crop, Florida’s agriculture could be at risk for extreme losses if this pest were to establish (FDACS 2013). Investigation on rearing of

B. hilaris is limited, making further studies necessary to successful rearing of a colony for research purposes in the future. Due to this risk of establishment in Florida, early research will aid in preventing the establishment of the pest. A rearing technique for a quarantine colony of B. hilaris is imperative to being able to complete these studies.

Materials and Methods

Colony Source

Appropriate permits were obtained prior to the shipment of live B. hilaris to the

Florida Department of Agriculture and Consumer Services (FDACS)-Division of Plant

Industry (DPI) Quarantine Facility in Gainesville, Florida. Initial packages and all rearing of insects were contained in the maximum-security room of the DPI-Quarantine Facility.

Initial shipments of 215 viable insects were received from Dr. John Palumbo on June 9 and 15, 2016. Insects were collected from the University of Arizona Agricultural Center

Yuma, AZ on broccoli (B. oleracea var. capitata) and shipped following the permit

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procedures. All shipment materials were autoclaved and disposed of following the removal of all insects.

Cages

Adult cages were made from one-gallon wide-mouth polyethylene terephthalate jars (25.6 cm x 14.7 cm diameter) (ULINE, USA, https://www.uline.com) (Fig. 4-1). Four

5 cm diameter holes were cut and evenly spaced around the sides of the jar. Nylon netting (off-white, 150x150 mesh) was cut to be slightly larger than the holes and a hot glue gun was used to attach the mesh to the lid on the outside. Excess fabric around the hole was on the outside of the cage so that insects could not crawl underneath and get stuck. Once the initial seal cooled and hardened, another layer of glue was added around the seal on the inner side of the lid to prevent insects from escaping or crawling between the mesh netting and the container. A hole was also added to the lid of the cage with about 1-2 cm from the edge of the lid and mesh was properly attached.

Immature cages were created from cylindrical plastic restaurant-style

Tupperware (Tupperware Corporation, Orlando, FL; www.tupperwear.com) containers measuring 6.7 cm in height and 11.4 cm in diameter (Fig. 4-2). Holes were cut in the lids with approximately 1-2 cm between the hole and the edge of the lid to ensure sealing. The mesh was secured in the same fashion as used for the adult cages.

Cages were frequently checked for breaches in the glue or cage walls as well as incomplete sealing containers to ensure escapes were not possible. Cages were created to be as secure as possible to prevent escape of B. hilaris. All individual cages are placed inside a Bug Dorm-2120F Insect Tent (BD2120F) (MegaView Science Co.,

Ltd.; www.bugdorm.com) for added security. The Bug Dorm had two entrance points including a zipper panel and an access hole with extended fabric. The access hole

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remained securely tied throughout the rearing process and the zipper was opened to remove and replace the sealed cages when the food was changed.

Conditions

The laboratory colony was maintained in the maximum-security room in the

FDACS-DPI quarantine facility. The room was regularly monitored for escaped insects using visual observation, yellow sticky traps, and sticky door mats.

The relative humidity was maintained at 50-65% and monitored with HOBO data loggers. Four dehumidifiers were placed within the room around cages to keep humidity levels below the upper limit. The temperature fluctuated between 23-27 °C depending on if the lights were on or off which were on a timer for a 14L:10D photoperiod.

Diet

Kale and cabbage were purchased from a local supermarket twice per week, washed thoroughly to remove dirt or any pesticide residues and dried to prevent excess moisture in the cages.

Rearing Procedure

Rearing procedures were periodically modified since the colony was established to facilitate insect survival and enhance quarantine conditions. All one-gallon adult cages were prepared prior to introduction of insects and labeled with the date the cage was created and the generation number. These large cages were lined with a folded paper towel and six cotton balls were added to the bottom of the cage as an oviposition substrate. A maximum of 30 freshly emerged and unmated adults were placed into each large cage with an average ratio of 15 males to 15 females using a fine tip paintbrush or gently by hand. Each cage received a large kale leaf that was pinned to the top of the cage with an insect pin and three pieces of cabbage cut to approximately 40 cm2. Old

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food was removed, thoroughly searched for insects, and new food replaced twice per week on Tuesdays and Fridays. Cotton balls, used as artificial oviposition substrates, were examined for eggs and transferred to small cages on days when food is changed and replaced as needed.

Small immature cages (6.7 cm by 11.4 cm diameter) were labeled with the collection date of the eggs and generation number. A maximum of 100 eggs that were laid on cotton balls were placed into each cage. Each nymphal cage was lined with a kim wipe folded to fit the bottom of the cage. Each nymphal cage received two pieces of cabbage 40 cm2 and half of a kale leaf pinned to the top of the cage with a pin. Upon reaching the 3rd instar, nymphs are separated into large one-gallon cages for a maximum of 50 3rd-5th instars per cage to prevent overcrowding. As with the adult cages, large nymph cages receive a single kale leaf pinned to the top of the cage and three pieces of cabbage (40 cm2). Food was changed twice per week on the same schedule as adults with careful inspection of food prior to disposal. Once the nymphs emerged as adults, they were sexed and randomly sorted into new adult cages as previously described and labeled with the date of eclosion and generation number.

All rearing was performed within a white plastic bin to prevent accidental escapes of insects. Following all rearing, surfaces and dirty cages were cleaned with 90% ethanol. Materials for disposal were placed into a plastic bag and immediately frozen in a deep freezer to ensure no live insects remain. As needed, trash was removed from the freezer, autoclaved and disposed outside the quarantine facility.

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Experiment 1: Effect of Photoperiod on Egg Hatching

Bagrada hilaris typically lays eggs beneath the soil suggesting that photoperiod may be a factor in determining the hatching rate. The purpose of this experiment was to determine if a dark condition improves the rate and number of B. hilaris eggs that hatch.

A one-gallon plastic jar was maintained with 50 adults with 25 males and 25 females. Dead adults were removed and replaced as needed to maintain the number and sex ratio of adults. Old food was removed from this cage twice per week and replaced with new food following standard rearing procedures. For oviposition, six cotton balls were placed in the bottom of the cage daily and old cotton balls were removed and inspected for eggs before the light timer turned on every morning.

Eggs were counted, randomly divided in half, sorted into two identical immature cages (6.7 cm height and 11.4 cm diameter) and labeled with a replicate number (1-27).

A total of 654 eggs were collected and divided into the two treatments. For each replicate, half of the eggs were held in a box with dark conditions and half were placed into a box with light conditions. The boxes (30.5 cm by 25.4 cm by 38.1 cm) were constructed from cardboard banker boxes and lined with mesh to prevent escapes, but still allowed for ventilation within the box. Within each box, cages could be accessed through a Velcro flap-sealed opening in the mesh. Dark condition cages were placed in a ventilated box that does not contain any light sources to represent the 0L:24D cycle.

Light condition cages were placed into a nearly identical box that differs only in that there is light provided by a single LED, full spectrum light bulb linked to a timer that allows for a 14L:10D cycle. Both boxes remained closed except for when cages were added to the treatment box, or eggs were inspected for hatching. Boxes were monitored

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for temperature and RH using HOBO data loggers to ensure no differences occurred between the two treatments besides the light.

Egg cages were inspected daily by visual examination for nymph emergence.

The number of nymphs that emerged from each cage was recorded along with the number of days it took for each nymph to emerge. Nymphs were removed upon recording and cages were replaced into the respective treatment box until either all nymphs had hatched, or two weeks had passed.

Data was analyzed in R using a T-test to determine if there was a significant difference between the viability of eggs (i.e. proportion of eggs that hatched) in the light and dark treatments. Similar analysis was used for the length of development time for the eggs (i.e. the number of days it took for the eggs to hatch) between the treatments.

Experiment 2: Preference of Oviposition Substrate

No-choice

Three substrates were evaluated as an oviposition substrate; cotton balls, dental wicks, and kim tech wipes (www.kcprofessional.com). Cotton balls were selected due to their efficacy as an artificial oviposition substrate for other pentatomid colonies including

Euschistus heros (Fabricus) (Silva and Panizzi 2007). Three adult cages described above were set up to only contain a food source and each one a different oviposition substrate. Generation eight 5th instar nymphs were separated from the colony, sexed after adult eclosion, and randomly added to a treatment cage. Each cage received 10 males and 10 females of B. hilaris adults for a total of 20 insects per cage. Three replicates of each substrate were tested. Except for the substrate within the cage, all other standard colony rearing procedures were followed. Twice per week, all substrate and food were removed and replaced with fresh materials following rearing procedures.

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Old substrate was inspected for eggs which were counted and recorded. Dead adults were removed and recorded whenever food and substrate were changed. Eggs were collected and recorded until all female insects in each cage had died. Egg laying was evaluated for the full life of the adult female until death to account for differences in egg laying over the adult life. Data was analyzed in R using an ANOVA and a Tukey HSD procedure to compare replicate and substrate type.

Choice

Three adult cages (one-gallon modified plastic containers) were set up to contain equal portions of all three substrates (cotton balls, dental wicks, and kim wipes).

Procedures were identical to the no-choice test except that each cage contained equal proportions of all substrates. Twice per week, substrates were removed and inspected for eggs which were counted and recorded. Data was analyzed in R using an ANOVA and a Tukey HSD procedure to test the effect of replicate and substrate type on the number of eggs laid.

Results and Discussion

Experiment 1: Effect of Photoperiod on Egg Hatching

A non-significant difference (t=0.5757; df=52; p=0.5673) occurred between the percent of eggs that hatch in the dark (µ=0.690; SD=0.304) or light (µ=0.638;

SD=0.357) treatments (Fig. 4-3). This suggests that photoperiod does not influence the number of B. hilaris eggs that hatch. Contrarily, the average number of days until hatching was significantly longer in dark (µ=5.461; SD=±0.367) compared to light

(µ=5.056; SD=0.586) conditions (t=9.278; df=504; p<0.001) of the eggs that did hatch in both treatments (Fig. 4-4). The eggs developed in a shorter number of days in the light than in the dark treatment. Based on these results, rearing of eggs under normal colony

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conditions (14L:10D) is sufficient for egg development and a dark period is not beneficial to the speed or percent of hatching.

Experiment 2: Preference of Oviposition Substrate

No-choice

Using an ANOVA, substrate type has a significant effect on number of eggs laid by B. hilaris females (F=7.981; df=2; p<0.001) when the adults were provided only a single substrate. Moreover, a non-significant difference occurred between the mean number of eggs laid on each substrate between the three replicates (F=2.886; df=2, p=0.060) (Fig. 4-5). The highest number of eggs was seen on the cotton balls with a total of 1211 eggs throughout the three replicates. The lowest number of eggs were seen laid on the kim wipes with only 320 eggs total. Post hoc analysis using a Tukey

HSD procedure indicated that there is a significant difference in number of eggs laid between kim wipes and cotton balls (p<0.001) and between kim wipes and dental wicks

(p=0.008). However, there was not a significant different (p=0.720) between number of eggs laid on the cotton balls versus the dental wicks. Based on no-choice experiments, both cotton balls and dental wicks are adequate artificial oviposition substrates. On the other hand, kim wipes are not sufficient for oviposition by B. hilaris.

Choice

Given a choice between all three substrates, a significant effect occurred for oviposition on a substrate type (F=28.61; df=2; p<0.001) (Fig. 4-5). As with the no- choice test, a non-significant difference occurred between the mean number of eggs laid on each substrate between the three replicates (F=1.80; df=2; p=0.170). The highest number of eggs were laid on the cotton balls for a total of 1573 eggs over the three replicates, which was significantly different from both the dental wicks (p<0.001)

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and the kim wipes (p<0.001) as evaluated by a Tukey HSD procedure. Throughout all three replicates, only 141 eggs were laid on the dental wicks and 13 eggs were laid on the kim wipes. There was no significant difference between the number of eggs laid on the dental wicks and the kim wipes (p=0.829). Results indicate that cotton balls are the preferred artificial oviposition substrate when given a choice between the three substrates. Results of both the no-choice and choice tests propose that cotton balls are the preferred substrate and should be implemented in the rearing of B. hilaris as the artificial oviposition substrate.

Conclusions

The goal of laboratory rearing of insects is often to establish a sustainable colony, but identifying optimal conditions for a given insect species can be time consuming and multifaceted (Leppla 2008). Despite the importance of B. hilaris as a pest and the risk is poses to new areas, information on the rearing of B. hilaris is limited

(Taylor et al. 2015). Although rearing could aid in the ability to assess the risk of this species to new areas like Florida, unique protocols for rearing under quarantine conditions have not yet been documented for B. hilaris. Experiment results contribute to enhancing current rearing procedures for B. hilaris with special attention to quarantine requirements.

Photoperiod is often a cue for insects to determine environmental suitability for hatching, reproduction, diapause or other important life histories (Salis et al. 2018).

Given the unique oviposition behavior of B. hilaris below the soil, photoperiod may contribute to the hatching of nymphs from eggs (Taylor et al. 2014). However, experiment results suggest a period of darkness (0L:24D) does not significantly increase the number of nymphs that hatch from eggs and in fact, lengthens the

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development time. For this reason, standard rearing photoperiod conditions (14L:10D) is better for B. hilaris nymph emergence from eggs.

The most commonly used oviposition substrate for B. hilaris is sand, but prior to this research, cheesecloth and wool were the only artificial substrates to be evaluated

(Atwal 1959, Taylor et al. 2015). Because of the unsuitability and risks associated with sand in quarantine conditions, an effective artificial oviposition substrate could greatly benefit the ability to rear B. hilaris. Cotton balls are considered effective substrates for several species of stink bugs (Silva and Panizzi 2008), and based on experiment results, also appear suitable for B. hilaris. Of the three substrates tested, cotton balls appear to be the preferred artificial oviposition substrate for B. hilaris and can be utilized in future rearing protocols.

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Figure 4-1. Adult cage utilized in rearing of B. hilaris. Cage was made from clear plastic containers (ULINE, USA, https://www.uline.com). Holes were cut and covered with mesh for aeration on the lid and sides of the cage.

Figure 4-2. Immature cage utilized in rearing of B. hilaris. Cage was made from clear plastic Tupperware containers (Tupperware Corporation, Orlando, FL; www.tupperwear.com). Holes were cut and covered with mesh for aeration on the lid.

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Percent of Eggs Hatching in Dark and Light Conditions

100% 90% 80% 69.02% 70% 63.82% 60% 50% 40% 30% 20%

Percent Percent ofeggs that hatch 10% 0% 0L:24D 14L:10D Treatment

Figure 4-3. Percent of hatching of B. hilaris eggs in dark (0L:24D) and light (14L:10D) conditions.

Days to Hatch in Dark and Light Conditions

6 5.461 a 5.056 b 5

4

3

2

Average Average days tohatch 1

0 0L:24D 14L:10D

Treatment

Figure 4-4. Average number of days it took for B. hilaris eggs to hatch in dark (0L:24D) and light (14L:10D) conditions.

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Oviposition of B. hilaris on Artificial Substrates 1800 A 1600 1400 a 1200 a 1000 800 600 Number ofeggs 400 b 200 B B 0 Dental wicks Cotton ball Kim wipe Dental wicks Cotton ball Kim wipe No-choice substrates Choice substrates

Figure 4-5. No-choice and choice test evaluating the total number of eggs laid by B. hilaris on three artificial substrates; dental wicks, cotton balls, and kim wipes.

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CHAPTER 5 IMPLICATIONS AND FUTURE DIRECTIONS OF RESEARCH

In an increasingly globalized world, the introduction and establishment of invasive species in new areas continues to be an important issue causing severe economic impacts, decreased biodiversity, and human and animal health hazards. Plant biosecurity is a strategic approached aimed at preventing the spread of invasive species and mitigating the subsequent effects of the movement of non-native species around the world. Early detection of invasive species is often a key component of plant biosecurity to prevent the establishment of non-native species in new areas. Public education can greatly aid in early detection and successful mitigation of invasive species including quarantines and eradication efforts. Middle and high schools throughout Florida offer captive audiences that, with proper education, can positively impact the future of plant biosecurity efforts and invasive species management.

However, invasive species and plant biosecurity are topics not typically included in classroom curricula and students are often unaware of the impacts. Continued adaptation and implementation of the youth outreach program will enhance federal and state efforts for invasive species and plant biosecurity and expand the involvement of an informed public in these efforts.

Piezodorus guildinii is an example of an invasive species that established in the

United States in the 1970’s and has spread throughout the southeast including Florida.

Since the early 2000’s, P. guildinii began to cause economic damage in important leguminous crops like soybean (Glycine max) every year and continues to be a significant agricultural pest. Piezodorus guildinii is an excellent example of the limitations of past plant biosecurity efforts and the consequences of the establishment of

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an invasive species. Although a widely researched bugs, many laboratories continue to struggle with maintaining a viable colony of P. guildinii which has posed a challenge for both previous and future experiments.

Bagrada hilaris is an invasive agricultural pest that was recently introduced in the

United States. Since its initial introduction to California in 2008, B. hilaris is now established in New Mexico, Arizona, Nevada, Utah, and Texas. Although currently not established in Florida, B. hilaris could potentially cause severe impacts in Florida’s large and profitable fresh market cabbage (Brassica oleracea var. capitata) industry if it were to be introduced. Recent research on B. hilaris focuses on insect biology and potential control methods, but this research is largely concentrated in regions where B. hilaris is already established using wild caught specimens. Rearing protocols, particularly designed for quarantine conditions, will assist future endeavors and allow for better assessment of the risk B. hilaris poses to Florida as an invasive species.

Piezodorus guildinii and B. hilaris are examples of two closely related invasive species of concern to Florida and are strong examples of the past, present, and future of invasive species research. Rearing of P. guildinii and B. hilaris is valuable in understanding the organisms’ biology and effectively, this knowledge can enhance the speakers’ background knowledge of these two invasive species. In addition to facilitating future research on these specie, integration of experimental case studies into the youth outreach program like the research on P. guildinii and B. hilaris, can aid in student understanding of current invasive species research and the impacts this research has on their lives.

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BIOGRAPHICAL SKETCH

Morgan Pinkerton is from Ormond Beach, Florida graduating from Seabreeze

High School in 2013. She attended the University of Florida and obtained her undergraduate degree in biology with two minors in Spanish and anthropology in

December of 2016. In the spring of 2016, she began working as a laboratory technician in the Biosecurity Research and Extension laboratory where she developed an interested in entomology.

Morgan began a graduate research assistantship in entomology under Dr.

Amanda Hodges in the spring of 2017. She began a dual enrollment, while working on her master’s, in the Doctor of Plant Medicine (DPM) Program in the summer of 2017. As part of her assistantship, she reared the redbanded stink bug, P. guildinii, for shipment to Bayer Crop Sciences, now BASF (2018), and B. hilaris in a quarantine facility. As of

Fall 2017, she was accepted as a USDA-NIFA-NNF in plant biosecurity and will continue her degree as a fellow.

She led the DPM youth outreach program on invasive species and plant biosecurity in the 2017-2018 school year and continued to participate in outreach events throughout her program. She has also participated heavily in Florida First Detector workshops educating master gardeners, small farmers, garden staff, and nursery growers on the early detection and identification of invasive species. Additionally, she has been involved with the creation and orchestration of biological control and IPM workshops in Ecuador developing materials in both English and Spanish. She plans to continue her pursuit of the DPM degree and graduate in 2020. In the future, she aims to work in biosecurity and extension at both a local and global level.

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