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Direct and indirect effects of host plant selection on larval performance in the cabbage looper, Trichoplusia… Shikano, Ikkei 2009

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   DIRECT AND INDIRECT EFFECTS OF HOST PLANT SELECTION ON LARVAL PERFORMANCE IN THE CABBAGE LOOPER, TRICHOPLUSIA NI   by  Ikkei Shikano  BSc., University of British Columbia, 2006    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  The Faculty of Graduate Studies  (Plant Science)           THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  September 2009  © Ikkei Shikano, 2009      ii  Abstract   Generalist insects should possess the ability to rank potential host plants for the suitability of their offspring. The decision to oviposit on a host plant that is inferior for larval development could have significant consequences for their offspring. Host plant quality can affect larval development and survival directly via nutrients and/or defensive chemicals, as well as indirectly by influencing larval condition and consequently their susceptibility to pathogens. In this thesis I examine the relationship between host preference and plant quality for larval performance in the cabbage looper, Trichoplusia ni (Hubner). I also examine the relationship between host plant quality, insect condition, immune responsiveness and resistance to pathogens.  No-choice, two-choice, and multiple-choice experiments were performed to measure adult oviposition preference and neonate larval preference for six plant species. Two baseline and induced immune parameters, haemocyte numbers and haemolymph phenoloxidase (PO) activity, were estimated for larvae on two host plants, broccoli and cucumber. Haemolymph protein concentration was assessed as an indication of insect condition, and the susceptibility of larvae to T. ni single nucleopolyhedrovirus (SNPV) was used as a measure of disease resistance. Trichoplusia ni adults and larvae both preferred the same two plant species out of six that maximised larval performance. Larvae however, also correctly identified anise hyssop as a suitable host, whereas adults did not, indicating that larval diet breadth can be wider than adult host range. Larval development, survival and condition were much higher when larvae were reared on broccoli than on cucumber. Haemocyte numbers were significantly higher in broccoli- reared larvae, whereas PO activity was not. An immune challenge induced significantly elevated numbers of haemocytes for larvae reared on both hosts, but did not affect PO activity or protein concentrations. Susceptibility to T. ni SNPV was significantly greater in larvae reared on cucumber than on broccoli. These results clearly indicate that T. ni are capable of ranking the suitability of plants for larval performance. Larval development on an inferior host plant can affect both immune response and disease resistance indicating that bottom-up effects could be important in interactions between insects and entomopathogens.                    iii  Table of Contents  Abstract…...................................................................................................................................... ii Table of Contents ......................................................................................................................... iv List of Tables ................................................................................................................................ vi List of Figures.............................................................................................................................. vii Acknowledgements .................................................................................................................... viii Co-authorship Statement ............................................................................................................ ix 1 Introduction........................................................................................................................... 1 1.1 Host plant selection......................................................................................................1 1.2 Effects of host plant quality on larval performance.....................................................5 1.3 Thesis theme and objectives ........................................................................................9 1.4 Study insect................................................................................................................10 1.5 References..................................................................................................................11 2 Relationship between Adult and Larval Host Plant Selection and Larval Performance in the Generalist Moth, Trichoplusia ni ................................................................ 18 2.1 Introduction................................................................................................................18 2.2 Materials and methods ...............................................................................................21 2.21 Insects. ...................................................................................................................... 21 2.22 Plants......................................................................................................................... 21 2.23 Measuring oviposition response ............................................................................... 22 2.24 Measuring neonate response ..................................................................................... 23 2.25 Growth inhibition...................................................................................................... 25 2.26 Larval performance................................................................................................... 26 2.27 Statistical analyses .................................................................................................... 26 2.3 Results........................................................................................................................27 2.31 No-choice tests.......................................................................................................... 27 2.32 Two-choice tests ....................................................................................................... 28 2.33 Multiple-choice tests................................................................................................. 29 2.34 Growth inhibition...................................................................................................... 29 2.35 Larval performance on whole leaves ........................................................................ 30 2.4 Discussion..................................................................................................................32 2.5 Acknowledgements....................................................................................................37 2.6 References..................................................................................................................38 3 Indirect Plant-Mediated Effects on Insect Immunity and Disease Resistance in a Tritrophic System ................................................................................................... 45 3.1 Introduction................................................................................................................45 3.2 Materials and methods ...............................................................................................49 3.21 Plants and insects ...................................................................................................... 49 3.22 Larval performance................................................................................................... 49 3.23 Testing for condition, immunity and disease resistance ........................................... 49 3.24 Escherichia coli challenge ........................................................................................ 50 3.25 Phenoloxidase and protein assays............................................................................. 51 3.26 Haemocyte count ...................................................................................................... 52 3.27 Virus bioassay........................................................................................................... 52 3.28 Statistical analysis..................................................................................................... 53 3.3 Results........................................................................................................................53 iv  3.31 Insect performance.................................................................................................... 53 3.32 Insect condition: protein levels ................................................................................. 54 3.33 Humoral defence response: phenoloxidase............................................................... 54 3.34 Cellular defence response: number of haemocytes................................................... 55 3.35 Disease resistance: virus bioassay ............................................................................ 57 3.4 Discussion..................................................................................................................58 3.5 Acknowledgements....................................................................................................62 3.6 References..................................................................................................................63 4 Summary & Conclusions.................................................................................................... 68 4.1 Summary ....................................................................................................................68 4.2 Conclusions................................................................................................................75 4.3 References..................................................................................................................79                         v  List of Tables   Table 2.1    Larval acceptance and numbers of eggs laid by female moths in no-choice tests..... 27 Table 2.2    Number of eggs laid in different locations in no-choice tests on cabbage, tomato, and geranium. .................................................................................................................. 28 Table 2.3    Percent larvae on leaf disks, and percent eggs laid on leaves in a two-choice test against cabbage ......................................................................................................... 28 Table 2.4    Percent larvae on leaf disks, and percent eggs laid on leaves in a multiple-choice... 29 Table 2.5    Larval performance on whole leaves ......................................................................... 31 Table 3.1    Mean growth rate, pupal weight, and percent survival of T. ni larvae on broccoli and cucumber................................................................................................................... 54 Table 4.1    Comparison of immune factors in uninduced T. ni larvae fed on broccoli or cucumber and changes in immune factors following induction by E. coli challenge ................................................................................................................................... 72                              vi  List of Figures   Figure 2.1  Multiple-choice oviposition cage set-up .................................................................... 23 Figure 2.2  Neonate larval multiple-choice test set-up. ................................................................ 25 Figure 2.3  Growth of larvae reared on artificial diet treated with 1000ppm of crude plant extracts .......................................................................................................................30 Figure 3.1  Baseline and induced cellular and humoral defence responses of T. ni larvae reared on broccoli and cucumber......................................................................................... 56 Figure 3.2  Variation in infectivity of T. ni SNPV to T. ni larvae reared on two different host- plants ......................................................................................................................... 58                               vii  Acknowledgements   I take this opportunity to thank my supervisor Dr. Murray B. Isman for first providing me with a position to work in his laboratory as an undergraduate student, and subsequently as a graduate student. Without his help and encouragement I would likely not be in this position today. I would also like to thank him for financial support throughout my project.  Many thanks to Dr. Yasmin Akhtar who continually provided me with research advice and mentored me to be a scientist during my years at UBC. Her mentorship and friendship has been and continues to be very important to me.  I would also like to thank Dr. Judith H. Myers and Dr. Jenny S. Cory for their research advice and supervision during this project. I have developed a strong work ethic and gained substantial research experience due to their training.  My deepest appreciation goes out to Dr. Catherine Rankin and Dr. Sheila Fitzpatrick who supported and advised me through difficult times during my project.  Lastly, I would like to thank all of my colleagues and friends at UBC, Jerry Ericsson, Saber Miresmailli, Tara Moreau, Cristina Machial, Dan Badulescu, Veronica Robertson, Nancy Brard, Karmen Scott, Rita Seffrin, John Jiang, Andrea Stephens, Michelle Franklin, Michelle Tseng, and Kate Menzies for their help and support.  This project was in part funded by Natural Sciences and Engineering Research Council of Canada Post Graduate Scholarship-Masters (NSERC PGS-M).                      viii  Co­authorship Statement   Chapter 2  I expanded the initial research design proposed by Yasmin Akhtar. I reared all of the insects and performed all of the bioassays with some assistance from Yasmin Akhtar. I performed all of the statistical analyses and prepared the original manuscript which was edited by Murray B. Isman.  Chapter  3  I participated in expanding the initial research design proposed by Jenny S. Cory and Judith H. Myers. I reared all of the insects, performed the virus bioassays, and aided Jerry D. Ericsson in haemocyte counting and molecular analyses. I performed some statistical analyses which were significantly supplemented by Jenny S. Cory. I then prepared the original manuscript which was then revised and edited by Jenny S. Cory, Judith H. Myers, and Jerry D. Ericsson.          ix  1  1  Introduction   Adult females of many phytophagous insects evaluate host plant suitability for the development of their offspring. Host plant suitability is most often determined based on host chemistry or existing feeding pressures. For most lepidopterans, the ability of females to locate and oviposit on suitable host plants is especially important, as the neonate larvae of many species are incapable of finding new hosts (Feeny et al. 1983; Singer 1986). The oviposition site can influence the growth and development of the offspring, and consequently their susceptibility to pathogens. The nutritional and chemical properties of plants can directly and indirectly influence the interactions between insects and their pathogens (Duffey et al. 1995; Cory & Hoover 2006).  1.1  Host plant selection   Host plant selection is a complex process that involves many factors that may impact on larval fitness. It involves a three-link chain of events used for host plant finding and host plant acceptance. The first link is directed by cues from volatile plant chemicals, the second link by visual stimuli, and the final link from non-volatile plant chemicals (Finch & Collier 2000). When flying insects search for appropriate oviposition sites, they determine the suitability of the plants they are passing over by evaluating the volatile chemicals emanating from the plants. These volatiles do not necessarily give accurate directional information. Instead they provide enough stimulation to arrest the flight of insects and encourage them to land (Finch & Collier 2000). For example, the cabbage looper moth Trichoplusia ni responds to the odour of host plants with upwind-oriented flight toward the plant or odour source (Landolt 1989). Once the insects locate the vicinity of a host plant using volatile chemicals, they use visual stimuli to decide where to land. The decision to land via visual stimulation can be affected by leaf shape (Hirota & Kato 2001), leaf colour (Smallegange et al. 2006), the density, diversity and distribution of vegetation surrounding the host plants (Rausher & Papaj 1983), host abundance (Heisswolf et al. 2005), and the plant part selected (Holland et al. 2004). Upon landing, the insects will determine the suitability of the plant for oviposition based on the chemical constituents of the plant, such as nutrients and plant defence chemicals (Renwick 2001). The decision is seldom based on one or two key stimuli. Instead, it often involves evaluating a combination of many stimulatory and inhibitory plant chemicals acting together (Schoni et al. 1987). Physical characteristics of the leaf surface such as the presence or absence of hairs and/or the types of hairs also play a significant role in influencing host acceptance by phytophagous insects (Juniper & Southwood 1986; Andres & Connor 2003). Optimum oviposition theory, also known as the preference-performance hypothesis, predicts that the preference of ovipositing females should correlate with host suitability for offspring performance (Jaenike 1978) since females are expected to maximize their fitness by ovpositing on high quality hosts. Offspring performance in this case refers to offspring survival, development, and reproduction, while oviposition preference refers to the hierarchical ranking of plant species by ovipositing females (Thompson 1988). The preference-performance hypothesis has been investigated in many studies with some finding strong positive correlations (e.g. Singer et al. 1988; Kouki 1993; Nylin & Janz 1996; Nylin et al. 1996). However, because the choice of ovipositing females and the performance of offspring can vary under different ecological conditions and selection pressures (Thompson 1988), correlations between adult oviposition preference and offspring performance is often weak or non-existent (e.g. Auerbach & Simberloff 2  1989; Fox & Eisenbach 1992; Singer et al. 1994; Berdegue et al. 1998; Jallow & Zalucki 2003; Rajapakse & Walter 2007). Optimal foraging could explain the lack of correlation between preference and performance, as many phytophagous insects use plants for both larval and adult feeding (Stephens & Krebs 1986; Scheirs & De Bruyn 2002). This theory predicts that herbivorous adult insects should prefer to feed on host plants that provide the highest adult performance (ie., realised fecundity) (Stephens & Krebs 1986). For example, females of the grass miner fly Chromatomyia nigra oviposit where they feed, which is consequently best for adult, not offspring, performance. Thompson (1988) summarizes four other general hypotheses of selection pressures that could act, jointly or independently, to influence host selection behaviour and possibly explain some of the poor correlation between oviposition preference and offspring performance: the time hypothesis, the patch dynamics hypothesis, the parasite/grazer hypothesis, and the enemy-free space hypothesis. The time hypothesis suggests that females may sometimes oviposit on newly introduced novel plants that are unsuitable for larval development, because there has been insufficient evolutionary time for selection to reduce the tendency for females to oviposit on these plants or to increase the ability of larvae to develop and survive on them (Wiklund 1975; Chew 1977; Legg et al. 1986). For example, Pieris napi females in the Rocky Mountains of Colorado oviposit on two introduced species that are fatal to larvae, Chorispora tenella and Thlaspi arvense, because they have glucosinolate profiles that are simlar to their indigenous host plants (Chew 1977; Rodman & Chew 1980). The patch dynamics hypothesis predicts that females will oviposit disproportionately more on hosts that they encounter more often. Thus, geographic variation in the abundance of 3  potential hosts can result in variations in host use and preference between populations (Wiklund 1981). As the structure of plant populations is not static, the probability of encountering a particular plant species may increase or decrease. Consequently, larval development and oviposition on a particular plant species may be ideal at one stage in local succession of a plant community, but that same plant species may become rare or less suitable for larval development at subsequent stages in local succession. Thus, the same plant species may not be consistently favoured, disrupting the relationship between preference and performance (Thompson 1988). The parasite/grazer hypothesis assumes that grazers (i.e., herbivores that can use more than one host plant to complete development) are likely to exhibit less host plant oviposition preference than parasitic herbivores (i.e., herbivores that complete development on one host plant)(Thompson 1982). Grazing insects may not follow the preference-performance hypothesis for many reasons. Females may prefer to oviposit on plants that are best for egg survivorship, even though survivorship and development in later instars may be better on other plant species. Or, if early larval development is not better on any particular host, females may not show any preferences. Some insects do not even lay eggs on any larval host plant. Furthermore, both the adults and immatures can express preferences for plant species (Thompson 1988). In some grazing insects, mixed-species diets are required for optimum development. In other words, survival and development would be poor on any one plant species, but significantly improves when reared on two or more plants species (MacFarlane & Thorsteinson 1980). The enemy free space hypothesis predicts that herbivorous insects may prefer poorer quality host plants if those hosts provide greater protection from natural enemies than higher quality hosts (Fox & Eisenbach 1992). The fitness gain resulting from that protection must outweigh any reduced fitness as a result of slower development on the poorer quality host 4  (Berdegué et al. 1996). The foraging success of natural enemies can be affected by morphological traits of host plants, such as foliar pubescence (Elsey & Chaplin 1978; Obrycki et al. 1983; Treacy et al. 1986; McAuslane et al. 1995) glandular trichomes (Obrycki & Tauber 1984; van Haren et al. 1987; Kauffman & Kennedy 1989; van Lenteren et al. 1995) waxy leaf surface (Eigenbrode et al. 1995; Eigenbrode et al. 1996), and plant architecture (Grevstad & Klepetka 1992) by either impeding or facilitating parasitoid and predator movement. Volatile chemicals released by plants can also affect foraging by natural enemies. Plants release specific herbivore-induced volatiles when infested by herbivorous insects (reviewed by Cortesero et al. 2000). Species vary in their chemical composition, and consequently differ in their ability to attract natural enemies of their herbivorous pests. For example, the parasitic wasp Diadegma semiclausum was equally attracted to brussel sprouts infested by its host Plutella xylostella and uninfested mustard plants (Bukovinszky et al. 2005). Plants can also influence the interactions between insects and their pathogens (Duffey et al. 1995; Cory & Hoover 2006). Plant architecture and leaf surface chemistry can affect pathogen persistence, the effect of an insect’s saliva on pathogen viability, and many direct interactions within the insect gut that can inhibit or enhance the infectivity of pathogens (e.g. Ali et al. 2004; Musser et al. 2005; Raymond et al. 2005). Both inter and intra-specific variation in plant quality have been shown to alter the levels of mortality and speed of kill by baculoviruses (e.g. Forschler et al. 1992; Ali et al. 1998; Raymond et al. 2002; Cory & Myers 2004)  1.2  Effects of host plant quality on larval performance    Host plant selection, in most Lepidoptera, is the responsibility of the ovipositing female as larvae are relatively immobile (Singer 1986). In fact, the site of emergence is so vital that the 5  choice of oviposition site can influence all aspects of larval performance (Resetarits 1993). Survival and development of the offspring is dependent on the quality of their host plant, with quality in this context referring to the nutritional and defensive compounds that positively or negatively influence insect performance (Awmack & Leather 2002). As nutritional components directly influence development, many herbivores will consume increased amounts of plant tissue to compensate for low nutrient concentration (Slansky & Feeny 1977; Price et al. 1980; Fajer 1989; Loader & Damman 1991). Defensive compounds found in poor or non-host plants are often toxic, antifeedant and/or repellent, and negatively affect insect performance (Awmack & Leather 2002). Negative effects of poor plant quality can include slower development, reduced adult biomass, and reduced fecundity (Awmack & Leather 2002), and can make the insect more susceptible to attack by parasitoids and pathogens (reviewed by Cory & Hoover 2006). On the other hand, chemicals found in suitable host plants have been extensively studied and shown to directly interfere with entomopathogen infection in the larval midgut (Cory & Hoover 2006). Many of these chemicals are allelochemicals, such as phenols and polyphenolics, which include tannins. They can cause baculoviruses to aggregate, as well as inactivate via the effects of redox cycling (e.g. Felton & Duffey 1990; Hoover et al. 1998). The main barrier for baculoviruses in the insect immune response is the midgut (Wilson et al. 2001) followed by cellular and humoral immune responses (Cory & Myers 2003). Studies with a recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) , Helicoverpa zea (a less susceptible species), and Heliothis virescens (a highly susceptible species) showed that the initial number of infection foci in the midgut was the same in the two insect species. Interestingly, the number of infection foci decreased in H. zea, suggesting that larvae were somehow able to clear the infections (Washburn et al. 1996). Haemocyte 6  aggregations, often containing capsules, were observed around infected tracheoles in association with decreasing infection foci. These capsules were identical to those attributed to the cellular immune response. The use of immunosuppressors reduced this response confirming the role of the cellular immune system (Washburn et al. 1996). Melanism has been associated with enhanced immune activity and disease resistance (Wilson et al. 2001). Melanin is a crucial component of insect defence; its biosynthesis is initiated by phenoloxidase (PO) (Hartzer et al. 2005). PO participates in three physiologically important biochemical processes: insect cuticle sclerotization (Sugumaran 1998), encapsulation and melanization of pathogens (Söderhäll et al. 1990; Ashida & Brey 1995; Gillespie et al. 1997), and wound healing (Lai-Fook 1966; Ashida & Brey 1998). For example, the African armyworm, Spodoptera exempta, exhibited an association between greater resistance to NPV infection and higher levels of PO activity in the haemolymph (Reeson et al. 1998). During encapsulation, the pathogens are enclosed in layers of haemocytes which then die, melanize, and harden.  This results in the isolation of the pathogen from the insect’s active circulation (Gillespie et al. 1997). Another important component of the humoral defense response is the production of microbial pattern recognition proteins and antimicrobial peptides from the fat body. Since they are also present on the surface of haemocytes, they are important in the cellular response as well. Drosophila injected with microorganisms differentially expressed genes encoding antibacterial and antifungal peptides (Lemaitre et al. 1997). Microbial pattern recognition proteins may include, hemolin, lectins, LPS-binding protein, peptidoglycan recognition protein, gram-negative bacteria recognition protein, and the thioester-containing protein αTEP1 (Bulet et al. 1999; Schmidt et al. 2001).  Hemolin is believed to have an important role in immune recognition and 7  modulation of defensive responses in Hyalophora cecropia and Manduca sexta (Gillespie et al. 1997). The cellular and humoral defense response is always active at a baseline rate, even in the absence of a pathogen (Gillespie et al. 1997). It is expected that this baseline rate would also differ if insect development takes place on host plants with differing nutritional qualities and defence chemicals. Evidence shows that both cellular and humoral defence responses can be induced to higher levels following pathogen attack (Gillespie et al. 1997). Thus, haemocyte numbers, antimicrobial protein levels, and PO activity are all expected to increase (Lemaitre et al. 1997; Wilson et al. 2001; Eleftherianos et al. 2006).  Host plants with poor nutritional quality or harmful defensive chemicals may result in less investment in immune factors resulting in higher susceptibility to pathogens or cause physiological changes that may influence the entry of pathogens via the mid-gut or the cuticle. The indirect effects of plant quality on insect susceptibility to pathogens have not been studied as extensively as the direct effects. In particular, those effects acting prior to pathogen exposure or after infection has been initiated.  For example, an infected insect’s likelihood of survival can be significantly influenced by the diet of the insect after pathogen challenge. In fact, the insect may have the ability to influence its own survival by altering its diet to take in more protein (Lee et al. 2006). Diet can also alter the structure of the peritrophic matrix (PM) in larval Lepidoptera. Such a change can have a significant effect on baculovirus infectivity (Plymale et al. 2008). Studies relating natural host plant quality to immunity and pathogen resistance are rare, although studies on the autumnal moth, Epirrita autumnata (Borkhausen) have demonstrated that intra-specific variation in host plant quality can alter immune defence in pupae (Klemola et al. 2007). 8  In conclusion, adult females of phytophagous species can oviposit preferentially based on many factors that can influence larval performance. The decision to oviposit on a host plant that is inferior for larval development could have consequences for their offspring. Larval development can be affected directly by host plant quality such as by nutrient concentration and defensive chemicals. Host plants can also negatively affect larval performance indirectly by influencing larval condition and consequently their susceptibility to parasitoids and pathogens.  1.3  Thesis theme and objectives   The overall theme of this thesis is to examine how accurately female T. ni are able to rank and oviposit on different plant species according to host plant quality, as determined by larval performance. I then examine whether the negative developmental effects as a result of rearing on an inferior host plant will affect larval immune response and larval susceptibility to a pathogen.  The major questions investigated in this thesis are: 1) Does host plant quality directly affect larval performance? 2) Are adult females and neonate larvae capable of ranking plant species according to larval performance? 3) Does the number of choices available influence the accuracy of adult and neonate choices? 4) Does larval host plant affect baseline and induced immune parameters such as haemocyte numbers, protein concentration, and phenoloxidase activity? 5) Does larval host plant quality influence susceptibility to T. ni single nucleopolyhedrovirus (T. ni SNPV)?  9  1.4  Study insect   The cabbage looper Trichoplusia ni Hubner belongs to the family Noctuidae of the order Lepidoptera. It is a major pest on cruciferous plants, but as it is a generalist insect, attacking plants as diverse as potato, tomato, bean, lettuce, spinach, nasturtium, and carnation. Larvae chew leaves, and can sometimes cause serious defoliation (Cranshaw, 2004). Adults are of moderate size with a wingspan of about 3 - 3.5 cm. It has a short life cycle of approximately 24 – 33 days from egg to adult. They are commonly found throughout North America, and thrive best in warmer climates. Adults are known to undergo annual migrations over long distances (Cranshaw, 2004).                         10  1.5  References   Ali M. I., Felton G. W., Meade T. & Young S. Y. (1998) Influence of interspecific and intraspecific host plant variation on the susceptibility of heliothines to a baculovirus. Biological Control 12: 42-49.  Ali M. I., Young S. Y. & McNew R. C. (2004) Host plant influence on activity of Bacillus thuringiensis Berliner against lepidopterous pests of crops. Journal of Entomological Science 39: 311-317.  Andres M. R. & Connor E. F. (2003) The community-wide and guild-specific effects of pubescence on the folivorous insects of manzanitas Arctostaphylos spp. Ecological Entomology 28: 383-396.  Ashida M. & Brey P. T. (1995) Role of the integument in insect defense: pro-phenol oxidase cascade in the cuticular matrix. Proceedings of the National Academy of Sciences of the USA 92: 10698-10702.  Ashida M. & Brey P. T. (1998) Recent advances in research on the insect prophenoloxidase cascade. In: Molecular mechanisms of immune responses in insects (eds. P. T. Brey & D. Hultmark) pp. 135-172. Chapman & Hall, London.  Auerbach M. & Simberloff D. (1989) Oviposition site preference and larval mortality in a leaf- mining moth. Ecological Entomology 14: 131-140.  Awmack C. S. & Leather S. R. (2002) Host plant quality and fecundity in herbivorous insects. Annual Review of Entomology 47: 817-844.  Berdegue M., Reitz S. R. & Trumble J. T. (1998) Host plant selection and development in Spodoptera exigua: do mother and offspring know best? Entomologia Experimentalis et Applicata 89: 57-64.  Berdegué M., Trumble J. T., Hare J. D. & Redak R. A. (1996) Is it enemy free space? - The evidence for terrestrial insects and fresh water arthropods. Ecological Entomology 21: 203-217.  Bukovinszky T., Gols R., Posthumus M. A., Vet L. E. M. & van Lenteren J. C. (2005) Variation in plant volatiles and attraction of the parasitoid Diadegma semiclausum (Hellen). Journal of Chemical Ecology 31: 461-480.  Bulet P., Hetru C., Dimarcq J. C. & Hoffmann D. (1999) Antimicrobial peptides in insects: structure and function. Developmental and Comparative Immunology 23: 329-344.  Chew E. S. (1977) Coevolution of pierid butterflies and their cruciferous food plants. II. The distribution of eggs on potential food plants. Evolution 31: 568-579.  11  Cortesero A. M., Stapel J. O. & Lewis W. J. (2000) Understanding and manipulating plant attributes to enhance biological control. Biological Control 17: 35-49.  Cory J. S. & Hoover K. (2006) Plant-mediated effects in insect-pathogen interactions. Trends in Ecology & Evolution 21: 278-286.  Cory J. S. & Myers J. H. (2003) The ecology an evolution of insect baculoviruses. Annual Review of Ecology, Evolution and Systematics 34: 239-272.  Cory J. S. & Myers J. H. (2004) Adaptation in an insect host-plant pathogen interaction. Ecology Letters 7: 632-639.  Cranshaw, W. (2004) Garden Insects of North America: The Ultimate Guide to Backyard Bugs. Princeton University Press, Princeton, NJ.  Duffey S. S., Hoover K., Bonning B. & Hammock B. D. (1995) The impact of host-plant on the efficacy of baculoviruses. In: Reviews in Pesticide Toxicology (eds. M. Roe & R. Kuhr) pp. 137- 275. CTI Toxicology Communications.  Eigenbrode S. D., Castagnola T., Roux M. B. & Steljes L. (1996) Mobility of 3 generalist predators is greater on cabbage with glossy leaf wax than on cabbage with a wax bloom. Entomologia Experimentalis et Applicata 81: 335-343.  Eigenbrode S. D., Moodie S. & Castagnola T. (1995) Predators mediate host-plant resistance to a phytophagous pest in cabbage with glossy leaf wax. 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Thus, optimal oviposition theory (Jaenike, 1978) predicts that oviposition preference should correlate with host plant suitability for the development of offspring, since females are expected to oviposit on high quality hosts to maximize their fitness (Awmack & Leather, 2002). This relationship between adult oviposition choice and offspring performance has been investigated by many studies using this notion (e.g. Courtney, 1981; Damman & Feeny, 1988; Legg et al., 1986; Nylin & Janz, 1993; Poykko, 2006; Rausher, 1982; Rausher & Papaj, 1983; Singer, 1984; Thompson, 1983; Wiklund, 1975). However, because the relationship between host choice and larval performance varies under different ecological conditions and selection pressures (Thompson, 1988), a positive correlation between adult oviposition choice and offspring performance is not always observed (e.g. Auerbach & Simberloff, 1989; Berdegue  1 A version of this chapter has been submitted for publication. Shikano, I., Akhtar, Y. & Isman, M.B. Relationship between adult and larval host plant selection and larval performance in the generalist moth, Trichoplusia ni. 18  et al., 1998; Fox & Eisenbach, 1992; Jallow & Zalucki, 2003; Rajapakse & Walter, 2007; Singer et al., 1994) The decision by a female to oviposit on a particular host plant is a complex process that involves many factors. One of the major factors is the chemical constituents of the host plant such as nutrients and secondary chemicals (Renwick, 2001). Suitability of a host plant is not always decided based on one or two key stimuli, but often involves evaluating a combination of many stimulatory and inhibitory plant chemicals acting together (Schoni et al., 1987). Physical plant characteristics, such as leaf size and texture also play a major role in influencing host acceptance by phytophagous insects (Harris & Miller, 1988; Miller & Strickler, 1984). Plant physical characteristics have been shown to significantly affect host selection behaviour in several studies using phytophagous fly species (Harris & Miller, 1983, 1984; Harris & Rose, 1990). Other factors involved in host selection that can potentially impact larval fitness include the density, diversity and distribution of vegetation surrounding host plants (Rausher & Papaj, 1983), host damage (Rostas & Hilker, 2002), host age (Leite et al., 2001), host abundance (Heisswolf et al., 2005), and the plant part selected (Holland et al., 2004). The quality of a host plant can significantly affect the growth and reproduction of phytophagous insects. Quality in this context refers to the nutritional and defensive compounds that positively or negatively affect the performance of herbivorous insects (Awmack & Leather, 2002).  Nutritional compounds such as nitrogen and carbon can directly limit the optimum growth of insects (Bernays & Chapman, 1994). Lower nutritional quality can result in  longer developmental time, lower adult biomass, and lower fecundity (Awmack & Leather, 2002). Defensive compounds have toxic, antifeedant and/or repellent properties that can negatively affect development (Awmack & Leather, 2002). Removal of these compounds from poor host 19  plants can result in faster development and lower mortality (Bosio et al., 1990). Low nutrient concentration and/or the presence of defensive compounds can also affect an insect’s interactions with its natural enemies. For example, it can make them more susceptible to successful attack by parasitoids or pathogens by altering insect condition (Cory & Hoover, 2006; Shikano et al., in press), and/or increase the length of exposure to predators and parasitoids by extending the host’s development time (Herrebout et al., 1963; Loader & Damman, 1991; Moran & Hamilton, 1980). Trichoplusia ni is an important pest of cruciferous plants, but also damages plants as diverse as potato, tomato, pea, lettuce, spinach, nasturtium, carnation, and various ornamentals (Cranshaw, 2004). It has been shown to respond to host plant odours with upwind oriented flights to the plant or odour source (Landolt, 1989). The final decision to oviposit or not is likely made based on chemosensory stimulation through tarsal receptors, as a non-volatile plant latex from Hoodia gordonii applied to the surface of cabbage leaves deters oviposition by T. ni moths (Chow et al., 2005). In the present study, I investigated the ability of both adults and larvae of a generalist insect, T. ni., to accurately rank six different plant species according to larval development. I also compared oviposition preferences to neonate larval acceptance and preference to determine whether the adult host range matched that of larval diet breath. Adult preferences were measured by the proportion or number of eggs laid on individual leaves, and larval preferences were measured by the proportion of neonate larvae choosing to feed on leaf disks. As I was unable to use whole plants for oviposition experiments, I cut leaves at the base of the stem just prior to the node. I realize that this could affect volatile emissions by the leaves, and potentially have an effect on adult host selection behaviours.  20  2.2  Materials and methods  2.21  Insects.   Trichoplusia ni (Lepidoptera: Noctuidae) were obtained from established laboratory colonies maintained for > 50 generations. T. ni  larvae were reared on artificial diet (No. 9795, Bioserv Inc., Frenchtown, NJ) supplemented with finely ground  alfalfa and vitamins (No. 8045, Bioserv Inc.), in a growth chamber at 24ºC and 16:8 LD photoperiod. Adult moths were provided with a 10% sucrose solution accessed by a cotton wick in a sealed Styrofoam cup.  2.22  Plants. Cabbage (Brassica oleracea; var. Stonehead), pak choi (Brassica rapa; var. Mei Qing Choi), and green bush bean (Phaseolus vulgaris; var. Spectacular) plants were routinely grown in 18-cell plastic flats, while tomato (Solanum lycopersicum; var. Redstone VF), anise hyssop (Agastache foeniculum) and geranium hybrid (Pelargonium x hortum; var. Multibloom Red) plants were routinely grown (for bioassays) in 1.89 litre plastic pots. All plants were grown in a mixture of sandy loam soil and peat moss (4:1) in the Horticulture Greenhouse at the University of British Columbia, Vancouver, BC, Canada. To avoid confusion, the plants will be referred to by their common names hereinafter. Cabbage, pak choi, tomato, and geranium plants used for bioassays and insect rearing were 6-8 weeks old, bean plants were 3-5 weeks old, and anise hyssop plants were 10-12 weeks old. Cabbage and pak choi were selected for this study to compare whether T. ni showed differential preference for plants in the same family that are known host plants. Tomato and bean were selected because T. ni is a known pest of these crops, but these plants are not in the family Brassicaceae. Lastly, geranium and anise hyssop were selected as T. ni has no known association with these plants.  21  2.23  Measuring oviposition response. Pupae of T. ni reared on artificial diet were sexed and kept in separate 1L plastic containers until emergence. Styrofoam inserts (24 cm length, 16.5 cm diameter) used for shipping 4 liter glass bottles were used as oviposition cages as described by Akhtar and Isman (2003). Pairs of moths (one male and one female) along with 10% sucrose solution (accessed via a cotton wick) were introduced into each cage. Leaves for the following experiments were placed into a plastic cup filled with water, then introduced into the cage. All leaves were rinsed with water prior to introduction. 1) No-choice tests. Oviposition preference for each plant was assessed by placing a single leaf (25 cm2) of one of the six plants into each cage. The number of eggs on the leaf was counted after 48 hrs. The numbers of cages set up were 39, 41, 47, 48, 45, and 47 for cabbage, pak choi, bean, tomato, anise hyssop, and geranium respectively. 2) Egg distribution. To determine whether low preference for some plants in the no-choice test was a result of egg laying on the walls of the cage rather than on the leaves, or whether the adults were retaining their eggs, all eggs – on and off the leaf – were counted. The inside walls of the oviposition cages were lined with white paper towel. I selected three plants that spanned the entire range from highly, to moderately, to least preferred (cabbage, tomato, geranium respectively). One leaf of one of these three plants was placed into each cage. After 48 hrs, the eggs on the leaf, the paper towel, the sugar solution container, and the plastic cup holding the leaf were counted. As both T. ni eggs and paper towels are white, the eggs on the paper towel were made visible by soaking the paper towels in water to make them transparent, then placing them on a black surface. The number of cages set up for each plant was 32. 22  3) Two-choice tests. Oviposition preference for each plant was compared against T. ni’s natural host plant, cabbage. One cabbage leaf and one leaf of one of the other five plants, both with a surface area of approximately 25 cm2 in size were placed into each cage. The numbers of eggs on each leaf were counted after 48 hrs. The numbers of cages set up were 45, 50, 50, 39, and 41 for pak choi, bean, tomato, anise hyssop, and geranium respectively. 4) Multiple-choice tests. Oviposition preference was measured by placing one leaf (25 cm2) from each of the six plant species in one cage (Fig. 2.1), for a total of 32 cages. The eggs on the leaves were counted after 48 hrs, and the proportion of eggs on each leaf were calculated.   Figure 2.1 Multiple-choice oviposition cage set-up.  2.24  Measuring neonate response. 1) No-choice tests. Neonate larvae were provided with a single leaf disk (1.77cm2) in the center of a 5cm diameter plastic Petri dish to determine the acceptability of that plant as a suitable host All leaf disks were cut from leaves that were rinsed with water. Twenty freshly hatched (<8 h old) neonate larvae were placed around the leaf disk (~1cm away). The lid was then placed on 23  the Petri dish, and the dish placed in a large plastic container lined with wet paper towels. The container was then placed in a growth chamber at 24°C and 16:8 LD. Acceptance was assessed after 24 hrs. As not all twenty larvae were located on the leaf disk, acceptance of the leaf disk as a food source was assessed based on observed feeding. The leaf disk was determined to be accepted if the larvae had changed colour from grey to green, which indicated consumption of the foliar material. A total of three Petri dishes were set up. 2) Two-choice tests. Acceptability of each plant was measured by comparing neonate larval preference for each of these plants to the natural host plant, cabbage. One cabbage leaf disk (1.77cm2) and one leaf disk from one of the other plants were placed in a 5 cm diameter plastic Petri dish approximately 1.5 cm away from each other. Twenty neonate larvae were placed in between the two leaf disks and the dishes were placed in a growth chamber as described above. The location of the neonates was recorded after 24 hrs. A total of nine Petri dishes were set up. 3) Multiple-choice tests. Six leaf disks, one from each plant were placed in a ring pattern along the edge of a 5 cm Petri dish (Fig. 2.2). Twenty neonate larvae were placed in the center of the ring and the dishes were placed in a growth chamber as described above. The location of the neonates was recorded after 24 hrs. A total of twelve Petri dishes were set up.        24   Figure 2.2 Neonate larval multiple-choice test set-up.  2.25  Growth inhibition Crude extracts of the plants were made by extracting foliage with MeOH using of a magnetic stirrer for 48 h. The extracts were vacuum filtered through Whatman filter paper No. 1 and evaporated in a rotovapor (Leatemia & Isman, 2004). Effect of crude extracts of the six plants on larval growth was determined as described in Akhtar and Isman (2004). To produce 20 g of treated artificial diet, a methanolic solution of extract was applied onto 3.5 g of dry artificial diet and allowed to dry in a fumehood for approximately 30 min. Following evaporation of the solvent, a mixture of boiling water (16 ml) and agar (0.5 g) was added to the diet and mixed thoroughly. Control diet was prepared with methanol alone. Once the diet cooled, each 20 g piece of diet was cut into 20 equal sized pieces. The pieces of diet were then placed into individual compartments of a plastic assay tray along with one freshly hatched neonate larva (<8 h old) (n=20). All larvae were individually weighed after 10 days. Mean larval weight from each treatment was presented as a percentage of the controls.  25  2.26  Larval performance   Each plant was assessed for its suitability for T. ni growth and survival. Approximately 2 g of whole leaves from each plant were gently folded into 20 individual 60 mL plastic cups. Each leaf was rinsed with water prior to introduction. One neonate larva was placed into each cup. Four pin-sized holes were opened on the lid of each cup for ventilation. The cups were placed in a growth chamber at 24ºC and 16:8 LD photoperiod. The cups were cleaned and the leaves changed every two days, and once per day from the fifth instar until pupation. The larvae were individually weighed after seven days. Larval weight after seven days, number of days until pupation, and pupal weight were all expressed as a percentage of those values for larvae reared on cabbage. Mortality was recorded throughout, and pupae were weighed two days after the onset of pupation (termination of feeding).  2.27  Statistical analyses   All comparisons of means between plants in no-choice, two-choice, and multiple-choice tests for larvae and adults were analyzed by Kruskal-Wallis one-way nonparametric ANOVA (Statistix 8). Measures of larval performance were analyzed by a general linear model ANOVA (general AOV/AOCV). Significances between means were obtained by Tukey’s tests. For mean larval weight, pupal weight, and days to pupation, the data from each replicate were pooled since there were no significant differences between replicates (Larval weight F2, 222 = 0.16, P = 0.86; Pupal weight F2, 160 = 0.95, P = 0.39; Days to pupation F2, 166 = 0.31, P = 0.73). An alpha level of 0.05 was used for all analyses.  26  2.3  Results  2.31  No­choice tests    Larvae accepted any leaf disk they were given in no-choice tests, except for a few larvae on geranium. Adult oviposition showed a ranking of plants with adults ovipositing the most on cabbage and pak choi leaves, followed by tomato and bean leaves. Anise hyssop and geranium leaves had the fewest eggs (Table 2.1; Kruskal-Wallis test: Larval acceptance, H [df = 5] = 16.88, P < 0.001; Number of eggs, H [df = 5] = 75.11, P < 0.001). To determine whether the reduced numbers of eggs laid were a result of females retaining their eggs or choosing to lay eggs elsewhere, I counted eggs laid on the leaves as well as on the walls of the cage and the plastic containers containing the leaf and sugar solution. I selected three plants that ranked highly preferred, moderately preferred, and least preferred (cabbage, tomato, and geranium respectively) in the previous no-choice oviposition test. I found that the total numbers of eggs laid by female moths did not differ significantly between the cages of the three plants. Females laid a significantly higher proportion of their eggs on cabbage leaves compared to tomato and geranium (Table 2.2; Kruskal-Wallis test: On leaf, H [df = 2] = 18.07, P< 0.001; Off leaf, H [df = 2] = 7.16, P = 0.03; Total, H [df = 2] = 0.05, P = 0.97).  Table 2.1 Larval acceptance and numbers of eggs laid by female moths in no-choice tests.   Cabbage Pak Choi Bean Tomato Anise hyssop Geranium Percent larvae on leaf disk 100.0 ± 0.0 a 100.0 ± 0.0 a 100.0 ± 0.0 a 100.0 ± 0.0 a 100.0 ± 0.0 a 91.7 ± 1.7 a Number of eggs on leaf 15.5 ± 3.2  a 14.9 ± 3.0 a 9.6 ± 1.4 ab 10.6 ± 1.8 ab 4.0 ± 0.9 b 2.9 ± 1.8 c  * Means ± SE within a row with different letters indicate significant differences (Kruskal-Wallis test, P < 0.05).  27  Table 2.2 Number of eggs laid in different locations in no-choice tests on cabbage, tomato, and geranium.   Cabbage Tomato Geranium On leaf 16.5 ± 2.5 a 8.2 ± 1.8 b 3.7 ± 0.9 b Off leaf 12.1 ± 2.2 a 20.2 ± 3.2 ab 22.8 ± 3.1 b Total number of eggs laid 28.6 ± 3.7  a 28.3 ± 3.9 a 26.5 ± 3.3 a  * Means ± SE within a row with different letters indicate significant differences (Kruskal-Wallis test, P < 0.05).  2.32  Two­choice tests   The results indicate that pak choi was equally preferred to cabbage in both larval and adult choice tests, whereas bean leaves were moderately chosen by both larvae and adults. There was a discrepancy in the larval and adult choices when evaluating preference for tomato and anise hyssop. Larvae significantly preferred anise hyssop over tomato, whereas adults preferred tomato over anise hyssop. Geranium was the least preferred by both larvae and adults (Table 2.3; Kruskal-Wallis test: Larval preference, H [df = 4] = 28.94, P < 0.001; Adult preference, H [df = 4] = 86.27, P < 0.001).  Table 2.3 Percent larvae on leaf disks, and percent eggs laid on leaves in a two-choice test against cabbage.   Pak Choi Bean Tomato Anise hyssop Geranium Larval choice (%) 50.6 ± 6.1  a 29.4 ± 4.7 ab 7.8 ± 3.0 b 34.4 ± 5.7 a 6.7 ± 2.9 b Oviposition choice (%) 53.6 ± 4.0  a 38.9 ± 4.9 a 35.1 ± 4.5 a 9.1 ± 3.4 b 7.9 ± 2.7 b  * Means ± SE within a row with different letters indicate significant differences (Kruskal-Wallis test, P < 0.05). 28  2.33  Multiple­choice tests   When presented with leaf disks from all six plants, the majority of larvae were found on cabbage, pak choi, and anise hyssop disks, followed by bean. There were significantly fewer larvae on tomato and geranium disks. Adult moths laid significantly higher proportions of eggs on cabbage and pak choi leaves when presented with six leaves simultaneously. Moderate proportions of eggs were laid on geranium, tomato, and bean, while anise hyssop received the fewest (Table 2.4; Kruskal-Wallis test: Larval preference, H [df = 5] = 52.53, P < 0.001; Adult preference, H [df = 5] = 54.45, P < 0.001).  Table 2.4 Percent larvae on leaf disks, and percent eggs laid on leaves in a multiple-choice test.   Cabbage Pak Choi Bean Tomato Anise hyssop Geranium Larval choice (%) 30.5 ± 3.5  a 29.2 ± 4.3 a 16.9 ± 1.9 a 0.4 ± 0.4 b 22.6 ± 3.5 a 0.4 ± 0.4 b Oviposition choice (%) 38.4 ± 5.7  a 33.4 ± 4.8 a 6.4 ± 1.9 b 9.7 ± 3.6 b 1.5 ± 0.7 c 10.6 ± 2.7 b  * Means ± SE within a row with different letters indicate significant differences (Kruskal-Wallis test, P < 0.05). 2.34  Growth inhibition   Mean weights of larvae reared on artificial diet containing crude extracts of tomato and geranium were significantly lower than those reared on extracts of the other plants (Fig. 2.3; Kruskal-Wallis test: Growth inhibition, H [df = 5] = 41.76, P < 0.001).      29  020 40 60 80 100 120 Cabbage Pak Choi Beans Tomato Hyssop Geranium G ro w th  re la tiv e to  c on tro l ( % ) a  ab  ab  bc  c  a    Figure 2.3 Growth of larvae reared on artificial diet treated with 1000ppm of crude plant extracts. Growth calculated as percent of growth relative to larvae reared on control diet. Mean growth ± SE with different letters indicate significant differences (Kruskal-Wallis test, P < 0.05). 2.35  Larval performance on whole leaves   When larvae were reared on whole leaves of the plants, the mean weight of larvae reared on pak choi was the highest relative to the weight of larvae reared on cabbage. Larvae on anise hyssop leaves had the second highest weight, followed by larvae on bean, and lastly by those on tomato (Table. 2.5; Larval weight F3, 221 = 168.5, P < 0.001). None of the larvae in the three replicates survived for seven days on geranium. In fact, none of the larvae survived passed the first larval instar. Number of days to reach pupation was calculated as more than or less than the number of days taken by larvae reared on cabbage to reach pupation. Larvae reared on pak choi reached pupal initiation significantly earlier than on the other plants and reached pupal initiation 30  slightly before cabbage-reared larvae. Anise hyssop-reared larvae were the next to pupate, followed by bean, then tomato (Days to pupation F3, 165 = 124.3, P < 0.001). Pupal weight was highest in pak choi and anise hyssop-reared larvae, and was equal to those reared on cabbage. Tomato-reared larvae weighed significantly less, but weighed more than those reared on bean (Pupal weight F3, 159 = 85.2, P < 0.001). Survival of larvae until successful pupal formation was greatest on cabbage and pak choi, followed by anise hyssop, then tomato and bean. No larvae survived to pupation on geranium (Larval survival F5, 14 = 61.1, P < 0.001). Bean-reared larvae showed the poorest survival during the pupal stage. However, this difference was not statistically significant (Pupal survival F4, 12 = 2.9, P = 0.07).  Table 2.5 Larval performance on whole leaves.  Cabbage Pak Choi Bean Tomato Anise hyssop Geranium Larval weight after 7 days (%) - 106.9 ± 4.8 a (60) 33.7 ± 2.8 c (50) 11.6 ± 0.9 d (56) 47.0 ± 2.6 b (59) - Days to pupation (± days) - -0.6 ± 0.1 a (56) +2.7 ± 0.4 c (31) +4.9 ± 0.2 d (36) +1.2 ± 0.1 b (46) - Pupal weight (%) - 100.6 ± 1.9 a (56) 59.4 ± 2.5 c (26) 83.5 ± 2.2 b (35) 103.8 ± 1.6 a (46) - Larval survival (%) 92.0 ± 2.5 a (100) 93.3 ± 4.4 a (60) 43.3 ± 6.0 c (60) 58.3 ± 8.8 bc (60) 76.7 ± 1.7 ab (60) 0.0 ± 0.0 d (60) Pupal survival (%) 100.0 ± 0.0 a (92) 100.0 ± 0.0 a (56) 83.1 ± 10.6 a (26) 97.8 ± 2.2 a (35) 100.0 ± 0.0 a (46) -  *Larval and pupal weight, and days to pupation are calculated relative to larvae reared on cabbage. Means ± SE (N) within a row with different letters indicate significant differences (Tukey’s test P < 0.05).   31  2.4  Discussion   The results indicate that both neonate larvae and adult moths identified and significantly preferred their two natural host plants from the family Brassicaceae, cabbage and pak choi, in all choice and no-choice tests. Bean leaves were moderately preferred in all tests for both larvae and adults. The only major discrepancy in choices between adults and larvae were between tomato and anise hyssop. Tomato leaves were moderately preferred by adults but not larvae, while anise hyssop leaves were preferred by larvae but not adults. The low preference of adults and larvae for geranium leaves is indicative of the lack of survivability on this plant. When presented with no choice, larvae accepted leaf disks from any plant, with the exception of a few larvae on geranium. Counting all eggs laid in the cage in the no choice oviposition test revealed that the preference for the plant influenced the location of the eggs laid, but not the total number.  The low preference for tomato by larvae appears to be related to growth inhibitory compounds found in these plants. Crude extracts of tomato significantly reduced the growth of T. ni larvae. Even when reared on whole leaves, larvae reared on tomato showed the slowest development and increased time to pupation. This slower development may be attributed to secondary chemicals that may have toxic or antifeedant properties or be of lesser nutritive quality. Plants can reduce the amount of nutrients available to herbivores by producing proteinase inhibitors, tannins, lignins, phenolic resins, silica, a hardened cuticula, spines, or by lowering nutrient content of tissues (Augner, 1995). The slower growth and prolonged larval stage can consequently lengthen the  exposure time of larvae to predators and parasitoids (Moran & Hamilton, 1980).  Although larval development was slower on tomato than bean, adult oviposition choices indicate almost identical preference between the two plants in three separate experiments. This is 32  likely due to the fact that while development time on tomato was significantly slower, pupal weight was significantly higher suggesting that there is a trade-off between the two developmental outcomes when selecting an oviposition site. Larger pupal size is indicative of higher fecundity in Lepidoptera (Awmack & Leather, 2002). Thus, selecting an oviposition site that increases offspring fecundity would ensure higher fitness for the ovipositing female. However, if a slower development time will potentially prolong exposure of her offspring to more predation and parasitism, than a plant that reduces offspring fecundity may become equally attractive if development time could be reduced.  Adult oviposition preference in two-choice and no-choice tests appears to correlate with offspring performance, with the exception of anise hyssop. In a multiple- choice test however, a high proportion of eggs were laid on geranium leaves which were the least preferred in the other tests. This “mistake” made by the females could be attributed to numerous factors. There is a distinct possibility that the presence of leaves from six plants being placed in one cage could result in a mixing of volatile chemicals. Thus, volatiles from some plants might “mask” those of others (Perrin & Phillips, 1978; Tahvanainen & Root, 1972). This “masking” effect has been observed in numerous studies using the cabbage root fly, where the fly will land only on the host when no other vegetation is present, but does not always land successfully on the host plant when other plants are present (Dempster, 1969; Finch & Edmonds, 1994; Kostal & Finch, 1994; Smith, 1976; Theunissen & den Ouden, 1980). However, the disruptive effect was successfully duplicated by green paper (Kostal & Finch, 1994; Ryan et al., 1980) and by plant models made from green cards (Kostal & Finch, 1994), suggesting that the flies will land on any green object in the vicinity of the host odour (Finch & Collier, 2000). 33  According to the ‘appropriate/inappropriate landings’ theory (Finch & Collier, 2000), when flying insects are in search of appropriate oviposition sites, they first utilize volatile chemicals emanating from the plants they are passing over to determine their suitability. The purpose of these volatiles is to provide enough stimulation to arrest the insects and encourage them to land, but not necessarily to provide accurate directional information. When host plants are not surrounded by other vegetation, insects will land on the host plant as there are no other green objects to land on. However, when other non-host vegetation is present, they will land in proportion to the relative areas occupied by their host plant and surrounding non-host plants. Upon landing, the insects will determine the amount of time they spend on the plant based on the acceptable or antagonistic stimuli received through their tarsal receptors. Once the insects reinitiate flight, they could be immediately stimulated again by volatiles after only a short distance, possibly landing on a host plant. Each landing on a suitable host plant will not necessarily provide sufficient stimulation for oviposition on the plant. Therefore, the complete system may involve finding and re-finding the host plant several times before enough stimulation is accumulated to induce oviposition. With this theory in mind, why then would an ovipositing female make a “mistake” when choosing between leaves from six different plants compared to choosing between two or having only one? As the theory suggests, T. ni are attracted to generalized plant odors that are redundant in many plant species. They are believed to follow these odors to the general vicinity of the plants (Landolt, 1993). Since an accumulation of acceptable stimuli are required to induce oviposition, when only one suitable host plant is present, landing on that plant will always elicit stimulation, thus resulting in oviposition. When only a poor host plant or non-host plant is present, the accumulation of stimuli will be slower resulting in fewer eggs laid in no-choice 34  oviposition tests. In a two-choice test, the likelihood of reaching the stimulus level required for oviposition when landing on a suitable host plant is greater than on a poor or non-host plant, because the stimulation elicited by the suitable host plant is much greater. When there are six plants to choose from, the chance of accumulating enough stimulation to oviposit on a poor or non-host plant increases since there is an increased probability of landing on them. Another well-studied hypothesis on host plant selection suggests that host plant choice in phytophagous insects may be limited by the amount of information they are able to process from the environment (Levins & MacArthur, 1969). In other words, there may be neural constraints resulting in a trade-off between diet breadth and the ability to differentiate among hosts (Bernays, 1998; Bernays & Wcislo, 1994; Janz & Nylin, 1997). Thus, the predominance of specialist insects may be a result of avoidance of confusion between good and poor host plants (Levins & MacArthur, 1969). Under this notion, generalist insects will be slower and/or less accurate in making decisions on individual oviposition sites due to their need to identify and often also rank many different plants (Nylin et al., 2000). Evidence from previous studies indicates that females of more specialized species and populations may make faster and/or more accurate decisions to accept or reject a plant as a suitable oviposition site (Bernays, 1998; Nylin et al., 2000). Experiments with a generalist whitefly, Bemisia tabaci, showed that oviposition performance was reduced when mixtures of host plants were presented as opposed to hosts presented  individually (Bernays, 1999). My results are consistent with this hypothesis as oviposition performance decreased when six leaves were presented as opposed to one or two. Other explanations would include leaf shape (Hirota & Kato, 2001), as cabbage, pak choi and geranium all have relatively round leaves with no sharp edges; whereas bean, tomato, and anise hyssop leaves all have pointed tips (tear drop-like shape). Moreover, it is also possible that 35  because I found a large proportion of eggs oviposited on the walls of the cages in the no-choice experiment, the eggs may have been oviposited onto geranium leaves instead of on the cage walls. My observations indicate an asymmetry between adult oviposition preference and larval performance on anise hyssop, where adults appear to have a narrower host range than larvae. Such asymmetry has been observed in several lepidopteran species (Berdegue et al., 1998; Bopp & Gottsberger, 2004; Liu & Liu, 2006; Thompson, 1988), as well as coleopterans (Ballabeni & Rahier, 2000; Eben & Lopez-Carretero, 2008; Pelletier et al., 1999), dipterans (Gratton & Welter, 1998), and hymenopterans (Digweed, 2006). For many of these species, it is likely that this asymmetry between adult preference and larval performance is limiting their ability to broaden their host range. However, as T. ni is a generalist with a wide host range, and has been shown to retain memory of larval food through metamorphosis (Akhtar & Isman, 2003; Chow et al., 2005; Shikano & Isman, 2009), it has significant evolutionary potential to utilize anise hyssop as a host plant. In conclusion, larval preference and adult oviposition preference correlated well with larval performance with the exception of tomato for larvae and anise hyssop for adults. It appears that larvae have a wider host range than the range oviposited on by adult moths, since larvae accepted and fed on all plants in no-choice experiments. Adults were more susceptible to laying eggs on an unsuitable host when presented with many leaves at one time, possibly due to neural constraints. The next step would be to examine whether the same preferences and larval performances are observed on whole plants, as well as in the presence of predators and/or parasitoids.  36  2.5  Acknowledgements   This project was supported by a Discovery grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to MBI and an NSERC Post Graduate Scholarship-Masters (NSERC PGS-M) to IS.                               37  2.6  References   Akhtar, Y. & Isman, M. B. (2003) Larval exposure to oviposition deterrents alters subsequent oviposition behavior in generalist, Trichoplusia ni and specialist, Plutella xylostella moths. Journal of Chemical Ecology 29: 1853-1870. Akhtar, Y. & Isman, M. B. (2004) Comparative growth inhibitory and antifeedant effects of plant extracts and pure allelochemicals on four phytophagous insect species. Journal of Applied Entomology 128: 32-38. Auerbach, M. & Simberloff, D. 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(1976) Influence of crop backgrounds on aphids and other phytophagous insects on Brussels sprouts. Annals of Applied Biology 83: 1-13. 43  Tahvanainen, J. O. & Root, R. B. (1972) The influence of vegetational diversity on the population ecology of a specialized herbivore, Phyllotreta crucifera (Coleoptera: Chrysomelidae). Oecologia 10: 321-346. Theunissen, J. & den Ouden, H. (1980) Effects of intercropping with Spergula arvensis on pests of Brussels sprouts. Entomologia Experimentalis et Applicata 27: 260-268. Thompson, J. N. (1983) The use of ephemeral plant parts on small host plants: how Depressaria leptotaeniae (Lepidoptera: Oecophoridae) feeds on Lomatium dissectum (Umbelliferae). Journal of Animal Ecology 52: 281-291. Thompson, J. N. (1988) Evolutionary ecology of the relationship between oviposition preference and performance of offspring in phytophagous insects. Entomologia Experimentalis et Applicata 47: 3-14. Wiklund, C. (1975) The evolutionary relationship between adult oviposition preferences and larval host plant range in Papilio machaon L. Oecologia 18: 185-197.             44  3  Indirect Plant­Mediated Effects on Insect Immunity and  Disease Resistance in a Tritrophic System2   3.1  Introduction    The growth and reproduction of phytophagous insects is largely dependent on the quality of their host plant. Host plant quality refers to the components of a host plant, such as the nutritional and defensive compounds that positively or negatively affect the performance of herbivorous insects (Awmack & Leather 2002). Herbivores may increase their total consumption of plant tissue to compensate for low nutrient concentration, either by spending more time feeding as opposed to resting, or by prolonging their complete development (Slansky & Feeny 1977; Price et al. 1980; Fajer 1989; Loader & Damman 1991). Defensive compounds can have toxic, antifeedant and/or repellent properties that directly affect development (Awmack & Leather 2002). Differences in plant quality can also affect an insect’s interactions with its natural enemies.  For example, the increased time spent feeding extends the window of opportunity for predator or parasitoid attack; the slow-growth, high-mortality hypothesis (Herrebout et al. 1963; Moran & Hamilton 1980; Loader & Damman 1991; Williams 1999). It may also alter insect condition, making them more vulnerable to challenge by insect parasitoids and pathogens (Cory & Hoover 2006; Caron et al. 2008). Studies of plant-mediated effects on the natural enemies of herbivores’ have primarily been focused on parasitoids, including the demonstration of a complex array of interactions wherein plants have been shown to attract parasitoids through the release of volatiles, thereby  2A version of this chapter has been accepted for publication. Shikano, I., Ericsson, J.D., Cory, J.S., & Myers, J.H. (In Press) Indirect plant-mediated effects on insect immunity and disease resistance in a tritrophic system. Basic and Applied Ecology. 45  manipulating their own defence (e.g. Turlings et al. 1990; Steinberg et al. 1993; Geervliet et al. 1994; Takabayashi et al. 1995; van Poecke & Dicke 2002). It is becoming apparent that the direct and indirect effects of plants on resource quality have increasingly wide ramifications; for example, recent work has shown that plant quality affects food web structure (Bukovinszky et al. 2008). While entomopathogens cannot be directly influenced by plant signalling, as far as I am aware (but see Hountondji et al. 2005), plants can still influence the interactions between insects and their pathogens in a range of both direct and indirect ways (Duffey et al. 1995; Cory & Hoover 2006). These include the effects of leaf surface chemistry and plant architecture on pathogen persistence, the effect of an insect’s saliva on pathogen viability, and a range of direct interactions that can inhibit or enhance the infectivity of pathogens within the insect gut (e.g. Ali et al. 2004; Musser et al. 2005; Raymond et al. 2005). Most evidence has been gathered from studies on a group of insect viruses, the baculoviruses; but other pathogen groups such as fungi and bacteria can also be affected (Cory & Myers 2003; Cory & Hoover 2006). It is well- established that both inter and intra-specific variation in plant quality can alter the outcome of infection by baculoviruses directly, in terms of both levels of mortality and speed of kill (e.g. Forschler et al. 1992; Ali et al. 1998; Raymond et al. 2002; Cory & Myers 2004). The majority of virus-insect studies have focused on interactions in the larval mid-gut, where it is clear that phytochemicals interfere directly with entomopathogen infection (Cory & Hoover 2006). Many plant allelochemicals, including phenols and polyphenolics, such as tannins, have a (usually) negative interaction with baculoviruses causing virus aggregation, virus inactivation via the effects of redox cycling, or oxidative stress damage on insect gut cells (e.g. Felton & Duffey 1990; Hoover et al. 1998). Fewer studies have addressed the influence of plant quality on insect susceptibility more indirectly either before pathogen exposure or after infection 46  has been initiated. For example, host plants with poor nutritional quality may result in less investment in immune factors resulting in lower resistance to pathogens or cause physiological changes that may influence the entry of pathogens via the mid-gut or the cuticle. Very recent work has shown that diet alters the structure of the peritrophic matrix (PM) in larval Lepidoptera, a change that can have a profound influence on baculovirus infection (Plymale et al. 2008). Another study has demonstrated that the diet that the insects consume after pathogen (baculovirus) challenge can significantly alter the likelihood of host survival, and more intriguingly, the possible capacity of an insect to influence this by altering its diet to take in more protein (Lee et al. 2006; Povey et al. 2009). Studies relating natural host plant quality to immunity and pathogen resistance are rare; however, research on the autumnal moth, Epirrita autumnata (Borkhausen) has demonstrated that variation in quality among individual birch trees can alter immune defence in pupae (Klemola et al. 2007).  In addition, a recent study showed that although host plant effects altered immune effectors such as phenoloxidase activity in a lepidopteran, Plutella xylostella (L.), they did not affect parasitoid survival (Karimzadeh & Wright 2008). When phytophagous insects are reared on host plants with differing nutritional qualities and defence chemicals, it is expected that the baseline defence response would also differ. Invasion by a pathogen can induce the cellular and humoral defence response to higher levels (Gillespie et al. 1997), suggesting that haemocyte numbers, antimicrobial protein levels, and PO activity will increase (Lemaitre et al. 1997; Wilson et al. 2001; Eleftherianos et al. 2006). Therefore, a host plant with poor nutritional qualities may result in less investment in immune factors and overall lower immune responsiveness of insect herbivores, whereas toxic or antifeedant properties of defence chemicals can either weaken the immune response or can 47  sometimes be sequestered by the insect and used as protection against natural enemies (Awmack & Leather 2002). In this study I investigate the influence of host plant quality on insect condition prior to pathogen challenge. In particular, I focus on the effect of host plant species on different measures of immunity and then see whether bottom-up effects can influence the resistance of the host to challenge with a baculovirus. The immune factors most responsible in the first line of defence against parasites and pathogens are the cuticle (in the case of pathogens such as fungi that enter by this route) and the midgut, which is the main barrier for baculoviruses and other groups of entomopathogens (Keddie et al. 1989; Hoover et al. 2000). Both the cellular and humoral immune responses have been implicated in resistance to baculoviruses (Cory & Myers 2003). My target herbivore is the cabbage looper, Trichoplusia ni (Hübner). T. ni is a generalist feeder and has a diet that includes various cruciferous plants, peas, tomatoes, cucumber, celery, certain ornamentals, and many weeds. The two host plants chosen for this study were broccoli and cucumber. These plants provide very different quality diets for T. ni and I speculated that the growth rate, and thus host condition, would differ between T. ni reared on the different host plants, which would in turn affect resistance to pathogens. Due to the complexity of the immune system, measurements of a single immune parameter is unlikely to reveal overall immune defence ability (Blount et al. 2003). Thus, I examined the effects of the two host plants on the baseline and induced cellular and humoral defence responses found in T. ni haemolymph via the numbers of haemocytes, protein levels, and PO activity.  48  3.2  Materials and methods  3.21  Plants and insects   Broccoli, Brassica oleracea, cv. ‘Green Sprouting’, and long English cucumber, Cucumis sativus, cv. ‘Marketmore’ were planted in 4 litre plastic pots containing a mixture of sandy loam soil and peat moss (4:1) and grown under greenhouse conditions at the University of British Columbia, Canada.  Leaves from 8-10 week old plants were used in the experiments.  Cabbage looper, T. ni eggs were obtained from a laboratory colony maintained for > 50 generations on a wheat-germ based diet (Janmaat & Myers 2003).  3.22  Larval performance   After egg hatch individual neonate larvae were placed into 60 mL plastic cups on leaves of either broccoli or cucumber. The leaves of both plants were rinsed in 0.2% bleach solution for 5 min then rinsed again with copious amounts of water before use. Previous attempts to rear larvae on unbleached cucumber leaves were unsuccessful. Each cup received three to four torn pieces of leaves (~ 50 cm2) and was placed in a temperature controlled room at 27 °C and 16:8 LD photoperiod. The leaves were changed and the cups were cleaned or replaced every two days. The larvae were weighed four and eight days after egg hatch and again at pupation, and the level of survivorship was assessed.  3.23  Testing for condition, immunity and disease resistance   The larvae were reared until the fourth instar on the two host plants, as described above, except that they were reared in groups of 10. Just prior to the fourth instar, the insects reared on each plant were randomly divided into treatment groups. In the first experiment the larvae were 49  divided into two groups; one to measure phenoloxidase (PO)/protein levels and the second to estimate resistance to a pathogen (a baculovirus). In the second experiment the insects were divided into three groups; one to assess haemocyte number, another for PO/protein measurements and the third to estimate pathogen resistance. In the second experiment, the first two groups were divided further; one half was given a bacterial challenge prior to bleeding and the other half was not (see below). A bacterial challenge was included as a positive control for immune system stimulation of a haemolymph-based response. This standard challenge is known to induce a fat body-mediated response (systemic), and would be independent of any host-plant mediated changes in the midgut that may also influence virus infectivity (e.g. Gorman et al. 2004). By including this standard challenge, a more sensitive measure of haemolymph-based immunity could be obtained. As growth rate was much slower on cucumber, larvae spent different times on the host plant to reach the same instar and weight. All larvae were weighed at the fourth instar to confirm that they had reached similar size; this is important, as smaller larvae will be more susceptible to virus infection. Detailed descriptions of each stage of the experiment are given below.  3.24  Escherichia coli challenge    Escherichia coli (K12 strain) was grown over night in Luria-Bertani culture broth at 37°C under agitation at 150 rpm. A 2ml aliquot was taken from this culture and then centrifuged at 2500g for 4 min. The supernatant was discarded leaving a moist bacterial pellet. The tip of a needle was dipped into this E. coli pellet and then late third instar larvae were pierced on the ventral side between the middle prolegs. They were left on their respective food treatments to 50  recover and moult for 24 h. The subsequently moulted early fourth instars were then subjected to haemolymph extractions.  3.25  Phenoloxidase and protein assays    Fifteen early fourth instar larvae were randomly selected from each food treatment and weighed prior to haemolymph extraction. A 5 μl haemolymph sample was added to 100 µl of ice-cold Dulbecco’s phosphate buffered saline (DPBS, Sigma-Aldrich). These buffered samples were kept on ice until all the sampling was complete, and were subsequently frozen at -25 ºC to rupture the haemocytes. The samples were thawed on ice and centrifuged prior to the PO and protein assays.  PO assay.  Fifty µl of each cell-free sample were placed in individual wells on a 96-well microplate and 150 µl of dopamine hydrochloride solution (20mM in DPBS) was added to each sample. The microplate was then placed in a spectrometer for 40 min at a wavelength of 492 nm, and absorbance measurements were taken every 30 seconds. For each sample, the enzymatic rate of PO which is represented by the change in optical density per minute (slope) during the linear phase of the reaction was recorded.  Protein assay.  The total protein concentration was determined using a Bradford assay. Briefly, 5 µl of each haemolymph-DPBS sample were placed in individual wells on a 96-well microplate along with 200 µl of Bradford reagent dye (BioRad protein kit, BioRad Inc.). A dilution series of bovine serum albumin (BSA) ranging from 0 to 2 mg/ml was included on the same microplate to establish a standard curve of known protein concentrations for comparison. 51  After 15 min incubation at room temperature, the microplate was then placed in the spectrometer and an endpoint reading at 595 nm was taken for each sample. These absorbance readings were extrapolated to the BSA standard curve to determine the total protein concentration of each sample  3.26  Haemocyte count    Twenty early fourth instar larvae were randomly selected from each food treatment and weighed prior to haemolymph extraction. Haemolymph was extracted from each larva by excising one of the middle prolegs and removing a 3 µl sample from the wound site with a pipette. The 3 µl sample was immediately mixed with 10 µl of ice-cold Schneider’s Insect medium (pH 7.2) supplemented with 5 mM calcium chloride, and 5mM sodium bicarbonate (Sigma-Aldrich). This entire sample was then immediately loaded onto an Improved Neubauer haemocytometer, and the number of haemocytes per microlitre of haemolymph was determined.  3.27  Virus bioassay    Early fourth instar larvae (< 24 hours after moult) from each host plant were randomly assigned to T. ni SNPV doses. In the first trial, only three viral doses of 200, 800, and 4000 occlusion bodies (OBs) per larva were tested (n = 20/ dose/ plant). In the second trial viral doses of 187, 375, 750, 1500, 3000, and 6000 OBs per larva were included (n = 24/ dose/ plant). A control of distilled water was used for each experiment. The viral dose was applied in 2 µl of distilled water to a 200 μl plug of artificial diet. This small amount of diet ensured complete ingestion of the viral dose within 24 h. Larvae from each host plant were weighed and starved for 4-5 h to clear the gut before feeding on the viral dose. After 24 h they were transferred into 52  individual 22 mL plastic cups and reared in a growth chamber at 25 °C and 16:8 LD photoperiod on 5 mL of artificial diet until death or pupation. The bioassay was monitored until all surviving larvae had pupated. All pupae successfully emerged as adult moths (data not shown).  3.28  Statistical analysis    Data for larval weight, haemocyte numbers, phenoloxidase levels and protein concentrations were first checked for normality and transformed if necessary. General linear models were used to compare the various response variables; for haemocyte numbers, phenoloxidase levels and protein concentrations; larval weight at the time of bleeding was also used as a covariate. Mortality data were analysed using generalized linear models, using a binomial error structure and a logit link function (JMP 6, SAS). In both cases, all factors and their interactions were fitted initially and non-significant terms removed sequentially to produce the final minimal model. The LD50 (median lethal dose) was determined via probit analysis using the EPA probit analysis program version 1.5.  3.3  Results  3.31  Insect performance   The growth rate, calculated using the weight of larvae recorded at four and eight days of development, showed that larvae reared on broccoli grew more than three times faster than larvae reared on cucumber (F1, 29 = 97.2, P < 0.0001, square root transformation) (Table 3.1). The mean weights of broccoli-reared pupae were not significantly different from those reared on cucumber (F1, 18 = 1.46, P = 0.24). Survival until pupation was approximately three times higher 53  on broccoli than cucumber (Table 3.1). Together, these results indicate that broccoli is a better host plant for T. ni development.  Table 3.1 Mean growth rate, pupal weight, and percent survival of T. ni larvae on broccoli and cucumber. Host plant Growth rate ± SE (mg/day) (n) Pupal weight ± SE (mg) (n) Survival (%) Broccoli 48.16 ± 0.70 (16) 197.05 ± 8.73 (15) 93.75 Cucumber 14.98 ± 0.44 (15) 176.70 ± 12.55 (5) 31.25  3.32  Insect condition: protein levels   Protein concentration per unit haemolymph was significantly higher in larvae reared on broccoli than on cucumber in both experiments (first: F1,27 = 52.89, P < 0.0001; second:F1,56 = 56.3, P < 0.0001) (Fig. 3.1A). Protein concentration also increased considerably with increasing larval weight (first: F1,27 = 22.17, P < 0.0001; second: F1,56 = 68.5, P < 0.0001). E. coli challenge did not alter the protein concentrations (F1,55 = 0.05, P = 0.82).  3.33  Humoral defence response: phenoloxidase   PO activity in the first trial showed higher activity in larvae reared on cucumber than on broccoli (F1,28 = 4.69, P = 0.039). The second experiment showed a similar trend with cucumber- reared larvae showing slightly higher mean PO activity than broccoli-reared larvae (Fig. 3.1B); however, this difference was not statistically significant (F1,50 = 2.34, P = 0.13). In order to get an overall picture of the effect on PO, the two experiments were combined, leaving out the E. coli challenge treatment. This confirmed that PO activity was higher in larvae fed cucumber 54  (F1,55 = 7.00, P = 0.011) (as well as being significantly higher in the first experiment, F1,55 = 24.54, P < 0.0001). Larval weight had no influence on PO activity (log10 wt; F1,54 = 0.36, P = 0.55). When larvae were challenged with E. coli in the second experiment there was no significant increase in PO levels (F1,50 = 2.22, P = 0.14). Larvae reared on cucumber also had higher PO per unit protein than those on broccoli (first: F1,28 = 11.68, P = 0.002; second: F1,52 = 23.95, P < 0.0001; Fig. 3.1C). This indicates that the amount of PO in cucumber-reared larvae makes up a substantially larger portion of the total protein concentration, than in broccoli-fed insects. Larval weight had no influence on the levels of PO/protein (first: F1,27 = 0.77, P = 0.39; second: F1,50 = 2.77, P = 0.1). Larvae challenged with the E. coli showed an increasing trend in PO/ protein, although this was not significant (F1,50 = 3.14, P = 0.08).  3.34  Cellular defence response: number of haemocytes    In the second experiment, the cellular defence response was also measured by counting the number of haemocytes (Fig. 3.1D). Larvae reared on broccoli had significantly more haemocytes than those reared on cucumber (F1,77 = 18.24, P < 0.0001). Furthermore, E. coli challenge induced a significant increase in the numbers of haemocytes in larvae reared on both host plants (F1,77 = 7.76, P = 0.007). Insects from both host plants responded similarly to bacterial challenge (interaction: F1,75 = 0.58, P = 0.45). Larval weight had no influence on the haemocyte number (F1,76 = 2.15, P = 0.15).    55   0 0.5 1 1.5 2 2.5 3 Broccoli Cucumber P O  a ct iv ity  (d O D  / m in ) a a a a 0 2 4 6 8 10 12 14 16 Broccoli Cucumber H ae m oc yt e co un t ( 10 3 µl -1 ) b   A  Ba 0 2 4 6 8 10 12 14 16 18 Broccoli Cucumber P ro te in  (µ g / µ l h ae m ol ym ph ) a a b    b 0 1 2 3 4 5 6 7 Broccoli Cucumber P O  / pr ot ei n (d O D  / m in  / m g)    a a b b (A) (B) (C) (D)  Figure 3.1 Baseline and induced cellular and humoral defence responses of T. ni larvae reared on broccoli and cucumber: A) haemolymph protein concentration (µg/µl), B) haemolymph PO activity, C) proportion of PO per unit protein (dOD/min/mg), and D) number of haemocytes per unit volume of haemolymph. Grey bars represent E. coli challenged larvae; white bars represent unchallenged larvae. Results represent mean values, and error bars represent the standard error of the mean (SEM). Bars with different letters and cases indicate significant differences between means (P < 0.05; PO and protein, n = 15; haemocytes, n = 20).  56  3.35  Disease resistance: virus bioassay   In the first trial, virus-induced mortality of larvae reared on cucumber was significantly higher than larvae reared on broccoli (χ2 = 24.02, df = 1,3, P < 0.0001). As expected, virus mortality increased with increasing virus dose (χ2 = 58.01, df = 1,3, P < 0.0001). The second, more comprehensive bioassay, confirmed the results from the initial trial (Fig. 3.2). Mortality of insects reared on cucumber was much higher than on broccoli host plants (χ2 = 35.92, df = 1,2, P< 0.0001), and again virus dose was highly significant (χ2 = 141.90, df = 1,2, P < 0.0001). The LD50 on cucumber for the second trial was 339 OBs (95% CI, 225, 458) and for broccoli-reared larvae the LD50 was 1272 OBs (95% CI of 991, 1635). Mean larval weight at the time of virus challenge had no effect on mortality (χ2 = 0.94, df = 1,4, P = 0.33), and there was no significant interaction between host plant and viral dose (χ2 = 0.46, df = 1,4, P = 0.50). There were no control deaths in either bioassay.            57  -5 -4 -3 -2 -1 0 1 2 3 4 5 2.0 2.5 3.0 3.5 4.0 Log10 dose (OBs) Lo gi t m or ta lit y ♦  Cucumber □  Broccoli  Figure 3.2 Variation in infectivity of T. ni SNPV to T. ni larvae reared on two different host- plants (Experiment 2). Points show the actual data (♦, cucumber; □, broccoli) and lines (dashed, cucumber; solid, broccoli) are the fitted models. Values of 0 or 100% are not represented in logits.  3.4  Discussion    Larval growth and development of herbivorous insects may be negatively affected by plants of poor nutritional quality or by plant defensive chemicals. Here I tested whether development on host plants of varying quality affected the innate immunity of an insect and its disease resistance. Insect performance studies clearly showed that broccoli was a more suitable host plant for the cabbage looper, T. ni than cucumber, both in terms of development rate and 58  survival. I compared the levels of three factors thought to be important in insect immunity, including defence against pathogens; phenoloxidase and protein levels in the haemolymph and the number of haemocytes. I then incorporated an immune challenge (E. coli infection) into the experiment to test whether the induced response of the insects was affected by host plant.  Haemocyte count was used as a measure of baseline and induced cellular defence responses. Haemocytes play important roles in insect immunity, but vary extensively in cell types and number between taxa. I detected greater numbers of haemocytes in broccoli-reared larvae than those fed cucumber, while the E. coli challenge induced increased numbers of haemocytes in larvae reared on both host plants. Haemocytes have been shown to be involved in the suppression of spread of baculovirus infection via an encapsulation and melanization response (Washburn et al. 1996). Greater numbers of haemocytes in broccoli-fed larvae should have coincided with higher levels of PO activity, because haemocytes produce some of the effector molecules for humoral immunity, including components of the PO cascade. However, I found that baseline levels of both PO and PO per unit protein tended to be higher in insects fed on the poorer host plant, cucumber. Levels of protein alone showed a different pattern with broccoli-fed insects having considerably higher levels, indicating that PO made up far less of the protein component in these insects. Bacterial challenge had no effect on either PO or protein levels in both host plant treatments, suggesting that the response is either ephemeral and was not detected using our sampling interval, or that immune-related proteins make up a small proportion of the total protein content of the haemolymph. A similar result for bacterial injection and protein levels has recently been found by Povey et al. (2009) using defined diets. The key test of the influence of host plant, in terms of disease resistance, is whether it alters the host’s susceptibility to infection. I examined this using an insect baculovirus, T.ni 59  SNPV.  This is a highly virulent pathogen, which will invariably result in host death if sufficient OBs are ingested and initiate systemic infection. It is already well established that plant phytochemicals can alter baculovirus infectivity by their interactions in the larval mid-gut (Cory & Hoover 2006). Therefore in the present study, the direct effect of phytochemicals was removed by exposing T. ni to the viral doses on an artificial diet, and by starving the larvae prior to the viral exposure to allow the expulsion of any plant material in the gut. The enhanced virus mortality observed in larvae reared on cucumber (compared to larvae reared on broccoli) should thus have resulted from the influence of the host plants on other insect-based factors prior to virus challenge. These effects are likely to result from three possible mechanisms; physical changes in the insects resulting from feeding on different plant substrates, reduced investment in other resistance mechanisms such as humoral and cellular defence and/or through active suppression of the immune system by plant defence chemicals that have been sequestered by the larva. Overall, broccoli proved to be the more suitable host plant for T. ni in terms of larval development and virus resistance than cucumber. Protein concentration and haemocyte numbers were positively associated with T. ni resistance to T. ni SNPV, whereas PO activity was not. Several studies have found correlations between PO activity and susceptibility to pathogens, including baculoviruses, in insects (Nigam et al. 1997; Reeson et al. 1998; Wilson et al. 2001). The fact that the amount of PO in the haemolymph of cucumber-reared larvae made-up a substantially larger portion of their total protein concentration than in broccoli-reared larvae, might indicate that proteins, other than PO, provided broccoli-reared larvae with greater resistance to T. ni SNPV. Alternatively, the larger pool of protein resources available for cell division may have increased the insects’ capacity to slough infected midgut cells more quickly. It 60  is also interesting that higher numbers of haemocytes were also associated with greater larval resistance. Haemocytes, in concert with the melanization response, have been shown to form the basis of resistance to fatal infection in another lepidopteran- baculovirus system (Trudeau et al. 2001), and thus it would not be surprising to find haemocyte number strongly associated with baculovirus resistance in general. My experiments do not show cause and effect, but they do indicate that bottom up effects can cascade through multi-trophic systems and that their effects include interactions with entomopathogens. It is possible that the poor nutritional quality or adverse effects from defence chemicals in cucumber may have resulted in less investment in disease resistance, via the immune system. Cucumber phloem has been found to contain many antioxidant defence proteins, as well as proteinase inhibitors that interfere with digestion (Walz et al. 2004). This suggests that the defensive compounds in cucumber may play a major role in weakening the condition of larvae. Alternatively, larvae reared on cucumber may not have assimilated nutrients from artificial diet as efficiently as larvae reared on broccoli after viral exposure. Preconditioning of larvae of the salvinia moth Samea multiplicalis on low-nitrogen diet resulted in less efficient assimilation of nitrogen when they were transferred to high-nitrogen diet, compared to those reared entirely on high-nitrogen diet (Taylor 1989). It is also possible that feeding on different host plants could have affected the physical barriers to infection, such as the peritrophic membrane (Plymale et al. 2008), instead of or in addition to investment in other resistance mechanisms. This will only be elucidated by further study. Here I have shown that T. ni larvae can vary in their susceptibility to nucleopolyhedrovirus when fed on different food plants and that larvae feeding on the better food plant were more resistant and had higher haemocyte concentrations. Other Lepidoptera also 61  vary in susceptibility to viral infection with developmental stage, later instars are more resistant (Hoover et al. 2002), and families and populations of Lepidoptera can vary in resistance to virus (Cory & Myers 2009). This variation among the susceptibility of individuals can be influenced by morphological factors such as the thickness of the peritrophic membrane (Plymale et al. 2008), the receptors on the midgut cells that allow virions to penetrate cell membranes, and the rate of sloughing of infected cells from the midgut (Keddie et al. 1989). Humoral and cellular immune responses are also likely to play a role (Strand 2008), and thus the elevated haemocyte counts of the T. ni larvae fed on the better food plant in our experiments may be directly related to their higher resistance to infection. Haemocytes have been shown to be involved in variation in the susceptibility to AcMNPV between Heliothis virescens and Helicoverpa zea (Trudeau et al. 2001).  In the more resistant H. zea, foci of viral infection in the trachea were melanized by haemocytes and circulating haemocytes also removed free virions from the haemolymph. I conclude from my study that the basis of the variation in the susceptibility to viral infection of larval T. ni is likely to be associated to haemocyte numbers and this may increase following an immune response challenge such as exposure to bacteria.  3.5  Acknowledgements    I thank Karmen Scott, Christal Nieman, Jing Niu, and Shoshana Turner for colony rearing and technical assistance.  I also thank Dr. Patricia Schulte for the use of her spectrophotometer.  This research was funded by NSERC.   62  3.6  References   Ali M. I., Felton G. W., Meade T. & Young S. Y. (1998) Influence of interspecific and intraspecific host plant variation on the susceptibility of heliothines to a baculovirus. Biological Control 12: 42-49.  Ali M. I., Young S. Y. & McNew R. C. 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K. (2001) Melanism and disease resistance in insects. Ecology Letters 4: 637-649.                    67  4  Summary & Conclusions  4.1  Summary   The major objective of this thesis was to examine how accurately female T. ni are able to rank and oviposit on different plant species according to host plant quality, as determined by larval performance. I then examined whether the negative developmental effects as a result of rearing on an inferior host plant would affect larval immune response and larval susceptibility to a pathogen. I believe that the questions have been answered by the studies conducted in this thesis.  1) Does host plant quality directly affect larval performance?  Larval survival and development were significantly influenced by the host plant they were reared on. I found that larval growth was significantly faster on the cruciferous species, cabbage and pak choi in the first study, and in broccoli in the second.  Nutritional components of host plant quality such as nitrogen and carbon have been extensively studied and determined to influence larval performance. For example, high concentrations of soluble carbohydrates can dilute other nutrients in plant tissues, and will consequently either negatively affect insect development or force the insects to increase their consumption rates (Bartlet et al. 1990). Douglas fir (Pseudotsuga menziesii) or artificial diets with high sucrose concentrations were found to negatively affect population growth rates of the Western spruce budworm (Choristoneura occidentalis) (Clancy 1992). Minerals and vitamins are also important for larval development (Awmack & Leather 2002). Rearing larvae of the Gypsy moth (Lymantria dispar) on artificial diets containing low concentrations of vitamins, minerals, or proteins resulted in longer development times and lower pupal weights than larvae 68  reared on control diets. This presumably occurred because the conversion efficiency of digestive enzymes decreased (Lindroth et al. 1991).  Plant defence chemicals can also have direct negative effects on insect performance. For instance, Trichoplusia ni reared on cellulose diets containing the nutritional extract of Solidago altissima achieved high survival, indicating that nutritional quality of S. altissima is sufficient for T. ni growth. However, the addition of defensive chemicals of S. altissima to the diets resulted in nearly 100% mortality. This demonstrates that plant defence chemicals can play a direct and significant role in insect performance (Bosio et al. 1990). Nevertheless, it is important to mention that plant defence chemicals do not always negatively affect insect performance, and can vary depending on the herbivore species (Awmack & Leather 2002). The generalist moth, Lymantria dispar, developed significantly slower on an artificial diet containing iridoid glycosides, whereas the specialist butterfly, Junonia coenia, developed faster on the glycoside- containing diet than on a glycoside-free diet (Bowers & Puttick 1988).  2) Are adult females and neonate larvae capable of ranking plant species according to larval performance?  Females oviposited on the two cruciferous plants, cabbage and pak choi, which were the best host plants for larval performance. They ranked tomato and bean next, followed by anise hyssop and geranium, except in the multiple-choice test where geranium was ranked equally to tomato and bean. Larvae ranked cabbage, pak choi, and anise hyssop as their most preferred host plants, followed by bean, then tomato and geranium in the two-choice and multiple-choice tests. They accepted all of the plants, except for a few larvae on geranium, when given no choice. Anise hyssop proved to be a suitable host which larvae correctly identified whereas adults did not. 69   Studies investigating the preference-performance hypothesis have yielded contradictory results, with some finding strong correlations (e.g. Singer et al. 1988; Kouki 1993; Nylin & Janz 1996; Nylin et al. 1996; Rajapakse & Walter 2007)) and others finding weak or no correlations (e.g. Auerbach & Simberloff 1989; Fox & Eisenbach 1992; Singer et al. 1994; Berdegue et al. 1998; Jallow & Zalucki 2003; Rajapakse & Walter 2007). Rajapakse et al. (2006) examined whether Helicoverpa armigera Hübner recognizes a suite or template of chemicals that are common to many plants. Gas chromatography linked to a mated female H. armigera electroantennograph (EAG) was used to screen the headspace volatiles of a primary host plant (pigeon pea), three other hosts (tobacco, cotton and bean) and two non-host plants (lantana and oleander). Eight electrophysiologically active compounds in pigeon pea headspace were identified in relatively high concentrations. The other host plants and non-host plants had a smaller subset of these compounds, all at relatively lower concentrations than pigeon pea. In a follow-up study, Rajapakse & Walter (2007) found that cotton and another host plant, common sowthistle, were equally suitable for larval H. armigera development as pigeon pea, but received far fewer eggs in oviposition experiments. Thus, stimulation from volatiles of some host plants can make them primary targets for moths in search of hosts while the other host plants are incidental or secondary targets.  3) Does the number of choices available influence the accuracy of adult and neonate choices?  Larvae accepted any leaf disk they were given in no-choice tests. In two- and multiple- choice tests, the rankings of leaf disks were very similar. Adult preferences were similar in the no-choice and two-choice tests. However, when provided six choices, adults oviposited a higher 70  proportion of eggs on geranium than would have been expected based on the results of the no- choice and two-choice tests. The fact that larvae accepted any leaf disk when given no choice, is in agreement with Dethier’s (1954) notion that if situated on a putatively unsuitable host plant, many insects will tend towards wider polyphagy when threatened by starvation. Many deterrent plant chemicals can be harmless (Bernays & Graham 1988) and adaptive at non-toxic concentrations (Glendinning & Gonzalez 1995). However, the results show that given the option to feed on a more palatable host in close proximity, T. ni neonate larvae are capable of making an appropriate choice. The error made by ovipositing T. ni in the multiple-choice test may have been due to a limit in the amount of information they were able to process (Levins & MacArthur 1969). It has been hypothesized that because generalist insects need to identify and often also rank many different plants, they will be slower and/or less accurate in making decisions on individual oviposition sites (Nylin et al. 2000). The generalist whitefly, Bemisia tabaci, exhibited reduced oviposition performance when mixtures of host plants were presented as opposed to hosts presented individually (Bernays 1999). Thus, T. ni could be displaying signs of neural constraint as they face increasing numbers of plants to evaluate as potential oviposition sites.  3) Does larval host plant quality affect baseline and induced immune parameters such as haemocyte numbers, protein concentration, and phenoloxidase activity?  Broccoli-reared larvae had significantly higher haemocyte numbers and protein concentration than cucumber-reared larvae, whereas phenoloxidase (PO) activity did not differ. Haemocytes for larvae reared on both host plants were induced to significantly higher numbers 71  after immune challenge. However, the immune challenge did not affect PO activity or protein concentrations (Table 4.1).  Table 4.1 Comparison of immune factors in uninduced T. ni larvae fed on broccoli or cucumber and changes in immune factors following induction by E. coli challenge (induced).   Immune factors Broccoli Cucumber Protein Higher Lower PO activity Equal Equal  Uninduced  Haemocytes Higher Lower Protein No change No change PO activity Slightly increased Slightly increased Induced Haemocytes Significantly increased Significantly increased  Viral susceptibility Lower Higher   The tissues most responsible in the first line of defence against parasites and pathogens are the cuticle (in the case of pathogens that enter by this route, such as fungi) and the midgut, which is the main barrier for baculoviruses (Wilson et al. 2001). Both the cellular and humoral immune responses have been implicated in resistance to baculoviruses (Cory & Myers 2003). Helicoverpa zea (a less susceptible species), and Heliothis virescens (a highly susceptible species) initially showed equal numbers of AcMNPV infection foci in the midgut. However, the number of infection foci decreased in H. zea, suggesting that they were somehow able to clear the infections (Washburn et al. 1996). Decreasing infection foci were associated with haemocyte aggregations, often containing capsules around infected tracheoles. These capsules were 72  indistinguishable from those attributed to the cellular immune response. The role of the cellular immune system was confirmed as the use of immunosuppressors reduced this response (Washburn et al. 1996). Phenoloxidase (PO) plays a crucial role in insect defense as it initiates the biosynthesis of melanin (Hartzer et al. 2005). By initiating melanin biosynthesis, PO participates in three physiologically important biochemical processes: insect cuticle sclerotization (Sugumaran 1998), encapsulation and melanization of pathogens (Söderhäll et al. 1990; Ashida & Brey 1995; Gillespie et al. 1997), and wound healing (Lai-Fook 1966; Ashida & Brey 1998). The African armyworm, Spodoptera exempta, exhibited an association between higher levels of PO activity in the haemolymph and greater resistance to NPV infection. Several other studies have also found correlations between PO activity and susceptibility to pathogens in insects (Nigam et al. 1997; Reeson et al. 1998; Wilson et al. 2001). Microbial pattern recognition proteins and antimicrobial peptides also play an important role in the humoral defense response. Drosophila injected with microorganisms differentially expressed genes encoding antifungal and antibacterial peptides (Lemaitre et al. 1997). Hemolin is considered to be an important antimicrobial protein involved in immune recognition and modulating defensive responses in Hyalophora cecropia and Manduca sexta (Gillespie et al. 1997).  4) Does larval host plant quality influence susceptibility to T. ni single nucleopolyhedrovirus?  Susceptibility to T. ni SNPV was significantly greater in larvae reared on cucumber than for those on broccoli (Table 4.1). Thus, the more suitable host plant for larval development is associated with greater resistance to viral infection. Whether the greater suitability of broccoli as a host plant over cucumber is due to differences in nutritional quality between the plants or due 73  to the presence or absence of defensive compounds was not investigated in this study. However, T. ni have demonstrated trenching behaviour on cucumber leaves to cut-off the flow of phloem sap, avoiding the exudates containing putatively noxious or neuroactive chemicals (Dussourd 1997, 2003). Furthermore, cucumber phloem has been found to contain many antioxidant defence proteins, as well as proteinase inhibitors that interfere with digestion (Walz et al. 2004), suggesting that the defensive compounds in cucumber may play a major role in weakening the condition of larvae. The concept that slower development will result in prolonged exposure to a pathogen (Awmack & Leather 2002) is not relevant in this study because larvae on both plants were exposed to the same viral doses for the same amount of time. However, the host plant upon which an entomopathogen is ingested can still significantly affect the levels of insect mortality (Duffey et al. 1995; Kouassi et al. 2001; Ali et al. 2004), possibly due to the presence of plant factors with protein-binding properties that enhance the effectiveness of entomopathogens (Sivamani et al. 1992). In fact, the efficacy of baculoviruses differs significantly depending on the plant species ingested with the virus for many insect species (reviewed by Cory & Hoover 2006).  In the present study, the effect of phytochemicals on the efficacy of T. ni SNPV was minimized by exposing T. ni to the viral doses on an artificial diet, and by starving the larvae prior to the viral exposure to allow the expulsion of any plant material in the gut. Therefore, the high mortality observed in larvae reared on cucumber likely resulted from poor nutritional quality leading to reduced investment in immune factors or through active suppression of the immune system by plant defence chemicals. Additionally, larvae reared on cucumber may not have assimilated nutrients from artificial diet as efficiently as larvae reared on broccoli after viral exposure. Preconditioning of larvae of the salvinia moth Samea multiplicalis on low-nitrogen 74  diet resulted in less efficient assimilation of nitrogen when they were transferred to high-nitrogen diet, compared to those reared entirely on high-nitrogen diet (Taylor 1989). Thus, less efficient nutrient assimilation after viral exposure may have adversely affected the immune response of cucumber-reared larvae.  4.2  Conclusions   Overall, I found that both adult and neonate larvae of T. ni are capable of ranking host plants based on suitability for larval performance. As larvae were able to correctly identify anise hyssop as a suitable host, whereas adults were not, I believe that larval diet breadth is wider than that of adult host range. This study, however, has some limitations. The cutting of leaves at the base of the stem for oviposition experiments would have modified the release of volatile chemicals from the leaves (e.g. Mattiacci et al. 1994; Arimura et al. 2005; Mithofer et al. 2005). The use of single leaves instead of whole plants neglects the possible effects of host plant size on host plant selection. The plant vigour hypothesis predicts that females should prefer to oviposit on large and vigorously growing host plants and that larvae should perform best on these plants (Price 1991; Heisswolf et al. 2005). Furthermore, as larval rearing was conducted on whole leaves rather than on whole plants, larvae did not encounter any induced plant defences (e.g. Van Dam et al. 2001; Dalin & Bjorkman 2003) When adults were faced with an increasing number of choices, they were more likely to make an error and oviposit on an unsuitable host. Such an error could be attributed to neural constraints, whereby generalist insects are believed to be slower and/or less accurate in making decisions of individual oviposition sites due to their need to identify and often also rank many different plants (Nylin et al. 2000). 75   I then examined the consequences of larval development on an inferior host plant. Larvae reared on cucumber, compared to those reared on broccoli, exhibited lower survival and growth rate, as well as some immune parameters in the haemolymph. Protein concentration and haemocyte numbers were associated with T. ni immune responsiveness to T. ni SNPV, whereas PO activity was not. The higher mortality caused by T. ni SNPV in larvae reared on cucumber suggests that larvae are less resistant to pathogens on a developmentally inferior host plant. Thus, I predict that fatal viral infections may occur more frequently on plants that are poorer hosts for certain insect species. On the contrary, differences in palatability among different host plants can influence the acquisition of a lethal pathogen dose (Cory & Hoover 2006). Larvae of the gypsy moth Lymantria dispar were more susceptible to NPV on white oak than on red oak. Yet, they consumed more leaf material on red oak and thus ingested more virus. The greater susceptibility on white oak and the higher dose of virus on red oak resulted in the same infection rate between the two host plants (Dwyer et al. 2005). Therefore, although broccoli-reared larvae were less susceptible to SNPV at fixed doses compared to larvae reared on cucumber, larvae on broccoli ate at a faster rate and consumed more plant material (personal observation), hence the faster development. If the virus was applied to the host plants rather than on artificial diet, larvae on broccoli may potentially ingest more virus and negate the stronger immune responsiveness, thus resulting in the same infection rate as larvae on cucumber. Unfortunately, studying the application of pathogens directly to host plants potentially introduces many other factors influencing infection rate; these may include the presence of plant factors with protein-binding properties that heighten the effectiveness of pathogens (Sivamani et al. 1992), or the adaptation of a pathogen to a particular host plant that may enhance the efficacy of the pathogen when ingested in conjunction with that plant (Raymond et al. 2002; Cory & Myers 2004). 76  My finding that the efficacy of T. ni SNPV varies significantly depending on the larval host plant has significant implications for pest management. This suggests that the application rates of microbial and viral biological control agents used to protect crops can be appreciably adjusted depending on the suitability of the crop for the pest insects. Furthermore, variation in susceptibility and immune parameters could have important consequences not just for insects in nature, but also for laboratories studying the efficacy of microbial insecticides, and laboratories investigating the cellular and molecular characteristics of insects. These observations necessitate the standardization of insect diet and rearing protocols between labs. Uses of different host plant species, cultivars, or varieties, as well as differences in the proportions of nutrients used to make artificial diets may very well contribute to the significant variation in results between labs. Future research will focus on host plant selection, larval performance, and disease resistance on whole plants. More specifically, plants with genetic variants that lack the production of certain chemicals. For example, Warbrick-Smith et al. (2006) used Arabidopsis mutants that had differing ratios of protein and carbohydrates. Ingestion of high levels of the macronutrient selenium (Se) has been found to increase resistance of insects to viral infection (Shelby & Popham, 2007). This can be important as fertilization methods such as foliar fertilizer applications are used as a means for Se biofortification of crops for human consumption. Such applications could result in elevated pest resistance to biocontrol agents (Shelby & Popham, 2007). Thus, investigating the effects of altered nutritional balance and/or the presence or absence of certain secondary chemicals could provide insight into insect immunity as well as suggest genetic variants of plants that will improve the efficacy of biological control agents.  In conclusion, the choice of oviposition site by a female T. ni can be a choice of life history for her offspring. The site of emergence can affect larval survival, development, and 77  susceptibility to natural enemies. 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