Novel Control Strategy for Bagrada Hilaris (Burmeister)

NOVEL CONTROL STRATEGY FOR BAGRADA HILARIS (BURMEISTER)

A Thesis

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

In Plant Science

Tasien L. Somé

2020

SIGNATURE PAGE

THESIS: NOVEL CONTROL STRATEGY FOR BAGRADA HILARIS (BURMEISTER)

AUTHOR: Tasien L. Somé

DATE SUBMITTED: Fall 2020

Department of Plant Science

Dr. Aaron Fox ______Thesis Committee Chair Plant Science

Dr. Anna Soper ______Assistant Professor Plant Science

Dr. Dawn Calibeo ______Pre-Commercial Development Gowan Company, Yuma, AZ

ACKNOWLEDGEMENTS

I would like to present my gratitude to all my advisors, Dr. Aaron Fox, Dr. Anna Soper, and Dr. Dawn Calibeo for their continuous availabilities, knowledge and support for my

Master thesis study and research. I am honored to receive the guidance, and benefited from the diverse education background of all these great professors.

In addition, I would like express my thanks to Dr. Robert Green for guiding me through all my study trials and sharing all his statistics and data analytical knowledge that contributed to the success of the research.

I would also like to thank Dr. Valerie Mellano, the chair of the Plant Science department,

Dr. Shelton Murinda, the chair of Animal & Veterinary Sciences department and Dr.

Oliver Li, Nutrition & Food Science department for their supports.

For, I want to express my gratitude to Dr. David Morgan and all his staffs from CDFA for making a section of the greenhouse and some equipment available for this study.

I would like to especially thank Dr. Lisa Kessler, the interim dean and Dr. Peter Kilduff the vice dean of The Don B. Huntley College of Agriculture for their supports.

Lastly, I want to thank Dr. Thomas Perring form UCR and all his staffs for rearing the insects throughout the entire research period.

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ABSTRACT

California and Arizona farmers are the major growers of cole crops in the United

States, and broccoli is considered to be economically one of their most important cash crops. Broccoli is grown organically and conventionally in California and Arizona. In

2008, a new invasive insect, Bagrada hilaris, was discovered in Los Angeles. Bagrada. hilaris quickly became a major pest for California and Arizona cole crop growers.

Extensive damage was reported and B. hilaris required innovative pest management strategies. Conventional growers effectively used synthetic pesticides to keep B. hilaris damage levels low. However, there were fewer options for organic growers to control the pest. Naturally, plants use secondary metabolites as a defense mechanism to protect themselves from herbivores. Promoting broccoli secondary metabolites as a defense mechanism could benefit organic broccoli growers and decrease pesticides usage for all growers. There is evidence from the literature that organically managed crops produce higher concentrations of secondary metabolites, such as glucosinolate (GLs). We hypothesized that organically grown brocccoli would be less attractive to B. hilaris due to higher concentrations of secondary metabolites. A greenhouse Choice Study was designed to investigate B. hilaris feeding activities on three treatments of broccoli seedlings (Brassica oleracea var. italica). The three treatments were organically fertilized seedlings (Organic), seedlings fertilized with synthetic fertilizer (Synthetic), and a control treatment where seedlings only received water (Control). The experimental design used was a randomized complete block design with multiple No-choice and Choice trials. In the No-Choice trials, adult B. hilaris were offered only one treatment, either Organic,

Synthetic, or Control. In the Choice trials, adults B. hilaris were offered all three

treatments. Observations of B. hilaris behavior and broccoli conditions were recorded at

30 minutes, and 1, 2, 4, 8, 12, 24 and 48 hours from the time of release of the insects.

During each recording time, the following observations were made: the number of B. hilaris on the seedlings, the number of leaves injured per cage, and the number of plants showing injury. At the end of each trial, at 48 hours, the percent leaf area damage and the percentage of dead seedlings were recorded. Photosynthetic activity and temperature inside the cages in the greenhouse were measured and analyzed. Glucosinolate concentrations in the seedlings were tested from one Choice trial. Overall, B. hilaris did not consistently prefer one treatment over the other in our study. Only in one of the

Choice trials were the Synthetic seedlings clearly preferred by B. hilaris feeding. While our results may not provide clear guidance for broccoli growers, our results show there is opportunity for further investigation.

TABLE OF CONTENTS

Signature Page………………………………………………………………...ii

Acknowledgements………………………………………………………...... iii

Abstract………………………………………………………………………iv

1.Introduction…………………………………………………………………1

1.1 Bagrada hilaris origins and life cycle……...………………………...……3

1.2 Bagrada hilaris feeding activities..……………………………...………...4

1.3 Economic losses……………………………….………………………….4

1.4 Transplant versus direct seeding….……………………………..………..6

1.5 Conventional field management methods of (brassica oleracea var. italic)

………………………………………………………………………………...7

1.6 Organic field management protocol for USDA.……………..…………...7

1.7 Organic field management methods of (brassica oleracea var. italic)……8

1.8 Benefits of organically grown broccoli………………………………...…9

1.9 Brassicaceae defense compounds…………………………………………9

2. Objective and Hypothesis…………………………………………………14

3. Materials and Methods……………………………………………………17

3.1 Glucosinolate analysis…………………………………………………...20

3.2 Temperature and photosynthetically activities radiation analysis (PAR)..

………………………………………………………………………….……21

4. Statistics Analysis…………………………………………………………22

5. Results…………………………………………………………………….24

5.1 Choice study results for season 1 and 2.………………………………..24

5.2 No-Choice study results for season 1 and 2……………….…………....26

5.3 Laboratory analysis of glucosinolate (GLs) concentration results………28

5.4 Temperature and photosynthetically activities radiation results………...28

6. Discussion…………………………………………………………...... 30

Conclusion…………………………………………………………………...36

References……...... 38

Appendix A (Tables)………………………………………………...... 48

Appendix B (Figures)………………………………………………………..60

vii

1. Introduction

Insects from Pentatomidae family (order Hemiptera) are commonly known as stink bugs. Many are phytophagous, but some are predators such as the ones belonging to the subfamily (Asopinae) (Azim, 2011; Raupp et al., 2019). Pentatomidae feeding behaviors are often broad and unpredictable (Torres-Acosta and Sánchez-Peña, 2016) but some are very specific. Bagrada hilaris (Burmeister) is an important pest on agricultural crops (Torres-Acosta and Sánchez-Peña, 2016). Bagrada hilaris origins are thought to be from Africa, Asia (Guarino et al., 2018), and some parts of Europe (Huang et al., 2013;

Reed et al., 2015). Bagrada hilaris was discovered in Southern California around the year

2008 as a pest in the Los Angeles area. Since then, it has migrated to different regions of the state (Matsunaga, 2014). In the past decade, B. hilaris has become an invasive pest in

California and Arizona, especially on Brassicaceae crops (Palumbo and Carrière, 2015).

Bagrada hilaris is not host specific because it feeds on a wide range of other crops including different varieties of wild plants. It was also found to be feeding on grass family and taproot plants (Reed et al., 2013). In general, young seedlings are the most vulnerable to phytophagous insects like B. hilaris, because the insect normally damages the epical meristem and stunts the plant growth (Hodges, and LeVeen, 2018; Azim,

2011). Bagrada hilaris as a stink bug, possesses mouthparts that pierce and suck plants during feeding sessions (Kuhar et al., 2015; Azim, 2011). The insect pest feeds on every part of the Brassicaceae plant such as leaves, stems, seeds and flowers by piercing and sucking (Huang et al., 2013). Bagrada. hilaris feeding damage produces stunting, scorched leaves, wilt, and lesions on plants. As result, the plants either die (Plantwise,

2012), or, if the plants survive, crops produced are often unmarketable (Palumbo and

Carriere, 2015). Furthermore, one study has reported that insect damage in fields is concomitant with the pests’ distribution in those fields locations (Lillian et al., 2018).

Therefore, damage caused by insect pests in small and large farms worldwide have been a major concern.

Numerous research projects have details the life-cycle of B. hilaris. The population of B. hilaris seems to peak in the desert region during Fall, Spring and peak in

Coastal regions during the Summer. These are the times when temperature is around twenty-nine degrees Celsius and there is an abundance of food supply (Grettenberger et al., 2016; (Hodges and LeVeen, 2018; Palumbo 2015, Huang et al., 2013). This insect pest prefers undisturbed fields where they can congregate in groups of hundreds (Hodges and LeVeen, 2018). Bagrada hilaris’ population tends to be dense on host plants

(Palumbo and Carriere, 2015), because of the overlapping generation. Optimal condition for B. hilaris reproduction are at 29.40C daytime temperature with 82.0% moisture level, and 8.40C nighttime temperature with 24.06% moisture level (Bharat et al., 2018).

Bagrada hilaris generally lays eggs beneath foliage, cotyledons, and on the surface of soil next to the host plants (Hodges and LeVeen, 2018). Its eggs have light yellowish coloration (Hodges and LeVeen, 2018). Additionally, B. hilaris often lays eggs in batches of ten eggs (Bundy et al, 2012). Under optimal conditions, the first instar will normally hatch within three to five days from the time the eggs are laid (Hodges and LeVeen,

2018; Huang et al., 2013). Bagrada hilaris’ eggs can be easily transported in the soil to different areas through infested equipment after tillage (Matsunaga, 2014). Bagrada hilaris was observed to overwinter as adults, and prefers to mate in warmer temperatures, around 82 degrees Fahrenheit (Huang et al, 2013).

Adult B. hilaris measures about 5-7 mm in length with mostly black color and some distribution of white and orange colors on its shielded back (Bealmear et al.,

(2012). Bagrada hilaris (figure 1- a) resembles its stink bug relative commonly known as the harlequin bug (Murgantia histrionica) (figure 1-b) (Huang et al., 2013). However, their color schemes and design on their shielded backs are very different (Bealmear, et al., 2016). Murgantia histrionica has a small looking plus sign located in the center of its shielded back which Bagrada hilaris lacks (LeVeen et al, 2018). Murgantia histrionica originates from Central America and Mexico (Huang et al., 2013), and it is specialized on

Brassicaceae plants generally with eight to ten leaves (Guarino et al., 2018).

1.1.Bagrada hilaris origins and life cycle

The native range of B. hilaris include Africa, Asia, India (Reed et al, 2013), and in the Middle East (Ganjisaffar et al., 2018). Bagrada hilaris’ life cycle is composed of five stages: eggs, three nymphal stages, and the imago, which is the mature stage of an insect (Halbert et al., 2010). The nymphs and adults occupy the same living environment.

In general, female insects select hosts that provide better food source for their offspring during oviposition (Gripenger et al, 2010). Oftentimes, female polyphagous insects host selection for food source and oviposition are broad and less efficient than monophagous female insects, which is specific (Gripenger et al., 2010). This may account for female B. hilaris (polyphagous) pattern of eggs laying (Reed et al, 2013) to appear sometimes scattered as well as grouped around the ground (Facknath et al., 2005) and under plants materials, and fewer than 15 eggs at the time (Reed et al., 2013). The overlapping generation allows B. hilaris to reproduce and populate an area faster (Torres-Acosta and

Sánchez-Peña, 2016).

1.2. Bagrada hilaris feeding activities

Bagrada hilaris displays some preferences when food choices are abundant (Reed et al., 2013). Bagrada hilaris is known for damaging a good portion of many Cole crops

(Cary, 2009), in different regions of the western U.S. (Huang et al., 2013). Bagrada hilaris possess a very particular mouthpart that encases four needles, which they use to inject fluids into various plant parts to be digested (Joseph, 2015). After digesting the mixed fluid and plant parts through sucking, the feeding process causes damage to plant cells, which result in stunted plant growth (Joseph, 2015). Bagrada. hilaris feeding on seeds can also result in seedlings damping off (Zavala et al., 2015). The most apparent symptoms from B. hilaris feeding activities on host plants leaves are lesions and stippling

(LeVeen et al., 2018). Bagrada hilaris polyphagy allows the insect to survive in many environments. For example, in Fall and Spring when cool season crops are grown including most cole crops, B. hilaris activities and population increase (Ramzi Samar and

Vahid Hosseininaveh, 2010). During Summer when grass family crops such as corn, and sorghum are grown, B. hilaris populations are able to survive on these crops. However,

Brassicaceae family crops such as broccoli, cabbage, kale, and radishes remain the most preferred host plants for B. hilaris (Piubelli et al., 2018, Joseph, 2015).

1.3. Economic losses

Pest damage on commercial agricultural crops causes enormous economic losses worldwide. The United States alone incurred about $40 billion in crop and forestry economic losses due to pests and pathogens over the past 20 years (Paini et al., 2016).

The U. S government has mobilized efforts and resources to control pests and disease infections in the agriculture sector, yet, over 15% of crops still remain unmarketable due

to pest damage (Chen, X. and Roeber, R., 2005). Bagrada hilaris in California and

Arizona may have been the leading cause of 90% of cole crop damage during certain recent years (Reed et al, 2013, Grasswitz, T. R. 2016). The Cole crops market is over $1 billion for California and Arizona (USDA-NASS, 2011). In 2009, 60% of cole crop seedlings were damaged by B. hilaris (USDA-NASS, 2011). Bagrada hilaris feeding activities were also reported to have caused damage on ornamental plants in California

(Reed et al., 2013). The ornamental plant industry is very lucrative for California growers with 11.2 percent of the total agriculture revenue for the state (Carman, H and Rodriguez,

A. M, 2004), and could be severely impacted by B. hilaris damages (Palumbo et al,

2013). Palumbo, (2015), suggested that between 2010 and 2014, California and Arizona growers were projecting that B. hilaris infestation rate on their combined cole crops acreage to be greater than 80%. Of all the cole crops, broccoli was the most affected with an estimated 10% mean plant damage (Piubelli et al., 2018; Palumbo, 2015). As of 2017,

B. hilaris populations across the western U.S. have declined. The insect is still an intermittent pest, but it is not causing the same amount of damage or concern as it once was (T. Perring personal communication November, 2019). It is unknown why this pest’s populations have dropped so dramatically, but it may be due to a delayed response from natural predators or a novel parasitoid being inadvertently introduced (Reed et al., 2013).

Currently, farmers use various integrated pest management (IPM) techniques to manage

B. hilaris populations, especially insecticides and cultural practices (Grasswitz, T. R.,

2016). Cultural practices include the removal of weedy vegetation that can serve as overwintering and breeding grounds. Another important cultural approach is the use of transplanting crops as seedlings instead of direct seeding in the fields. In recent years,

transplanting has become the most ideal approach for California and Arizona farmers that grow commercial cole crops. Arizona growers agreed that transplanted broccoli was much less susceptible to insect attack than direct seeded broccoli (Halbert et al., 2010).

1.4. Transplant versus direct seed

Transplanting is the process of moving seedlings grown in controlled environment

(i.e greenhouses) into a field for the completion of its lifecycle. Directly sown crops complete their entire life cycle in the field, from germination to harvest. Crops with fibrous root structure are much more suited for transplant than taproot crops (Leskovar et al, 1993)., Numerous studies determined that transplanted crops uniformly develop while avoiding early biotic and abiotic factors that could hinder their establishment and growth

(Balfanz, 2012; Katz, 2002; Leskovar et al, 1993). Transplanted crops have other potential benefit such as earlier harvest than direct seeded crops (Balfanz, 2012; Fanadzo et al, 2009; Katz 2002; Leskovar et al, 1993;). However, transplanted crops are sometimes subjected to transplant shock because their roots were conditioned to develop in a predetermine boundary in the greenhouse (Johnny’s Selected Seeds, 2017).

Furthermore, Shumaker, (1969), observed that direct seeded cabbage had an increase in head-weight compared to transplanted cabbage. Ultimately, direct seeding is cheaper than transplanted crops (Johnny’s Selected Seeds, 2017, Shumaker, 1969). Cole crop farmers used to always direct seed their crops to save money. However, because the seedling stage for Cole crops are more vulnerable to B. hilaris, (Reed et al., 2013), farmers have switched to transplanting these crops (Halbert et al., 2010).

1.5. Conventional field management methods of Brassica oleracea var. italic

California grows many commercial varieties of broccoli from early to late harvest such as Captain, Everest, Imperial, Emerald Pride, Gypsy, Pinnacle, Packman, Diplomat,

Premium Crop, and Windsor. However, the hybrid Italian green or calabrese (Harper et al., 2012), is also commonly grown in California (Strange et al., 2010). Broccoli is a cool season crop that requires high nutrient input for adequate crop yield (Harper et al., 2012,

LeStrange et al., 2010). Soil profile plays an important role in crops nutrients demand. In the Central and South coast of California commercial growers have been known to over apply nutrients, which has led to groundwater pollution (Rosenstock et al., 2013).

Typically, conventional broccoli growth requires amending the soil with phosphorus at a rate of 40 -80 pounds per acre (45 -90 kg/ha), (Strange et al., 2010). Farmers also apply potassium to optimize soil fertility and balance nutrient levels. When applying potassium after harvest, the ideal amount ranges from 100 to 140 pounds per acre or 112 to 157 kg per hectare (Harper et al., 2012, Chun et al., 2015, LeStrange et al., 2010). The seasonal application of nitrogen for broccoli is about 180 to 240 pounds per acre (202 to 270 kg per hectare) (LeStrange et al., 2010). Conventional broccoli production requires a balanced nutrient approach and optimal growth space. For example, “Marathon F1” broccoli seeds were reported to use 150 kg of nitrogen (N), 60 kg of phosphate (P2O5) and 200 kg of potassium (K2O) per hectare, with a planting density of 2.08 per square meter, (Cockshull, 2011).

1.6. Organic field management protocols for USDA

In the U.S. organic production must use USDA approved protocols to become certified. Certified organic fields are required to use natural derived fertilizers and

amendments, such as manure, compost, or leguminous cover crops. Any pesticide use in the organic field is typically derived from natural ingredients and must be from the

USDA approved list of inputs. Conventional field management methods do not have the same restrictions and typically use synthetic fertilizer and pesticides inputs (USDA,

2015). The conventional management methods are not bound to any USDA certification protocol, except when the inputs are on the Environmental Protective Agency (EPA) banned lists. For example, the use of DDT is banned by EPA in the U.S.A. and many other countries.

1.7. Organic field management methods of (Brassica oleracea var. italic)

Organically managed farms require diverse approaches for all aspects of production, from amending the soils, to the seed selection, to rotation planning, in order to maintain the organic integrity and certification. It is often difficult to organically manage a field because of these complexities and the restrictions on inputs described above (USDA, 2015). Moreover, organically managed field requires innovative pest management methods, such as pest resistant seed varieties, to withstand various biotic and abiotic conditions (Lammerts van Bueren et al., 2002). Often, organically managed farms use compost and manure to amend the soil and fertilize their crops. In an organically managed field, most crops require 30 pounds of organic compost for every

100 square feet of space (Vinje, 2014). Broccoli is an annual cool-season crop that grows well under optimal sunlight and water supply and it thrives in well-drained soil with ideal fertility levels (Vinje, 2014).

1.8. Benefits of organically grown broccoli

Consumer choices for healthy food has spiked the demand for organically grown vegetables around the world and including the demand for organic broccoli (Verkerk et al., 2008; Zapata et al, 2012). Some studies suggest the compound sulforaphane in broccoli may possibly prevent the proliferation of cancer cells (Jeffery and Araya, 2009;

Barcelo et al, 1996). Numerous studies have reported the health benefits of broccoli to be associated with its content of bioactive compound property such as glucosinolates (GLs), flavonoids, and vitamins C, E, A as well as antioxidants (Zapata et al, 2012; Verkerk et al, 2008; Jeffery and Araya, 2009). Organically grown broccoli is often subjected to stresses (Lammerts van Bueren, 2002, Zapata et al, 2012), which increase its production of secondary metabolites as a defense mechanism (Ballhorn et al, 2011). Therefore, organically grown conditions may lead to higher production of secondary metabolites, such as antioxidants, which is beneficial for human health (Zapata et al, 2012).

Cockshull (2011), reported that “Marathon F1” broccoli seeds grown under organic cultivation practice with an application of 20,000 kg of cow manure per hectare and non- application of herbicides, or insecticides in the field produce broccoli with higher content of Glucorapharin (glucosinolate in broccoli (GLs)) and antioxidant than conventional ones. Some studies have reported higher concentration of glucosinolate under organically managed field (Herr et al., 2013).

1.9. Brassicaceae defense compounds

Both organic and conventional broccoli field managed methods promote the concentration of secondary metabolite defense compounds such glucosinolates (GLs)

(Bednarek et al., 2009; Zapata et al, 2012), and phenolic use by the plants for protection

again phytophagous insects (Goodger et al., 2013). Glucoraphanin, or glucosinolates

(GLs), a secondary metabolite, is a bioactive compound found in Brassicaceae plants such as broccoli, cabbage, kale, cauliflowers, horseradish, rapeseed, and mustard

(Soledade et al, 2010; Gimsing and Kirkegaad, 2009; Jeffery and Araya, 2009, Verkerk et al., 2009). When the GLs are chemically broken down, they form different compounds.

GLs are composed of b-thioglycoside, N-hydroxysulphates known as (Z)-N- hydroximinosulphate esters or S-glucopyr- anosyl thiohydroximates, and a side chain R with a Sulphur attached b-D-glucopyranose moiety (Bone and Mils, 2013, Klaiber et al.,

2013, Verkerk et al., 2009). Klaiber et al., 2013 and Herr et al., 2013, suggested that a sulphate group is known to be balanced by a potassium cation. Interestingly, GLs could be identified as aliphatic, indole by it the side chain R, or aromatic (Herr et al., 2013).

Aliphatic GLs is defined as a chemical compound that may not contain any aroma, while aromatic GLs contains strong aroma, and indole GLs is a chemical compound associated with better taste (Klaiber et al., 2013, Herr et al., 2013). (Figure 2) shows the byproduct of GLs after chemical decomposition. (Table 1), lists the common names of GLs and the associated chemical names of R-groups. GLs are known to be a nitrogen based defense mechanism in Brassicaceae plants (Klaiber et al., 2013). After the adenosine triphosphate

(ATP) hydrolysis process, new toxic compounds (isothiocyanates, thiocyanates, nitriles, epithionitriles, etc.), many associated with mustard oil, are formed after molecular synthesis to protect plants from phytophagous insect pest feeding (Rodriguez –Hernandez et al, 2014, Klaiber et al., 2013, Herr et al., 2013), (Figure 3). Mustard oil

(isothiocyanates, sulphoraphane) was reported in other experiments to have an anti-viral, anti-bacterial and anti-fungal property (Conrad et al., 2006, Winter, A. G. and Rings-

Willeke, L. 1958). GLs concentration were reported to be found in various parts of many plants depending on the cultivar (Verkerk et al., 2008), including plants in the

Brassicaceae family (Hsu et al., 2009). For example, Klaiber et al., 2013, reported higher accumulation of GLs in Ethipian Kale (Brassica Carinata) seeds than the leaves. Another study conducted on radish suggested a greater concentration of GLs in the cotyledons than the roots, but the roots had more GLs than the leaves as the crop matured (Klaiber et al, 2013). The concentration of GLs in crops depends on how the crop is managed and this can subsequently influence crop-pest dynamics. For example, one study suggested that long-term application of nitrogen based synthetic fertilizers could reduce plants secondary metabolites (Klaiber et al, 2013). These secondary metabolites (GLs) are critical to plants’ natural defense strategies. However, some specialist insects have evolved to neutralize the toxins better than generalist insects. For example, a study reported that when plant tissue is injured due to insect feeding activities, it triggers the plant GLs stock to react with other compounds such as myrosinase and produce nitriles and isothiocyanates that are known to be very toxic and repels generalist phytophagous insects (Beekwilder et al., 2008). Another study found that when broccoli plant cell tissues are damaged during insect’s attacks, they produce secondary metabolites (GLs) as a defense mechanism which are toxic to some insects. Numerous studies have suggested that higher concentration of GLs may suppress lepidopteran insect feeding and, even decreasing the life cycle and development of certain insects (Onyilagha et al., 2004;

Beekwilder et al, 2008). However, some specialist insects have evolved an ability to sequester and neutralize the toxins. Harlequin bug (Morgantia histrionica) is a

Brassicaceae specialist and may be sequestering GLs compounds, but B. hilaris is a

generalist herbivore and is not expected to be attracted to GLs compound (Aliabadi et al.,

2002).

Some studies report benefits associated with organically managed fields on plant resistance to pests. For example, numerous studies report that organically fertilized soil with manure harbor less insect pests than synthetically fertilized soil (Alyokhin et al.,

2004; Eigenbrode and Pimentel, 1988; Feber et al., 1997; Hartmann et al., 2015). Many of these studies pointed to, the Soil Mineral Balance Hypothesis first described by Phelan et al, (1996) as a mechanism. The hypothesis postulates that the increase reliance on soil organic matter and microbial activity associated with organic farming may serve as a way to ensure optimal balanced nutrient distribution for plants (Beanland et al, 2003, Johannes and Larry, 2003, , Alyokhin et al., 2004). This optimal nutrient availability in the soil promotes plant growth and ultimately resistance to pests and diseases (Van et al., 2009,

Hartmann et al, 2015, Clancy et al, 1988, Alyokhin et al.,2004). Conversely, the absence of organic matter and microbial activity in soil could lead to either excess or deficient nutrient uptake by plant, which in turn, lead to plant inability to resist pests and diseases

(Altieri & Nicholl, 2003, Alyokhin et al., 2004, Clancy et al, 1988). It was reported that inadequate nutrient distribution in the soil can permit plant to grow, but it can also promote the reduction of plant defense mechanism (primary and secondary metabolites), therefore exposing plants to phytophagous pest attack (Alyokhin et al., 2004). A study was done to compare the presence and the density of Colorado potatoes beetle larvae on potato fields with two soils management techniques, synthetic fertilizers and raw cow manure. The study concluded that the field under raw cow manure amendment recorded fewer numbers and lower density of the Colorado potatoes beetle larvae than the field

under synthetic fertilizer treatment (Alyokhin et al., 2004). Another study also reported an increase of European corn borer (Ostrinia nubilalis), oviposition in fields under synthetic fertilizer management compared to organic (manure) management (Phelan et al., 2000; Phelan et al. 1995; Barbercheck, 2011). In one study, European corn borer reported on the synthetic fertilizer field was eighteen times greater than that of the organically managed field (Phelan et al., 1995). The results of this study and others suggest organic field management may be a key factor influencing the reduction of pest presence and activities (Phelan et al., 2000, 1995, 1996, Staley et al, 2010, Hartmann et al., 2015). Numerous other studies assessed the relationships between organic/conventional fertilizers and various agricultural pest activities. Simon et al.

(2010) conducted a field study to assess the presence of aphids and their natural enemies on both organic and conventional fertilized barley cereal crop fields. The results of the study suggested that conventional fields harbor more pests than organic fields (Simon et al., 2010). Subsequent studies suggested that synthetic fertilizers in the field may have negative effect on resistance by reducing concentration of secondary metabolites (GLs), leading to the proliferation of pests in the field (Evans, E. W., and Youssef, N. N. 1992,

Simon et al, 2010; Chun et al.,2015).

2. Objective and Hypothesis

Choice test is a method used to assess experimental organisms’ feeding behavior in relation to host plants. Insects use unique biological mechanisms to identify their host plant and secondary metabolites often play a key role in this process. Huang et al.,

(2014) conducted research on the host preference of B. hilaris (Hemiptera: Pentatomidae) on cruciferous seedlings including broccoli and ornamental plants. The main objective of the research was to determine B. hilaris feeding behavior in relation to the host plants, so that the most preferred host plant could be used as a trap crop in the future (Huang et al,

2014). The research was conducted in the greenhouses in Yuma and Coachella Valleys

(Huang et al., 2014). Mesh cages were used to house the insects inside the greenhouses.

The insects (B. hilaris) selected for this specific experiment were all adults, male and female, (Huang et al., 2014). It is essential to mention that these insects were put under stress (starvation), before they were released on the to host plants (Huang et al, 2014), because starvation may influence host preference. All the relevant data for their feeding activities was captured for 48 hours (Huang et al, 2014). Three set of trials were conducted on Brassicaceae plants for data collection (Huang et al., 2014). This experiment is one of the few known to captured data on B. hilaris host preference (Huang et al., 2014). The study revealed that cruciferous seedlings were preferred by B. hilaris

(Huang et al., 2014). The results also estimated that radish appeared to be the highly preferred host plant compared to all the other potential hosts including broccoli (Huang et al, 2014). However, a follow-up study on broccoli seedling vulnerability, found broccoli leaf stage four was the most preferred host plant for B. hilaris feeding activity (Huang et al, 2014). This experiment also indicated that ornamental plants such as sweet alyssum,

were the least preferred host for B. hilaris (Huang et al, 2014). The overall results of the experiment suggested that Brassicaceae could be planted as traps crops to divert B. hilaris from main crops (Huang et al, 2014). This study has some limitations because the experiment was done in a controlled environment, and the pests had undergone starvation before being exposed to the host plants. Controlled environment is subject to manipulation and the results may be different from field obtained data (Knolhoff and

Heckel, 2013). Insect life stage and mobility in controlled environment may also change the feeding behavior.

A similar study was conducted on host plant preference of M. histrionica

(Hemiptera: Pentatomidae). The study was conducted in two environments, field and cage and some of the crops used for the study included mustard, rapeseed, rapini, arugula, and bean (Wallingford et al., 2013). The purpose of the experiment also was to evaluate ways to control M. histrionica with trap crops through their feeding behaviors on Cole crops (Wallingford et al., 2013). The results suggested that Brassicaceae plants can be used as trap crops to control the pest. In summary, the general purpose of both studies were to identify the most preferred host plant by either M. histrionica, or B. hilaris through choice test. The results from both studies concluded that both insects are attracted to the Brassicaceae family. We will use a choice-test method to determine if

Bagrada hilaris prefers organically or conventionally grown broccoli seedlings. We hypothesized that B. hilaris will prefer conventionally grown broccoli over organically grown broccoli. From our literature review we predicted that organic broccoli contains more secondary metabolite compounds such as Glucosinolate (GLs), that defend the plant

by repelling insects during plant cell tissue injury. The results of our study will inform growers about the potential cultural practice that may improve management of this pest.

3. Materials and Methods

Field location and seasons

Trials were conducted from February through 2020 in the greenhouse at

California State Polytechnic University, Pomona (Cal Poly Pomona) (34o 03’02.9” N

117o 49’21.6” W). The greenhouse was equipped with a ventilation system to ensure air circulation. Inside the greenhouse, we used twelve mesh insect cages (75cm x 75 cm x

115cm) from (Abris company, Guangdong China) to conduct all the trials.

Broccoli cultivar, soil substrate, treatments and seeding plugs

Brassica oleracea var. italic is a commercial crop grown both organically and conventionally in California and Arizona where the crop has been susceptible to B. hilaris feeding activities. Brassica oleacea var. italic seeds of commercial grade from

(Johnny’s Selected Seeds, 955 Benton Avenue Winslow, Main 04901), were directly sown in soil substrate inside trays containing 42 miniature cells. The sizes of the trays and pots are as follow: tray dimension, 55’’x 42” and each pot dimension is 4” x 2.6”x

3.5”. Sunshine #4 aggregate plus mix, by (Sungro Horticulture Company, 770 silver street Agawam, MA 01001-2907 United States of America) was used as the soil substrate for all treatments. Under the conventional managed method, “Instant VIGORO 18-18-21

Water Soluble Plant Food” from Vigoro Corporation (Agriculture Chemicals 225 North

Michigan avenue Suite 2500 Chicago, IL 60601) was used as synthetic fertilizer to amend the soil substrate during seedling stage. Under the organic method, “DR. Earth

Home Grown 3-2-2 Organic Pump and Grow” (Dr. Earth Inc., P.O Box 460 Winters, CA

95694) was used to amend the soil substrate at the seedling stage. The control treatment consisted of growing seedlings in Sunshine #4 soil substrate with no fertilizers applied.

All treatments had an equal number of 70 seedlings grown per treatment per Trial. The seedlings were kept in the greenhouse where they received 20ml of water (control) or

20ml of water mixed with fertilizers (synthetic or organic) per seedling, three times per week throughout the growing season of 4 weeks per trial. Adult mating pairs of B. hilaris were used for each trial (Reed et al, 2013). The insects were reared by University of

California Riverside Entomology Department insectary. The insects were brought to the choice test area a day before the trial started. Each pair of insects were subjected to a single specific feeding period (48hr), per trial.

Measurement and illumination devices

The quantum PAR (photosynthetically active radiation) meter (Hydro Farm company Petaluma, California) was used to evaluate the photosynthesis activity of the broccoli seedlings during each trial. A GENRAL 6:1 Infrared thermometer (The Seeker.

General IRT102) from (General Tools & Instruments, New York, NY and Montreal

Canada) was used to measure temperature inside cages. LED illuminator LUMENS 120

MAX 1362066 from L G Sourcing Inc. Wilkesboro, North Carolina was used to illuminate the interiors of the cages and inspect seedlings injuries during nighttime data recordings.

Brassica oleracea var. italic (broccoli) managed under Organic, Synthetic and

Control methods were used to conduct our No-Choice and Choice Study trials. There were total of four sets of trials: one No-Choice Study in February, 2020 and one Choice

Study March, 2020 (hereafter “Season 1”) and one No-Choice Study April, 2020 and one

Choice Study May, 2020 (hereafter “Season 2”).

No-Choice Study:

Twelve cages were used for the No-Choice Study. Four cages contained only organic grown broccoli seedlings (four seedlings per cage). Another four cages contained only conventional grown broccoli seedlings (four seedlings per cage). The last four cages contained the control treatment, only broccoli grown with water (four seedlings per cage). Four mating pairs of B. hilaris were placed in each cage in the morning (9am). In total, the No-Choice Study had 96 insects (equal number of adult mating: 48 males and 48 females) and 48 broccoli seedlings (16 Organic seedlings, 16

Synthetic seedlings and 16 Control seedlings). For the No-Choice Study, a randomized block design was used to account for a temperature gradient in the greenhouse (warmer on the east side of the greenhouse). Four blocks, each with one cage of each treatment, were placed in a row along a greenhouse bench.

Choice Study

Twelve cages were also used for the Choice Study trials. Inside each cage, we placed 12 seedlings in a circle with a sequence of Organic, followed by Synthetic followed by Control. Seedlings were placed along the wall of the cage 2 cm apart from each other and 30 cm from the center of the cage where the insects were released (Huang et al, 2013). The Choice Study trial had a total of 144 mixed seedlings (48 Organic, 48

Synthetic and 48 Control broccoli seedlings) and 96 insects (48 adult males and 48 females) in the cages.

All the insects were kept in our experimental facility and were not fed (food or water) for one day before the trial (Huang et al., 2013). Observations, data collection and measurements were done at 30 min, and 1, 2, 4, 8, 12, 24 and 48hr from the time of

release of B. hilaris in the cages as described in (Huang et al., 2013). During nighttime

(12h), we used a LED lamp to illuminate the cages in order to visually collect data

(Huang et al., 2013). We investigated all the host plants during each trial to determine five major variables according to the insects feeding preference. The five major variables evaluated during each trial were the seedlings Attractiveness (number of insects on the seedlings); seedlings Acceptance (number of plants showing injury); seedlings

Susceptibility (number of leaves injured per cage); dead plant (percentage of seedling death after 48 hours.); area damage (percentage of total leaf area damage after 48 hours)

(Huang et al., 2013). The symptom of seedling damage was evaluated by presence of small white stipples and lesion of the leaves (Palumbo, J. C., and Natwick, E. T. (2010).

The seedling death was assessed by the presence of small white stipple and lesion on all the entire leave of the seedling and the arrested development of the seedling’s epical meristem (Huang et al., 2013).

3.1. Glucosinolate analysis

Seedlings from one Trial, the second Choice Study (May 2020) were analyzed for secondary metabolites. Glucorapharin compound was isolated in a professional laboratory (Eurofins Lab) from the entire fresh broccoli seedlings (leaves, stems and roots) of 4 weeks of age. Fresh broccoli seedlings (18) of all three treatments (6 Organic,

6 Control and 6 Synthetic) were taken on May 14, 2020 at 2:34PM in their entirety

(entire plants in the growing pots and substrates) into Eurofins Lab, (Eurofins Food

Chemistry Testing US, Inc. 2951 Saturn Street Brea, Ca 92821) for determination of

Glucorapharin (glucosinalate (GLs)) concentration. In order to isolate Glucorapharin compound, Eurofins Lab used the procedure described in the research of (Clarke et al.,

2011, Schonoholf et al., 2007) with some minor modification done by Eurofins Lab (Y.

Hong personal communication May, 2020).

3.2. Temperature and photosynthetically active radiation (PAR) Analysis

Temperature and PAR were measured three times inside the cages during each trial period at the same exact time (12pm PST) to determine whether the sunlight and the temperature had an effect on the trials in each season. Temperature and PAR were taken one day before the trial, the first day of the trial and one day after the trial. The means of these three numbers were used for analysis. The measurement tools described above were used for that purpose (The quantum photosynthetically active radiation meter and The

GENRAL 6:1 Infrared thermometer).

4. Statistical Analysis

Data met the requirements for analysis of variance (ANOVA). ANOVA for choice and no-choice studies was performed using a General Linear Model procedure to test main effects of fertilizers treatment (with three levels; synthetic, organic, and control) for response variables (attractiveness, susceptibility, acceptance; and dead plant and area damage only at the last measurement time) in a randomized complete block design with twelve replications for the choice study and four replications for the no-choice study.

Two individual seasons and eight measurement times for choice and no-choice studies were analyzed separately. The statistical software was SAS 9.4 (SAS Institute, Statistical

Analysis System, Cary, North Carolina). For the choice and no-choice studies, means were compared by using a Fisher’s protected LSD test and one degree-of-freedom contrasts, respectively.

Additionally, ANOVA for choice and no-choice studies was performed to test main effects of location of replications for response variables [temperature and photosynthetic active radiation (PAR)] in a randomized complete block design. Two individual seasons for choice and no-choice studies were analyzed separately. For each season during choice and no-choice studies, temperature and PAR were measured at each replication (block) during three consecutive days at the same day time during each study. For ANOVA, the three measurement days were considered replications and replications were considered location. For the No-Choice Study, an average temperature and PAR for the three fertilizers treatments within each block was calculated. For the Choice and No-Choice

Studies, location means were compared by using a Fisher’s protected LSD test.

Additionally, for the Choice Study, a contrast procedure was used to compare means of

locations 1 to 6 to locations 7 to 12. (R. Green personal communication May, 2020).

5. Results:

Three hundred eighty- four total observations were made during the four trials (eight observations per trial on twelve cages multiplied by four trials). Three hundred eighty- four B. hilaris insects and three hundred eighty- four broccoli seedlings were used during these four trials. The average temperature over all four trials ranged from 75 to 87 degrees Fahrenheit and average photosynthetic activity radiation (PAR) ranged from

40µmol*m-2 s-1 to 171 µmol*m-2 s-1 . The average temperature during February (Season 1

No-Choice Test) was 87oF and PAR was 40µm µmol*m-2 s-1. The average temperature during March (Season 1 Choice Study) was 75oF and PAR was 40 µmol*m-2 s-1. The average temperature during April (Season 2 No-Choice Study) was 81oF and PAR was

171 µmol*m-2 s-1. The average temperature during May (Season 2 Choice Test) was 81oF and PAR was 37 µmol*m-2 s-1. Many of the months had low PAR values due to unseasonably cloudy weather during much of spring 2020 in Southern California.

Because of the month-to-month discrepancies, each trial was analyzed separately.

5.1. Choice study results for Season 1 and 2

Overall, during March 2020 (hereafter “Season 1”) Choice Study, there were no clear trends or differences between treatments. Attractiveness and Susceptibility were all statistically the same at all time periods. Similarly, between 30 minutes and 12hr, the mean Acceptance of broccoli seedlings to B. hilaris feeding activities under all treatments was not significantly different (see Table 5 & Figure 8 Season1) and (see table 5A &

Figure 8A Season1). However, between 24hr and 48hr, the mean Acceptance (average number of plants showing injury at this time-period) under Control treatment was significantly lower than the Organic or Synthetic treatments (24hr, Acceptance P=

0.0076), (48hr, Acceptance P=0.0034) (see Table 5A & Figure 8A Season1). Mean

Acceptance of Control was 0.7b at 24 hours while the mean for Organic was 1.8a, and at

48hr, mean Acceptance for Control was 0.7b while the mean for Synthetic was 1.9a and the mean for Organic was 2.2a. But there was no significant difference between the mean

Acceptance of broccoli grown under Organic versus Synthetic treatment at any other time period during the Season 1 Choice Study. Additionally, at the end of the Study (48hr), the mean Percent Plant Mortality (dead plant), and the mean percentage leaves area damage of broccoli seedlings under all three treatments were not significantly different (see Table

5A & Figure 8A Season1).

In May 2020 (hereafter “Season 2”) Choice Study, there was a clear trend where the Synthetic treatment consistently had higher values for Attractiveness, Susceptibility, and Acceptance than the other two treatments. Similarly, at the end of the Study (48hr)

Dead Plant and Area Damaged were all significantly higher for the Synthetic treatment.

At the beginning of the Study, at both the 30 mins and 1hr observations, the mean

Attractiveness of broccoli seedlings to B. hilaris adults were significantly higher in the

Synthetic treatment than the Organic and Control treatment (30 mns P= 0.0038; 1hr

P=0.0003), and this trend did continue for subsequent time periods. At 30 minutes the mean Attractiveness for Synthetic was 2.7a while Organic and Control were 0.8b and

0.7b respectively. Similarly, at 1 hour the mean Attractiveness for Synthetic was 3.2a while Organic and Control were 0.3b and 1.1b respectively. Organic and Control

Attractiveness were not significantly different for either time period. This trend continued for all subsequent observations except the last one (48 hours) when Attractiveness was not significantly different between any of the treatments. There was one sporadic

significant difference between Organic and Control treatments at the 2-hour observation

(see Table 6 and Figure 9 Season2). At the beginning of the Season 2 Choice Study, there was an emerging trend in Susceptibility where Organic was lower than the other treatments. This did not appear until the 1hr observation (P=0.0437) when mean

Susceptibility for Synthetic, Organic, and Control treatments were 0.03a, 0.00b, and

0.06a respectively (see Table 6 & Figure 9 Season2). This significant trend continued and strengthened for the next two observations (2hr and 4hr). Susceptibility for the Organic treatment continued to be lower than other two treatments, but it was not significant for any of the later observations (8hr, 12hr, 24hr, and 48hr) (see Table 6A & Figure 9A

Season2). Mean Acceptance was consistently higher in the Synthetic treatment during

Season 2 Choice Study after the first two observations (30 min and 1hr). At 2hr, the mean

Acceptance was significantly lower (P=0.0153) under the Organic treatment (mean

=0.1b) than Synthetic treatment (mean = 1.0a) (see Table 6 & Figure 9 Season2). This trend continued at 4hr and expanded to include the Control treatment. Mean acceptance

(P<.0001), was significantly higher for Synthetic compared to Organic and Control treatments at 4 hrs and this continued for all subsequent observations (8hr, 12hr, 24hr, and 48hr). Additionally, at 48hr, the mean Dead Plant and the mean Area Damage was significantly higher in the Synthetic Treatment compared to Organic and Control and there were no differences between Organic and Control Treatments (see Table 6 & 6A and Figure 9 and 9A).

5.2. No-Choice study results for season 1 and 2

During February 2020 (hereafter, “Season 1”) No-Choice Study there were no differences in Attractiveness or Susceptibility between any of the treatments for most of

the trial. Only at 48 hours was there a significant difference in Attractiveness (P= 0.0156) where Organic was higher than the other treatments, but this seems to be an anomaly when looking at the rest of the results (see Table 7 & Figure 10 and Table 7A & Figure

10A season 1). Similarly, at 2hr, mean Susceptibility of broccoli seedlings to B. hilaris under the Organic treatment was significantly lower than Control but not Synthetic

(P=0.0134), but this trend did not continue for any subsequent observations (see Table 7

& Figure 10 and Table 7A & Figure 10A Season 1).

During the Season 1 No-Choice Study Acceptance was consistently lower in the

Organic Treatment throughout most of the trial. Starting at 2hr, the mean Acceptance of broccoli seedlings to B. hilaris feeding activities under Organic Treatment was significantly lower than both Synthetic and Control treatments (P=0.0105). The mean

Acceptance for the Organic Treatment at 2hrs was 0.7b while the mean Acceptance for

Synthetic and Control were both 2.0a (see Table7 & Figure 10). During subsequent observations, Acceptance in the Organic treatment was significantly lower than the

Synthetic treatment but not the Control treatment up until the last observation at 48hrs when all means were statistically equal (see Table 7 & Figure 10 and Table 7A & Figure

10A Season 1). At the end of the Season 1 No-Choice Test the mean percentage of dead plants was significantly lower for the Control treatment compared to the Synthetic treatment (P=0.0054). The mean Dead Plant for Organic was numerically lower than

Synthetic and numerically higher than Control, but there was no statistically significant difference between Organic and the other two treatments (see Table 7A & Figure 10A

Season 1).

The April 2020 (hereafter, “Season 2”) No-Choice study had no significant differences between treatments for Attractiveness, Susceptibility, Acceptance, Dead

Plant, or Area Damage (see table 8 & Figure 11, table 8A & figure 11A season 2). The only exception was Susceptibility at the 48hour observation when the Organic Treatment was significantly lower than the Control Treatment (see Table 8A & Figure 11A Season

2). However, this one significant value did not seem indicative of any trend.

5.3. Laboratory analysis of glucosinolate (GLs) concentration results

The six seedlings from each treatment sent for chemical analysis all had different masses. The total mass for the Synthetic, Organic, and Control treatments were 8.0781g,

5.5045g, and 1.2823g, respectively. Glucorapharin (glucosinolate found in broccoli) concentration in broccoli seedlings was the highest in the Control Treatment. (37.1 mcg/g of glucorapharin). The Organic Treatment had the second highest concentration (17.8 mcg/g of glucorapharin). The Synthetic treatment had the lowest concentration

(<18.0mcg/g of glucorapharin) (see Table 2).

5.4. Temperature and photosynthetically active radiation (PAR) results

Temperature effects on the location of cages was significant for the Season 1

Choice Study (P= 0.0296) (see Table 3 & Figure 6 Season 1). The subsequent LSD

Fisher’s separation of means showed a general trend of higher temperatures in the first locations and lower trends in the last locations. Furthermore, the contrast of Locations 1-

6 vs 7-12 was also significant. The blocking in our experimental design accounted for these temperature differences. There was no significant difference of PAR across locations for the Season 1 Choice Study (P=0.1874) therefore no follow-up comparison was made (see Table 3 & Figure 4 Season 1). Temperature effects on the location of

cages was not significant for the Season 2 Choice Study (P= 0.3995) (see Table 3 and

Figure 6 Season 2). However, the probability of the PAR effects on location during the

Season 2 Choice Study was significant (P=0.0086) (see Table 3 & Figure 6 Season 2).

However, follow-up procedures show no real trend for PAR across locations and the contrast of Locations 1-6 vs 7-12 was not significant (P=0.5272). Temperature effects on the location of cages was significant for the Season 1 No-Choice Test (P= 0.0433).

Subsequent mean separation procedures showed Location 1 had higher mean temperature compared to Location 3 and Location 4 but not Location 2 (see Table 4 & Figure 7

Season 1). The blocking in our experimental design accounted for these temperature differences. There were no significant differences between locations for PAR during the

Season 1 No-Choice study (P=0.9404) (see Table 4 & Figure 5 Season 1). The Season 2

No-Choice Study had no significant differences for temperature (P=0.1921) nor photosynthetic active radiation (PAR) (P=0.2046) (see Table 4 & Figure 7 Season 1 and

Table 4& Figure 5 Season 2).

6- Discussion

The overall study did not show strong statistical differences between the treatments. In a previous study, broccoli seedlings were reported to be attractive and susceptible to B. hilaris feeding activities (Huang et al., 2013), but it was not clear whether fertilizer treatment would affect this relationship. Our results show that fertilizer treatment has no major effect on the interaction between B. hilaris and broccoli seedlings. However, there were some emerging trends between Organic and Synthetic treatments. Throughout our study, there were numerous time periods when response variables for the Organic treatment was significantly lower than the Synthetic treatment, and there were no instances where the Synthetic treatment was lower than the Organic or

Control treatments.

Results from the Season 2 Choice Study most closely followed our prediction that

B. hilaris would be more attracted to the Synthetic treatment. In the May 2020 (Season 2)

Choice Study, there was a clear trend where the Synthetic treatment consistently had higher values for Attractiveness, Susceptibility, and Acceptance than the other two treatments. Similarly, at the end of the Study (48hr) Dead Plant and Area Damaged were all significantly higher for the Synthetic treatment. We expected the Synthetic treatment to be preferred by B. hilaris throughout the Seasons and Trials due to lower glucosinolate concentrations.

Glucorapharin (glucosinolate found in broccoli) concentrations in the broccoli seedlings from May 2020 (Season 2) Choice Study were tested in a professional laboratory for all three treatments. The laboratory results determined that the Control treatment had the highest concentration of glucosinolate followed by the Organic

treatment. The Synthetic treatment had the lowest glucosinolate concentration of all the treatments. Consequently, the Season 2 Choice Test and laboratory analysis results nearly matched our expectations of organically grown broccoli seedlings to be less desired by

B.hilaris than synthetically grown broccoli seedlings due to differences in defense- compound concentrations. Glucosinolate concentration in broccoli seedlings from the

No-Choice Tests and the Season 1 Choice Test were not determined due to budgetary constraints, but this would have been a valuable piece of information for this experiment.

We do not know if the glucosinolate concentrations in the seedlings from the other

Studies would have matched the laboratory results from the Season 2 Choice Study. If the concentrations were different that may explain why the results from the other tests did not match the Season 2 Choice Study results.

Other studies of broccoli seedlings under different fertilizer treatments demonstrated that organically managed broccoli has increased glucosinolate concentrations compared to synthetically managed broccoli (Boccia et al., 2004; Zapata et al, 2012). Bowers (1990) indicated that glucosinolate concentration in plants may play an important defense role against insect herbivore attacks. However, short-term crop management may not impact glucosinolate concentrations. One study suggested that long-term application of nitrogen based synthetic fertilizers could reduce plant secondary metabolites (Klaiber et al, 2013). Our study may not have produced similar results because it was short-term in scope; the soils were only managed differently for four weeks before the experiment began. A similar study done with soils from long-term managed organic and synthetic fields may have produced different results from our greenhouse study.

Other research studies have reported a link between soil management and pest management known as the Soil Mineral Balance Hypothesis, this theory postulates that plant secondary metabolites are more abundant in organically grown crops because the nutrients are more “balanced” in organic soils. “Balanced”, here, means that diverse array of nutrients are slowly released into the soil from amendments rather than single nutrients immediately available from fertilizers. The Soil Mineral Balance Hypothesis postulates that the increase reliance on soil organic matter and microbial activity associated with organic farming may serve as a way to ensure optimal balanced nutrient distribution for plants (Beanland et al, 2003, Johannes & Larry, 2003, Alyokhin et al., 2004). For example, two studies reported that organically managed soil fertilized with manure harbor less insect pests than synthetically fertilized soil (Hartmann et al.,2015), Alyokhin et al., 2004). Our research here on organically managed broccoli may not refute the Soil

Mineral Balance Hypothesis because we used a soluble organic fertilizer that was quickly available to the broccoli seedling. Many organic farmers use soil amendments like compost or green manures to build-up soil organic matter over time rather than using organically approved quick-uptake fertilizers. With amendments, nutrients are incrementally available at lower rates over a long-term. For example, Vinje (2014) recommends specific compost applications to optimize soil microbial activity. Kleber et al. (2015) point out that the soil organic matter these amendments help form have complex relationships with the mineral portion of the soil, the soil microbiome, and abiotic conditions. Fertilizers, even organic ones, do not form these complex relationships because they are only available for a short period of time.

In our experiment, liquid organic fertilizer (DR. Earth Home Grown 3-2-2 Organic Pump and Grow) was used as a source of nutrients for the organic broccoli seedlings, and this is not typically used in a field setting. Since liquid Organic fertilizer and Synthetic fertilizer were used at proportion as source nutrients for broccoli seedlings in our experiment, both treatments may have had the same impact on the broccoli seedlings. While the Organic and Synthetic treatments may not have been dissimilar enough to produce significantly different results, the Control treatment was very different. The Control Treatment only received water and the only nutrients available were those naturally in the potting-mix. Bagrada. hilaris feeding activities on the Control treatment were significantly lower than both Organic and Synthetic in many trials, seasons and time- periods of our study. The Control treatment was often the least desirable to B. hilaris feedings activities. Furthermore, the Control treatment had the highest concentration of glucosinolate in the one instance where we tested for the secondary metabolite. The soil Mineral Balance Hypothesis described by Phelan et al, (1996) might have played an important role in balancing the nutrients in the Control treatment leading to an increase in secondary metabolites such glucosinolate in these broccoli seedlings. This may indicate that water only treated broccoli seedlings could be a more resilient host than fertilized seedlings. Seasonal effects may have also influenced our results and impacted B. hilaris feeding activities on broccoli seedlings. The average temperature during Season 2 Choice Study was 82 degrees Fahrenheit. This matches the optimal temperature condition for B. hilaris activities (Bharat et al, 2018), and may be one reason we saw the predicted results in this one trial. While these same temperatures occurred in Season 2 No-Choice when predicted results were not met, PAR may have had some impact in this difference. The Season 2 Choice Study had an average PAR of 37.7 µM*m-2*sec-1, while Season 2 No-

Choice Study had an average PAR of 172 µM*m-2*sec-1 . Many studies show that herbivory is influenced by light intensity (Mole and Waterman, 1988; Dudt and Shure, 1994; Jansen and Stamp, 1997; Roberts and Paul, 2006; Izaguirre et al. 2007). However, our results may not match the expectation that as light intensity increases herbivory decreases. Increased light intensity typically leads to increased plant quality and an increased production of secondary plant metabolites, reducing herbivory. A cursory analysis of our data does not seem to show decreased B. hilaris herbivory in the Season 2 No- Choice Study, when PAR was higher, compared to the Season 2 Choice Study. Light intensity may also affect insect behavior. For example, a greenhouse study suggested that ultraviolet lights modified aphids’ host finding ability (Dader et al, 2017). The intrinsic set up of these trials may be another reason the results from Season 2 Choice Study and Season 2 No-Choice Study did not match. The Choice Study exposed insects to multiple treatments per cage while the No-Choice Study exposed insects to only one treatment per cage. According to Murray et al, (2010) both Choice and No- Choice Studies have the potential to produce a false negative results when the period of exposure is too short, however, it is always ideal to use both methods in combination in general choice studies. One of our study limitations was that the insects had undergone starvation before being exposed to the host plants which may produce artificial results (Huang et al, 2014). Furthermore, studies were conducted in the greenhouse, and this controlled environment may produce data different from field condition (Knolhoff & Heckel, 2013). Our results do not provide clear guidance to farmers on novel cultural management strategies to improve B. hilaris pest management. However, the Control treatment in our study typically had lower B. hilaris numbers, and this may indicate that low fertilizer applications or delayed fertilizer applications on young seedlings may have pest management benefits. Further research in this area is necessary to determine if this is

a viable strategy. Continued research will be required to determine the relationship between fertilizers treatments, broccoli seedlings, and B. hilaris.

Conclusion

Since our study investigated the relationship of different fertilizers treatment on broccoli seedlings in response to B. hilaris feeding activities, therefore, the overall conclusion did not show any statistical difference between the Synthetic and Organic fertilizer treatments. But, the results of the study suggested that the Control treatment had lower numbers B. hilaris than the Synthetic or Organic treatments. Furthermore, our study results from the Season 2 Choice Study and from the laboratory analysis of glucosinolate nearly matched our expectations that organically grown broccoli seedlings would be less desired by B. hilaris feeding activities than synthetically grown broccoli seedlings due to differences in defense- compound concentrations. However, the results of all the other trials in our study did not match the Season 2 Choice Study results, and we did not analyze the seedling defense-compounds from any other trial.

Many studies report a correlation between glucosinolate accumulation in crops and organic field management methods. Our results did not support these previous studies but that may have been due to the nature of our experiments. Our soils were only managed organically for a short period of time and we used liquid organic fertilizer rather than an amendment like manure. Future studies should address these limitations. Instead of using potting soil, future studies could use soil from fields that have been managed organically and conventionally for years to grow their broccoli seedlings. Similar future studies could compare broccoli seedlings with an organic amendment like composted manure, to broccoli seedlings fertilized with synthetic fertilizer. Another limitation of our study was the greenhouse we worked in. The results from the greenhouse environment are difficult to extrapolate to fields conditions. Future studies should be

validated with outdoor field-scale experiments where B. hilaris activity is assessed in organic and conventional fields. Finally, our greenhouse did not fully control all environmental factors and future studies should be conducted in spaces where temperature and PAR can be completely standardized. Even though the overall results were not statistically different, this study contributed to the general understanding of the complex interactions between pest, crop, and fertilizer.

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Appendix A

Table 1: Lists of GLs & chemical names of R-groups. Fahey et al (2002). Common names of Aliphatic (GLs) Chemical names of R-groups Sinigrin 2-Propenyl Gluconapin 3-Butenyl Glucobrassicanapin 4-Pentenyl 2(R)-2-Hydroxy-3-butenyl Progoitrin

2(S)-2-Hydroxy-3-butenyl Epiprogoitrin 2-Hydroxy-4-pentenyl Gluconapoleiferin 3-Methylthiopropyl Glucoibervirin 4-Methylthiobutyl Glucoerucin 4-Methylthio-3-butenyl Dehydroerucin 3-Methylsulphinylpropyl Glucoiberin Glucoraphanin 4-Methylsulphinylbutyl Glucoraphenin 4-Methylsulphinyl-3-butenyl Glucoalyssin 5-Methylsulphinylpentenyl Glucoerysolin 3-Methylsulphonylbutyl

Indole(GLs)

Glucobrassicin

4-Hydroxyglucobrassicin 4-Mercaptobutyl 4-Methoxyglucobrassicin 3-Indolylmethyl Neoglucobrassicin 4-Hydroxy-3-indolylmethyl Aromatic (GLs) 4-Methoxy-3-indolylmethyl Glucotropaeolin 1-Methoxy-3-indolylmethyl Gluconasturtiin Benzyl

2-Phenylethyl

Table: 2 48

0.3 0.5 0.8 0.2 a 0.0 a 0.0 a 0.5 a 1.0 a 1.7 a 0.0 b 0.1 b 0.2 b 0.5 b 0.4 ab 0.6 ab 0.1346 0.0526 0.0153 <.0001

Acceptance

P =0.05.

0 0.03 0.06 0.08 0.01 a 0.00 a 0.00 a 0.06 a 0.09 a 0.12 a 0.10 a 0.00 b 0.01 b 0.03 b 0.1346 0.0437 0.0310 0.0253 0.03 ab 0.07 ab Time 1 hour Time 2 hour Time 4 hour Susceptib ility

Time 30 minutes

nt, Fisher's protected LSD test,

ess

tiven 1.4 1.5 1.5 1.4 3.2 a 3.0 a 0.2 c 2.9 a 0.8 b 0.7 b 0.3 b 1.1 b 1.4 b 0.8 b 0.5 b 2.7 a 0.0038 0.0003 0.0002 0.0004 Attrac

letters are not significantly differe

he sa

by t

llow

ns fo

) ) ) )

lum P P P P (

cts effe cts ( effe cts ( effe cts ( nd co effe me a

e ti OVA

AN e sam

treatment

ent ent ent ent

c c c c rs

mary of mary of ANOVA mary of ANOVA mary of ANOVA trol trol trol trol ans in th tilize Treatm Treatm Treatm Treatm Me Table: 5 Choice Study, Season hours) Table: 1 4 Season minutes 5 (30to Study, Choice Fer Syntheti Organic Con Mean Sum Syntheti Organic Con Mean Sum Syntheti Organic Con Mean Sum Syntheti Organic Con Mean Sum z

Table: 5 A. Choice Study, Season 1 (8 hours to 48 hours).

Fertilizers treatment Attractiveness Susceptibility Acceptance Dead plant Area damage Time 8 hour

z Synthetic 2.9 a 0.17 a 2.2 a Organic 1.4 b 0.06 a 0.7 b Control 0.5 b 0.13 a 0.6 a Mean 1.6 0.12 1.1 Summary of ANOVA effects (P) Treatment 0.0026 0.0748 0.0006 Time 12 hour Synthetic 1.3 a 0.17 a 2.2 a Organic 1.1 ab 0.06 a 0.6 b Control 0.2 b 0.13 a 0.6 b Mean 0.9 0.12 1.1 Summary of ANOVA effects (P) Treatment 0.0413 0.0748 0.0002 Time 24 hour

Synthetic 2.2 a 0.22 a 2.6 a Organic 0.9 a 0.08 a 0.8 b Control 0.4 a 0.20 a 0.9 b Mean 1.2 0.16 1.4 Summary of ANOVA effects (P) Treatment 0.0006 0.1130 0.0001 Time 48 hour Synthetic 1.6 a 0.31 a 3.1 a 3.2 a 3.2 a Organic 1.3 a 0.14 a 1.3 b 1.9 b 1.9 b Control 0.4 a 0.20 a 1.0 b 2.0 b 2.0 b Mean 1.1 0.22 1.8 2.4 2.4 Summary of ANOVA effects (P) Treatment 0.0793 0.0710 <.0001 0.0131 0.0084 z Means in the same time and columns follow letters by the are same not significantly different, Fisher's protectedP=0.05. LSD test,

Table: 6 Choice Study, Season 2 (30 minutes to 4 hours)

Attractiveness

z Acceptance 2.1 a SyntheticFertilizers treatment

2.1 a Susceptibility Organic 1.2 a Time 30 minutes 0.8 a Control 1.8 0.06 a 0.6 a

Mean 0.03 a 0.2 a

Treatment 0.3762 0.5 0.02 a ) Summary of ANOVA effects ( P 0.04

1.8 a 0.1585

Synthetic 1.5 a 0.1816 Organic 1.2 a Time 1 hour 0.9 a

Control 1.5 0.6 a 0.06 a MeanSummary of ANOVA effects ( 0.03 a 0.4 a

0.6230 0.03 a 0.6 Treatment ) P 0.04

1.1 a 0.2737 Synthetic 1.7 a 0.2914 Organic 1.0 a Time 2 hour 1.0 a Control 1.2 0.07 a 0.7 a Mean 0.4 a Summary of ANOVA effects ( 0.05 a 0.4869 0.03 a 0.7 Treatment ) P 0.05

1.0 a 0.1585 Synthetic 1.6 a 0.2984 Organic 1.1 a Time 4 hour 1.0 a

Control 1.2 0.07 a 0.7 a

0.05 a 0.5 a Mean 0.6296 z 0.04 a 0.7 SummaryMeansTreatment in ofthe ANOVA same time effects and columns ( ) follow by P 0.06 Fisher's protected LSD test,

same the letters are not significantly different, 0.3052 0.4320 P=0.05.

2.4 3.2 a 1.9 b 2.0 b Area damage 0.0084

Dead plant 3.2 a 1.9 b 2.0 b 2.4 0.0131

Acceptance Time 8 hour 2.2 a 0.7 b 0.6 a 1.1 0.0006 Time 12 hour 2.2 a 0.6 b 0.6 b 1.1 0.0002 Time 24 hour 2.6 a 0.8 b 0.9 b 1.4 0.0001 Time 48 hour 3.1 a 1.3 b 1.0 b 1.8 <.0001

P =0.05.

Susceptibility 0.17 a 0.06 a 0.13 a 0.12 0.0748 0.17 a 0.06 a 0.13 a 0.12 0.0748 0.22 a 0.08 a 0.20 a 0.16 0.1130 0.31 a 0.14 a 0.20 a 0.22 0.0710

letters are not significantly different, Fisher's protected LSD test,

Attractiveness 2.9 a 1.4 b 0.5 b 1.6 0.0026 1.3 a 1.1 ab 0.2 b 0.9 0.0413 2.2 a 0.9 a 0.4 a 1.2 0.0006 1.6 a 1.3 a 0.4 a 1.1 0.0793

Mean Mean Mean Mean Table: 6 A. hours).2 Season hours48 (8 to Study, Choice Treatment Treatment Treatment Treatment Control Control Control Control Organic Organic Organic Organic Synthetic Synthetic Synthetic Synthetic Fertilizers treatment Means in the same time and columns follow by z Summary of ANOVA effects ( P ) Summary of ANOVA effects ( P ) Summary of ANOVA effects ( P ) Summary of ANOVA effects ( P )

0 0 0 NA NA NA NA 1.0 0.5 a 1.2 a 2.0 a 2.0 a 2.5 a 0.7 b 1.2 b 2.2 ab 0.3432 0.6253 0.1737 0.3554 0.0070 1.0000 0.0070 0.0105 0.0338 0.6036 0.0710 0.0723 Acceptance Acceptance Acceptance Acceptance

than 0.05.

0 0 0 NA NA NA NA 0.06 0.03 a 0.10 a 0.18 a 0.15 a 0.12 a 0.25 a 0.06 b 0.4344 0.2558 0.0812 0.1897 0.0708 0.0708 0.0046 0.0134 0.5984 0.1553 0.0720 0.1564 0.12 ab Susceptibility Susceptibility Susceptibility Time 30 minutes Susceptibility Time 1 hour Time 2 hour Time 4 hour

z ferent by use of one degree-of-freedom contrasts. 4.7 a 4.5 a 3.2 a 4.5 a 3.7 a 3.5 a 4.0 a 4.2 a 4.7 a 4.2 a 5.2 a 3.2 a 0.3545 0.4352 0.8727 0.5896 0.5418 0.8045 0.7114 0.8149 0.7084 0.5778 0.8510 0.8400 0.7119 0.7119 0.4680 0.7513 Attractiveness Attractiveness Attractiveness Attractiveness

treatment treatment treatment

Table: 4 Studyminutes (30to hours) 7 1 Season No-Choice Means followed by the same letter in time and column are not significantly dif Fertilizers treatment Synthetic Organic Control Contrast ( P ) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment Fertilizers Synthetic Organic Control Contrast ( P ) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment Fertilizers Synthetic Organic Control Contrast ( P ) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment Fertilizers Synthetic Organic Control Contrast ( P ) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment z When P = 0.05 or less for contrasts, means are significantly different, even if probability of the ANOVA treatment effect is greater

a 3.2 3.5 a 2.5 a 0.7917 0.4391 0.3116 0.5502 Area damage

a 0 b 1.5 0.7 ab 0.0781 0.0054 0.0781 0.0156 Dead plant

a a 2.7 a 2.7 a 3.0 3.5 3.0 a 3.0 a 1.7 b 1.7 b 2.2 b 2.2 ab 2.2 ab 2.7 ab 0.0498 0.2666 0.2666 0.1250 0.0498 0.2666 0.2666 0.1250 0.0293 0.3794 0.1066 0.0723 0.3794 0.3794 1.0000 0.5787 Acceptance Acceptance Acceptance Acceptance

a a 0.23 a 0.17 a 0.31 a 0.23 a 0.17 a 0.31 a 0.34 0.26 a 0.44 a 0.47 0.45 a 0.48 a 0.3494 0.2416 0.0600 0.1465 0.3494 0.2416 0.0600 0.1465 0.4643 0.3412 0.1195 0.2673 0.9125 0.9125 0.8263 0.9742 Susceptibility Susceptibility Time 8 hour Time 12 hour Susceptibility Time 24 hour Susceptibility Time 48 hour

y use of one degree-of-freedom contrasts.

5.2 a 4.5 a 3.7 a 3.7 a 5.2 a 5.2 a 5.0 a 5.2 a 7.0 a 3.5 b 3.0 b 5.2 a 1.0000 0.6088 0.6088 0.8284 1.0000 0.2143 0.2143 0.3430 0.8236 1.0000 0.8236 0.9647 0.0144 0.6438 0.0080 0.0156 Attractiveness Attractiveness probability of the ANOVA treatment effect is greater than 0.05.

Attractiveness Attractiveness

treatment treatment treatment

Table: 48 (8 No-Choice to 7A. Study, hours).1 Season hours When P = 0.05 or less for contrasts, means are significantly different, even if Means followed by the same letter in time and column are not significantly different b Fertilizers treatment Synthetic Organic Control Contrast ( P) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment Fertilizers Synthetic Organic Control Contrast ( P) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment Fertilizers Synthetic Organic Control Contrast ( P) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment Fertilizers Synthetic Organic Control Contrast ( P) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment z

ab 0 0 0 0 b NA NA NA NA 0.7 a 0.5 a 0.7 a 1.2 a 1.2 a 1.0 a 1.2 a 0.2 0.3794 0.1066 0.0293 0.0723 0.6253 0.1737 0.3432 0.3554 0.7163 1.0000 0.7163 0.9091 Acceptance

Acceptance Acceptance Acceptance

0 0 0 0 a NA NA NA NA 0.03 0.06 a 0.04 a 0.06 a 0.10 a 0.09 a 0.09 a 0.10 a 0.3104 0.3104 0.0686 0.1664 0.7036 0.1769 0.3013 0.3494 1.0000 0.7911 0.7911 0.9506 Time 4 hour Susceptibility Susceptibility Time 30 minutes Susceptibility Susceptibility Time 1 hour Time 2 hour

-of-freedom contrasts. t effect is greater than 0.05.

z 4.0 a 4.5 a 3.7 a 3.7 a 4.0 a 4.0 a 3.7 a 4.2 a 4.2 a 4.7 a 4.2 a 3.7 a 0.8848 0.6662 0.7726 0.9006 1.0000 0.8497 0.8497 0.9744 0.8296 0.8296 0.6687 0.9053 0.7517 1.0000 0.7517 0.9303 Attractiveness Attractiveness Attractiveness Attractiveness

treatment

Table: 4 Studyminutes (30to hours). 8 2 Season No-Choice When P = 0.05 or less for contrasts, means are significantly different, even if probability of the ANOVA treatmen Means followed by the same letter in time and column are not significantly different use of one degree Fertilizers treatment Synthetic Organic Control Contrast ( P ) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment Fertilizers Synthetic Organic Control Contrast ( P ) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment Fertilizers treatment Synthetic Organic Control Contrast ( P ) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment Fertilizers Synthetic Organic Control Contrast ( P ) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment z

3.2 a 2.2 a 3.2 a 0.2666 1.0000 0.2666 0.4219 Area damage

3.2 a 2.2 a 3.2 a 0.2666 1.0000 0.2666 0.4219 Dead plant

1.5 a 1.0 a 1.5 a 1.5 a 1.2 a 1.5 a 1.7 a 1.2 a 1.7 a 3.0 a 2.2 a 2.5 a 0.4198 1.0000 0.4198 0.6297 0.6973 1.0000 0.6973 0.8966 0.3794 1.0000 0.3794 0.5787 0.3587 0.5322 0.7517 0.6232 Acceptance Acceptance Acceptance

Acceptance

0.14 a 0.09 a 0.12 a 0.14 a 0.11 a 0.12 a 0.14 a 0.11 a 0.19 a 0.38 a 0.17 b 0.2969 0.7739 0.4322 0.5325 0.5331 0.7922 0.7131 0.8083 0.5952 0.3860 0.1854 0.3800 0.4940 0.1421 0.0520 0.1204 0.23 ab Susceptibility Time 8 hour Susceptibility Time 12 hour Susceptibility Time 48 hour Time 24 hour Susceptibility

z 4.0 a 2.2 a 1.0 a 3.5 a 1.7 a 2.5 a 4.5 a 2.7 a 1.7 a 2.5 a 2.7 a 3.7 a 0.7517 0.0940 0.0595 0.1163 0.0930 0.5713 0.2119 0.2039 0.0835 0.8420 0.1196 0.1577 0.5380 0.4175 0.8349 0.6806 Attractiveness Attractiveness Attractiveness Attractiveness

treatment

When P = 0.05 or less for contrasts, means are significantly different, even if probability of the ANOVA treatment effect is greater than 0.05. Means followed by the same letter in time and column are not significantly different use of one degree-of-freedom contrasts. Fertilizers treatment Synthetic Or ganic Control Contrast ( P) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment Fertilizers Synthetic Organic Control Contrast ( P) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment Fertilizers treatment Synthetic Organic Control Contrast ( P) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment Fertilizers treatment Synthetic Organic Control Contrast ( P) Synthetic vs. organic Synthetic vs. control Organic vs. control Summary of ANOVA effects ( P ) Treatment z Table: 8A. No-Choice Study Season 2 (8 hours to 48 Table: hours).2 Season No-Choice 8A. Studyhours(8 to

Appendix B

Bagrada hilaris physiologically resembles its stink bug relative commonly known as the harlequin bug (Murgantia histrionica) (b)

(a) (b)

Figure 2: Byproduct of GLs after chemical decomposition. Image credited to Bartnik et. al, (2017), “The enzymatic hydrolysis of glucosinolate and their main degradation products”.

Figure 3: The Mustard Oil Bomb. Glucosinolates and myrosinase are stored in separate cell compartiments. Break down of the cell, e.g. by chewing, cutting or heating, leads to the release of glucosinolates and myrosinase. Myrosinase causes hydrolytic cleavage of glucosinolates as exemplified for glucoraphanin present in high concentration in broccoli. The resulting mustard oil is the isothiocyanate sulforaphane (Herr et al., 2013)

Figure: 6. Season 1 and 2 Choice Study Temperature.

Figure: 7. Season 1 and 2 No-Choice Study Temperatures