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A study of insecticidal synergy of plant essential oil constituents against Trichoplusia ni Tak, Jun-Hyung 2015

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A STUDY OF INSECTICIDAL SYNERGY OF PLANT ESSENTIAL OIL CONSTITUENTS AGAINST TRICHOPLUSIA NI  by  Jun-Hyung Tak  B.Sc., Seoul National University, 2003 M.Sc., Seoul National University, 2005   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Plant Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   April 2015  ©  Jun-Hyung Tak, 2015 ii  Abstract  Chemical compositions of plant essential oils can be affected by many environmental and biological factors.  Understanding the role of individual constituents and their interactions to overall insecticidal bioactivity is prerequisite to the use of essential oils as an alternative to conventional insecticides.  In the present study, the chemical compositions of Rosmarinus officinalis (rosemary), Thymus vulgaris (thyme) and Cymbopogon citratus (lemongrass) essential oils were analyzed by gas chromatography-mass spectrophotometry (GC-MS), and relationships between chemical composition and toxicity of the constituents, and synergistic interactions of the major constituents of the oils were evaluated against third instar larvae and an ovarian cell line of the cabbage looper, Trichoplusia ni, via different application methods.  To explore underlying mechanisms of synergy, penetration through the insect cuticle and inhibitory activities on three detoxifying enzyme systems were investigated.  The most abundant constituents of rosemary, thyme and lemongrass oils were 1,8-cineole, thymol and citral, respectively, and their overall contributions to in vivo and in vitro toxicity varied according to the application methods, as did their designation as major active principles.  A weak correlation between insecticidal activity and cytotoxicity was observed, indicating limitation of insect cell cultures as a screening tool for novel insecticides.  Several synergistic interactions were found among the major constituents of each oil, including 1,8-cineole+camphor, thymol+p-cymene and citral+limonene. Analysis by GC-MS showed a significant penetration-enhancing effect of topically applied camphor by 1,8-cineole in their synergistic binary mixture.  1,8-Cineole was more toxic than camphor when applied topically to larvae, but a bioassay via injection revealed greater toxicity of camphor than 1,8-cineole.  A bioassay combining injection and topical application confirmed the increased iii  penetration of both compounds when mixed, showing the same bioactivity with higher amounts applied individually.  A similar pattern of enhanced penetration of insecticides through the cuticle of T. ni in other synergistic combinations was observed as well.  Lowered surface tension and increased solubility along with the interaction between essential oil constituents and the lipid layer of the insect’s cuticle may explain their enhanced penetration.  Although some mild enzyme inhibitory activities were observed in essential oil-treated larvae, no correlation was observed between detoxicative metabolism and synergistic toxicity.   iv  Preface  Led by the intellectual guidance and support of my supervisory committee, Prof. Murray B. Isman, Prof. Eduardo Jovel, Prof. Jörg Bohlmann and Dr. Saber Miresmailli, this research was designed, conducted and analyzed by me.  The initial and final drafts of this thesis were prepared by me, with extensive and devoted editing by Prof. Isman. The research in Chapter 2 has been submitted to two different peer-reviewed journals, currently in the review process;  Jun-Hyung Tak, Eduardo Jovel, and Murray B. Isman*, Comparative and synergistic activity of Rosmarinus officinalis L. essential oil constituents against the larvae and an ovarian cell line of the cabbage looper, Trichoplusia ni (Lep., Noctuidae)  Jun-Hyung Tak, Eduardo Jovel, and Murray B. Isman*, Contact, fumigant and cytotoxic activities of thyme and lemongrass oils against larvae and an ovarian cell line of the cabbage looper, Trichoplusia ni (Lepidoptera: Noctuidae) I conducted all experiments and analyzed data, and the manuscripts were a collaborative effort between me and Prof. Isman.  Prof. Jovel provided cell lines and supported cytotoxicity assays. Versions of chapter 3 and 4 will be submitted for publication shortly. v  Table of Contents  Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ...........................................................................................................................v List of Tables ................................................................................................................................ ix List of Figures ............................................................................................................................... xi List of Abbreviations ................................................................................................................. xiii Acknowledgements .................................................................................................................... xiv Dedication ................................................................................................................................... xvi Chapter 1: Introduction ................................................................................................................1 1.1 Plant essential oils: origin, variations and usage ............................................................ 1 1.2 Bioactivity and advantages of essential oils ................................................................... 4 1.3 The cabbage looper, Trichoplusia ni .............................................................................. 9 1.4 Research trends in the bioactivity of essential oils in insects ....................................... 10 1.5 Insecticidal mode-of-action and detoxification of essential oils and plant secondary metabolites ................................................................................................................................ 13 1.6 Synergistic interactions of essential oil constituents .................................................... 17 1.7 Research objectives ....................................................................................................... 18 Chapter 2: Comparative toxicities of essential oil constituents against larvae and an ovarian cell line of Trichoplusia ni ..............................................................................................20 2.1 Introduction ................................................................................................................... 20 2.2 Materials and methods .................................................................................................. 21 2.2.1 Chemicals .................................................................................................................. 21  vi  2.2.2 Gas chromatography-mass spectrophotometry ......................................................... 22 2.2.3 Insect and cell line maintenance ............................................................................... 23 2.2.4 Topical application assay and fumigant assay against 3rd instar larvae ................... 23 2.2.5 Cytotoxicity assay on the cell line ............................................................................ 25 2.2.6 Comparative activity of individual constituents ....................................................... 26 2.2.7 Evaporation control of thymol and p-cymene and their fumigant activity ............... 26 2.2.8 Statistics .................................................................................................................... 28 2.3 Results ........................................................................................................................... 29 2.3.1 Chemical compositions of essential oils ................................................................... 29 2.3.2 Insecticidal and cytotoxic activities of individual constituents ................................ 32 2.3.3 Comparative toxicity of the constituents .................................................................. 37 2.3.4 Evaporation control and fumigant activity ............................................................... 43 2.4 Discussion ..................................................................................................................... 44 Chapter 3: Synergistic interactions of the major constituents of three plant essential oils and their insect cytotoxicity and cuticular penetration as potential mechanisms of synergy    ........................................................................................................................................................50 3.1 Introduction ................................................................................................................... 50 3.2 Materials and methods .................................................................................................. 51 3.2.1 Chemicals .................................................................................................................. 51  3.2.2 Insect and cell line maintenance ............................................................................... 51 3.2.3 Synergistic interactions of the four major constituents of essential oils via topical application assay ................................................................................................................... 52 3.2.4 Cytotoxicity of the synergistic combinations from contact toxicity ......................... 53 3.2.5 Sample preparation for cuticle penetration analysis ................................................. 54 vii  3.2.6 GC-MS analysis of cuticle penetration ..................................................................... 55 3.2.7 Injection assay using 5th instar larvae ...................................................................... 56 3.2.8 Mixed application of 1,8-cineole and camphor ........................................................ 57 3.2.9 Insecticidal activity of metabolites of citral and limonene ....................................... 57 3.2.10 Surface tension and contact angle measurement ...................................................... 58 3.2.11 Statistics .................................................................................................................... 58 3.3 Results ........................................................................................................................... 58 3.3.1 Interactions among the four major constituents ........................................................ 58 3.3.2 Cytotoxicity of synergistic combinations ................................................................. 64 3.3.3 Cuticular penetration of the 1,8-cineole and camphor mixture ................................ 67 3.3.3.1   GC-MS analysis ................................................................................................... 67 3.3.3.2   Injection assay and mixed application ................................................................. 69 3.3.3.3   Surface tension and contact angle ....................................................................... 72 3.3.4 Cuticular penetration of the thymol and p-cymene mixture ..................................... 73 3.3.4.1   GC-MS analysis ................................................................................................... 73 3.3.4.2   Injection assay ..................................................................................................... 74 3.3.4.3   Surface tension and contact angle ....................................................................... 75 3.3.5 Cuticular penetration of the citral and limonene mixture ......................................... 76 3.3.5.1   GC-MS analysis ................................................................................................... 76 3.3.5.2   Insecticidal activity of the metabolites ................................................................ 78 3.3.5.3   Surface tension and contact angle ....................................................................... 78 3.4 Discussion ..................................................................................................................... 79 Chapter 4: Effects of three essential oils and their major constituents on detoxifying enzymes and interactions with enzyme inhibitors in Trichoplusia ni......................................88 viii  4.1 Introduction ................................................................................................................... 88 4.2 Materials and methods .................................................................................................. 90 4.2.1 Chemicals .................................................................................................................. 90  4.2.2 Insect maintenance .................................................................................................... 91 4.2.3 Enzyme preparation .................................................................................................. 91 4.2.4 Enzyme assay ............................................................................................................ 92 4.2.5 LD50 determinations of major constituents and enzyme inhibitor mixtures ............. 94 4.2.6 Influence of pre-treatment of enzyme inhibitors on insecticidal activity ................. 94 4.2.7 Statistics .................................................................................................................... 95 4.3 Results ........................................................................................................................... 95 4.3.1 Enzyme activity ........................................................................................................ 95 4.3.2 Effect of enzyme inhibitors ....................................................................................... 99 4.3.3 Effect of pre-treatment with enzyme inhibitors ...................................................... 102 4.4 Discussion ................................................................................................................... 105 Chapter 5: Summary and discussion .......................................................................................110 5.1 Insecticidal activity of essential oil constituents ......................................................... 110 5.2 Synergistic interactions and mechanisms ................................................................... 112 5.3 Future directions ......................................................................................................... 116 Bibliography ...............................................................................................................................120 Appendices ..................................................................................................................................143 Appendix A: Chemical structures of the major constituents of essential oils tested .................143 Appendix B: Standard curves ....................................................................................................144   ix  List of Tables  Table 1.1 List of essential oils and active constituents of registered (in Canada) and exempted (in the USA) commercial insecticides ............................................................................................. 9 Table 1.2 Numbers of publications on essential oil research based on major orders of insects and other arthropods (2004-2014) .................................................................................................. 12 Table 2.1 Chemical constituents of three essential oils ................................................................ 30 Table 2.2 In vivo insecticidal activities of rosemary oil and individual constituents to 3rd instar T. ni larvae by topical application and in vitro cytotoxicity to T. ni ovarian cells ....................... 34 Table 2.3 In vivo contact and fumigant toxicities of thyme oil and individual constituents to 3rd instar T. ni larvae, and in vitro cytotoxicity to T. ni ovarian cells ........................................... 35 Table 2.4 In vivo insecticidal activities of lemongrass oil and individual constituents to 3rd instar T. ni larvae by topical application and in vitro cytotoxicity to T. ni ovarian cells ................... 36 Table 3.1 Synergistic interactions of four major constituents of rosemary essential oil in 3rd instar larvae of T. ni by topical application .............................................................................. 60 Table 3.2 Synergistic interactions of four major constituents of thyme essential oil in 3rd instar larvae of T. ni by topical application ........................................................................................ 62 Table 3.3 Synergistic interactions of four major constituents of lemongrass essential oil in 3rd instar larvae of T. ni by topical application .............................................................................. 63 Table 3.4 Cytotoxicity of selected synergistic combinations from topical application assay ...... 64 Table 3.5 GC-MS quantifications of 1,8-cineole and camphor in rinsed and extracted solutions at different observation times ....................................................................................................... 68 Table 3.6 Comparison of the insecticidal activities of 1,8-cineole and camphor in the topical and injection assays against 3rd and 5th instar larvae of T. ni ........................................................ 70 Table 3.7 Comparative toxicity of mixed application assay against 5th instar larvae of T. ni ..... 71 x  Table 3.8 Surface tension and contact angle measurement of rosemary oil and major constituents .................................................................................................................................................. 72 Table 3.9 GC-MS analysis of the cuticle penetrations of individual and mixture of thymol and p-cymene in 3rd instar larvae of T. ni .......................................................................................... 73 Table 3.10 Comparison of LD50 values of thymol, p-cymene and their mixture via different administration methods ............................................................................................................ 74 Table 3.11 Surface tension and contact angle measurement of thyme oil and their major constituents ............................................................................................................................... 75 Table 3.12 GC-MS analysis of the cuticle penetrations of individual and mixture of citral and limonene in 3rd instar larvae of T. ni ....................................................................................... 77 Table 3.13 Insecticidal activities of the major metabolites of citral and limonene ...................... 78 Table 3.14 Surface tension and contact angle measurement of lemongrass oil and their major constituents ............................................................................................................................... 79 Table 4.1 In vivo enzyme activity of esterases, glutathione S-transferases and cytochrome P450s following treatment of larvae with selected essential oils and their major constituents .......... 97 Table 4.2 Insecticidal activities of the major constituents of rosemary oil and their combination when mixed with enzyme inhibitors in third instar T. ni larvae by topical application ......... 100 Table 4.3 Insecticidal activities of the major constituents and their combination of thyme oil when mixed with enzyme inhibitors on third instar T. ni larvae by topical application ........ 101 Table 4.4 Insecticidal activities of the major constituents and their combination of lemongrass oil when mixed with enzyme inhibitors on third instar T. ni larvae by topical application ........ 102 xi  List of Figures  Figure 1.1 Trends in the importation and export of essential oils in Canada ................................. 4 Figure 1.2 Literature survey of essential oils associated with insect and arthropod species from WoS (2004-2014) ..................................................................................................................... 12 Figure 1.3 Research trends in essential oils on two orders of insects ........................................... 13 Figure 2.1 Fumigation assay chamber system .............................................................................. 24 Figure 2.2 A schematic of the fumigation chamber system and heated evaporation assay .......... 28 Figure 2.3 GC-MS chromatograms of three essential oils used in this study ............................... 31 Figure 2.4 Toxicity of the individual major constituents of rosemary oil and their binary mixture .................................................................................................................................................. 37 Figure 2.5 Toxicity of the individual major constituents of thyme oil and their binary mixture . 38 Figure 2.6 Toxicity of the individual major constituents of lemongrass oil and their binary mixture. .................................................................................................................................... 39 Figure 2.7 Compound elimination assays of the major constituents of rosemary essential oil via different application methods ................................................................................................... 40 Figure 2.8 Compound elimination assays of the major constituents of thyme essential oil via different application methods ................................................................................................... 41 Figure 2.9 Compound elimination assays of the major constituents of lemongrass essential oil via different application methods ................................................................................................... 42 Figure 2.10 Evaporation of thyme oil and major constituents at two different temperatures ...... 43 Figure 2.11 Fumigant activity of thyme oil following different evaporation times ..................... 44 Figure 3.1 Validation of cytotoxic synergy between citral and limonene in an ovarian cell line of T. ni .......................................................................................................................................... 65 Figure 3.2 Morphological observations of T. ni cells when treated with individual citral and limonene and a mixture thereof ................................................................................................ 67 xii  Figure 3.3 Penetration rate of the compounds when applied individually or when mixed in 3rd instar larvae of T. ni ................................................................................................................. 69 Figure 3.4 Cuticle penetration of citral and limonene .................................................................. 77 Figure 3.5 Applied status comparison of 1,8-cineole and camphor solutions on black cotton fabric ........................................................................................................................................ 87 Figure 4.1 Relative influence of three essential oils and their major constituents on esterases in third instar larvae of the cabbage looper .................................................................................. 98 Figure 4.2 Relative influence of three essential oils and their major constituents on glutathione S-transferases in third instar larvae of the cabbage looper .......................................................... 98 Figure 4.3 Relative influence of three essential oils and their major constituents on cytochrome P450s in third instar larvae of the cabbage looper ................................................................... 99 Figure 4.4 Contributions of enzyme inhibitors by different application methods to the insecticidal activity of individual major constituents of rosemary oil and their mixture on third instar larvae of T. ni ......................................................................................................................... 103 Figure 4.5 Contributions of enzyme inhibitors by different application methods to the insecticidal activity of the individual major constituents of thyme oil and their mixture on third instar larvae of T. ni ......................................................................................................................... 104 Figure 4.6 Contributions of enzyme inhibitors by different application methods to the insecticidal activity of the individual major constituents of lemongrass oil on and their mixture third instar larvae of T. ni ......................................................................................................................... 105 Figure A.1 Major consitutents of rosemary oil ........................................................................... 143 Figure A.2 Major consitutents of thyme oil ................................................................................ 143 Figure A.3 Major consitutents of lemongrass oil ....................................................................... 143 Figure B.1 Florescence intensity standard curve of viable T. ni ovarian cells ........................... 144 Figure B.2 Standard curves of 1,8-cineole and camphor for GC-MS quantification ................. 144 Figure B.3 Standard curve for protein quantification via Bradford assay .................................. 145  xiii  List of Abbreviations  AChE acetylcholineesterases ANOVA analysis of variance Bt Bacillus thuringiensis (Berliner) CDNB 1-chloro-2,4-dinitrobenzene CL confidence limit DEM diethyl maleate DMSO dimethyl sulfoxide DTT dithiothreitol EA ethacrynic acid EDTA ethylenediaminetetraacetic acid EPA US Environmental Protection Agency GABA gamma-aminobutyric acid GC-MS gas chromatography-mass spectrophotometry GRAS generally recognized as safe  GST glutathione S-transferases IC50 inhibitory concentration 50% LC50 or LC95 lethal concentration 50% (or 95%) LD50 or LD95 lethal dose 50% (or 95%) P450s cytochrome P450 dependent monooxygenases PBO piperonyl butoxide PMRA Pest Management Regulatory Agency PMSF phenylmethanesulfonyl fluoride SC stratum corneum SD standard deviation SE standard error TMBZ 3,3′,5,5′-tetramethylbenzidine TPP triphenyl phosphate WoS Web of Science    xiv  Acknowledgements  First and foremost, I want to thank my supervisor, Professor Murray B. Isman for his great guidance and assistance, showing me the exemplary mindset and insight how to become a great scientist and supervisor.  It has been a great honor to learn so many lessons from him.  My deepest appreciation goes to my co-supervisor, Professor Eduardo Jovel, for his sincere support and care not only in academic but also in personal.  His warm heart gave me a great deal of strength to endure the entire course.  My research committee members have been admirable examples of great scholars, as Professor Joerg Bohlmann guided my research with his keen and wise scientific perspectives and through his lecture, and Dr. Saber Miresmailli shared his vivid experience as a graduate student as well as a postdoc fellow, helped me to set my paths for not only my research but also my future career. Another mentor of mine, Dr. Yasmin Akhtar, taught me the responsibility and perseverance for a scientist as well as a lecturer must have, also guided my research and the techniques I needed.  To my current and former lab mates, Dr. Mahnaz Khanavi, Dr. Rita Seffrin, Dr. Zhili Jiang, Dr. (soon-to-be) Sepideh Tahriri, Dr. Hoda Kabiri, Dr. Leandro Prado Ribeiro and Pedram Laghaei, thank you for your help and I wish your great success on your career. The staff members in labs and the faculty, Nancy Brard, Zyta Abramowski, Lina Madilao, Sylvia Leung, Lia Maria Dragan, Roxana Quinde and Rebecca Lee, helped my research and my doctoral student life to become a great experience.  I owe particular thanks to Shelley Small, who has been my sincere friend and a big supporter. xv  There are so many people that I must give my appreciation back home in South Korea who supported and helped me and my research.  Professor Young-Joon Ahn, my academic supervisor in master’s course, led me get into this fascinating world of botanical insecticides.  Along with him, Professor Si-Hyeock Lee and Professor Yeon-Ho Je from Seoul National University, and Mr. Sug-Youn Chang and Mr. Jeong-Rae Lee from LG Household and Health Care gave me very precious advices on my graduate life and my research.  Dr. Soon-Il Kim, Dr. Kyu-Sik Chang and Mr. Jung-Hwan Bae supported me and concerned my research together with me as if it was their own work. I also want to show my gratitude to my old buddies, Andy Xuan, Sung-Hoon Cho, Ohne Lee, Danny Park, Chang-Sik Ham, Jung-Hyun Choi, Ki-Tae Kim, Sang-Wook Park, Youngseob Shin, Jae-Hwa Lim, Nam-Seo Son, and HyoSeon Kim for the good memories.  Funding for this project was made possible thanks to the UBC Four Year Doctoral Fellowship. xvi  Dedication    I dedicate this thesis to my wife, Joeng-Hwa Lee,  who has been, and will be, my best friend and companion in my life,  and to our greatest achievment, Jin-Hyun.  1  Chapter 1: Introduction For centuries, plant essential oils or plant extracts have been widely used in traditional medicine, aromatherapy, as flavors in food, as perfumes, as preservatives and biological agents.  Insecticidal activities of plant secondary metabolites have also been recognized for a long time.  For example, in the book of Dongui Bogam, which literally means ‘Mirror of Eastern Medicine’, published by Korean royal physician, Heo Jun in 1613 and considered one of the most important treatises in the medicinal history in Eastern Asia, several plant extracts including those from cinnamon, cocklebur, wormwood, and rock pine are noted to control insect pests.  1.1 Plant essential oils: origin, variations and usage Plant essential oils are the products obtained from hydrodistillation, steam distillation, dry distillation, or mechanical cold pressing of plants, mostly from plants belonging to just a few families including the Myrtaceae, Lauraceae, Lamiaceae and Asteraceae (Regnault-Roger et al. 2012).  They consist primarily of terpenoid compounds, especially monoterpenes and sesquiterpenes.  Although terpenes are the most diverse class of organic compounds in plants, they have a common biosynthetic origin, derived from five-carbon units of isopentenyl diphosphate (IPP).  Terpenes are classified by the number of IPP units in their structure, as monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) or polyterpenes [(C5)n carbon atoms, where n > 8] (Gershenzon and Croteau, 1991).  Steps in the biosynthesis of all terpenoids are; (a) synthesis of the fundamental precursor IPP; (b) repetitive additions of IPP to form a series of prenyl diphosphate homologs; (c) modification of allylic prenyl diphosphates 2  by specific terpenoid synthases to yield terpenoid skeletons; and (d) secondary enzymatic modifications to the skeletons to give functional properties.  Monoterpenes mainly originate in cell plastids, created via the methylerythritol phosphate pathway (Croteau et al. 2000).  A well characterized plant model system of monoterpene biosynthesis is peppermint (Mentha  piperita).  The biosynthesis of p-menthane is initiated from ()-limonene, which is formed by ()-limonene synthases. Limonene undergoes subsequent modifications by enzymatic reactions including oxidations, reductions, isomerizations and conjugations (Ahkami et al. 2015).  The basic principles of monoterpene biosynthesis are likely the same or similar in other essential oil-producing plants.  For example, both in peppermint and spearmint (M. spicata), cytochrome P450 monooxygenases (P450s) mainly perform the same role in hydroxylations or epoxidations involved in oxygen atom introduction to terpene skeletons, but at different allylic positions. In peppermint, ()-limonene undergoes a P450-dependent hydroxylation, catalyzed by ()-limonene 3-hydroxylase, to form ()-transisopiperitenol, which is later diversified to many other monoterpenes such as (+)-pulegone, ()-menthone, or ()-menthol.  In spearmint, the process is shorter, as P450 limonene 6-hydroxylase introduces oxygen at the other allylic postion of the same parent compound, ()-limonene, to produce ()-trans-carveol, which can be oxidized to ()-carvone (Croteau et al. 2000). The production of essential oils by plants as well as their subsequent accumulation, emission or secretion is usually associated with specialized anatomical and cell structures, including glandular trichomes, secretory cavities of leaves and glandular epidermis of flower petals (Croteau et al. 2000).  Essential oils can be obtained from other plant parts, including buds, seeds, 3  twigs, bark, fruits or roots (Burt 2004).  There is an immense scientific literature reporting chemical compositions and variations of constituents in essential oils, but unfortunately, many reports are primarily descriptive, failing to explain the underlying factors responsible for variation.  However, Rios-Estepa et al. (2008) showed chemical variation can be mathematically simulated in a controlled environment by adjusting the stress levels that not only modifies yields of essential oils (possibly resulting from different numbers of biosynthetically active glandular trichomes) but also the compositions of specific monoterpenes.  Moreover, Mahmoud and Croteau (2001) reported that the quality (composition of specific monoterpnenes) and the quantity (yields) of essential oils can be genetically engineered in transgenic peppermint, indicating that production of essential oils is governed by biochemical reactions.  These findings encourage investigators to provide more detailed information, such as climate conditions, temperature variations, duration of sunlight and other possible abiotic conditions that can affect the biosynthesis of essential oils when they report geographic or seasonal variations in essential oils for determining the most suitable cultivation conditions for essential oil bearing crop plants. In Canada, the primary markets for essential oils are in the flavour and fragrance industries, which include soft drink, food, and cosmetic companies, along with niche markets such as the aromatherapy industry (Agriculture and Agri-Food Canada 2013).  As shown in Figure 1.1, production and consumption of essential oils in Canada are stable but gradually increasing annually, with the majority being used in cosmetics, household and personal care products.  Use of essential oils for consumer products in Canada is regulated by Health Canada, under several legislative acts, according to their purpose and any claims they make, such as the Food and Drug Act, Canadian Cosmetic Act, and Pest Control Products Act.  For example, if a soap product 4  contains essential oils and the purpose of the oil is strictly to add fragrance, then the soap will be considered as a cosmetic product, and the oils as simple fragrance ingredients.  But if the product claims to treat eczema or psoriasis by therapeutic actions of the essential oils in the soap, then the oils will be regarded as active ingredients and the product as a drug or natural health product.  These fall into different categories with different regulations to meet, requiring standardized clinical trials to ensure both safety and efficacy.  Figure 1.1. Trends in the importation and export of essential oils in Canada.  ‘Others’ include crude oils, concentrates and aqueous distillates of essential oils, and terpenic by-products of essential oils (resource: Statistics Canada 2014).  1.2 Bioactivity and advantages of essential oils There is extensive scientific literature on biological activities of essential oils with a wide variety of foci.  It has long been recognized that some essential oils possess insecticidal, antibacterial, antifungal, antiviral, and antioxidant activities, as well as pharmaceutical and therapeutic 5  potentials.  Bioactivities of essential oils in many disciplines are well-documented (Burt 2004; Isman 2006; Edris 2007; Bakkali et al. 2008; Lang and Buchbauer 2012; Vergis et al. 2013). In general, the use of essential oils as biological agents can have several advantages.  First, compared to synthetic chemicals, most essential oils pose little if any harmful environmental impact, as their synthesis is driven by plant photosynthesis, and many essential oils are easily bio-degradable.  Therefore, no serious environmental accumulation or side effects can be expected compared to many synthetic pesticides such as DDT, which brought numerous and unexpected problems in soil, for wildlife, and even in humans.  Second, essential oils have a long history of safe use by humans. Although there are a few exceptions (Regnault-Roger et al. 2012), most have a very long history of use as culinary spices, medicines and fragrances.  Also, a considerable number of essential oils are on the GRAS (generally recognized as safe) list of the US Food and Drug Administration (Burt 2004), which led to their reduced registration as pesticides and the costly toxicological evaluation normally required for conventional commercialization of a pesticide.  Third, essential oils can show synergistic effects with conventional agents such as drugs or pesticides.  They may have different modes-of-action from conventional insecticides (Tong and Bloomquist 2013, Chang et al. 2012), or may enhance the effect of drugs by increasing their penetration (Williams and Barry 1991).  The synergy may help to reduce the use of the primary agents which can be beneficial in many ways, including slowing or delaying the development of resistance.  Last, essential oils may be more readily accepted by consumers as pesticides, especially for indoor use. There are also several disadvantages of essential oils as pesticides, but most can be overcome or bypassed.  First, many essential oils tend to be highly volatile.  This lessens their long-term 6  residual effects in field applications or protection time against blood-sucking insects (Fradin and Day 2002).  However, this volatility of essential oils enables their use as fumigants in closed environments such as greenhouses (Kim et al. 2014) or storage facilities (Loni and Panahi 2014), for post-harvest protection (Camele et al. 2012).  To extend protection times, studies of slow release formulations or carriers have been widely conducted (Chang et al. 2006; Kim et al. 2012).  Second, essential oils are complex and variable in their composition, reducing consistency in bioactivity, efficacy and understanding of their actions.  Chemical compositions of any particular essential oil can vary due to genetic differences and many other biotic and abiotic factors (Ghnaya et al. 2013) leading to undesirable variation in toxicity (Isman et al. 2008).  In addition to qualitative and quantitative variation in active constituents, there is also the potential for interactions between constituents that can include synergistic or antagonistic responses (Miresmailli et al. 2006).  Therefore, a comprehensive understanding of constituents and their relative contributions to bioactivity will ensure that an essential oil is used optimally.  Third, essential oils are considered relatively expensive compared to some conventional pesticides.  However, in some affluent countries and regions including Canada, the USA and the European Union, there is growing interests in safer alternatives for pest management, particularly for industrial applications or on high-value crops, driving increased use of botanical pesticides (Isman 2008).  In contrast, in less developed or ‘poor’ countries, even conventional (imported) pesticides can often be too expensive to use, and local plants can serve as a resource to reduce or replace synthetic insecticides (Isman 2008).  Last, essential oils can impact the sensory attributes of food or consumer products.  Although many essential oils are used for fragrance, because high concentrations can be required for adequate biological activity, they can lead to a bad perception in terms of odor or taste.  However, there is a wide selection of candidate oils from which to 7  choose that have acceptable or pleasant characteristics and required bioactivity (Goulas and Kontominas 2007; Kostaki et al. 2009).  Formulation research can control the release of essential oils and this may help to reduce any unpleasant perception. For current use of essential oils as commercial insecticides, the USA is the only country that exempts these from registration, if they meet certain criteria.  The US Environmental Protection Agency (EPA) does not review or require registrations of pesticides that satisfy List 25(b) of the Federal Insecticide, Fungicide, & Rodenticide Act, regarding them as minimum risk pesticides.  About half of the 31 active ingredients so listed are known to possess insecticidal activity, including cinnamon, citronella and thyme oils.  All other developed countries have strict drug and pesticide registration processes and responsible agencies including these in Canada pose a significantly more difficult entry barrier than the US-EPA.  For example, the EPA allows not only the use of oils but also the use of their active ingredients of some essential oils to be exempted such as clove, garlic or rosemary oils.  However, in Canada, crude essential oils and their active constituents have to be registered separately, since they are not considered as the same (Table 1.1).  Whereas only a few essential oil-based insecticides are being sold in Canada, due to easier commercialization (by exemption) in the USA, greater numbers of botanical insecticides are currently being distributed in that market.  Not only the number of commercialized products but also their application targets and selections of active ingredients differ greatly between the two countries.  The majority of registered insecticides in Canada based on essential oils are insect repellents using citronella, garlic, or soybean oils as well as p-menthan-3,8-diol and verbenone as their active ingredients, whereas in the USA, applications of essential oils as active ingredients 8  are much broader, including for control of agricultural pests such as thrips, aphids, whiteflies and mite species (Regnault-Roger 2013).  As for the selections of ingredients, for example, in Canada, a blend of two essential oils needs to be re-registered with clinical data of the blended product even though both individual essential oils were registered as insecticides previously, but in the USA, manufacturers can choose the essential oils freely as long as all the ingredients are on the exemption list.  Many of essential oil-based insecticides in the USA contain a blend of two or more essential oils.  This blending trend might be somehow helpful to maintain the efficacy of an insecticide since the variation of chemical composition of essential oil can be compensated by other essential oils, sometimes producing synergistic effects.  But at the same time, it increases the importance of understanding the roles of each ingredient and their interactions to ensure the selection of components for the optimal performance of the product.      9  Table 1.1. List of essential oils and active constituents of registered (in Canada) and exempted (in the USA) commercial insecticides  Canada USA registered (exempted) essential oils camphor oil citronella oil eucalyptus oil garlic oil lemon oil geranium oil pine needle oil soybean oil thyme oil wintergreen oil  cedar oil cinnamon oil* citronella oil clove oil* garlic oil* geranium oil lemongrass oil mint oil peppermint oil* rosemary oil* thyme oil* registered (exempted) active constituents d-limonene p-menthan-3,8-diol thymol verbenone eugenol geraniol* 2-phenethyl propionate  regulatory authority Health Canada** US EPA * indicates exempt active ingredients that are also exempt from pesticide residue tolerance requirements. ** Pest Management Regulatory Agency (PMRA).  1.3 The cabbage looper, Trichoplusia ni The cabbage looper, Trichoplusia ni Hübner, belongs to the family Noctuidae.  It is widespread, occurring throughout the Americas from the Canadian Nearctic area to Brazil, as well as East Africa, India and some Asian countries (Robinson et al. 2010).  It is one of the major pests of cruciferious crops including cabbage, broccoli, and cauliflower, and may also be found on beets, 10  celery, lettuce, peas, spinach, tomatoes and flowers, including carnations and nasturtiums (Natural Resources Canada 2014).  It takes about a month in warm weather to complete its life cycle and a single female can produce 300 to 600 eggs.  Prior to the 3rd instar, larvae feed on the undersides of leaves; during the 4th and 5th larval instars, they consume three times their own body weight daily (Natural Resources Canada 2014).  Like many other insect and arthropod pests, efforts to control the cabbage looper with conventional insecticides have led to resistance of this species to many pesticides including organophosphate and carbamate insecticides (Leibee and Capinera, 1995).  Moreover, resistance to the microbial insecticide, Bacilus thuringiensis in greenhouse populations in British Columbia has been reported (Janmaat and Myers 2003), and possibility of resistance to an insect pathogenic virus has also been suggested (Milks and Myers 2003).  1.4 Research trends in the bioactivity of essential oils in insects The majority of published studies on the biological activity of essential oils to insects focus on their acute toxicity or repellence of the oils to agricultural or public health pests, and this subject attracts more attention each year.  As shown in Figure 1.2, a literature survey conducted following the method of Isman and Grieneisen (2014) using the Web of Science (WoS) indicates the rapid increase in publications on the bioactivity of essential oils against insect and arthropod species.  Over the last 10 years, on average about three quarters of papers on essential oil bioactivity in insects focused on acute effects including toxicity and repellent activity (2,637 publications), and the remainder (987 publications) plus 14% of the publications on acute 11  toxicity (376 publications, 1,363 in total) were on physiological effects including feeding deterrence, and inhibition of growth and oviposition.  Among insect and arthropod species involved in this research, allowing for overlaps, the order Diptera had the highest frequency (705 publications), followed by Coleoptera and Acari (Table 1.2).  This trend may be associated with the characteristics of essential oils.  Since many essential oils are highly volatile, using them as repellents to mosquitoes or ticks might be a valuable strategy.  Also, this volatile property allows essential oils to be adopted as fumigants, targeting many Coleopteran stored product pests.  For their use as a tool for integrated pest management or organic farming, effects of essential oils on agricultural pests in the orders Lepidoptera and Hemiptera as well as Acari have also been featured.   12   Figure 1.2. Literature survey of essential oils associated with insect and arthropod species from WoS (2004-2014).  Search was conducted with the keywords for ‘acute effect’: (essential oil or terpene*) and (repell* or insecticid* or larvicid* or acaricid*), ‘chronic effect’: ‘acute effect’ + (or deterr* or antifeed* or growth regulat* or oviposit*), and ‘synergy’: ‘acute and chronic effect’ + (and synerg*) on 24 Dec 2014.  The total number of publications was 3,604, ‘acute effect’ had 2,637, and ‘synergy’ had 120. A total of 376 publications overlapped in ‘acute’ and ‘chronic’ effects.  Table 1.2. Numbers of publications on essential oil research based on major orders of insects and other arthropods (2004-2014) order number of publicationsa search wordb major target of research Diptera 705 ‘full’ + (and diptera*) mosquitoes, housefly Coleoptera 585 ‘full’ + (and coleoptera*) stored product pests Acari 420 ‘full’ + (and acari*) two-spotted spider mites, ticks Lepidoptera 222 ‘full’ + (and lepidoptera*) armyworms, cutworms Hemiptera 167 ‘full’ + (and hemiptera* or heteroptera* or homoptera*) aphids, stinkbugs a Search was conducted in 24 Dec 2014 using WoS database. b ‘full’ indicates the search query: (essential oil or terpene*) and (repell* or insecticid* or larvicid* or acaricid* or deterr* or antifeed* or growth regulat* or oviposit*).  01002003004005006002004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014Number of WOS articles acute effect chronic effect synergy13  In terms of the biological activities demonstrated in essential oil research with insects, trends in subjects vary with the orders of insects studied.  In comparing 30 recent publications on each of the Diptera and Lepidoptera, almost half of the studies in each order focused on the acute toxicity of essential oils, but a significant portion also focused on repellent activity in the Diptera, but feeding deterrent or growth inhibition was a major subject of research in the Lepidoptera (Figure 1.3).  Figure 1.3. Research trends in essential oils on two orders of insects.  Data were analyzed for 30 publications each in 2013-2014 for Lepidoptera, and in 2014 for the Diptera.   1.5 Insecticidal mode-of-action and detoxification of essential oils and plant secondary metabolites Compared to extensive studies and understanding of the modes-of-actions of many synthetic and a few botanical insecticides such as neem, pyrethrum, or rotenone, we still know little about the modes-of-action of essential oils.  Understanding the mode-of-action of novel insecticides is 14  important not because it has scientific significance but because it can lead to the development of a new class of insecticides.  For example, whereas some classes of insecticides such as organochloride and organophosphate insecticides were de novo chemically synthesized, pyrethroid and neonicotinoid insecticides were developed by chemical engineering of plant natural product precursors.  Studies on the structure-activity relationships of monoterpene compounds revealed some interesting and common characteristics related to efficacy against insect and arthropod pests (Rice and Coats 1994; Tak et al. 2006), and histopathological observations have shown some physical alterations or damage to exterior and interior insect systems (Abdel-Shafy et al. 2009; Kumar et al. 2012).  In addition, the observation of behavioral responses in essential oil-treated insects suggests rapid neurotoxic effects, such as convulsions or uncontrolled movements, leading to flaccid paralysis or moribund status (Isman et al. 2011).  These findings provide a foundation for better understanding mode-of-action, but not many biochemical studies have been conducted on essential oils or their major constituents thus far.  This might be due to the wide variety of monoterpenes in terms of their skeleton structures (i.e., cyclic, bicyclic, acyclic, or aromatic) and functional groups (i.e., hydrocarbon, ketone, alcohol, or aldehyde), or complex compositions and bioactivities of essential oils (i.e., bioactivities of many oils can be produced by interactive effects of more than two constituents) making it difficult to focus on specific target site. Nevertheless, the biochemical and physiological studies of mode(s)-of-action of essential oils led to some possible targets including acetylcholineesterases (AChE), octopamine receptors and gamma-aminobutyric acid (GABA) receptors (Rattan 2010; Regnault-Roger et al. 2012).  More importantly, there is evidence that many essential oils as well as their active constituents have 15  multiple modes-of-actions.  For example, 1,8-cineole has been reported to have inhibitory activity on AChE (Abdelgaleil et al. 2009), but also it is associated with octopamine (Zhukovskaya 2007) and GABA receptors (Tong and Coats 2012).  Likewise, thymol binds to GABA receptors (Tong and Coats 2012) as well as inhibiting the octopaminergic system in vitro (Bonnafé et al. 2014).  Eugenol blocks octopamine receptor binding sites (Enan 2001), and can disrupt the cell membrane physically (Devi et al. 2010).  These indicate that the bioactivities of essential oils are possibly due to complex mixtures of constituents which have multiple effects on multiple targets. Throughout the long and ongoing evolutionary arms race between insects and plants, each party has developed their own survival and defense strategies.  Some herbivores have managed to evade or neutralize plant defenses, even utilize them as biological cues in host finding or intraspecific communication, or sometimes sequestering and transforming them into their own defensive agents against predators (Gershenzon and Croteau 1991).  In many cases, these adaptations of insects involve enzymatic detoxification.  For example, the tobacco hornworm, Manduca sexta, can metabolize nicotine produced by the plants of the genus Nicotiana through induction of P450s enzyme levels, giving this insect a unique opportunity to monopolize a toxic plant as its own dietary source in a less competitive environment (Snyder et al. 1993).  An extraordinary example might be the chemical ecology between pine trees and pine bark beetles.  Pine beetles respond to pine monoterpenes as attractive kairomones, then further utilize these compounds in pheromone biosynthesis for interspecies communication; P450s are involved in the process.  This complex interaction is well documented (Seybold et al. 2006). 16  At the same time, the successful inhibition of enzymatic response in insects can lead to enhanced toxicological or physiological effects.  Several reports have shown inhibitory activity of essential oils on insect and human P450s in vivo as well as in vitro, associated with increased toxicity or repellent activity (Ganzera et al. 2006; Ramirez et al. 2012; Bräunlich et al. 2013; Tong and Bloomquist 2013).  In particular, an ethyl acetate extract of Piper nigrum showed significantly (11.6-fold) enhanced toxicity when mixed with pyrethrum in Drosophila melanogaster (Jensen et al. 2006a), concomitant with a regulating effect on P450s activity (Jenson et al. 2006b).  In terms of detoxification, in addition to P450s, two other types of enzymes, esterases and glutathione S-transferases (GST), are also considered important, not only because they are involved in the metabolism of xenobiotics but also because these three enzymes systems are significantly related to resistance development to insecticides (Li and Berenbaum 2007).  Inhibition of esterases or GST by plant secondary metabolites, such as tannins (Juntheikki and Julkunen-Tiitto 2000), lignans, flavonoids (Wang et al. 2014), phenols or other compounds (Yu and Abo-Elghar 2000) suggest their potential as new insecticide synergists.  In particular, taxifolin, a flavonoid derived from Picea mariana, Pinus banksiana and Larix laricina, showed greater GST inhibitory activity than the standard GST inhibitor, diethyl maleate (DEM) on insecticide-resistant Colorado potato beetle, Leptinotarsa decemlineata in vitro (Wang et al. 2014).  To date, efforts to discriminate enzyme-induced resistance largely rely on synthetic synergists, such as piperonyl butoxide (PBO), a P450 inhibitor (Scott 1999), ethacrynic acid (EA) or DEM, GST inhibitors (Qin et al. 2013), and S,S,S-tributyl phosphorotrithioate or triphenyl phosphate (TPP), esterase inhibitors (Gao et al. 2014).   17  1.6 Synergistic interactions of essential oil constituents Identifying synergistic interactions among essential oil constituents or with other agents, and understanding the underlying mechanisms are important since they may point to new strategies for pest management with reduced use of conventional insecticides, determine optimal cultivation condition for source plants, or confirm beneficial combinations with other bioactive control agents.  Compared to the scientific interest in relating essential oil chemistry to insecticidal activity, research on blend effects has been far less explored.  As shown in Figure 1.2, despite the growing interests in essential oils as pesticides, numbers of publications on synergistic effects have remained somewhat static.  Synergy can sometimes be observed between essential oil constituents, as indicated by the superior bioactivity of an intact oil compared to that of any individual constituents.  This phenomenon has been observed in many essential oils with respect to antioxidant activity (Padmakumari et al. 2011), antimicrobial activity (Veras et al. 2012), acute toxicity to agricultural pests (Miresmailli et al. 2006; Jiang et al. 2009; Suthisut et al. 2011) and insect feeding deterrence (Suthisut et al. 2011; Akhtar et al. 2012).  Likewise, combinations of essential oils with other oils (Noosidum et al. 2014), antibiotic drugs (D’Arrigo et al. 2010; Santos et al. 2011), synthetic insecticides (Tong and Bloomquist 2013) or microbial insecticides (Chang et al. 2014) have shown synergistic effects or in some cases gave prolonged protection on blood-feeding mosquitoes compared to individual oils (Mukesh et al. 2014; Reegan et al. 2014). Mechanisms explaining synergy, especially in the entomological area have been even less studied, but research on resistance to synthetic insecticides can provide us some paths to explore.  In general, target site insensitivity, increased detoxification and decreased cuticular penetration 18  are considered to be the main resistance mechanisms to insecticides in insects (Scott 1999).  If a synergistic combination can interfere with at least one of these mechanisms, i.e., by enhancing target site sensitivity, decreasing detoxification or increasing penetration, this could lead to enhanced toxicity.  This is a reasonable assumption since many essential oils appear to have multiple modes-of-action and sites-of-action in insects (Isman et al. 2011), and some essential oils do not appear to share a common target site with conventional insecticides (Perumalsamy et al. 2010; Chang et al. 2012).  Also, some essential oils have been shown to inhibit detoxifying enzymes, resulting in synergy with synthetic insecticides (Tong and Bloomquist 2013).  Moreover, as both synthetic xenobiotics and plant-derived secondary metabolites are targets of metabolism by insects, insecticidial activity of essential oils and/or their major constituents could be significantly enhanced when detoxyfing enzymes are inhibited (Waliwitiya et al. 2009; Waliwitiya et al. 2012).  To date, increased cuticular penetration by essential oil constituents as a mechanism of synergy has not been reported, although thymol showed different patterns of penetration and toxicity when mixed with different carrier solvents including hydrocarbons and essential oils in the cabbage looper (Wilson and Isman 2006).   1.7 Research objectives The present study has three main goals; (1) to evaluate the toxicity of individual essential oil constituents and their contributions to overall insecticidal and cytotoxic activity, (2) to identify synergistic interactions among essential oil constituents, and (3) to elucidate the mechanism(s) underlying the observed synergy.  Larvae and an ovarian cell line of the cabbage looper were selected as test subjects to examine in vivo insecticidal activity and in vitro cytotoxicity, and 19  three essential oils - rosemary (Rosmarinus officinalis L.), thyme (Thymus vulgaris L.) and lemongrass (Cymbopogon citratus Stapf.) were chosen as test essential oils.  To evaluate the effects of individual constituents and their contributions, the research examined the following;  1) Chemical analyses of the three essential oils 2) Insecticidal and cytotoxic activities of individual constituents of essential oils 3) Evaluation of comparative toxicity by different application methods Regarding synergy, investigations of synergistic interactions of the major constituents, and two out of the three possible aspects mentioned above, were examined to understand how the synergy can be produced; 1) Assessment of synergistic interactions of the major constituents  2) Examinations of cuticular penetrations in synergistic combinations 3) Evaluations on detoxifying enzymes in synergistic combinations Physico-chemical studies of penetration and biochemical studies of detoxification were verified by further bioassays. 20  Chapter 2: Comparative toxicities of essential oil constituents against larvae and an ovarian cell line of Trichoplusia ni  2.1  Introduction General concerns for the negative environmental and human health effects attributed to the widespread use of conventional insecticides have stimulated an exponential increase in research on botanical insecticides.  Within that field of research, growing interest in plant essential oils is an unambiguous trend.  Over the last dozen years, the percentage of research papers focusing on the insecticidal activity of plant essential oils among all papers on botanical insecticides has increased from 8.4% in 2000 to 22.8% in 2012 (Isman and Grieneisen 2014).  Relative to the large numbers of reports on the toxicity of plant essential oils to insects, only a handful of essential oils have been commercialized as pesticides.  One of the main obstacles to commercialization may be registration.  Although many essential oils are used in consumer products including perfumes and cosmetics, and many essential oils pose little threat to the environment or to human health (Isman 2006), their use as insecticides is very strictly regulated in many countries (Regnault-Roger et al. 2012).  More importantly, essential oils are often very complex mixtures with vast numbers of constituents, and chemical composition can be affected by the conditions of the source plant.  For example, in a previous report on the cabbage looper, Trichoplusia ni Hübner and the armyworm, Pseudaletia unipuncta Haworth, ten commercial rosemary oils showed 5.7- and 2.2-fold differences in toxicity, respectively (Isman et al. 2008). 21  Variations of chemical composition by seasonal or geographic differences, growth conditions and extraction methods in rosemary (Rosmarinus officinalis L.) (Elamrani et al. 2000; Serrano et al. 2002; Salido et al. 2003), thyme (Thymus vulgaris L.) (Senatore 1996; Hudaib et al. 2002; Asllani and Toska 2003) and lemongrass (Cymbopogon citratus Stapf.) essential oils (Zheljazkov et al. 2011; Desai et al. 2014; Moncada et al. 2014) are well documented, along with their insecticidal activity (Lee et al. 2001; Yang et al. 2004; Waliwitiya et al. 2009; Machial et al. 2010; Jiang et al. 2012), repellence (Prajapati et al. 2005; Diaz-Montano and Trumble 2013; Zhang et al. 2013), and physiological impact such as larval growth or feeding deterrence (Jiang et al. 2012; Zhang et al. 2013) as well as acaricidal activity (Miresmailli et al. 2006; Sertkaya et al. 2010).  The objectives of the present study were to (1) determine the chemical compositions of three essential oils, (2) determine the insecticidal activity of the individual constituents in larvae of the cabbage looper and cytotoxicity to an ovarian cell line, and (3) investigate the overall contribution of individual compounds via in vivo and in vitro test methods.  In addition, (4) the effect of controlled evaporation of an essential oil and its effect on fumigant activity were examined.  2.2 Materials and methods 2.2.1 Chemicals Essential oils of rosemary (Intarome Fragrance & Flavors Corp. Norwood, NJ, USA), thyme and lemongrass (Ungerer & Company, Lincoln Park, NJ, USA) were obtained from EcoSafe Natural 22  Product Inc. (Saanichton, BC, Canada).  Pure standard compounds of essential oil constituents were purchased from Sigma-Aldrich (St Louis, MO, USA) and Thermo Fisher Scientific (Waltham, MA, USA).  AlamarBlue dye, Express Five SFM medium, gentamicin and L-glutamine were purchased from Life Technologies (Carlsbad, CA, USA).  2.2.2 Gas chromatography-mass spectrophotometry Major constituents of thyme and lemongrass essential oils were analyzed on an Agilent 6890A/5973N gas chromatograph mass spectrometer (Agilent Technologies Canada Inc., Ottawa, ON, Canada) operating in electron ionization mode fitted with a SGE SolGel-wax 30 m × 0.25mm ID, 0.25 m thickness fused silica column by a liquid injection method.  The injection was performed with a volume of 0.2 L in a split mode (50:1).  The oven temperature setup was programed for 40 ºC for 2 min, increased to 250 ºC at an increase of 15 ºC min-1 and held for 5 min.  The total run time was 21 min, and helium was used as a carrier with 0.9 mL min-1 of flow.  Data were analyzed using Enhanced Chemstation software and constituents were identified by matching spectra against the Wiley09/Nist08 MS library.  An artificial essential oil based on the gas chromatography-mass spectrophotometry (GC-MS) analysis was prepared, and the types and concentrations of individual constituents were confirmed by re-analyzing the artificial oil with the same condition as above.  23  2.2.3 Insect and cell line maintenance Eggs of the cabbage looper, T. ni were obtained from the Great Lakes Forestry Centre, Natural Resources Canada (Sault Ste. Marie, ON, Canada).  The colony was reared on pinto bean-based artificial diet (Bio-Serv Inc., Frenchtown, NJ, USA) in the insectary at the University of British Columbia, Vancouver, BC, at 22-25 ºC and a 16:8 h LD photoperiod.  An ovarian cell line of T. ni (High Fivetm, Life Technologies) was grown in Express Five SFM medium, supplemented with 1 mM L-glutamine and 50 g mL-1 gentamicin.  For long term maintenance of the cell line, subculture was performed twice a week with a split ratio of 1:11 to 2:10 (cell suspension : new medium) and cells were allowed to grow at 27 ºC in an incubation chamber.   2.2.4 Topical application and fumigant assay against 3rd instar larvae A topical application method using third-instar larvae of the cabbage looper was employed using a Hamilton Microliter syringe attached to a repeating dispenser.  Each larva received 1 L of acetonic solution of the test compounds or acetone alone as the control.  Three replications of ten treated larvae were transferred into a 7 cm diameter Petri dish with 0.5 g of artificial diet, and kept under the same conditions of insect maintenance.  After 24 h, mortality was recorded; larvae were determined to be dead when no movement was observed by probing the insect with a fine brush. Four to six different concentration levels were used to estimate LD50 values.  24  For the fumigation assay, ten third instar larvae were transferred into a 5.5 cm diameter Petri dish with 0.5 g of artificial diet on a filter paper (4.25 cm diameter, Thermo Fisher Scientific) and covered with a ventilated lid (70 holes created by a heated syringe), and sealed with Parafilm (insect chamber).  Six different concentrations of 50 L of an acetonic solution of the essential oils and the individual constituents of thyme oil were applied onto another filter paper.  After 30 sec, the filter paper was attached to the bottom of a new Petri dish using adhesive tape (fumigant chamber), and both chambers were combined and sealed with Parafilm (Figure 2.1).  Since the diameter of holes was smaller than that of the larvae, and there was a gap between the treated filter paper and the inner lid, larvae were prevented from having direct contact with the treated filter paper, but were exposed to the evaporated compounds.  LC50 value was determined after 24 h, and all tests were repeated three times.    Figure 2.1. Fumigation assay chamber system.   25  2.2.5 Cytotoxicity assay on the cell line T. ni cells were collected 72 h after the subculture and diluted to a density of 2 × 105 cells mL-1 with fresh medium.  Fifty L of cell suspension was loaded into a 96-well plate (Thermo Fisher Scientific), and allowed to settle for 2 h in the incubation chamber.  Target compounds were dissolved in sterilized dimethyl sulfoxide (DMSO) and filtered with a nylon syringe filter with a pore size of 0.2 m (Thermo Fisher Scientific), and then the filtered solution was mixed with fresh medium (1:149, v/v).  Fifty L of treatment solution was applied to each well of the plate, with DMSO/medium solution alone applied to control cells.  After 48 h of incubation at 27 ºC as recommended by supplier, cells were inspected under Axiovert 35 inverted florescence microscope (Zeiss, Oberkochen, Germany) and the viability of cells was monitored by a fluorescent method.  Ten L of alamarBlue dye was added into each well, and fluorescence intensity was measured after 15 min of incubation using a Polarstar galaxy spectrophotometer (544 nm excitation and 590 nm emission, BMG Labtechnologies, Ortenberg, Germany).  Nine concentrations with three replications were used to determine the IC50 values of essential oil and constituents.  The intensity of the fluorescence was directly proportional to the number of metabolically active cells, and inversely proportional to the toxicity of the tested samples (Appendix B, Figure B.1).   26  2.2.6 Comparative activity of individual constituents The contributions of the two most abundant compounds of each essential oil to insecticidal activities of the oils were evaluated via topical application and fumigation assay methods.  A proportional amount of each compound and their mixture, as well as mixtures of the remaining constituents (>1% concentration in the parent oil as identified in GC-MS analyses) at the equivalent amounts at LD95 of the essential oil was applied using the methods described above.  Mortality was determined after 24 h, and the test was repeated three times.  To evaluate the contribution of individual constituents to overall activity of an essential oil, a compound elimination assay was performed similar to that previously described (Miresmailli et al. 2006).  An artificial essential oil was prepared by mixing the constituents following their natural proportions (full mixture), and a set of incomplete artificial oils was prepared by eliminating a single compound from each oil.  For topical application and fumigation assays, the artificial oils were used at the equivalent of LD95 and LC95 doses of essential oils, and double the IC50 value was applied in the cytotoxicity assay.  2.2.7 Evaporation control of thymol and p-cymene and their fumigant activity  The evaporation rates of thyme oil and the two major active compounds in fumigant toxicity, thymol and p-cymene were measured, as well as the effect of increased evaporation to fumigant toxicity.  To measure the speed of evaporation, 100 L of thyme oil, thymol and p-cymene solution (50% in acetone, w/v) was applied onto a 4.25 cm diameter filter paper with three replications, and the filter papers were weighed up to three hours following evaporation at room 27  temperature (22ºC).  Fumigant activity associated with enhanced evaporation was evaluated; an insect chamber which had ten 3rd instar larvae of the cabbage looper with artificial diet inside (5.5 cm diameter, same as above) was attached to the lid of a 7 cm diameter fumigation chamber (Figure 2.2).  Fifty L of thyme oil solution (50% in acetone, w/v) was applied to a 4.25 diameter filter paper, then the filter paper was transferred to the bottom of the fumigation chamber.  Solutions on filter papers were allowed to evaporate for 1, 5, 10, 15, 20 and 30 min at room temperature and then the lids (with insect chamber) were combined together and sealed with Parafilm.  The chambers with three replications of each evaporation time were incubated for 24 h at room temperature then larval mortality was determined. The evaporation speed as well as fumigant activity at room temperature were compared to those at higher temperatures.  The fumigant chamber was floated on a water bath heated to 45ºC, and the evaporation speed of thyme oil and thymol was measured.  Also, the fumigation bioassay was conducted under the same conditions as above but at a higher temperature (45ºC).  The inner temperature of the fumigation chamber was 35ºC when the lid was attached, to avoid damage to the insects by heat, the total heating time was set at 3 h.  After that, the fumigation chamber was removed from the water bath and incubated at room temperature until mortality was determined (24 h).  The heating plate for the fumigation chamber (2.5  6.5 cm) was a double-layered aluminum plate with a rectangular hole (1.5  6 cm) inside to avoid direct contact of water to the heating plate. 28   Figure 2.2. A schematic of the fumigation chamber system and heated evaporation assay.  A rectangular hole (1.5  6 cm) was drilled between the two aluminum plates (2.5  6.5 cm); the upper plate was heated indirectly by radiant heat.  2.2.8 Statistics One-way analysis of variance (ANOVA) was used to evaluate the comparative activity of mortality and cytotoxicity, and the means were compared using Tukey’s test.  The differences in fumigant activity at different temperatures were determined by t-tests, and probit analysis was used to calculate the LD50 and LC50 values.  IC50 values were determined by using GraphPad Prism 5 (Version 5.02, GraphPad Software Inc., La Jolla, CA, USA), all other analyses were conducted using StatPlus 2009 (version 5.8.4, AnalystSoft, Alexandria, VA, USA).  29  2.3 Results 2.3.1 Chemical compositions of essential oils Based on the GC-MS analysis of rosemary essential oil constituents, 1,8-cineole (37.6%) was the most abundant compound followed by camphor (20.2%), -pinene (15.7%), and camphene (7.9%) (Table 2.1).  These four major constituents comprised more than 80% of the total weight of the oil, all of the remaining individual constituents comprised less than 5% of the concentration, with a total of 97.7% of the constituents identified. In thyme oil, with 98.1% of total constituents identified, thymol (50.2%) comprised almost half of the oil, and p-cymene was the second most abundant compound (33.1%).  No other constituent accounted for more than 5% of the oil.  For lemongrass oil, more than 75% of the oil was identified as citral, a combination of the two isomeric forms known as geranial (or citral A, E-isomer) and neral (or citral B, Z-isomer).  Limonene was the second major constituent but its concentration was only 7.0%.  A total of 95.2% of the compounds were identified.    30  Table 2.1. Chemical constituents of three essential oils compound retention timea (min) peak area % rosemary oil thyme oil lemongrass oil -pinene 4.2 15.7   1.9  -fenchene 4.6   0.2   camphene 4.7   7.9   0.6  -pinene 5.2   4.3   0.2  myrcene 5.9    0.6  limonene 6.3   2.1   0.6   7.0 1,8-cineole 6.4 37.6   0.4  p-cymene 7.1   2.5 33.1  4-nonanone 7.6     1.3 methyl heptenone 7.7     1.0 -pinene oxide 8.2   0.5   linalool oxide 8.7    1.6  camphor 9.5 20.2   0.3  linalool 9.6   0.7   4.3   1.2 bronyl acetate 10.0   2.1   4-terpineol 10.2    0.2  t-caryophyllene 10.2     0.7 pinocarveol 10.6   0.3   iso-borneole 10.7   0.4   citral (neral) 10.8   30.3 -terpineol 10.9   1.7   1.0  borneol 10.9   1.7   0.8  geraniol formate 11.0     0.6 citral (geranial) 11.2   46.8 geranyl acetate 11.4     2.2 geraniol 12.0     2.5 caryophellene oxide 13.2    2.0   1.5 humulene oxide 13.6    0.3  thymol 14.3  50.2  total identified  97.7 98.1 95.2 a Retention times are given to show the order in which compounds were detected.  Compounds were identified based on MS patterns.  31   Figure 2.3. GC-MS chromatograms of three essential oils used in this study 32  2.3.2 Insecticidal and cytotoxic activities of individual constituents In third instar larvae of the cabbage looper, the LD50 value of rosemary oil was 215.8 g insect-1 (Table 2.2).  Among the rosemary oil constituents, -terpineol was the most potent (61.9 g insect-1), whereas camphene was inactive at the highest concentration treated (> 1,000 g insect-1).  Although 1,8-cineole showed activity comparable to the complete oil, none of the major compounds (> 5% of the oil) had LD50 values lower than that of the intact oil. In the in vitro cytotoxicity assay, the most active compound of rosemary oil was limonene (49.8 g mL-1), and several constituents including 1,8-cineole (> 1,000) and camphor (> 100) failed to show a cytotoxic effect even at the highest concentrations tested.  Among the thyme oil constituents (Table 2.3), thymol showed much greater contact toxicity than p-cymene (7.4-fold based on LD50), but p-cymene was more efficacious via fumigation than thymol (4.4-fold).  Although -terpineol and -pinene also showed considerable insecticidal activities in both topical and fumigation assays, their concentrations in the oil were relatively low (1.0% and 1.9%, respectively).  In general, the cytotoxicity trend of individual thyme oil constituents was more similar to that of contact toxicity than to fumigant activity.  Thymol was the most cytotoxic constituent, followed by -pinene, -terpineol, and limonene; whereas in contact toxicity, the activity order (from the most to less active) was thymol, -terpineol, limonene and p-cymene.  Caryophyllene oxide did not exhibit any toxic activity irrespective of application method. Whereas thyme oil was the most active essential oil in contact toxicity on 3rd instar larvae, lemongrass oil had the greatest cytotoxicity (Table 2.4).  Similar finding to the results for 33  rosemary oil; lemongrass oil was more toxic in vivo and in vitro than its major constituent citral, in spite of citral being applied at high concentrations.  Although geraniol was more toxic than lemongrass oil, it only occurred at a concentration of 2.5% in the oil.  With the same carbon backbone but different functional groups, geraniol and geranyl acetate, showed a major difference in contact toxicity (4-fold) and cytotoxicity (11-fold). 34  Table 2.2. In vivo insecticidal activities of rosemary oil and individual constituents to 3rd instar T. ni larvae by topical application and in vitro cytotoxicity to T. ni ovarian cells compound topical application cytotoxicity LD50a  (95% CLb) Slope ± SE 2 (dfc) IC50d (95% CL) Slope ± SE R2 (df) rosemary oil 215.8 (191.2 - 246.1) 5.0 ± 0.8 2.3 (4) 54.6 (47.4 - 62.9) 4.2 ± 1.2 0.95 (23) 1,8-cineole 229.6 (171.3 - 341.6) 5.2 ± 1.0 1.4 (3) >1000   camphor 471.3 (402.4 - 538.2) 4.8 ± 0.7 2.1 (2) >100   -pinene 501.1 (433.6 - 598.9) 2.5 ± 0.3 3.0 (4)    142.2 (128.6 - 157.4) 4.0 ± 0.8 0.98 (23) camphene >1000   >100   -pinene 537.5 (475.9 - 601.5) 4.8 ± 0.7 1.8 (2) >1000   p-cymene 242.5 (204.1 - 290.6) 4.1 ± 0.6 0.5 (2)     931.7 (713.1 - 1217.0) 8.5 ± 4.8 0.86 (35) bornyl acetate 248.1 (211.7 - 284.1) 4.6 ± 0.7 2.0 (2) >300   limonene 233.8 (158.5 - 367.6) 2.4 ± 1.2 2.4 (3) 49.8 (92.4 - 104.2) 4.2 ± 0.4 0.99 (23) -terpineol 61.9 (51.2 - 74.6) 4.0 ± 0.7 1.8 (2)   498.3 (371.2 - 669.1) 2.9 ± 0.9 0.92 (23) borneol 628.0 (519.4 - 764.6) 3.6 ± 0.7 0.7 (2) >100   a g insect-1. b CL denotes 95% confidence limit. c df denotes degree of freedom. d g mL-1. 35  Table 2.3. In vivo contact and fumigant toxicities of thyme oil and individual constituents to 3rd instar T. ni larvae, and in vitro cytotoxicity to T. ni ovarian cells compound topical application fumigation assay cytotoxicity LD50a (95% CLb) Slope ± SE 2 (dfc) LC50d  (95% CL) Slope ± SE 2 (df) IC50e  (95% CL) Slope ± SE R2 (df) thyme oil 54.0 (44.8-65.5) 2.3±0.2 1.1 (5) 200.8 (183.0-218.2) 6.0 ± 0.8 2.0 (5) 82.2 (65.48-103.1) 6.5 ± 2.3 0.94 (23) thymol 32.6 (26.2-40.2) 2.7 ± 0.3 3.8 (5) 549.6 (479.7-616.0) 4.5 ± 0.5 1.9 (6) 31.6 (25.90-38.63) 2.4 ± 0.5 0.96 (23) p-cymene 242.5 (204.1-290.6) 4.1 ± 0.6 0.5 (2) 125.8 (117.6-133.8) 10.6 ± 1.4 10.3 (6) 931.7 (713.1-1217) 8.5 ± 4.8 0.86 (35) linalool 189.5 (157.7-229.1) 3.6 ± 0.5 1.4 (3) 675.3 (455.3-1163.4) 1.2 ± 0.3 16.9 (11) 764.5 (420.1-1391) 2.7 ± 0.9 0.94 (23) caryophyllene oxide >1000   >2000   >100   -pinene 501.1 (433.6-598.9) 2.5 ± 0.3 3.0 (4) 181.8 (165.9-197.5) 5.7 ± 0.6 0.7 (8) 142.2 (128.6-157.4) 4.0 ± 0.8 0.98 (23) linalool oxide 307.6 (278.7-343.0) 5.3 ± 0.8 3.3 (2) 526.6 (483.9-577.1) 6.4 ± 1.0 0.7 (3) >400   -terpineol 61.9 (51.2-74.6) 4.0 ± 0.7 1.8 (2) 1506.7 (1093.7-2709.6) 1.5 ± 0.3 8.8 (7) 498.3 (371.2-669.1) 2.9 ± 0.9 0.92 (23) a g/insect. b CL denotes 95% confidence limits. c df denotes degree of freedom. d g/mL air. e g/mL.36  Table 2.4. In vivo insecticidal activities of lemongrass oil and individual constituents to 3rd instar T. ni larvae by topical application and in vitro cytotoxicity to T. ni ovarian cells compound topical application cytotoxicity LD50a  (95% CLb) Slope ± SE 2 (dfc) IC50d  (95% CL) Slope ± SE R2 (df) lemongrass oil 123.8 (101.4 - 154.1) 1.9 ± 0.2 1.3 (2) 19.4 (17.2 - 21.9) 3.5 ± 0.6 0.98 (23) citral 135.9 (106.2 - 175.2) 2.2 ± 0.3 1.7 (3) 28.5 (24.4 - 33.2) 3.2 ± 0.7 0.96 (23) limonene 233.8 (158.5 - 367.6) 2.4 ± 1.2 2.4 (3) 49.8 (47.2 - 52.5) 4.2 ± 0.4 0.99 (23) geraniol 100.8 (72.5 - 127.1) 2.1 ± 0.3 1.4 (4) 183.5 (160.4 - 209.9) 2.9 ± 0.5 0.97 (23) geranyl acetate 403.6 (354.0 - 463.0) 4.0 ± 0.6 0.1 (2) 2041.0 (64.9 - 64183) 2.7 ± 1.6 0.91 (23) caryophyllene oxide >1000   >100   4-nonanone 159.9 (147.1 - 171.9) 7.1 ± 1.1 2.2 (4) >500   linalool 189.5 (157.7 - 229.1) 3.6 ± 0.5 1.4 (3) 764.5 (420.1 - 1391) 2.7 ± 0.9 0.94 (23) methyl heptenone 640.2 (529.7 - 802.8) 2.9 ± 0.5 0.9 (3) 264.1 (77.8 - 896.8) 1.1 ± 0.8 0.91 (21) a g/insect. b CL denotes 95% confidence limits. c df denotes degree of freedom.. d g/mL. 37  2.3.3 Comparative toxicity of the constituents The proportional amounts of the two major constituents of rosemary oil (1,8-cineole and camphor) showed limited toxicity when applied at the LD95 and LC95 concentration of the oil in both application methods of topical application (446.7 g insect-1), and fumigation assay (175.8 mL-1 air, Figure 2.4).  However, when those compounds were mixed, the binary mixture showed significantly increased mortality, comparable to that of rosemary oil (p > 0.05).  The mixture of the remaining constituents did not show any substantial toxicity.    Figure 2.4. Toxicity of the individual major constituents of rosemary oil and their binary mixture. Compounds were applied at the equivalent amounts of LD95 and LC95 of essential oil via topical application and fumigation assay.  Error bars represent the standard error of the mean of three replicates of 10 larvae each.  Bars with the same letter indicate no significant differences (Tukey HSD test, p < 0.05); upper case letters refer to the topical assay, lower case letters to the fumigant assay.   38  Among the major constituents of thyme oil, thymol exhibited full toxicity when it was applied alone topically (at LD95 of thyme oil, 282.9 g insect-1, Figure 2.5).  In contrast, fumigant toxicity of thyme oil (LC95 = 375.7 g mL-1 air) can be reproduced by the additive toxicity of thymol and p-cymene, and both compounds showed the same degree of activity (p > 0.05).    Figure 2.5. Toxicity of the individual major constituents of thyme oil and their binary mixture. Compounds were applied at the equivalent amounts of LD95 and LC95 of essential oil via topical application and fumigation assay. Error bars represent the standard error of the mean of three replicates of 10 larvae each.  Bars with the same letter indicate no significant differences (Tukey HSD test, p < 0.05); upper case letters refer to the topical assay, lower case letters to the fumigant assay.    39  The fumigation assay of lemongrass oil gave an LC50 of 901.3 g mL-1 air.  Since the slope value was relatively low (1.3 ± 0.3), the test was conducted with double the amount of LC50, instead of the LC95 dose.  The insecticidal activity of lemongrass oil appears to be mostly attributable to the activity of citral (Figuge 2.6).  Although a slight increase of fumigant toxicity was observed with the binary mixture of citral+limonene, citral showed statistically the same insecticidal activity as the full mixture of the constituents (p > 0.05).  The citral+limonene combination produced the same mortality as the full mixture in both bioassays.    Figure 2.6. Toxicity of the individual major constituents of lemongrass oil and their binary mixture.  Compounds were applied at the equivalent amounts of LD95 of essential oil via topical application, and double the LC50 via fumigation assay. Error bars represent the standard error of the mean of three replicates of 10 larvae each.  Bars with the same letter indicate no significant differences (Tukey HSD test, p < 0.05); upper case letters refer to the topical assay, lower case letters to the fumigant assay.  40  In the compound elimination assay via topical application at LD95 of rosemary oil, there was a significant decrease in mortality when 1,8-cineole was eliminated from the full artificial oil (p < 0.01, Figure 2.7).  Although the mortality was decreased slightly when camphor was excluded, it was not statistically different (p < 0.05).  Based on the mortality observed via fumigation assay, 1,8-cineole and camphor were significantly more active than all the other constituents (p < 0.01).  In the cytotoxicity assay, the most significant decrease of activity occurred when -pinene was eliminated, along with bornyl acetate and borneol (p < 0.01).  When 1,8-cineole was missing from the lineup, a decrease of cytotoxicity from that of the full mixture was found (p < 0.05), but not significantly different with some of other inactive constituents including camphor (p > 0.05).  Figure 2.7. Compound elimination assays of the major constituents of rosemary essential oil via different application methods.  Topical application and fumigation assays were applied at the equivalent concentration of LD95 and LC95 of essential oil, whereas double the IC50 of rosemary oil was used in cytotoxicity assay.  Asterisks denote significant differences between the chemical compositions at p < 0.05 (*) and p < 0.01 (**) in one way ANOVA followed by the Tukey HSD test. 41  In the compound elimination assay with thyme oil, thymol was identified as the only active compound in the topical and cytotoxicity assays (Figure 2.8).  Thymol also decreased fumigant activity when absent from the full mixture of constituents (p < 0.05), but p-cymene produced the greatest decrease in fumigant toxicity when it was missing (p < 0.01).  Other major constituents did not generate any significant decrease in toxicity in the compound elimination assay.    Figure 2.8. Compound elimination assays of the major constituents of thyme essential oil via different application methods.  Topical application and fumigation assays were applied at the equivalent dose of LD95 and LC95 of essential oil, whereas double the IC50 of thyme oil was used in the cytotoxicity assay.  Asterisks denote significant differences between the chemical compositions at p < 0.05 (*) and p < 0.01 (**) in one way ANOVA followed by the Tukey HSD test.  42  Compared to the other two essential oils, the main constituents of lemongrass oil, citral was determined to be the sole component responsible for insecticidal activity as well as cytotoxicity in the compound elimination assay (Figure 2.9).    Figure 2.9. Compound elimination assays of the major constituents of lemongrass essential oil via different application methods.  Doses of LD95, double the LC50, and double the IC50 of lemongrass oil were selected for topical application, fumigation and cytotoxicity assays, respectively.  Asterisks denote significant differences between the chemical compositions at p < 0.05 (*) and p < 0.01 (**) in one way ANOVA followed by the Tukey HSD test.   43  2.3.4 Evaporation control and fumigant activity Over 3 h at room temperature, 18% of thymol evaporated based on the initially applied amount, and it took more than 12 h for half of the thymol to evaporate.  On the other hand, p-cymene evaporated rapidly, taking less than 20 min for complete evaporation (Figure 2.10).  On the heated evaporation plate, both thyme oil and thymol showed increased speed of evaporation, with thymol’s evaporation speed tripled.    Figure 2.10. Evaporation of thyme oil and major constituents at two different temperatures. Standard errors for each observation are smaller than the size of the symbols.  Consistent with the swift evaporation of p-cymene, the fumigant activity of thyme oil disappeared rapidly.  When the treated filter paper was transferred into the fumigation chamber 44  following different times of evaporation, insect mortality was stable only for the first 10 min (Figure 2.11).  Increased evaporations of the oil gave only five more min of efficacy (p < 0.05), but after that it produced decreased mortality as well, showing no significant difference to that at room temperature.   Figure 2.11. Fumigant activity of thyme oil following different evaporation times.  Trhichoplusia ni larvae were introduced after 1, 5, 10, 15, 20 and 30 mins of evaporation of treated filter paper at two different temperatures.  The asterisk denotes significant difference between the temperature conditions in the t-test (p < 0.05).  2.4 Discussion Plant essential oils often show greater insecticidal activity than any of their individual constituents (Hummelbrunner and Isman 2001; Jiang et al. 2009; Akhtar et al. 2012).  Rosemary and lemongrass oils are consistent with that phenomenon in that the oil showed stronger 45  insecticidal and cytotoxic effects compared to its major constituents tested individually.  In particular, the two major constituents (1,8-cineole and camphor) in rosemary oil showed a significant synergistic effect, both in contact and fumigant activity.  Also, the two main constituents of thyme and lemongrass oil, respectively, though additive, exhibited increased fumigant activity when they were combined, and comparable to the activity of their parent essential oils (Figure 2.5 and 2.6). The compound elimination bioassays pointed to 1,8-cineole as the major insecticidal constituent of rosemary oil based on topical administration, and 1,8-cineole and camphor as major insecticidal constituents via fumigation to the cabbage looper.  Although -terpineol had the lowest LD50 value when applied topically, its concentration in the oil is relatively small (1.7%), and elimination of it from the artificial mixture did not lead to a decrease in mortality, suggesting that its contribution to overall toxicity is minimal.  In a previous study of Miresmailli et al. (2006) on the toxicity of rosemary oil to the two-spotted spider mite, Tetranychus urticae, 1,8-cineole showed the greatest contribution to overall activity, followed by -pinene.  In another study of basil essential oil (Ocimum kilimandscharicum), the major constituent, camphor, exhibited not only high insecticidal activity but was also significant repellent and inhibited growth and development of four species of stored product beetles (Obeng-Ofori et al. 1998).  A similar trend of activity was found in the compound elimination assay on thyme oil.  In topical applications, thymol was determined to be the only active compound in thyme oil, whereas both thymol and p-cymene were active in the fumigation assay.  Interestingly, p-cymene was significantly more effective as a fumigant than thyme oil (Figure 2.5), consistent with its individual LC50 value in the fumigation assay (Table 2.3).  In a fumigation assay using the rice 46  weevil, Sitophilus oryzae, Lee et al. (2001) found a very similar result.  They reported that the LC50 value of thyme oil (63.9 L L-1 air) was almost double that of rosemary oil (30.5 L L-1 air), and p-cymene (25.0 L L-1 air) was more toxic than thymol (69.7 L L-1 air).  In the present study, fumigant LC50 values of thyme oil and rosemary oil were 200.8 and 107.4 g mL-1 air, respectively, with LC50 values of 125.8 and 549.6 g mL-1 air for p-cymene and thymol, respectively, an identical trend in fumigant toxicity. Nevertheless, compared to the direct exposure to a toxicant in an insect by topical application, a fumigation assay can show wide differences in response of insects based on the experimental method.  Many factors including evaporation time, size and structure of the test chambers, temperature, types of carrier solvent, evaporation medium and exposure area can affect the results of a fumigation assay.  There are several reports indicating greater fumigant activity of thymol compared to p-cymene (Regnault-Roger and Hamraoui 1995; Pavela 2010), or comparable activity between the two compounds (Park et al. 2008).  Choi et al. (2006) also reported better fumigant toxicity of thyme oil than that of rosemary oil toward Lycoriella mali adults.  Undoubtedly, differences in specific response of insect species can cause these inconsistencies in results.  The rapid evaporation speed of p-cymene could be another important cause for these differences.  When 50 mg of p-cymene was applied on a filter paper and left at room temperature, it took less than 6 min for half of the compound to evaporate (Figure 2.10).  Since I showed that the fumigant activity of thyme oil disappears in parallel to the rapid evaporation of p-cymene, and the exclusion of p-cymene showed the greatest decrease in activity from the compound elimination assay, I conclude that the fumigant activity of thyme oil can be mainly attributed to p-cymene.  With that in mind, it is important for investigators to pay 47  attention to experimental design and interpretation of their results from a fumigation assay if there is a discrepancy between those results with reported ones, especially if the target compound is highly volatile. Although thymol demonstrated noticeable insecticidal activity via topical application, its fumigant activity was not as good compared to other constituents.  One reason for the relatively weak fumigant toxicity may be the low vapor pressure of this compound, and its slow evaporation.  In the present study, the evaporation of thymol was accelerated at elevated temperatures.  The evaporation of thymol was tripled, as was the evaporation of thyme oil, but the mortality was not affected as much as expected.  One strategy to enhance efficacy of thyme oil or thyme oil-based formulations could be to slow down the evaporation of active compounds to prolong the insecticidal activity.  This can be achieved using nanotechnology, by impregnating or encapsulating the essential oil in porous materials or polymers, such as silica (Rani et al. 2014) or polyethylene glycol (González et al. 2014). Among the major constituents of lemongrass essential oils, citral was determined to be the main active compound irrespective of the method used.  Because of the high proportion of citral (77%), it might be easy to assume that the activity of the oil mainly comes from it.  However, I observed greater insecticidal activity as well as cytotoxicity of the oil compared to that with citral.  The second major constituent, limonene was less efficacious than citral, so the better efficacy of lemongrass oil than its major constituents in isolation suggests positive (synergistic) interactions between the constituents. Moreover, though not statistically different, citral showed 3-fold greater fumigant activity than limonene (Figure 2.6), and the elimination assay suggests a possible contribution of limonene to 48  overall activity (Figure 2.9).  Considering the concentration difference between citral and limonene (10-fold), limonene might have potent fumigant activity or at least might synergize the fumigant activity of citral. In terms of the relationships between in vitro cytotoxicity and in vivo insecticidal activity, for rosemary oil, neither 1,8-cineole, camphor nor their binary mixture had a potent cytotoxic effect on the T. ni cell line (IC50 > 1,000 g mL-1, > 100 g mL-1, > 1,000 g mL-1, respectively).  One possible reason for this could be the lower solubility of the compounds in the cytotoxicity assay system, because I used a relatively low concentration of DMSO as a carrier solvent, and without surfactant.  While it is possible that some of the test compounds were not fully solubilized at higher concentrations tested, in lesser concentrations the compounds would have been completely solubilized, yet those compounds still failed to exhibit any cytotoxicity.  Meanwhile, in the case of thyme oil and lemongrass oil, each of the major constituents of the two oils tested, thymol and citral exhibited good cytotoxicity along with insecticidal activity, as indicated in the compound elimination assay (Figure 2.8 and 2.9).  Insect cell systems have been investigated as a convenient tool to screen for insecticidal bioactivity, as a replacement for bioassays with live insects.  However, the rapid insecticidal action of plant essential oils to some insect species suggests one or more neurotoxic modes of action (Isman 2006).  It has been reported that certain biochemical targets such as octopamine (Enan 2005) or GABA (Tong and Coats 2010) receptors are associated with the insecticidal mode of action of monoterpene compounds.  Decombel et al. (2004) reported weak correspondence between in vivo insecticidal activity and in vitro cytotoxicity of synthetic neurotoxic insecticides.  Moreover, no direct association (high mortality to mites / high 49  cytotoxicity to an insect cell line) was found in a screening study of 67 plant extracts (Rasikari et al. 2005).  Cell culture systems may be useful in identifying prospective insecticides with modes-of-action based on disruption of primary metabolism, protein synthesis and other activities common to most animal cells, but not for substances that depend on receptors found only in the nervous system (i.e., those with a neurotoxic mode of action).  In this regard, studies using neuronal (or cell lines) or receptor-transfected cells might give us a clearer picture (Gross et al. 2014).  In this study, although some compounds in thyme oil showed weak but possibly correlated activities between in vivo and in vitro investigations, in general, no direct association was observed.  What can be gleaned from the present study is that if a certain compound shows notable cytotoxic effect on a cell line, it might be biologically active in vivo as well.  Some compounds that showed good cytotoxicity including thymol, citral, limonene and geraniol (< 50 g mL-1 of IC50 value) showed meaningful insecticidal activities in the topical bioassay.  Ultimately, what we see as toxicity of a certain compound is the result of a complicated set of physiological actions in the insect.  Since cytotoxicity only denotes certain modes-of-action, and doesn’t reflect the pharmacokinetics that determine how much of a xenobiotic reaches the target site inside the insect, it might need to be combined with other screening tools for a comprehensive understanding of insecticidal activity. In summary, the most abundant constituents of oils were associated with their insecticidal and cytotoxic activity but the types or the degree of association of the main bioactive constituent of each oil varied based on the test method.  Cytotoxicity might be a useful tool in screening of insecticidal compounds, but it can only give limited information.  50  Chapter 3: Synergistic interactions of the major constituents of three plant essential oils and their insect cytotoxicity and cuticular penetration as potential mechanisms of synergy  3.1 Introduction Not unlike the pharmaceutical activity of drugs in animals and humans, insecticidal activity is the result of a series of complex actions and counteractions between a toxicant and an insect’s tissues.  These complex dynamics of toxicity can be simplified into three categories-penetration, activation, and detoxification (Sun 1968).  For insecticidal activity to ensue, the toxicant must penetrate the insect’s integument and the membranes of target organs, but some portion of it may be metabolized and neutralized by insect defense mechanisms, originally evolved in response to natural toxicants in host plats, before reaching the target site(s).  Ultimately, the amount of toxicant reaching to the target site will determine the toxicity of the insecticide. Synergistic insecticidal activities have been observed in many cases, not only between constituents of essential oils, but also between essential oils (Ngamo et al. 2007), monoterpenoid compounds (Pavela 2010), synthetic insecticides (Pennetier et al. 2005) and between essential oils and synthetic compounds (Shaalan et al. 2005).  To show enhanced toxicity of a combination of toxicants (i.e., synergy), at least one of these categories needs to be affected, e.g., penetration is increased, the accumulated compound shows a higher level of activation, or less of the active compound is detoxified.  The study of insecticide synergy has mostly focused on biochemical aspects of activation or detoxification (Enan 2001; Young et al. 2005; Waliwitiya et al. 2012; 51  Tong and Bloomquist 2013), whereas enhanced penetration as a possible mechanism of synergy has been relatively less explored.  The objectives of the present study were to (1) evaluate the synergistic interactions of the major constituents of three essential oils to larvae of the cabbage looper via topical application, (2) investigate whether there is increased penetration of compounds in synergistic combinations, and (3) examine in vitro cytotoxicity in an ovarian cell line of T. ni of the selected combinations.  In addition, the possible mechanisms for increased penetration were examined.   3.2 Materials and methods 3.2.1 Chemicals Pure essential oil constituents and the reagents for cytotoxicity test used in the experiments were the same as noted in Chapter 2.  Standards of geranic acid and limonene-1,2-diol, the major metabolites of citral and limonene, respectively, were obtained from Sigma-Aldrich.  3.2.2 Insect and cell line maintenance Third and fifth instar larvae of the cabbage looper were used for experiments in the present study. The source and maintenance conditions of the larvae and cell line are described in Chapter 2.  52  3.2.3 Synergistic interactions of the four major constituents of essential oils via topical application assay To evaluate potential synergies among the four major constituents of each essential oil, mixtures were prepared following either the natural constituent ratio based on chemical analyses of the oils, or a 1:1 ratio of the major constituents.  Mixtures were applied topically to 3rd instar larvae, and their LD50 values were estimated after 24 h.  To determine the relationships of the mixtures, two statistical models were used to compare expected and observed LD50 values, Hewlett and Plackett’s model (Don-Pedro 1996) and Wadley’s model (Gisi et al. 1985). Based on Hewlett and Plackett’s calculation, the expected LD50 values (assuming additive interaction) are determined from the equation   (       ( ))  (       ( ))  (        ( ))     (        ( ))   where a is the proportion of compound A in the mixture, and LD50(a) is the LD50 of compound A. According to Wadley, theoretical LD50 values are calculated from                            ( )        ( )        ( )             ( )  The interaction between the observed and theoretical LD50 values are defined as                                53  The relationship between the constituents of the mixture is defined as either synergistic (where R > 1.5), additive (1.5  R > 0.5) or antagonistic (R  0.5) based on this model.  3.2.4 Cytotoxicity of the synergistic combinations from contact toxicity The possibility of cytotoxicity as a candidate for synergy mechanism was evaluated.  IC50 values of the binary mixtures of the two major constituents from each oil (1,8-cineole+camphor, thymol+p-cymene, and citral+limonene) at the synergistic ratios from the topical application (63:37, 1:1 and 1:1, respectively) were determined in an ovarian cell line from T. ni as described in Chapter 2.  A combination showing synergistic cytotoxicity (citral+limonene) was validated by a two-way titration (Tan et al. 2012).  A 9  9 concentration matrix of each compound (from 0 to 50 g mL-1) was prepared, and the cytotoxicity of each dose was examined.  The cellular morphology of T. ni cells after 2 d of incubation was also observed when treated individually with citral and limonene, as well as their binary mixture.  The integrity of treated and untreated cells was captured by an Olympus DP72 digital camera (Olympus, Center Valley, PA, USA) integrated with an Axiovert 35 inverted florescence microscope, and cell areas were measured using DP2-BSW software (Olympus, ver 2.2).  54  3.2.5 Sample preparation for cuticle penetration analysis Differences in penetration through the cuticle between individual compounds (1,8-cineole and camphor) and their binary mixture via topical application was monitored by GC-MS. Four groups of forty 3rd instar larvae of the cabbage looper in each treatment (total 12 groups) were topically applied with 1,8-cineole, camphor or the binary mixture of them at the equivalent amounts of 1,8-cineole (98.7 g) and camphor (56.9 g) at the LD50 for the mixture (155.6 g insect-1).  Treated larvae were transferred individually into 5 mL glass vials, and the caps were loosely fitted.  After 10, 30, 60, and 180 min, thirty live larvae from each group were selected and each larva was rinsed with 200 L of n-hexane (Optimatm, Thermo Fisher Scientific, Waltham, MA, USA) twice with gentle shaking for 30 sec.  Larvae were then pooled and ground using a tissue homogenizer for 1 min with 1 mL n-hexane.  Five mL of n-hexane was added and sonicated for 30 sec, the supernatant was transferred into a glass vial, and sonication was repeated again with another 6 mL of n-hexane.  The extracted and rinsed hexane solutions were kept sealed in a freezer overnight for analyis. In a similar manner, cuticle penetration of two other synergistic combinations, thymol+p-cymene and citral+limonene at 1:1 ratio (w/w), were analyzed by GC-MS.  Binary mixtures at the doses of LD50 in both combinations and the proportional amounts of each individual compound were topically applied (30 larvae in a group), and then each 20 live larvae from each group were rinsed then extracted with n-hexane as the same method above after 10 and 60 min.  For the thymol+p-cymene combination, three groups of larvae (thymol, p-cymene and thymol+p-cymene) were also incubated for 8 h after receiving an LD10 dose of the mixture.  Only the extracted 55  solutions were prepared for the GC-MS analyses of thyme oil and lemongrass oil constituents’ combinations.  3.2.6 GC-MS analysis of cuticle penetration  Rinsed and extracted hexane solutions of 1,8-cineole+camphor, from 3.2.5, were analyzed by GC-MS in pulsed splitless mode using an Agilent 6890A series GC system coupled to an Agilent 5973 Network MSD (70 eV).  The injection volume was 0.2 L, and the oven temperature setup was 40 ºC for 3 min, increasing at 25 ºC min-1 to 300 ºC then held for 5 min.  Total run time was 18.4 min, and an HP-5ms column (30m × 0.25 mm × 0.25 µm, Agilent) was selected with helium as carrier at 0.9 mL min-1 of flow.  Five µg mL-1 of -pinene was used as an internal standard, and to quantify the amount of 1,8-cineole and camphor, five concentrations of each compound were used to prepare standard curves (Appendix B, Figure B.2).  The analysis was repeated three times. For the analysis of extracted solutions from thymol+p-cymene and citral+limonene combinations, the same GC system, but a different column and temperature program was used.  A J&W DB-wax column (30m × 0.25 mm × 0.25 µm, Agilent) was used with a 1 L injection volume, and the oven temperature was set at 50 ºC for 3 min, to 230 ºC for 5 min with an increase of 30 ºC min-1, and run time was 14 min.  The scan mass range was 40 to 350, as mass spectra were matched against Wiley09/Nist08 MS library.  The peak areas of each compound were compared to that of the internal standard, -pinene for quantification. 56   3.2.7 Injection assay using 5th instar larvae To evaluate the effect of the cuticle as a barrier to penetration, an injection assay was conducted using 5th instar larvae of the cabbage looper.  Ten larvae were placed into an ice-cold beaker for 5 min to slow down their movement, and one L of an acetonic solution of test compounds (acetone alone for controls) was injected into the ventral body cavity close to the nerve cord using a microneedle (O.D. × I.D. = 0.47 × 0.21 mm) under a microscope.  Insects were transferred into 7 cm diameter Petri dishes and provided with 1g of artificial diet.  After 24 h, mortality was recorded and confirmed after 48 h.  Six to seven doses were used to determine LD50 values.  The LD50 values from injection were compared to those from topical administration against 3rd instar larvae from a previous study (Chapter 2) by using the equation;    (     (              ))  (                           )(     (                ))  (                           ) To calculate the average weight of 3rd instar larvae, ten larvae were grouped and weighed together with 5 replications (9.4 ± 1.6 mg insect-1), and twenty 5th instar larvae were weighed individually (238.4 ± 27.6 mg insect-1).   3.2.8 Mixed application of 1,8-cineole and camphor To confirm the penetration-enhancing effect of the mixture and its relationship to increased toxicity, and to examine whether pre-injecting one compound can affect the other compound’s 57  penetration rate, a mixed application assay was performed using 5th instar larvae.  In this test, the compounds were applied as follows: (1) a mixture of 1,8-cineole and camphor was topically applied (64:37), (2) a mixture of 1,8-cineole and camphor was injected (mixture ratio was calculated based on the average penetration rate of each compound in the mixture’s application, 41:59), (3) one compound was injected first and the other compound was topically applied (applied amount was determined based on the individual compound’s penetration rate), (4) an individual compound was either topically applied or injected.  Mortalities resulting from each test [(1), (2), and (3)] were compared to the sum of mortalities from individual application in the topical or injection assay (4).  All tests were repeated three times, and each test had ten larvae.  3.2.9 Insecticidal activity of metabolites of citral and limonene GC-MS analysis of cuticular penetration of citral and limonene revealed one major metabolite of each in the cabbage looper.  Insecticidal activity of the major metabolites, geranic acid and limonene-1,2-diol, from citral and limonene, respectively, was evaluated via topical application to 3rd instar larvae of the cabbage looper, and LD50 values at 24 h were determined.  3.2.10 Surface tension and contact angle measurement The physical properties of the three essential oils and their major constituents were investigated by measuring surface tensions and their contact angles on a wax layer.  Surface tensions of the essential oils as well as their individual major constituents or mixtures thereof were measured 58  using a tensionmeter (K100, KRŰSS GmbH, Hamburg, Germany) by the Wilhelmy plate method.  To measure the contact angle on a wax layer, beeswax specimens were prepared by dipping microscope slides into melted beeswax, and the specimens were air-dried overnight.  Test solutions were prepared using acetone (50%, v/v), and 3 L of solution was applied on the specimen and the contact angles were measured (DSA100, KRŰSS GmbH).   3.2.11 Statistics Probit analysis was used to determine LD50 values, and mean mortalities were compared by one-way ANOVA with Tukey’s post hoc analysis using StatPlus 2009 software.  IC50 values were determined by GraphPad Prism 5.   3.3 Results 3.3.1 Interactions among the four major constituents Insecticidal synergistic interactions between the four major constituents from each essential oil were investigated.  Among the major constituents of rosemary oil, the combinations following natural proportions of the essential oil composition showed several synergistic relationships; the most significant synergy based on Wadley’s determination was the mixture of camphor+-pinene+camphene (R = 3.82, Table 3.1).  A binary mixture of the two most abundant constituents, 1,8-cineole+camphor, produced a significantly lower LD50 than for the individual compounds.  A notable trend of synergy was found with camphor.  Apart from the full mixture of the four 59  compounds, all other combinations that included camphor as an element showed synergy (R > 1.5).  When the constituents were mixed at 1:1 ratio, camphor+camphene showed the greatest increase in toxicity (R = 2.28), followed by camphor+-pinene and camphor+-pinene+camphene.  No combination tested showed an antagonistic interaction.   60  Table 3.1. Synergistic interactions of four major constituents of rosemary essential oil in 3rd instar larvae of T. ni by topical application ratio (%) obs LD50b exp LD50c 1,8-cineole (215.8)a camphor (471.3) -pinene (501.1) camphene (>1000) H&Pd Wadleyd Rf Sg 63.4 36.6   155.6 308.0 267.7 1.72 S 72.0  28.0  201.0 294.4 255.0 1.27 A 83.9   16.1 222.7 340.6 245.1 1.10 A  59.8 40.2  242.2 483.2 482.7 1.99 S  75.0  25.0 311.8 603.4 542.9 1.74 S   66.9 33.1 337.6 665.7 600.2 1.78 S 50.8 29.4 19.8  187.9 346.5 294.8 1.57 S 56.5 32.7  10.8 191.6 384. 5 290.5 1.52 S 63.2  24.6 12.2 273.0 380.6 280.5 1.03 A  49.9 33.5 16.6 138.3 569.0 528.1 3.82 S 46.3 26.8 18.0 8.9 216.4 405.6 314.4 1.45 A 50.0 50.0   214.9 342.6 294.4 1.37 A 50.0  50.0  231.2 357.6 300.0 1.30 A 50.0   50.0 317.2 607.0 352.7 1.11 A  50.0 50.0  233.3 486.1 485.7 2.08 S  50.0  50.0 280.3 735.6 640.5 2.28 S   50.0 50.0 354.3 750.5 667.6 1.88 S 33.3 33.3 33.3  278.2 391.5 344.8 1.24 A 33.3 33.3  33.3 244.1 556.1 388.8 1.59 S 33.3  33.3 33.3 307.9 566.0 395.3 1.28 A  33.3 33.3 33.3 285.4 650.8 592.1 2.07 S 25.0 25.0 25.0 25.0 270.5 546.6 408.6 1.51 S a LD50 value of individual compound, g insect-1. b observed LD50, g insect-1. c expected LD50 based on each calculation model, g insect-1. d Hewlett and Plackett’s calculation of expected LD50, g insect-1. e Wadley’s calculation of expected LD50, g insect-1. f synergy ratio by Wadley’s calculation. g determination of interaction of the mixture based on Wadley’s determination method, when R > 1.5 : synergistic (S), 1.5  R > 0.5 : additive (A), R  0.5 : antagonistic interaction.   61  Of the combinations of thyme oil constituents at their natural proportions, insecticidal activities of the combinations seemed to be governed by thymol (Table 3.2), as all the combinations which had thymol showed relatively similar LD50 values to that of thymol.  Among the binary mixtures with thymol at the same ratio (50:50), only p-cymene synergized thymol.  Interestingly, both thymol and caryophyllene oxide showed increased toxicity when mixed individually with p-cymene [R = 1.78 (with thymol) and 3.13 (with caryophyllene oxide) or linalool (R = 1.39 and 1.88)] at the same proportions, but tertiary mixture of thymol+p-cymene (or linalool)+caryophyllene oxide, or the full mixture of those four compounds, did not exhibit any enhanced toxicity (R < 1). Similarly in lemongrass oil, citral in natural proportions failed to exhibit any synergistic interaction with other constituents, and citral+limonene at 1:1 was the only binary mixture which showed synergy based on Wadley’s determination (Table 3.3).    62  Table 3.2. Synergistic interactions of four major constituents of thyme essential oil in 3rd instar larvae of T. ni by topical application ratio (%) obs LD50b exp LD50c thymol (32.6)a p-cymene (242.5) linalool (189.5) caryophyllene oxide  (>1000) H&Pd Wadleye Rf Sg 63.0 37.0     33.5 110.2   47.9 1.43 A 92.8    7.2    27.7  43.9   34.6 1.25 A 96.1     3.9   34.6  70.3   33.8 0.98 A  88.3 11.7  247.4 236.3 234.8 0.95 A  93.6    6.4 292.4 290.9 254.8 0.87 A   65.8 34.2 274.1 466.7 262.1 0.96 A 60.0 35.3   4.7    32.8 114.0   49.7 1.52 S 61.4 36.1    2.5   32.9 132.5   49.1 1.49 A 89.4   7.0   3.6   25.1  82.4   35.9 1.43 A  83.2 11.0   5.7 308.4 279.6 245.8 0.80 A 58.6 34.5  4.6   2.4   32.4 135.4   50.8 1.57 S 50.0 50.0     32.3 137.5   57.4 1.78 S 50.0  50.0    40.1 111.0   55.6 1.39 A 50.0   50.0   76.5 516.3   63.1 0.82 A  50.0 50.0  149.6 216.0 212.7 1.42 A  50.0  50.0 124.6 621.2 390.3 3.13 S   50.0 50.0 169.5 594.7 318.6 1.88 S 33.3 33.3 33.3    50.4 153.3   75.5 1.50 S 33.3 33.3  33.3  85.3 420.8   84.5 0.99 A 33.3  33.3 33.3 106.7 403.3   81.9 0.77 A  33.3 33.3 33.3 164.0 472.5 291.3 1.78 S 25.0 25.0 25.0 25.0 108.5 366.1   97.3 0.90 A a LD50 value of individual compound, g insect-1. b observed LD50, g insect-1. c expected LD50 based on each calculation model, g insect-1. d Hewlett and Plackett’s calculation of expected LD50, g insect-1. e Wadley’s calculation of expected LD50, g insect-1. f synergy ratio by Wadley’s calculation. g determination of interaction of the mixture based on Wadley’s determination method, when R > 1.5 : synergistic (S), 1.5  R > 0.5 : additive (A), R  0.5 : antagonistic interaction.  63  Table 3.3. Synergistic interactions of four major constituents of lemongrass essential oil in 3rd instar larvae of T. ni by topical application ratio (%) obs LD50b exp LD50c citral (135.9)a limonene (233.8) geraniol (100.8) geranyl  acetate  (403.6) H&Pd Wadleye Rf Sg 92.1   7.9   127.8 143.7 140.6 1.10 A 96.8    3.2  131.0 134.8 134.5 1.03 A 97.2     2.8 104.7 143.5 138.5 1.32 A  72.4 27.6  196.2 197.1 171.5 0.87 A  74.6  25.4 218.8 277.0 261.8 1.20 A   52.8 47.2   92.8 243.8 156.1 1.68 S 89.4   7.7   2.9  103.5 142.4 139.0 1.34 A 89.7   7.7    2.6 101.8 150.5 143.0 1.41 A 94.2    3.1   2.7  96.1 142.2 137.0 1.43 A  58.1 22.1 19.8 164.0 238.0 193.5 1.18 A 87.2   7.5   2.8   2.5   95.0 149.0 141.3 1.49 A 50.0 50.0   115.0 184.9 171.9 1.50 S 50.0  50.0    87.4 118.4 115.8 1.32 A 50.0   50.0 105.9 269.8 203.4 1.92 S  50.0 50.0  149.0 167.3 140.9 0.95 A  50.0  50.0 239.7 318.7 296.1 1.24 A   50.0 50.0 161.5 252.2 161.4 1.00 A 33.3 33.3 33.3  129.0 155.3 140.6 1.09 A 33.3 33.3  33.3 171.6 255.2 214.8 1.25 A 33.3  33.3 33.3 102.0 211.3 153.4 1.50 S  33.3 33.3 33.3 175.0 243.6 181.8 1.04 A 25.0 25.0 25.0 25.0 146.4 218.6 166.5 1.14 A a LD50 value of individual compound, g insect-1. b observed LD50, g insect-1. c expected LD50 based on each calculation model, g insect-1. d Hewlett and Plackett’s calculation of expected LD50, g insect-1. e Wadley’s calculation of expected LD50, g insect-1. f synergy ratio by Wadley’s calculation. g determination of interaction of the mixture based on Wadley’s determination method, when R > 1.5 : synergistic (S), 1.5  R > 0.5 : additive (A), R  0.5 : antagonistic interaction.  64  3.3.2 Cytotoxicity of synergistic combinations Of the synergistic combinations determined above, three binary combinations from each essential oil were tested for cytotoxicity in the ovarian cell line of T. ni (Table 3.4).  If Wadley’s determination method is applied to thymol+p-cymene combination, the expected IC50 is 61.1 g mL-1, with a synergy ratio of 1.93, synergistic interaction.  On the other hand, if p-cymene is considered as a solvent with no activity, the expected IC50 value has to be double that of thymol, but the observed IC50 value of the mixture was 3-fold greater, defining it as antagonistic.  Since the binary mixture of citral and limonene showed lower IC50 values than either compound showed, only the citral+limonene combination was determined to be synergistic.  Table 3.4. Cytotoxicity of selected synergistic combinations from topical application assay compound IC50a 95% CLb compound IC50 95% CL synergy 1,8-cineole >1000  1,8-cineole+camphor (63:37)c >1000  - camphor >100  thymol   31.6  25.9 - 38.6 thymol+p-cymene (50:50) 96.9  82.9 - 113.3 - p-cymene 931.7 713.1 - 1217 citral  28.5  24.4 - 33.2 citral+limonene (50:50) 25.1 21.8 - 28.9 synergy limonene  49.8  47.2 - 52.5 a g mL-1, data from Chapter 2.  b CL denotes 95% confidence limit. c mixing ratio of each compound in combination (w/w).   65  To validate the synergy for cytotoxicity between citral and limonene, a two-way titration test was conducted with different doses (Figure 3.1).  In general, cell growth was inhibited in parallel with increasing concentration, and no dominant compound was identified, indicating comparable contributions to the cytotoxicity of the binary mixture.   Figure 3.1. Validation of cytotoxic synergy between citral and limonene in an ovarian cell line of T. ni.  Nine different doses of each compound (80 combinations + control) were applied by two-way titration experiment.  Values of each cell are the observed cytotoxicity at the specific mixture doses of each compound.   66  Significant cellular morphology changes were observed in the treatment of cells with citral, limonene and their binary mixture (Figure 3.2).  In cells exposed with citral, more particles (possibly cytoplastic materials) were observed in the medium and there was distinctive damage consisting of blurred and thinned cell membranes.  In contrast, limonene treatment led to cell enlargement, with swollen vacuoles, suggesting a different type of damage produced by the two compounds.  With the binary mixture of compounds, both swelled and blurred effects were observed.  The mean areas of cells in control, citral, limonene and citral+limonene were 412±189, 307±102, 676±225 and 551±190 m2 (±SD), respectively, confirming the enlargement of cell size by limonene.   67    Figure 3.2. Morphological observations of T. ni cells when treated with individual citral and limonene and a mixture thereof.  At the end of 2 d incubation, cells were photographed by an Olympus DP72digital camera fitted with inverted florescence microscope.    3.3.3 Cuticular penetration of the 1,8-cineole and camphor mixture 3.3.3.1 GC-MS analysis The residual (nonpenetrated) and penetrated amounts of 1,8-cineole and camphor in the cabbage looper were analyzed by GC-MS (Table 3.5).  Quantities in larval rinses (representing the 68  amount of material that failed to penetrate the cuticle) showed a decreasing pattern over time, whereas internal quantities (extracted) increased for 1 h after application, but decreased at 3 h post-treatment.  When the two compounds were mixed, there was a notable increase in penetration of camphor to more than double the amount seen from its individual application (from 15.9% to 36.2%, on average, Figure 3.3).  The average penetration rate of 1,8-cineole was also increased when mixed with camphor (from 10.3% to 14.3%, on average), but the rate of increase was lower.  The recovery rate of camphor was higher than that of 1,8-cineole in all analyses.  Table 3.5. GC-MS quantifications of 1,8-cineole and camphor in rinsed and extracted solutions at different observation times  amount (g mL-1 ± SD)  from rinse amount (g mL-1 ± SD) from extract recovery (%)a  1,8-cineole camphor 1,8-cineole camphor 1,8-cineole camphor individualb       10 min 16.7 ± 0.2 34.7 ± 0.2 12.2 ± 0.3 4.0 ± 0.2 28.6 70.8 30 min 14.5 ± 0.8 47.6 ± 0.7   8.7 ± 0.1   9.1 ± 0.8 22.9 103.9 60 min 11.8 ± 0.1 21.9 ± 0.1 14.8 ± 0.3 13.1 ± 0.2 26.3   64.1 180 min   7.3 ± 0.2 10.4 ± 0.1   5.9 ± 0.1   8.5 ± 0.2 13.1   34.7 mixturec       10 min 30.2 ± 0.2 33.3 ± 0.5 12.7 ± 0.2 9.67 ± 0.4 42.4   78.8 30 min 27.9 ± 1.2 68.8 ± 2.3 15.3 ± 0.4 21.5 ± 0.7 42.7 165.2 60 min 14.8 ± 0.1 32.8 ± 0.3 16.6 ± 0.3 26.1 ± 0.6 31.0 107.9 180 min 14.2 ± 0.4 29.8 ± 0.8 13.3 ± 0.3 21.7 ± 0.4 27.2   94.4 a % based on the initially applied amount. b data from the individual application of each compound. c data when the two compounds were applied in mixture.  69   Figure 3.3. Penetration rate of the compounds when applied individually or when mixed in 3rd instar larvae of T. ni.  Both compounds showed increased penetration rates when they were mixed, but the increased rate for camphor (15.9 to 36.2%, on average) was much greater than that for 1,8-cineole (10.3 to 14.3%).  Error bars represent the standard deviations of replications (n=3).  3.3.3.2 Injection assay and mixed application The toxicities of each compound by injection were examined and compared to those from topical administration (Table 3.6).  As mentioned in Chapter 2, 1,8-cineole had a lower LD50 value than camphor against 3rd instar larvae of the cabbage looper.  However, when injected into 5th instar larvae, camphor had a significantly lower LD50 than 1,8-cineole, with a toxicity ratio of 81.3, compared to 1,8-cineole with a ratio of 15.6.    70  Table 3.6. Comparison of the insecticidal activities of 1,8-cineole and camphor in the topical and injection assays against 3rd and 5th instar larvae of T. ni compound topical (3rd instar)a injection (5th instar) Rd nb LD50 (g insect-1) (95% CLc) nb LD50 (g insect-1) (95% CLc) 1,8-cineole 360 229.6 (171.3 - 341.6) 210 374.3 (301.0 - 476.4) 15.6 camphor 240 471.3 (402.4 - 538.2) 210 147.0 (109.9 - 200.1) 81.3 a data from Chapter 2. b number of insects used to determine LD50. c CL denotes 95% confidence limit. d toxicity ratio between injection assay and topical application assay calculated by the equation;    (     (              ))  (                           )(     (                ))  (                           )   Mortalities in a mixed application assay using 5th instar larvae of the cabbage looper were compared to separate topical administration and injection of individual compounds (Table 3.7).  As shown in groups A and C, all four applications of topical (1,8-cineole+camphor), injected (1,8-cineole+camphor), and topical+injected treatments at theoretically (topical) or actually (by injection) comparable penetrated doses showed no difference in toxicity (p < 0.05), nor did they differ from the sum of the mortalities from individual application of the compounds in both concentrations (between group A and B, p = 0.08 / C and D, p = 0.23).   71  Table 3.7. Comparative toxicity of mixed application assay against 5th instar larvae of T. ni groupa topical assay (g insect-1) injection assay (g insect-1) mortality (% ± SE) 1,8-cineole camphor 1,8-cineole camphor A 717.8b 414.4b   70 ± 12   102.7 150.0 77 ± 7 996.6c   150.0 87 ± 3  943.4c 102.7  87 ± 3 B 996.6    20 ± 10  943.4   40 ± 6     (60d)   102.7  13 ± 3    150.0 53 ± 9     (66d) C 358.9 207.2   17 ± 9   51.35 75.0 17 ± 3 498.3   75.0 20 ± 10  471.7 51.35  33 ± 3 D 498.3    3 ± 3  471.7   10 ± 6     (13d)   51.35  7 ± 3    75.0 27 ± 3      (34d) a group A and C: in combination assay, two compounds were mixed and topically applied or injected, whereas in separated assay, one compound was injected first and the other compound was topically applied. b in combination assay, the amounts for topical assay were determined based on the mixture’s penetration rate (1,8-cineole: 14.3%, camphor: 36.2%). c in separated application, the amounts for topical assay were based on the individual’s penetration rate (1,8-cineole: 10.3%, camphor: 15.9%). d sum of mortality of individual application.  72  3.3.3.3 Surface tension and contact angle The surface tensions and contact angles of the selected compounds and their combinations on a layer of beeswax were measured (Table 3.8).  Although no notable difference was found in surface tension measurements, the camphor solution displayed a relatively greater contact angle on beeswax than did the other materials.  1,8-Cineole had the lowest contact angle, and the mixture of 1,8-cineole and camphor had relatively similar angles to that of rosemary oil.  When camphor was mixed with all other constituents of rosemary oil except 1,8-cineole, a similar lower contact angle was observed.   Table 3.8. Surface tension and contact angle measurement of rosemary oil and major constituents  surface tension (mN m-1 ± SD) contact angle (º ± SD, 50% solution in acetone) rosemary oil 29.8 ± 0.09 22.7 ± 1.7 1,8-cineole 29.2 ± 0.05 19.5 ± 1.6 camphor  58.3 ± 2.4 1,8-cineole+camphora 30.0 ± 0.03 24.1 ± 1.0 (full mixture) – 1,8-cineoleb 29.3 ± 0.07 23.9 ± 1.0 a the ratio of the mixture of 1,8-cineole+camphor was 63:37 (w/w), their natural ratio in rosemary oil. b full mixture is the mixture of all rosemary essential oil constituents occurring at more than 1% concentration in the oil.  73  3.3.4 Cuticular penetration of the thymol and p-cymene mixture 3.3.4.1 GC-MS analysis In the GC-MS analyses of thymol and p-cymene, there was no difference in penetration after 10 min, whether the two compounds were mixed or individually applied (Table 3.9).  At 1 h, although the penetration of thymol was the same in both cases, the penetration of p-cymene showed a 5.5-fold increase when mixed with thymol.  In contrast, for a longer incubation to 8 h and a lower dose (LD10 of thymol+p-cymene), though the peak area ratios were very low, more than double the amount of thymol was identified when applied as a mixture.  Table 3.9. GC-MS analysis of the cuticle penetrations of individual and mixture of thymol and p-cymene in 3rd instar larvae of T. ni timea treatment dose (g insect-1)b peak area ratioc thymol p-cymene 10 min thymol 16.2  1.30  p-cymene 16.2   0.15 thymol+p-cymene 16.2+16.2  1.24  0.14 1 h thymol 16.2  1.39  p-cymene 16.2   0.02 thymol+p-cymene 16.2+16.2  1.39  0.11 8 h thymol 8.5 0.0057  p-cymene 8.5  0.001 thymol+p-cymene 8.5+8.5 0.0125 0.001 a incubation time before extraction. b for observations of 10 min and 1 h incubations, LD50 dose of thymol+p-cymene (1:1, w/w, 32.3 g insect-1) and the proportional amounts of individual compounds were applied, whereas in 8 h incubation, LD10 dose of thymol+p-cymene (17.0 g insect-1) was applied. c ratio of peak areas of thymol and p-cymene to that of internal standard, -pinene.   74  3.3.4.2 Injection assay As increased penetration was observed at different incubation times, and the GC-MS analyses revealed increased cuticular penetration of both compounds, the contributions of each compound to insecticidal activity of the binary mixture were examined via injection to 5th instar larvae (Table 3.10).  p-Cymene had a greater toxicity ratio than thymol or the binary mixture, indicating a greater difference in toxicity between the topical application and injection.  However, whereas the topical application of thymol+p-cymene was synergistic, the injection of the binary mixture failed to show a synergistic interaction.  From Wadley’s determination, the expected LD50 was 382.0 g insect-1, which was lower than the observed LD50 (534.8. g insect-1, synergy ratio = 0.71, i.e., additive), suggesting that enhanced toxicity of the thymol+p-cymene combination may be produced by enhanced penetration of thymol, as suggested by the GC-MS analysis at 8 h of incubation.  Table 3.10. Comparison of LD50 values of thymol, p-cymene and their mixture via different administration methods. compound topical (3rd instar)a injection (5th instar) Rd nb LD50 (g insect-1) (95% CLc) nb LD50 (g insect-1) (95% CLc) thymol 210 32.6 (26.2 - 40.1) 210 244.3 (186.1 - 357.6) 3.4 p-cymene 150   242.5 (204.1 - 290.6) 180   875.4 (687.9 - 1293.6) 7.0 thymol+p-cymene 180 32.3 (28.7 - 36.5) 180 534.8 (445.3 - 691.8) 1.5 a data from Chapter 2. b number of insects used to determine LD50. c CL denotes 95% confidence limit. d toxicity ratio between injection assay and topical application assay calculated by the equation;    (     (              ))  (                           )(     (                ))  (                           )  75  3.3.4.3 Surface tension and contact angle The contact angle measurement indicates p-cymene’s higher affinity to the wax layer than thymol or thyme oil (Table 3.11).  Thymol had a relatively higher contact angle than the essential oil, but the difference in surface tension between thyme oil and a full mixture minus thymol was not significant, indicating the presence of other constituents that have high surface tension as well.   Table 3.11. Surface tension and contact angle measurement of thyme oil and their major constituents  surface tension (mN m-1 ± SD) contact angle (º ± SD, 50% solution in acetone) thyme oil 30.7 ± 0.09 30.9 ± 1.8 thymol  38.3 ± 2.7 p-cymene  23.0 ± 1.5 thymol+p-cyemenea  29.2 ± 2.1 (full mixture) – thymolb 28.6 ± 0.03 25.5 ± 1.6 a the mixture ratio of thymol+p-cymene was 1:1 (w/w). b full mixture is the mixture of all rosemary essential oil constituents occurring at more than 1% concentration in the oil.   76  3.3.5 Cuticular penetration of the citral and limonene mixture 3.3.5.1 GC-MS analysis Unlike the chemical analyses of the other penetrated essential oil constituents, GC-MS analysis of citral and limonene revealed major metabolites (Table 3.12).  Two hydroxylated metabolites of citral, neric acid and geranic acid were identified, and five metabolites were identified from limonene, including limonene-1,2-diol as the major metabolite (66.2% on average of total metabolites).  Metabolism occurred rapidly, with more than 85% of citral and 67% of limonene converted within 10 min.  A slight decrease in total penetration of both compounds in the mixture was observed at 10 min, but increased penetration of total amounts as well as those of parent citral and limonene was detected for the mixture at the 1 h analysis (Figure 3.4).  Metabolic conversion rates were largely unchanged whether the compounds were applied individually or as a mixture.    77  Table 3.12. GC-MS analysis of the cuticle penetrations of individual and mixture of citral and limonene in 3rd instar larvae of T. ni timea treatmentb peak area ratioc conversion (%)d citral metabolites neral geranial neric acid geranic acid 10 min individual 0.04 0.02 0.14 0.49 90.9 mixture 0.04 0.02 0.06 0.28 85.0 1H individual 0.04 0.02 0.31 1.48 96.6 mixture 0.11 0.05 0.33 1.49 91.9 time treatment limonene metabolitese conversion (%) A B C D E 10 min individual 0.16 0.03 0.06 0.02 0.01 0.23 68.6 mixture 0.11 0.01 0.04 0.02 0.01 0.15 67.7 1H individual 0.09 0.01 0.05 0.02 0.02 0.19 75.9 mixture 0.25 0.03 0.08 0.03 0.01 0.27 62.1 a incubation time before extraction. b LD50 concentration of citral+limonene combination (1:1, w/w, 115.0 g insect-1) and their equivalent amounts of each compound were applied individually or as a mixture. c ratio of peak area of citral, limonene and their metabolites to that of internal standard, -pinene.  d the conversion rate was calculated by the equation; (sum of peak area of metabolites)/(sum of all peak area)100. e A: limonene oxide, B: carvone, C: trans-carveol, D: cis-carveol, and E: limonene-1,2-diol.    Figure 3.4. Cuticle penetration of citral and limonene.  After 1 h of incubation, the penetration of both compounds through the cuticle was enhanced when they were applied as a mixture. 78  3.3.5.2 Insecticidal activity of the metabolites Insecticidal activities of the major metabolites of citral and limonene were examined via topical application in 3rd instar larvae.  Whereas limonene-1,2-diol did not show any insecticidal activity, the major metabolite of citral, geranic acid (81.3% in average of total metabolites) showed comparable LD50 value to the parent, citral.   Table 3.13. Insecticidal activities of the major metabolites of citral and limonene compound LD50 (g insect-1) 95% CLa slope ± SE Rb geranic acid 103.1  62.2 - 156.9 2.6 ± 0.6 1.32 limonene-1,2-diol >1000 - - <0.23 a CL denotes 95% confidence limit. b toxicity ratio of LD50 values to those of the substrate compounds, citral (LD50: 135.9 g insect-1) and limonene (LD50: 233.8 g insect-1).   3.3.5.3 Surface tension and contact angle No apparent difference between citral and limonene was found in surface tension measurement, but limonene had a significantly lower contact angle than citral, also resulting in a lower contact angle for citral+limonene compared to citral (Table 3.14).  However, the difference of contact angles between citral and limonene (9.6º) was smaller than those of 1,8-cineole and camphor (38.8º) or thymol and p-cymene (15.3º). 79  Table 3.14. Surface tension and contact angle measurement of lemongrass oil and their major constituents  surface tension (mN m-1 ± SD) contact angle (º ± SD, 50% solution in acetone) lemongrass oil 32.2 ± 0.13 33.2 ± 1.5 citral 32.8 ± 0.03 35.9 ± 1.8 limonene 30.1 ± 0.05 26.3 ± 1.6 citral+limonenea 31.5 ± 0.08 30.3 ± 1.2 (full mixture) – citralb 25.6 ± 0.05 26.4 ± 1.6 a the mixture ratio of citral+limonene was 1:1 (w/w). b full mixture is the mixture of all lemongrass essential oil constituents occurring at more than 1% concentration in the oil.   3.4 Discussion Since plant essential oils obtained from ‘actual’ plants vary chemically based on numerous biotic and abiotic factors, the chemical profile of oils will not be uniform, except where oils from different sources are intentionally blended to achieve a consistent composition.  Variation in response of noctuid larvae based on variation in chemical composition of rosemary oil has been previously reported, with LD50 values ranging from 58.9 to 335.9 g insect-1 in T. ni, and 167.1 to 372.1 g insect-1 in the armyworm, Pseudaletia unipuncta Haworth (Isman et al. 2008).  This points to the importance of understanding the contributions of individual constituents and combinations thereof to overall insecticidal activity as a basis for standardization and quality control of commercial insecticides based on plant essential oils.  80  Interestingly, among the four major constituents of rosemary oil, camphor displayed the most consistent synergistic effects with other compounds.  Eleven out of 14 combinations that included camphor showed synergy, and the remaining 3 combinations showed at least 20% increases in insecticidal activities (Table 3.1).  In a similar study of synergy among 30 insecticidal compounds to Spodoptera littoralis, camphor exhibited synergy with 22 substances, and 9 of the mixtures with camphor showed the maximal synergic effect (Pavela 2014).  With the four major constituents of thyme oil, a synergy between thymol and p-cymene on larvae of the cabbage looper was observed.  This insecticidal synergy was also reported in S. littorali (Pavela 2014) and the house fly, Musca domestica as well (Pavela 2008).  In addition, as shown in Table 3.2, the synergy between thymol (or caryophyllene oxide) and p-cymene (or linalool, though additive to thymol but positive) disappeared in the tertiary mixture or the full mixture comprising thymol and caryophyllene oxide together.  The insignificant interaction between thymol and caryophyllene oxide (synergy ratio of 0.82) might be one reason for the observed disappearance of positive effects, but also the competitive uptake or interaction between thymol and caryophyllene oxide with other compounds may explain these negative effects.  As shown in Table 3.1 to 3.3, since not only the types of compounds but also their ratios are important factor to produce synergy, this change in ratio can lead to different interactions, disrupting the specific ratio which can produce enhanced toxicity. Several statistical models have been proposed to assess synergy, and in the present study we adopted two models, Hewlett and Plackett’s calculation and Wadley’s.  Some of the estimated LD50 values were similar, but in general, Wadley’s model was deemed to be the more conservative of the two.  One shortcoming of these two models is the time and effort required to 81  obtain the empirical LD50 values of the individual constituents as well as those of mixtures.  A somewhat more convenient model is Finney’s calculation (Trisyono and Whalon 1999). Expected mortality of a mixture can be calculated using the equation: E = Oa + Ob (1 – Oa), where E is the expected mortality, Oa and Ob are the observed mortalities of the individual compounds.  A chi-square value is obtained via: 2 = {(Om – E)2} / E, where Om is observed mortality of the mixture.  The calculated chi-square can be compared to a chi-square tabular value with df = 1 and  = 0.05, which gives 3.84.  If the chi-square is > 3.84, synergy is confirmed.  However, care must be used with this approach.  Being based on single concentrations or doses, this method is considerably less precise than models based on LD50 values, and depends on appropriate selection of the single doses used. In terms of the cytotoxicity assay, although Delgado et al. (2004) reported synergistic cytotoxicity between thymol and p-cymene in the gram-positive food-poisoning bacterium, Bacillus cereus, in the present study this binary mixture did not exhibit any synergistic interaction in a cell line derived from an insect, and only the citral+limonene combination showed enhanced cytotoxicity relative to the individual compounds.  As shown in Figure 3.2, the cytotoxic modes-of-action of these compounds may differ.  Treatment with citral seemed to cause direct damage to the cell membrane, whereas limonene seemed to enhance membrane permeability, resulting in enlarged cells (areas 1.6-fold compared to controls), specifically associated with vacuoles.  A higher dose of limonene caused cells to burst internally, with cells shrinking eventually (data not shown).  This dual effect observed in the application of the citral+limonene combination may explain the synergistic cytotoxicity.  Fields et al. (2010) suggested that increased permeability of the insect midgut cell membrane by soysaponin I may 82  lead to cysteine-rich pea albumins (PA1b) readily entering into the cells, as a possible mechanism of synergistic insecticidal activity.  Although there is lack of evidence that cytotoxicity is directly involved in the insecticidal actions of monoterpenoids, the synergistic cytotoxicity between citral and limonene may be related to increased penetration through the insect’s cuticle that was observed in this study. In the present study, I found reversed order of toxicity for camphor and 1,8-cineole between injection and topical assays (Table 3.6).  The enhanced insecticidal activity of camphor when bypassing the cuticular barrier combined with the observation of its increased penetration when admixed with 1,8-cineole (Figure 3.3) can explain the synergy between the two compounds.  This is clear when comparing toxicities of mixtures and individual applications (Table 3.7).  The sum of mortalities from topical applications of individual compounds (1,8-cineole: 997 g insect-1, camphor: 943 g insect-1) did not differ from the toxicity of the mixture which had lesser amounts of each compound (1,8-cineole+camphor: 718+414 g insect-1, p > 0.05), but when injected compounds in the same amounts produced comparable toxicity whether they were mixed or not (1,8-cineole: 103 g insect-1, camphor: 150 g insect-1, p > 0.05), indicating that toxicity of the mixture via injection can be expressed as the sum of individual toxicities via the same route.  These comparisons suggest that synergy between 1,8-cineole and camphor largely results from the increased penetration of the compounds, especially that of camphor in this particular combination.  In addition, pre-injected 1,8-cineole or camphor did not alter the penetration of the other compound; further confirming increased penetration as the mechanism of synergy (Table 3.7). 83  The GC-MS analyses of the penetration of thymol and p-cymene revealed relatively lower peak areas of p-cymene compared to those of thymol, even though the applied amounts of both compounds were the same (Table 3.9).  The higher affinity to a wax layer and the more hydrophobic property of the hydrocarbon, p-cymene may suggest stronger bonding to the wax, i.e., less penetration can occurr.  Another possible explanation is faster metabolism of p-cymene than thymol.  The peak area at 1 h of individual p-cymene showed a 7.5-fold decrease from the 10 min sample, whereas the area of thymol at 1 h was slightly increased from that at 10 min, suggesting different rates of metabolism between the compounds.  A longer incubation with a lesser amount applied, increased the amount of internal thymol.  Along with the LD50 values acquired via injection in 5th instar larvae, the increased internal amount of thymol could account for the enhanced toxicity of the mixture with p-cymene.  Two possible mechanisms for elevated amounts of thymol can be proposed; increased penetration of unpenetrated p-cymene or decreased metabolism of penetrated p-cymene.  The former hypothesis can be supported by the lower contact angle of the thymol+p-cymene combination compared to thymol due to p-cymene’s higher affinity to the wax.  The latter hypothesis will be discussed in Chapter 4.  A similar finding was observed for the major constituents of lemongrass oil.  Higher internal concentrations of both citral and limonene were found at 1 h extraction when applied as a mixture compared to when they were individually administrated.  Whereas the chemical analyses following penetration of the major constituents of rosemary and thyme oils failed to detect any metabolites, several metabolites from citral and limonene, the two major compounds of lemongrass oil, were identified.  A notable difference of metabolic rates was observed between the isomeric compounds of citral.  In the GC-MS analysis of lemongrass oil, geranial is more 84  prevalent than neral in the natural oil (46.8% : 30.3%, from Table 2.1), but a reverse order was found from the larval extract where the concentration of neral was double that of geranial (Table 3.12).  Since the peak areas of geranic acid were significantly greater than those of neric acid, geranial appears to be more susceptible to metabolism by the cabbage looper.  Citral can be metabolized to their acid forms (gernaic acid and neric acid), alcohols (geraniol and nerol) or others by rats and in plant seeds (Diliberto et al. 1990; Dudai et al. 2000).  The metabolites of limonene are also reported as carveol and perillyl alcohol by rat liver microsomes (Miyazawa et al. 2002), or uroterpenol and perillic acid by tobacco cutworm larvae (Miyazawa et al. 1998).   Although the metabolite of limonene did not produce any mortality in the cabbage looper, geranic acid had a similar LD50 value to that of citral.  Since the conversion rate of citral was more than 90%, it is possible that the toxicity of citral is actually a consequence of the toxicity of its metabolite.  From the study of structure-activity relationships, aldehydes are known to be more toxic than structurally similar alcohols or acids (Tak et al. 2006), so the increase in the amount of unmetabolized citral in the mixture relative to its individual application (2.5-fold) can directly contribute to the increase in toxicity of the mixture along with the increased amount of limonene (2.8-fold) in the mixture. Although no metabolite was detected in the present study of the major constituents of rosemary and thyme oils, the chemical analyses suggest the possibility of metabolism (i.e., decreased amounts of them observed with increasing incubation time).  1,8-Cineole was reported to be hydroxylated by the pyrgo beetle, Paropsisterna tigrina (Southwell et al. 1995) and Leichhardt’s grasshopper, Petasida ephippigera (Fletcher et al. 2000), and camphor was hydroxylated by the tobacco cutworm, Spodoptera litura (Miyazawa and Miyamoto 2004) when the insects were fed 85  diets containing the compounds.  Thymol was reported to be glycosylated by the tobacco cutworm and the cabbage looper (Passreiter et al. 2004), while no metabolism of p-cymene in an insect has been reported thus far, oxidative metabolisms by mammals including rats, guinea pigs (Walde et al. 1983) and koala (Boyle et al. 2010) have been observed.  In the present study, chromatographic investigations of penetration showed good correspondence with toxicity.  However, a new technique that can trace penetration, and discriminate enzymatic reactions might be required.  Since the introduced compounds can be readily detoxified by insects, what we observed might not be the full amount that penetrated, but the final result of complex metabolic interactions at the specific time of sampling.  To address this problem, it may be necessary to adapt an in vitro membrane model from the pharmaceutical field, but careful attention will be required to select appropriated materials and methods, which may be challenging.  As for the mechanism of enhanced penetration of binary mixtures, a change in physico-chemical properties might be one possible explanation.  Although it was not clear for the citral+limonene combination, acetonic solutions of 1,8-cineole+camphor and thymol+p-cymene had lower contact angles on beeswax than solutions containing individual compounds of the more toxic ones, camphor and thymol.  Since beeswax has a chemical composition similar to the wax layer of insect integument (Webb and Green 1945), we can assume that when the more toxic compound is mixed with a synergizing one, its spreadability or the affinity to the wax layer is increased, by lowering its surface tension compared to when it is individually administered, at least for these particular combinations. 86  Another possible hypothesis for increased penetration of camphor by 1,8-cineole might be through increased mobility.  When 100 mg of camphor was dissolved in acetone and applied to black cotton fabric, camphor recrystallized after the solvent evaporated (Figure 3.5).  When the same amount of camphor was applied on the fabric as a mixture with 1,8-cineole, no solid crystals were observed, indicating that the compounds can physically interact with each other, increasing the solubility of camphor.  The liquid phase of camphor will have higher mobility than the solid phase, and this can also contribute to the increased penetration rate.  If this increased solubility of camphor is one of the key factors in the mechanism of synergy, it might change the status of camphor from the synergizing or boosting agent to the subject or active agent being synergized by other elements of the mixture.  For example, camphor synergized -pinene, whereas -pinene did not show any penetration enhancing effect of drugs on human skin (Williams and Barry 1991).  This may be a consequence of different characteristics of human skin and insect integument, but at the same time this also may indicate different mechanisms underlying the penetration enhancing effect between the two membranes. In summary, several synergistic interactions between the major constituents of essential oils were determined, and different mixture ratios had different responses in their toxicity.  A citral and limonene combination at the same ratio showed synergistic activity in vivo and in vitro as well, indicating different cytotoxic modes-of-action.  Penetrations of the toxicants were increased by combining them, and additional bioassays supported the direct contributions to enhanced toxicity of these phenomena.  Lowered surface tension or increased solubility of the compound may explain the increased penetration through the insect cuticle.  87   Figure 3.5. Applied status comparison of 1,8-cineole and camphor solutions on black cotton fabric.  When 100 mg of camphor (50% in acetone) was applied on fabric, it was recrystallized making a white circle, whereas when the same amount of camphor was mixed with 1,8-cineole and applied (1,8-cineole : camphor = 63 : 37, 50% in acetone), there was no trace of solid crystals. 88  Chapter 4: Effects of three essential oils and their major constituents on detoxifying enzymes and interactions with enzyme inhibitors in Trichoplusia ni  4.1 Introduction Any insecticidal compound that can successfully enter into an insect’s body may be subject to detoxification by a series of enzymes in the insect.  The outcome of these enzymatic processes is to render lipophilic xenobiotics more hydrophilic, as more polar (water-soluble) products are more amenable to excretion.  Detoxification can often be considered as a two phase process.  Phase I reactions are comprised of oxidations, reductions, or hydrolyses.  The resulting product is sometimes sufficiently polar to be excreted directly, but more often this process provides a functional group that can be subsequently conjugated by highly polar sugars, amino acids, phosphate, or glutathione (phase II reactions) (Yu 2008a).  Among many enzymes and biochemical systems involved in detoxification of xenobiotics, cytochrome P450 dependent monooxygenases (P450s), esterases (in phase I reactions), and Glutathione S-transferases (GST) (phase II reactions) are considered the most important.  Their importance in detoxifications as well as in resistance development are well documented (Li and Berenbaum 2007; Yu 2008a).  Cytochrome P450 monooxygenases, also called mixed function oxidases, microsomal oxidases, polysubstrate monooxygenases or heme thiolate proteins, are involved in oxidation, and considered by far the most important among phase I reactions.  These are involved not only in the oxidation of xenobiotics, but also the physiological functions of insects through metabolism 89  (both activation and deactivation) of juvenile hormones, ecdysteroids, fatty acids and pheromones (Feyereisen 1999).  Owing to their genetic diversity and broad substrate specificity, P450s mediate resistance to many classes of insecticides, but at the same time, those characteristics create a challenge understanding their resistance mechanisms.  Many resistant strains of insects show higher constitutive levels of total P450s and elevated gene expressions (e.g., in the whitefly, Bemisia tabaci) (Guo et al. 2014). Esterases play a significant role in hydrolysis of insecticides containing ester linkages, such as organophosphates, carbamates, pyrethroids and some juvenoids (Yu 2008a).  A resistant strain of the western flower thrips, Frankliniella occidentalis showed 2.8- and 1.5-fold higher enzyme activities in P450s and esterases than a susceptible strain (Scott 1999).  Similarly, resistance to lambda-cyhalothrin was reported in a predatory lady beetle, Eriopis connexa was a sonsequence of 4.16-fold greater esterase activity based on -naphthol formation (Rodrigues et al. 2014). Among phase II reactions in insects, glutathione conjugation is performed by a group of enzymes known as glutathione S-transferases.  These enzymes catalyze the conjugation of electrophilic substrates with reduced glutathione (Yu 2008a).  They are known to be related to organophosphorous insecticides resistance directly via O-dealkylation and O-dearylation pathways, and are similarly associated with all major classes of insecticides (Enayati et al. 2005). One fast and easy way to determine the involvement of a specific enzyme system in insecticide resistance and to potentially mitigate resistance is through the use of enzyme inhibitors together with the insecticides.  The involvement of P450s in the metabolism of target insecticides can be confirmed via combined application with the P450s inhibitors sesamex or piperonyl butoxide 90  (PBO) (Scott 1999). Although not commercially significant, piperonyl butoxide can also inhibit the activity of esterases, which are far better inhibited by triphenyl phosphate (TPP) (Gao et al. 2014).  Likewise, the activity of GST can be constrained when insects are treated with ethacrynic acid (EA) (Qin et al. 2013). Plant-produced secondary metabolites can potentially induce or inhibit the metabolism of insects through direct interactions with detoxicative enzyme systems (Lee 1991).  In the previous chapter, enhanced penetration of active compounds was proposed as one possible mechanism of synergy.  Not only may cuticular penetration of the active compounds be increased, but inhibitions of insect detoxicative metabolism could contribute the enhanced toxicity of target compounds.  In this chapter, the possibility of enzymatic inhibition as a candidate mechanism for synergy of selected combinations of essential oil constituents is explored.  Also, the contributions of selected enzyme inhibitors to the insecticidal activity of the constituents and oils were investigated.  4.2 Materials and methods 4.2.1 Chemicals The same three essential oils of rosemary, thyme and lemongrass from Chapter 2 were used as well as the major constituents of these oils.  Reagents and enzyme inhibitors used in enzyme assay were purchased from Sigma-Aldrich (bovine serum albumin, Bradford reagent solution, cytochrome C from equine heart, dithiothreitol [DTT], Ethylenediaminetetraacetic acid [EDTA], fast blue RR salt, -naphthol, -naphthyl acetate, phenylmethanesulfonyl fluoride [PMSF], 91  reduced glutathione and TPP), Thermo Fisher Scientific (1-chloro-2,4-dinitrobenzene [CDNB], 3,3′,5,5′-tetramethylbenzidine [TMBZ] and PBO), and MP Biomedicals, LLC (EA, Santa Ana, CA, USA).  4.2.2 Insect maintenance Third instar larvae of the cabbage looper were used for experiments in the present study.  The source and maintenance conditions of the larvae and cell line are described in Chapter 2.  4.2.3 Enzyme preparation Whole body enzyme extractions were prepared from pre-treated insects.  For the assay of esterases and GST, three replications of forty 3rd instar larvae of T. ni were topically treated at the LD20 dose of each essential oil as determined in Chapter 2.  The LD20 doses of the synergistic combinations of the two major constituents of each oil were obtained from Chapter 3 (mixture ratio of 1,8-cineole:camphor = 63:37, thymol:p-cymene = 1:1, citral:limonene = 1:1, w/w) and the equivalent amounts of individual compounds at those doses were also applied topically.  After 6 h of incubation, three sets of 20 live larvae were collected into 2 mL microcentrifuge tubes from each treatment and homogenized in 500 L of homogenizing buffer (1 mM EDTA, 0.1 mM DTT, 0.5 mM PMSF and 10% glycerol in 0.1 M sodium phosphate buffer, pH 7.5) in ice-cold conditions.  Another 500 L of homogenizing buffer was added and the solution was centrifuged at 13,000  g for 20 min in a Sorvall Legend Micro 17 R centrifuge (Thurmo Fisher 92  Scientific).  From each tube, 800 L of supernatant was carefully collected and diluted 4-fold with 2.4 mL of 0.1M phosphate buffer, pH 7.5 and used as the enzyme source. For the P450s assay, the LD20 dose of each essential oil or the proportional amount of the major constituent were topically applied to one hundred 3rd instar larvae.  After 6 h, thirty live larvae were collected and homogenized by using a glass-Teflon homogenizer with 10 vertical passes in 2 mL of homogenizing buffer, and another 18 mL of phosphate buffer (0.1 M, pH 7.5) was added and centrifuged at 10,000  g for 20min at 4ºC using a Beckman Coulter Avanti J-E centrifuge (Beckman Coulter Canada, LP., Mississauga, ON) with a JA-20 fixed-angle rotor.  The supernatant was collected and centrifuged again at 100,000  g for 1 h using a Beckman Coulter Optima L-100XP ultracentrifuge with a 70Ti rotor.  The supernatant was discarded and the pellet was resuspended with 1.5 mL of homogenizing buffer. The same topical application method and enzyme preparations were conducted with TPP (50 g mL-1), EA (100 g mL-1) and PBO (50 g mL-1), and they served as positive controls for inhibition of esterases, GST and P450s, respectively.  All enzyme assays was conducted immediately without freezing as soon as the extracted enzyme solutions were prepared, and each assay was repeated three times.  4.2.4 Enzyme assay Esterase enzyme activity was measured as described by Scott et al. (2010) with slight modification.  Briefly, 1 mL of 30 mM -naphthyl acetate in acetone was mixed with 99 mL of 93  0.1 M phosphate buffer, pH 7.5 to prepare the substrate solution.  Twenty L of enzyme solution was mixed with 200 L of substrate solution in a 96-well plate and incubated for 5 min at room temperature.  Fifty L of fast blue RR salt solution in distilled water (10 mg mL-1) was added and the absorbance was measured at 450 nm using a Polarstar galaxy spectrophotometer (BMG Labtechnologies, Ortenberg, Germany).  A standard curve was prepared using -naphthol for quantification of the final product. For glutathione S-transferase enzyme activity, CDNB was used as a substrate (Scott et al. 2010). Fifty L of enzyme solution was transferred into each well of a 96-well plate, and then mixed with 50 L of 0.1 M phosphate buffer (pH 7.5) and 100 L of substrate solution (4 mL of 10 mM reduced glutathione in water mixed with 1 mL of 10 mM CDNB in methanol).  Absorbance was measured every min for 10 min at 320 nm without incubation.  The GST activity was determined using the CDNB extinction coefficient of 0.00503 M-1, and expressed as pmol min-1 mg protein-1. The assay procedure to determine P450s activity was adopted from Tiwari et al. (2011) with minor modification.  Forty L of enzyme solution was mixed with 200 L of 0.05% TMBZ solution (10 mg of TMBZ in 5 mL methanol with 15 mL of 0.25 M sodium acetate buffer, pH 5.0) and 60 L of 50 mM potassium phosphate buffer, pH 7.2 in a 96-well plate.  Twenty-five L of 3% hydrogen peroxide was added and incubated for 2 h at room temperature.  Absorbance was measured at 630nm, and cytochrome C from equine heart was used to produce a standard curve. 94  Protein quantities of enzyme solutions were measured at 620 mm as following the Bradford method (Bradford 1976).  A series of concentrations of bovine serum albumin was prepared to determine the standard curve (Appendix B, Figure B.3).  All enzyme assays were repeated three times.  4.2.5 LD50 determinations of major constituents and enzyme inhibitor mixtures Potential contributions of enzyme inhibitors to the insecticidal activities of major constituents of the oils were investigated.  LD50 values for the two major compounds of each essential oil as well as their binary mixtures were determined when they were mixed with enzyme inhibitors. Six to ten different concentrations of test solutions in acetone were prepared by mixing with EA, PBO or TPP at a 1:1 ratio (w/w).  The bioassay was conducted as described in Chapter 2.   4.2.6 Influence of pre-treatment of enzyme inhibitors on insecticidal activity The effects of enzyme inhibitors to insecticidal activity of the essential oil constituents were compared when they were administered in advance of the toxicants, administered with the target compounds as mixtures, or applied simultaneously at different sites on insects via topicall application.  The inhibitor solutions were prepared with 100 mg of EA, 50 mg of TPP and PBO in one mL acetone, and the test solutions were prepared to provide LD50 doses of each compound as well as their mixtures.  For pre-treatment, ten 3rd instar larvae were treated with 0.5 L of an inhibitor solution on the dorsum in the abdominal region.  After 6 h, 0.5 L of test solution was 95  applied ventrally at approximately the same site on the abdomen of larvae (1/2 of LD50, since the LD50 was determined with 1 l).  For mixtures, one L of binary mixture of inhibitor solution and test solution (1:1, v/v) was applied ventrally to larvae, whereas for simultaneous application on different sites, each of 0.5 L of inhibitor solution and test solution was applied dorsally and ventrally on the abdomen, respectively.  All tests were repeated three times, and mortality was determined after 24 h of incubation.  4.2.7 Statistics Mortality and enzyme activity were subjected to ANOVA and means were compared using Tukey’s test, and probit analysis was used to calculate the LD50 values using StatPlus 2009 (version 5.8.4, AnalystSoft, Alexandria, VA, USA).   4.3 Results 4.3.1 Enzyme activity  Effects of enzyme activity on esterases, glutathione S-transferases and cytochrome P450s from the three essential oils, synergistic combinations of their major constituents, as well as from individual compounds were evaluated (Table 4.1).  In the case of esterases, only lemongrass oil and limonene showed statistical differences (p < 0.05) from the control, but within the group, no differences were observed in the three essential oils (for example, rosemary oil, 1,8-cineole, 96  camphor and 1,8-cineole+camphor showed no difference).  TPP-treated larvae showed significant differences to all other test groups including the control. Regarding GST, EA strongly inhibited activity showing notable difference to all other treatments, while some treatments produced mild inhibitory activity (p < 0.05) including the combinations of 1,8-cineole+camphor, thymol+p-cymene and citral+limonene.  Although some treatments showed inhibitory activity compared to controls, there were no statistical differences within the test groups.  In the assay of P450s, because PBO treatment alone showed a significant difference to the control and none of the essential oils or their major constituents showed inhibitory activity, no further investigation was conducted.  The relative activities of each treatment to that of the controls are shown in Figures 4.1 through 4.3.  In general, lemongrass oil and its constituents showed slightly greater inhibitory effects than other essential oils on esterases and GST, but no difference on P450s.    97  Table 4.1. In vivo enzyme activity of esterases, glutathione S-transferases and cytochrome P450s following treatment of larvae with selected essential oils and their major constituents   esterases (nmol/min/mg  protein) GST (pmol/min/mg  protein) P450s (pmol/min/mg  protein) control   681 ± 34 1.73 ± 0.03 1.97 ± 0.07 rosemary oil   593 ± 30 1.35 ± 0.12 1.78 ± 0.09 1,8-cineole   523 ± 48 1.56 ± 0.07 1.92 ± 0.17 camphor   746 ± 10 1.25 ± 0.01 - 1,8-cineole+camphor (63:37)   644 ± 30   1.12 ± 0.06* - thyme oil   702 ± 32   1.04 ± 0.08* 2.15 ± 0.09 thymol   801 ± 16   1.02 ± 0.04* 2.26 ± 0.12 p-cymene 690 ± 8 1.25 ± 0.01 - thymol+p-cymene (1:1) 710 ± 6   1.10 ± 0.03* - lemongrass oil  498 ± 8*   0.92 ± 0.30* 2.09 ± 0.17 citral  537 ± 35   1.13 ± 0.15* 2.30 ± 0.07 limonene    510 ± 65*   0.84 ± 0.23* - citral+limonene (1:1)  514 ± 52   1.11 ± 0.10* - TPP (50 g insect-1)      82 ± 9** - - EA (100 g insect-1) -     0.14 ± 0.02** - PBO (50 g insect-1) - -     0.92 ± 0.05** * significantly different from the control (Tukey HSD test, p < 0.05). ** significantly different from all other test compounds (Tukey HSD test, p < 0.05).  98   Figure 4.1. Relative influence of three essential oils and their major constituents on esterases in third instar larvae of the cabbage looper.  Values represent as means ± SE (n=3), and the asterisk denotes significant differences to control at p < 0.05 in one way ANOVA followed by Tukey’s multiple comparisons test.   Figure 4.2. Relative influence of three essential oils and their major constituents on glutathione S-transferases in third instar larvae of the cabbage looper. Values represent as means ± SE (n=3), and the asterisk denotes significant differences to control at p < 0.05 in one way ANOVA followed by Tukey’s multiple comparisons test. 99   Figure 4.3. Relative influence of three essential oils and their major constituents on cytochrome P450s in third instar larvae of the cabbage looper. Values represent as means ± SE (n=3), and the asterisk denotes significant differences to control at p < 0.05 in one way ANOVA followed by Tukey’s multiple comparisons test.  4.3.2 Effect of enzyme inhibitors   To understand relationships of specific detoxifying enzymes to the insecticidal activities of the major compounds, the contributions of selected enzyme inhibitors to toxicity were examined.  LD50 values of major rosemary oil constituents, 1,8-cineole, camphor and their synergistic combination (63:37, w/w) when they are mixed with TPP, EA and PBO were described in Table 4.2.  A major increase in toxicity of camphor was found when it was mixed with EA and PBO.  On the other hand, antagonistic effects were observed in the combinations of TPP with 1,8-cineole and 1,8-cineole+camphor.  Mixing the terpenes with EA or PBO showed more than 100  doubled toxicity when they were combined with individual compounds, but the contributory effect on the mixture of 1,8-cineole+camphor was slightly less at 1.6- and 1.5-fold, respectively.  Table 4.2. Insecticidal activities of the major constituents of rosemary oil and their combination when mixed with enzyme inhibitors in third instar T. ni larvae by topical application compound LD50 (g insect-1) 95% CLa slope ± SE Rb 1,8-cineole 229.6 171.3 - 341.6 5.2 ± 1.0  camphor 471.3 402.4 - 538.2 4.8 ± 0.7  1,8-cineole + camphor (63:37) 155.6 138.0 - 174.2 4.6 ± 0.5  1,8-cineole + TPP 765.9 649.0 - 947.3 2.5 ± 0.3 0.6 camphor + TPP 645.1 569.9 - 730.7 3.7 ± 0.5 1.5 1,8-cineole + camphor + TPP 549.2 489.8 - 622.2 3.7 ± 0.4 0.6 1,8-cineole + EA 210.1 176.3 - 252.0 2.6 ± 0.4 2.2 camphor + EA 236.0 171.3 - 380.9 3.0 ± 0.7 4.0 1,8-cineole + camphor + EA 190.4 162.1 - 222.9 2.9 ± 0.4 1.6 1,8-cineole + PBO 200.6 153.9 - 253.1 1.7 ± 0.2 2.3 camphor + PBO 187.8 121.2 - 268.0 1.9 ± 0.4 5.0 1,8-cineole + camphor + PBO 201.7 127.1 - 305.5 3.1 ± 0.6 1.5 a CL denotes 95% confidence limit. b relative toxicity was calculated by (LD50 of the compound without inhibitor) / (1/2 of LD50), since the mixture ratio was 1 : 1 (target compounds (or their mixtures) : enzyme inhibitor).   Table 4.3 represents the activity of thymol and p-cymene when they were mixed with enzyme inhibitors.  Compared to the increased toxicity of rosemary oil constituents by the enzyme inhibitors (except TPP and 1,8-cineole), the inhibitors were less effective on toxicity of thyme oil constituents.  Rather, the effects of TPP and PBO to thymol were antagonistic, and EA had no influence on toxicity.  Interestingly, all relative toxicity values were slightly reduced in the thymol+p-cymene mixture compared to those for individual thymol (TPP : 0.3 to 0.1, EA : 1.1 to 101  0.9, and PBO : 0.5 to 0.4).  EA and PBO significantly enhanced toxicity of p-cymene, but when p-cymene was mixed with thymol, this enhancement disappeared.  Table 4.3. Insecticidal activities of the major constituents and their combination of thyme oil when mixed with enzyme inhibitors on third instar T. ni larvae by topical application compound LD50 (g insect-1) 95% CLa slope ± SE Rb thymol   32.6 26.2 - 40.2 2.7 ± 0.3  p-cymene 242.5 204.1 - 290.6 4.1 ± 0.6  thymol + p-cymene (1:1)   32.3 28.7 - 36.5 4.6 ± 0.6  thymol + TPP 229.7 195.4 - 272.0 3.4 ± 0.4 0.3 p-cymene + TPP 552.5 489.3 - 647.7 3.9 ± 0.6 0.9 thymol + p-cymene + TPP 522.8 427.1 - 652.3 2.0 ± 0.3 0.1 thymol + EA   57.4 48.2 - 68.3 2.7 ± 0.3 1.1 p-cymene + EA 124.6 108.4 - 140.6 5.0 ± 0.7 3.9 thymol + p-cymene + EA   71.4 60.0 - 86.0 2.6 ± 0.3 0.9 thymol + PBO 145.0 121.6 - 179.4 2.1 ± 0.2 0.4 p-cymene + PBO 207.5 177.8 - 246.4 3.6 ± 0.5 2.3 thymol + p-cymene + PBO 146.1 117.6 - 182.0 1.8 ± 0.2 0.4 a CL denotes 95% confidence limit. b relative toxicity was calculated by (LD50 of the compound without inhibitor) / (1/2 of LD50), since the mixture ratio was 1 : 1 (target compounds (or their mixtures) : enzyme inhibitor).   As for other pairs of essential oil constituents, TPP also showed a mitigating effect on the toxicity of the major constituents of lemongrass oil, citral and limonene (Table 4.4).  In particular, the combination of limonene and TPP was inactive even at the highest dose tested.  The contributions to toxicity by EA and PBO seemed to be relatively evenly distributed compared to the other two essential oils.  102  Table 4.4. Insecticidal activities of the major constituents and their combination of lemongrass oil when mixed with enzyme inhibitors on third instar T. ni larvae by topical application compound LD50 (g insect-1) 95% CLa slope ± SE Rb citral 135.9 106.2 - 175.2 2.2 ± 0.3  limonene 233.8 158.5 - 367.6 5.3 ± 1.2  citral + limonene (1:1) 115.0   99.0 - 133.2 3.4 ± 0.6  citral + TPP 467.6 256.8 - 696.1 3.6 ± 0.9 0.6 limonene + TPP >1000   ND citral + limonene + TPP 648.4 577.5 - 724.7 3.6 ± 0.4 0.4 citral + EA 114.3 97.9 - 132.2 4.3 ± 0.6 2.4 limonene + EA 375.5 249.2 - 565.7 4.0 ± 1.3 1.2 citral + limonene + EA   75.6 53.6 - 95.3 2.4 ± 0.4 3.0 citral + PBO 161.0 130.6 - 190.8 3.7 ± 0.5 1.7 limonene + PBO 211.6 151.7 - 264.8 2.3 ± 0.4 2.2 citral + limonene + PBO 146.1 118.0 - 175.6 3.0 ± 0.4 1.6 a CL denotes 95% confidence limit. b relative toxicity was calculated by (LD50 of the compound without inhibitor) / (1/2 of LD50), since the mixture ratio was 1 : 1 (target compounds (or their mixtures) : enzyme inhibitor).  4.3.3 Effect of pre-treatment with enzyme inhibitors The contributions of selected enzyme inhibitors by different application methods to the insecticidal activity of the major constituents were compared.  Compounds and enzyme inhibitors were applied as a mixture, or applied on different body sites simultaneously, or enzyme inhibitors were pre-applied 6 h prior to application of the insecticides.  In general, when the enzyme inhibitors and test compounds were applied separately, mortality was greater than when applied together as a mixture.  Moreover, although there was a slight increase in toxicity, pre-application of enzyme inhibitors did not alter toxicity compared to the separate application of inhibitors and test compounds.  103  For the major constituents of rosemary oil, 1,8-cineole and camphor or their mixture, EA did not show any difference whether applied together or separately, but when TPP and PBO were applied separately mortality increased.  In particular, the effect of TPP on camphor showed a significant difference between the treatment methods (Figure 4.4).  PBO produced the greatest increases in toxicity for both compounds as well as their mixture compared to the other inhibitors.  In terms of the two separated applications, there was no apparent effect of incubation.  Figure 4.4. Contributions of enzyme inhibitors by different application methods to the insecticidal activity of individual major constituents of rosemary oil and their mixture on third instar larvae of T. ni.  Error bars represent the standard error of the mean of three replicates of 10 larvae each.  The asterisk denotes significant differences between the application methods at p < 0.05 in one way ANOVA followed by Tukey’s multiple comparisons test.  A similar trend was found for thymol and thymol+p-cymene, showing significantly increased toxicity when any of the three enzyme inhibitors were applied independently, but not for p-104  cymene.  All of the inhibitors boosted the toxicity of thymol to a similar degree, but TPP was not very effective on the thymol+p-cymene mixture, nor on p-cymene by itself.  Isolated applications of PBO and EA showed significant differences compared to the mixed applications with thymol and thymol+p-cymene.   Figure 4.5. Contributions of enzyme inhibitors by different application methods to the insecticidal activity of the individual major constituents of thyme oil and their mixture on third instar larvae of T. ni.  Error bars represent the standard error of the mean of three replicates of 10 larvae each.  The asterisk denotes significant difference between the application methods at p < 0.05 in one way ANOVA followed by Tukey’s multiple comparisons test.  In the comparison between the major constituents of lemongrass oil, citral was more affected by enzyme inhibitors than was limonene.  TPP and PBO showed notable differences between the application methods on the toxicity of citral (p < 0.05), but in limonene and citral+limonene, no 105  statistical differences were found.  Among the enzyme inhibitors, PBO was the most effective synergizing agent.  Figure 4.6. Contributions of enzyme inhibitors by different application methods to the insecticidal activity of the individual major constituents of lemongrass oil on and their mixture third instar larvae of T. ni.  Error bars represent the standard error of the mean of three replicates of 10 larvae each.  The asterisk denotes significant difference between the application methods at p < 0.05 in one way ANOVA followed by Tukey’s multiple comparisons test.  4.4 Discussion In the present study, I found that synergistic effects between the major constituents of three essential oils cannot be explained by inhibition of detoxifying enzymes, at least for esterases, GST, or P450s.  However, all of the compounds tested as well as mixtures thereof showed potentials for enhanced efficacy when the detoxification process is suppressed. 106  There are many reports of plant essential oils and botanical extracts inhibiting detoxifying enzymes leading to increases in biological activity or they can directly contribute to synergy of other insecticides.  For example, Piper sarmentosum essential oil and its major compound, myristicin, inhibited larval growth and development and were inhibitory to esterases and GST of Brontispa longissima (Qin et al. 2010).  Ramirez et al. (2012) demonstrated significant correlations between inhibition of P450s by several plant essential oils and their repellency to the yellow fever mosquito, Aedes aegypti.  Another study on the same mosquito species revealed that several essential oils including sesame oil and black pepper oil inhibited P450s, and showed synergistic insecticidal activity with the conventional insecticide carbaryl (Tong and Bloomquist 2013).  Similarly, ethanol extracts of the leaves of Melia azedarach and Jatropha gossypifolia inhibited P450s and esterases in the fall armyworm, Spodoptera frugiperda, and synergized toxicity when mixed with the pyrethroid insecticide, cypermethrin (Bullangpoti et al. 2012). Since plant-derived secondary metabolites can affect not only the three detoxifying enzymes tested but also a variety of other biochemical processes, there remains the possibility that the synergistic interactions of selected compounds could be explained by inhibition or induction of other enzymatic reactions.  When Artemisia annua leaf extracts and Lavandula angustifolia essential oil were applied to the lesser mulberry pyralid, Glyphodes pyloalis, the activity of digestive enzymes including -amylase and protease were decreased, whereas higher levels of GST and esterase activity were induced in response to the treatment (Khosravi et al. 2011; Yazdani et al. 2013).  Likewise, botanicals can be associated with glutathione peroxidase, superoxide dismutase antioxidant or glutathione reductase (Ahmad and Pardini 1990; Lee 1991).  However, the involvement or contribution to the insecticidal activity of those digestive or 107  antioxidant enzymatic systems is likely of less consequence than the discussed detoxifying enzymes.  The best evidence for this comes from the many studies indicating that resistance of insects to synthetic insecticides is usually associated with higher levels or increased production of detoxifying enzymes (Wilson 2001).  For instance, throughout sub-Saharan Africa, resistance to pyrethroid insecticides in mosquito species has been demonstrated to be related to elevated enzyme levels of esterases, GST and P450s (Coetzee and Koekemoer 2013), and resistance to metaflumizone, a voltage-dependent sodium channel blocker in beet armyworm, Spodoptera exigua was achieved through elevated esterase levels (Tian et al. 2014).  These survival strategies of insects indirectly imply the important contributions of these enzymes to insecticide resistance.  It is not surprising to find significant participation of detoxifying enzymes in the fate of plant-derived compounds in insects.  A majority of the major constituents of selected essential oils tested in the present study were reported to be metabolized through phase I detoxification processes.  Oxidation of 1,8-cineole to (+)-2-hydroxycineole by the pyrgo beetle, Paropsisterna tigrina (Southwell et al. 1995), limonene by the tobacco cutworm (Miyazawa et al. 1998), and citral (Berenbaum 1995) and p-cymene (Boyle et al. 2000) by mammals have been reported.  Likewise, camphor can be hydroxylated by the tobacco cutworm (Miyazawa et al. 2004).  Hydroxylated thymol was found in human urine as well, but phase II associated metabolites including thymol glucuronide and thymol sulphate were also identified (Thalhamer et al. 2011).  On the contrary, in the tobacco cutworm and the cabbage looper, another phase II process (glycosylation) was observed, with the major metabolite of thymol identified as thymol 3-O- -glucoside, a combined form with a hexose-type sugar (Passreiter et al. 2004). 108  Among those detoxifying enzymes mentioned, P450s are considered to play a key role in plant-insect interactions (Després et al. 2007).  In this study, the inhibition of P450s by PBO tended to produce the greatest increases in toxicity of individual compounds and their combinations, consistent with previous reports that oxidation is the most common metabolic process for detoxication of plant secondary metabolites.  However, other enzyme inhibitors, though not implicated in the metabolism of plant secondary metabolites to date, might also contribute to the increased toxicity.  This could be due either to cross-inhibition by the enzyme inhibitors or their involvement in metabolism not yet identified.  For example, although not strong, PBO inhibited not only P450s, but esterases as well, synergizing toxicity of insecticides on resistant strains through elevated levels of esterases (Young et al. 2006; Bullangpoti et al. 2012).  But this phenomenon requires further investigation as PBO might simply contribute to toxicity of the insecticides regardless of the type of resistance by inhibiting P450s.  More knowledge and supporting evidences are required to understand the full involvement of enzyme inhibitors and interpret them correctly.  For instance, the toxicity of thymol was increased significantly when mixed with EA, which is generally regarded as a GST inhibitor.  However, thymol was previously reported to be metabolized mainly through glycosylation in the cabbage looper (Passreiter et al. 2004), suggesting that either EA can inhibit other enzymes involved in phase II detoxification, or thymol can also be detoxified by GST.  Moreover, other compounds also showed increased mortality when specific enzymes, whose involvement in detoxication is unclear, were inhibited.  Amongst the major constituents tested, toxicity of camphor was influenced the most by inhibitors, and limonene the least.  In the GC-MS analyses of their penetration in Chapter 3, I observed that 109  the metabolism of limonene started almost immediately (< 10 min), and because the metabolite, limonene-1,2-diol, was inactive, I expected to see decreased toxicity.  Limonene metabolism may be complex in the cabbage looper, with less dominant involvement of a single metabolic process, relative to that for other compounds.  In this study, some mild enzyme inhibitory activities were observed, but were inconsistent with detoxicative metabolism as a mechanism of synergy among essential oil constituents.  For the particular synergistic interactions I have observed, inhibition of metabolism appears to be largely irrelevant or a minor factor at most.  Further investigation is warranted however, as there are some inconsistent earlier findings.  For example, Kumrungsee et al. (2014) reported induced GST activity in the diamondback moth, Plutella xylostella, when 1,8-cineole and thymol were applied both in vivo and in vitro.  On the other hand, Ramirez et al. (2012) reported that thymol can inhibit P450s prepared from rabbit liver microsomes by 86.9%, and Waliwitiya et al. (2012) found that thymol inhibited mosquito P450s, although only at the highest concentration tested (100 mg L-1) and longest exposure time (16 h).  Neither induced activity of GST by 1,8-cineole and thymol nor inhibition by thymol on microsomal protein of the cabbage looper were observed in this study, although thymol showed inhibition of GST activity.  My results could be due to a relatively short exposure time (6 h) and the low dose applied (LD20), or by different response patterns among insect species, which require further study.  Nevertheless, what is interesting is the relatively lower enhancement of toxicity by the enzyme inhibitors to the binary mixtures of the major oil constituents compared to the toxicity of individual ones, and the differences in mortality between the application methods for inhibitors.    110  Chapter 5: Summary and discussion   5.1 Insecticidal activity of essential oil constituents In the present study, the chemical compositions of rosemary, thyme and lemongrass oils were analyzed and the in vivo insecticidal and in vitro cytotoxic activities of their major constituents were evaluated.  Also, the contributions of each constituent to overall activities of the oils were examined.  Based on the application methods, the bioactivities of essential oils were produced either by the actions of the most abundant constituents or by combined effects of the major compounds.  For example, contact toxicities following topical administration with thyme and lemongrass oil were achieved through the individual activities of thymol and citral, respectively, whereas the fumigant toxicities of the oils were produced by the binary mixtures of thymol/p-cymene and citral/limonene, respectively. Cytotoxicity assays are potentially a convenient tool for screening novel insecticidal agents, but in the present study, only weak correspondence with in vivo contact toxicity was observed, consistent with some earlier reports (Decombel et al. 2004; Rasikari et al. 2005).  Such assays need to be combined with other screening tools or else caution should be exercised in extrapolating to insecticidal action, since cytotoxicity may not be the main mode-of-action in insects for many essential oils.  The rapid action of essential oils against some insect species is indicative of a neurotoxic mode of action, with some evidence of interactions with the neuromodulator octopamine and GABA-gated chloride channels (Isman 2006).  Also, several 111  essential oils and monoterpenes have been shown to inhibit AChE (Abdelgaleil et al. 2009; Qin et al. 2010). Of the major constituents of the oils in the present study, Zhukovskaya (2007) found that 1,8-cineole can stimulate the response of pheromone sensitive sensilla of American cockroaches, Periplaneta americana, receptor cells which are modulated by octopamine.  Bonnafé et al. (2014) reported that treatment with thymol led to decreased gene expression of an octopamine receptor in the honeybee, Apis mellifera and Price and Berry (2006) found some similarity in action between citral and octopamine, as both reversibly increased the frequency of spontaneous foregut contractions (at lower dose of citral) in cockroaches.  Thymol significantly increased 36Cl- uptake induced by GABA in American cockroach nerve cord (Tong and Coats 2010), and decreased wing beat frequency of the blowfly, Phaenicia sericata, showing a similar pattern to that observed with GABA (Waliwitiya et al. 2010), indicating positive modulation of GABA receptors by thymol.  Moreover, in a study of housefly GABA receptors, 1,8-cineole, thymol and camphor showed indications of significant binding to insect GABA receptors, but their binding pattern to [3H]-TBOB, the picrotoxin binding site, was different, suggesting camphor has a different site-of-action than the other two compounds (Tong and Coats 2012).  1,8-Cineole showed inhibitory activity towards both human and insect AChE (Perry et al. 2000; Savelev et al. 2003; Abdelgaleil et al. 2009), whereas camphor, p-cymene and limonene did not (Miyazawa et al. 1997; Miyazawa and Yamafuji 2005; López and Pascual-Villalobos 2010). On bovine erythrocyte AChE the mixture of 1,8-cineole and camphor had an antagonistic effect (Savelev et al. 2003).  112  5.2 Synergistic interactions and mechanisms Plant essential oils have been regarded as a fascinating research subject by many disciplines because of their wide range of bioactivities including insecticidal, antimicrobial, therapeutic and medicinal effects.  The activity of an essential oil will sometimes depend on that of its major constituent, but the opposite is also possible, i.e., the overall activity of the oil cannot be explained by the sum of the activities of individual constituents, indicating synergistic or antagonistic effects.  Recent research on Lippia sidoides oil showed that although thymol constituted 85% of the oil, the intact oil showed antimicrobial activity superior to that of pure thymol (Veras et al. 2012).  Similar reports of partial or incomplete activity of individual major constituents of essential oils on microorganisms and insects suggest synergy with minor constituents of those oils (Hendry et al. 2009; Jiang et al. 2009; Vilela et al. 2009; Birkett et al. 2011; Akhtar et al. 2012).  Synergy of essential oils can be also found between essential oils (Fu et al. 2007) or terpene compounds (Vuuren and Viljoen 2007; Gallardo et al. 2012), even in their interactions with synthetic antimicrobials (Amber et al. 2010), insecticides (Tong and Bloomquist 2013), or other natural organic and inorganic materials such as amphotericin B (Silva et al. 2011), Bacillus thuringiensis (Chang et al. 2014), diatomaceous earth (Islam et al. 2010) and vanillin (Kwon et al. 2011; Kim et al. 2012). In the present study, several synergistic interactions (in terms of larval toxicity) among the major constituents of each essential oil were found, including between the two most abundant compounds (1,8-cineole and camphor / thymol and p-cymene / citral and limonene).  In particular, 1,8-cineole and camphor showed limited toxicity individually applied following their natural proportions in the oil, but binary mixtures were synergistic in both topical and fumigant 113  bioassays to the cabbage looper.  1,8-Cineole, the major constituent of rosemary and eucalyptus (Eucalyptus globus) oils, showed interesting combinatory effects in several areas.  Synergistic bioactivity was reported when it was mixed with aromadendrene (Mulyaningsih 2010), chlorhexidine digluconate (Hendry et al. 2009), limonene (Vuuren and Viljoen 2007), or other minor constituents of essential oils (Vilea et al. 2009; Mulyaningsih et al. 2010).  Likewise, the synergistic effect of camphor with other compounds has also been reported (Viljoen et al. 2003; Maggi et al. 2011), and Pavela (2014) recently reported the notable insecticidal boosting effect of camphor with other terpene compounds.  Despite vigorous efforts to understand the mechanism, we still know little about how synergy is produced.  From the pharmaceutical point of view (Wagner and Ulrich-Merzenich 2009; Langeveld et al. 2014), the synergy mechanism was suggested as (1) a multi-target effect in which compounds target different sites, (2) pharmacokinetic or physicochemical effects on improved solubility or bioavailability, or (3) interactions of agents with resistance mechanisms.  Since plant essential oils are usually complex mixtures of compounds, they can be expected to have multiple modes of action, and increased penetration by enhanced solubility might also be involved as the constituents often have a wide range of polarities.  In the present study, enhanced penetration of compounds in binary mixture through the insect cuticle was observed, and bioassays support that phenomenon as a possible synergy mechanism in these particular combinations.  Penetration-enhancing effects of terpene compounds have been reported in the pharmaceutical field.  Although the chemical compositions and physical constructions of human (or mammalian) skin and the integument of insects are quite different, they share the same biological functions of 114  protecting the body from xenobiotics and reducing evaporative water loss.  The principal barrier to topical drug delivery in humans is the stratum corneum (SC), a complex mixture of lipids embedded in an intercellular matrix of dense dead cells (Williams and Barry 1991; Aqil et al. 2007).  On the other hand, the insect integument can be considered a two-phased structure, with lipophilc (epi- and exocuticles containing lipids, lipoprotein and protein) and hydrophilic (endocuticle, a chitin-protein complex) layers (Yu 2008b).  It is generally accepted that hydrocarbon or nonpolar terpenes are considered good penetration enhancers of lipophilic drugs in human skin, whereas polar terpenes provide better enhancement of hydrophilic ones (Williams and Barry 1991).  In particular, some monoterpene compounds including 1,8-cineole, limonene and thymol were proven to be promising penetration enhancers of several drugs including curcumin (Fang et al. 2003), 5-fluorouracil (Gao and Singh 1997), mefenamic acid (Heard et al. 2006), melatonin (Kanikkannan et al. 2004) and zidovudine (Narishetty and Panchagnula 2005) either in vivo or in vitro.  The penetration-enhancing mechanism of 1,8-cineole and thymol was suggested to be achieved by making the SC lipids less ordered (i.e., increasing lipid fluidity) (Gao and Singh 1997; Narishetty and Panchagnula 2005).  Insecticide penetration enhancers have been relatively less explored than pharmaceutical ones, but it has been observed that some resistant strains of insects have a thickened cuticle layer that could delay the penetration of insecticides as one resistance mechanism (Ahmad et al. 2006; Lin et al. 2012).  In a previous report of from this laboratory, the pharmacokinetic fate of thymol with different carriers including 1,8-cineole and rosemary oil was examined using radiolabelled thymol as a tracer, but there were no apparent trends related to the synergies observed (Wilson and Isman 2006).  The present study may elucidate the question of insecticidal synergy mechanism in terms of cuticular penetrations more clearly using simpler test methods. 115  Although no inhibitory activity on detoxifying enzymes from synergistic combinations or even the essential oils themselves was found in the present study, it cannot be ruled out as a possible synergy mechanism for essential oils.  Plant essential oils and terpene compounds showed some promise in addressing current insecticide resistance problems.  Some essential oils can markedly inhibit resistant strains of gram-negative pathogenic bacteria, such as Klebsiella pneumonia (Orhan et al. 2011) and Escherichia coli (Si et al. 2008).  Other reports show that although essential oils were less potent than conventional insecticides against susceptible cockroaches, the oils showed consistent toxicity to resistant strains as well, indicating their different modes-of-action (Chang et al. 2012).  Tong and Bloomquist (2013) observed inhibitory activities of essential oils on detoxifying enzymes including P450s and esterases, which are well-known to be related to the development of resistance to conventional pesticides, as well as showing synergistic toxicities with synthetic pesticides to the yellow fever mosquito, Aedes aegypti.  Since most essential oils are complex mixtures, they themselves might also impede the ability of insects or other organisms to develop resistance.  For example, when pure azadirachtin, the major insecticidal constituent from the Indian neem tree, Azadiracta indica, or a refined neem seed extract at the equivalent amount of azadirachtin were repeatedly sprayed on the green peach aphid, Myzus persicae, only the pure compound-treated line showed 9-fold resistance to the compound after 40 generations (Feng and Isman 1995).  Diffused selection of resistance by blending with other constituents instead of a single active ingredient can be a quite useful strategy which is not limited to botanical insecticides.  Transgenic broccoli having two different Bacillus thuringiensis (Bt) toxins showed slower development of resistance in lepidopteran insects than to a transgenic variety having a single Bt toxin (Gershenzon and Dudareva 2007).  116  Although more evidence is necessary, based on a comprehensive understanding of synergy interactions and their mechanisms, proper utilization of synergy may result in reducing the required amounts of biological agents, but also delaying resistance development in insect pest populations.  5.3 Future directions Plant essential oils tend to have complex chemical compositions, and these can vary due to many factors.  When using essential oils, investigators are encouraged to ensure that oils they use are representative in general (for example, obtained from many sources and artificially mixed) and make an effort to chemically characterize the oils, allowing for comparisons with previous (or future) reports.  This fundamental information, along with understanding the bioactivity of individual constituents and their roles in overall activity, is vital to determining the merits of further research on efficacy, formulation or commercialization as botanical insecticides. In terms of insecticidal synergistic activity of essential oil constituents, the present study revealed a penetration-enhancing effect as a synergy mechanism for the first time.  Although I made my best effort to be thorough, I have several other ideas for further research to validate this finding as well as other topics related to synergy research; these are outlined below.    117  1) Fast and reliable method to determine synergy Since synergistic interactions were determined through comparisons based on preceding determinations of LD50 values of individual compounds and their mixtures, this is a time- and effort-consuming task.  Although a simpler statistical model was introduced in Chapter 3 (i.e., Finney’s calculation), it has several limitations and requirements. Development of a more reliable and less restrictive method would be beneficial for synergy screening.  2) Development of an in vitro penetration model The penetration monitoring was conducted in vivo in the present study.  Since there were some indications of metabolism of compounds even at the earliest time of observation (for example, citral, limonene and p-cymene at 10 min), avoidance of metabolic processes through development of an in vitro cuticle model would give a clearer picture with which to examine this penetration-enhancing effect of synergistic combinations.  3) Quantitative research on metabolism Along with the development of an in vitro cuticle model, further study of metabolic processes involved in synergistic combinations is also important.  Particularly for the thymol+p-cymene combination, for example, the present study failed to determine with certainty whether the increased amounts of p-cymene at 1 h and thymol at 8 h in the binary mixture were due to increased penetrations or inhibited metabolism accomplished by the other compounds that may 118  have penetrated into the larvae.  A quantitative study of metabolic processes could help to elucidate the interpretation of these observations.  4) Characterization of the penetration-enhancing effect Although increased solubility of camphor was evident when in the binary mixture with 1,8-cineole, and it increased penetration through the cuticle of the cabbage looper along with enhanced affinity for a wax layer, other combinations such as the binary mixture of citral and limonene showed increased penetration of both compounds.  This suggests the presence of other biological and chemical reactions, warranting further investigations of penetration.  Research on physical bonding, chemical solubilization and optical observation will facilitate our interpretation of results of analyses of actual quantities of toxicants penetrating the cuticle.  5) Screening of detoxification inhibitions by essential oils As shown in Chapter 4, not only synthetic insecticides, but also essential oils and their constituents can show enhanced toxicity when the detoxifying enzymes of insects are successfully inhibited.  The screening and selection of natural and promising enzyme inhibitors can aid the development of not only safer alternatives to chemical insecticides and enzyme inhibitors, but also more efficacious botanical insecticides.   119  6) Mode-of-action studies Finding new modes-of-action of essential oils or determining their target sites were not main goals of in this study.  However, synergy can be also accomplished by different modes or sites-of-action, so this area can be another fascinating research subject.  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Major constituents of rosemary oil  CH3CH3CH3OH CH3CH3CH3 CH2OHCH3CH3CH3 CH3CH3CH2CH3 O thymol p-cymene linalool caryophyllene oxide  Figure A.2. Major constituents of thyme oil  CH3CHOCH3CH3  CHOCH3CH3CH3 CH3CH3CH2 CH3CH3CH3OH CH3CH3CH3O CH3O (geranial) (neral) limonene geraniol geraniol oxide      citral     Figure A.3. Major constituents of lemongrass oil 144  Appendix B: Standard curves   Figure B.1. Florescence intensity standard curve of viable T. ni ovarian cells.  In the range from 5.6  104 to 7.4  105 cells mL-1, it showed good correspondence between the number of cells and florescence intensity (R2 = 0.9716).   Figure B.2. Standard curves of 1,8-cineole and camphor for GC-MS quantification.  145   Figure B.3. Standard curve for protein quantification via Bradford assay.   

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