PHOTOSYNTHETIC AND GLYCOALKALOID RESPONSES OF POTATO (Solanum tuberosum L.) TO COLORADO POTATO BEETLE (Leptinotarsa decemlineata Say) DEFOLIATION by Courtney Louise Pariera Dinkins A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Entomology MONTANA STATE UNIVERSITY Bozeman, Montana November, 2006 © COPYRIGHT by Courtney Louise Pariera Dinkins 2006 All Rights Reserved ii. APPROVAL of a thesis submitted by Courtney Louise Pariera Dinkins This thesis has been read by each member of the thesis committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style, and consistency, and is ready for submission to the Division of Graduate Education. Dr. Robert K.D. Peterson Approved for the Department of Land Resources and Environmental Sciences Dr. Jon Wraith Approved for the Division of Graduate Education Dr. Carl A. Fox iii. STATEMENT OF PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a master’s degree at Montana State University-Bozeman, I agree that the Library shall make it available to borrowers under the rules of the Library. If I have indicated my intention to copyright this thesis by including a copyright notice page, copying is allowable only for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for permission for extended quotation from or reproduction of the thesis in whole or in parts may be granted only by the copyright holder. Courtney Louise Pariera Dinkins November, 2006 iv. ACKNOWLEDGMENTS I would like to extend my thanks and gratitude to my advisor, Dr. Robert K. D. Peterson, for his support, encouragement, guidance, and overwhelming generosity and patience. Dr. Peterson’s consideration for my academic, professional, and personal development has been extremely appreciated, helpful, and encouraging. I feel privileged to have had this opportunity to work and grow under his guidance. I would also like to thank my supervisory committee, Dr. David K.Weaver, Dr. Andreas Fischer, Dr. Tulio B. Macedo, and Dr. Robert K. D. Peterson, for providing useful advice and direction during my program. For their collaborative research and assistance I would like to extend my gratitude to Norma Irish, David Baumbauer, Dr. Jim Gibson, Qu Qing, Dr. Jarrett Barber, and Dr. Jim Robison-Cox. Special thanks to Team COBRA: Leslie Shama, Ryan Davis, Marjolein Schat, Dr. Paula Macedo, and Jerome Schleier, and the Sawfly Lab: Megan Hofland, Jeanie Peterson, Norma Irish, Gavin Peck, Micaela Butler, Oscar Perez, Godshen Robert, Zhitan Sun, and Jenny Perez for their assistance, patience, laughter, and support. I thank Dr. David Weaver for providing equipment and use of laboratory space. For their assistance and support, I thank the faculty and staff of Land Resources and Environmental Sciences and the former Entomology Department. Thank you to my husband, Jonathan, who has believed in me, listened with an open ear, and has been a sturdy shoulder to lean on. I would also like to extend a special thank you to my parents, Mike and Debbie Pariera, and in-laws, Jerry and Terry Dinkins, for their overwhelming encouragement over the years. This research project was supported by the Montana Agricultural Experiment Station and Montana State University. v. TABLE OF CONTENTS LIST OF TABLES........................................................................................................... viii LIST OF FIGURES .............................................................................................................x ABSTRACT...................................................................................................................... xii 1. INTRODUCTION ...........................................................................................................1 Photosynthesis..................................................................................................................2 Stomatal Conductance .....................................................................................................5 Transpiration ....................................................................................................................5 Insect Injury Guilds..........................................................................................................6 Insect Injury Guilds and Photosynthetic Rates ................................................................7 Sources, Sinks, and Photosynthetic Rate .........................................................................9 Potato .............................................................................................................................11 Colorado Potato Beetle ..................................................................................................13 Colorado Potato Beetle Injury and Potato Response .....................................................14 Potato Biochemical Response to Colorado Potato Beetle Injury...................................15 Glycoalkaloids ...............................................................................................................17 Health Risks ...................................................................................................................19 Risk Assessment ............................................................................................................21 2. GAS EXCHANGE RESPONSES OF POTATO TO COLORADO POTATO BEETLE DEFOLIATION.............................................................................................23 Abstract ..........................................................................................................................23 Introduction....................................................................................................................24 Injury Guilds ...............................................................................................................24 Effects of Insect Injury on Photosynthetic Rates........................................................25 Secondary Compounds and Photosynthetic Rates......................................................26 Colorado Potato Beetle Injury and Potato Response ..................................................28 Objective ........................................................................................................................29 Material and Methods ....................................................................................................29 Plant Material..............................................................................................................29 Colorado Potato Beetles..............................................................................................30 Larval Leaf Defoliation Study – 2005 ........................................................................31 Adult Leaf Defoliation Study – 2005..........................................................................31 Manual Defoliation Study – 2004...............................................................................33 Manual and Colorado Potato Beetle Defoliation Study – 2005..................................34 Statistical Analyses .....................................................................................................35 Results............................................................................................................................35 Larval Colorado Potato Beetle Leaf Defoliation Study – 2005..................................35 Adult Colorado Potato Beetle Leaf Defoliation Study – 2005 ...................................39 vi. TABLE OF CONTENTS—CONTINUED Manual Defoliation Study – 2004...............................................................................43 Manual and Colorado Potato Beetle Defoliation Study – 2005..................................47 Discussion ......................................................................................................................53 3. QUANTITATIVE HUMAN DIETARY RISK ASSESSMENT ASSOCIATED WITH GLYCOALKALOID RESPONSES OF POTATO TO COLORADO POTATO BEETLE DEFOLIATION ............................................................................58 Abstract ..........................................................................................................................58 Introduction....................................................................................................................59 Glycoalkaloids ............................................................................................................61 Health risks .................................................................................................................62 Objective ........................................................................................................................64 Material and Methods ....................................................................................................65 Plant Material..............................................................................................................65 Glycoalkaloid Analysis...............................................................................................66 Sample Preparation .................................................................................................66 Eurofins’ Preparation of Standards.........................................................................67 Eurofins’ Sample Preparation .................................................................................68 Eurofins HPLC-ELSD ............................................................................................69 Cellular Proliferation Analysis ...................................................................................69 Inhibition of Cellular Proliferation Assay...............................................................69 Sample Preparation .................................................................................................69 Cell Maintenance ....................................................................................................70 Treatment Preparation.............................................................................................70 Evaluation of Cellular Proliferation........................................................................70 Statistical Analyses .................................................................................................71 Risk Assessment .........................................................................................................71 Problem Formulation ..............................................................................................71 Hazard Identification and Dose-Response Relationships .......................................72 Selection of Toxic Endpoint ...................................................................................74 Exposure Assessment..............................................................................................75 Risk Characterization..............................................................................................76 Results............................................................................................................................77 Glycoalkaloids ............................................................................................................77 Toxicity .......................................................................................................................81 Risk Characterization..................................................................................................87 Discussion ......................................................................................................................91 vii. TABLE OF CONTENTS—CONTINUED 4. CONCLUSION............................................................................................................101 LITERATURE CITED ....................................................................................................102 APPENDIX A: Tuber Yield of Manual and Colorado Potato Beetle Defoliation Study 2004 and 2005 .......................................................................................................108 viii. LIST OF TABLES Table ............................................................................................................................. Page 1. ANOVA for Larval Colorado Potato Beetle Leaf Defoliation Experiment (2005).....................................................................................................................36 2. ANOVA for Adult Colorado Potato Beetle Leaf Defoliation Experiment (2005).....................................................................................................................40 3. ANOVA for Manual Defoliation Experiment (2004).............................................44 4. ANOVA for Manual and Colorado Potato Beetle Defoliation Experiment (2005).....................................................................................................................48 5. Mean Glycoalkaloid Concentrations in Potatoes from Experiment 1 ....................78 6. ANOVA for Glycoalkaloid Concentrations of Potatoes from Experiment 1 .........79 7. ANOVA for Glycoalkaloid Concentrations from Potatoes of Control, High Manual Defoliated, and High Colorado Potato Beetle Plants Experiment 2.........80 8. ANOVA for IC50s of Potatoes from Experiment 1.................................................82 9. Mean IC50 Concentrations of Potatoes from Experiment 1 ....................................83 10. Exposure, Percentage of Toxic Dose, and Risk Quotient for the Acute Exposure of Human Subgroups to Glycoalkaloids in the Inner Tissue of Tubers from Control Plants....................................................................................88 11. Exposure, Percentage of Toxic Dose, and Risk Quotient for the Acute Exposure of Human Subgroups to Glycoalkaloids in the Inner Tissue of Tubers from Manually Defoliated Plants...............................................................89 12. Exposure, Percentage of Toxic Dose, and Risk Quotient for the Acute Exposure of Human Subgroups to Glycoalkaloids in the Inner Tissue of Tubers from High Colorado Potato Beetle Defoliated Plants................................90 13. Acute Risk Quotients (RQ) for Human Subgroups Exposed to Glycoalkaloids in Tubers .......................................................................................92 14. Analysis of Variance Table for Tuber Yield of Manual and Colorado Potato Beetle Defoliation Study – 2004...............................................................111 ix. LIST OF TABLES—CONTINUED Table ....................................................................................................................... Page 15. Analysis of Variance Table for Tuber Yield of Manual and Colorado Potato Beetle Defoliation Study – 2005...............................................................112 x. LIST OF FIGURES Figure ............................................................................................................................ Page 1. Photosynthetic Rates of the Larval Colorado Potato Beetle Leaf Defoliation Study (2005) on Cal Red Plants.............................................................................37 2. Photosynthetic Rates of the Larval Colorado Potato Beetle Leaf Defoliation Study (2005) on Russet Burbank Plants ................................................................38 3. Photosynthetic Rates of the Adult Colorado Potato Beetle Leaf Defoliation Study (2005) on Cal Red Plants.............................................................................41 4. Photosynthetic Rates of the Adult Colorado Potato Beetle Leaf Defoliation Study (2005) on Russet Burbank Plants ................................................................42 5. Photosynthetic Rates of the Manual Defoliation Study (2004) on Upper Leaves of Russet Burbank Plants...........................................................................45 6. Photosynthetic Rates of the Manual Defoliation Study (2004) on Lower Leaves of Russet Burbank Plants...........................................................................46 7. Photosynthetic Rates of the Manual and CPB Defoliation Study (2005) on Upper Leaves of Cal Red Plants ...................................................................…49 8. Photosynthetic Rates of the Manual and CPB Defoliation Study (2005) on Lower Leaves of Cal Red Plants.......................................................................50 9. Photosynthetic Rates of the Manual and CPB Defoliation Study (2005) on Upper Leaves of Russet Burbank Plants...........................................................51 10. Photosynthetic Rates of the Manual and CPB Defoliation Study (2005) on Lower Leaves of Russet Burbank Plants ..........................................................52 11. Glycoalkaloid Concentrations for the Skin of Potatoes Harvested from Control Plants (0% defoliation), High Manual Defoliation (∼90% defoliation), and High Colorado Potato Beetle Defoliation (∼90% defoliation) for Experiments 1 and 2...............................................................................................84 12. Glycoalkaloid Concentrations for the Inner Tissue of Potatoes Harvested from Control Plants (0% defoliation), High Manual Defoliation (∼90% defoliation), and High Colorado Potato Beetle Defoliation (∼90% defoliation) for Experiments 1 and 2 .....................................................................85 xi. LIST OF FIGURES—CONTINUED Figure ............................................................................................................................ Page 13. Mean IC50s (Inhibitory Concentration, 50%) from Potatoes of Control, High Manual Defoliated, and High Colorado Potato Beetle Plants Experiment 1..........................................................................................................86 14. Manual and Colorado Potato Beetle Defoliation Study- 2004 and 2005: Yield of Tubers from Cal Red and Russet Burbank Plants Across all Defoliation Levels................................................................................................113 xii. ABSTRACT Photosynthetic and glycoalkaloid responses of potatoes (Solanum tuberosum L.) to varying levels of Colorado potato beetle (Leptinotarsa decemlineata Say) and manual defoliation were measured on ‘Cal Red’ and ‘Russet Burbank’ plants. No alteration in photosynthesis was observed on the remaining tissue of an injured leaf for Cal Red and Russet Burbank leaves defoliated by larval Colorado potato beetles nor for Russet Burbank leaves defoliated by adult Colorado potato beetles. No significant differences were observed between actual Colorado potato beetle and manual defoliation for both varieties. In both of the whole-plant defoliation studies, defoliation level consistently did not result in increased or decreased gas exchange parameters of individual leaves compared to undefoliated controls. There was no evidence of delayed leaf senescence in defoliated treatments. Plants defoliated by Colorado potato beetles had a significantly greater production of glycoalkaloids than in control and in manually defoliated plants for both skin and inner tissue. There was also a 32.6% and a 36.8% glycoalkaloid increase in skin and inner tissue of tubers from plants defoliated at high levels by Colorado potato beetles in comparison to control plants. Although a significant difference in glycoalkaloid concentration was not observed among the treatments in subsequent experiments, the skin and inner tissue of tubers from plants defoliated at high levels by Colorado potato beetles increased by 18.9% and 12.7% in comparison to tubers from control plants. For the experiments where glycoalkaloid concentrations were measured, there was a significantly greater concentration of glycoalkaloids in the skin versus the inner tissue of potatoes, in addition, for the initial glycoalkaloid experiment, the concentration of tuber extract required to reduce Chinese hamster ovary cellular proliferation by 50% was 10 times less for the skin versus the inner tissue. The dietary risk assessments revealed that the glycoalkaloid concentrations within the inner tissue of tubers from control plants, manually defoliated plants, and high Colorado potato beetle defoliated plants exceeded the toxic endpoint for all human subgroups at less than the 99.9th percentile of exposure. 1 INTRODUCTION Insect herbivory is a fundamental feature of most terrestrial ecosystems, and it can profoundly affect agroecosystems. Foliage feeding insects are estimated to reduce crop yields from between 5% and 30% (Mattson and Addy, 1975), and during insect outbreaks, it is estimated that more than 70% of the primary biomass may be removed (Cyr and Pace, 1993). Although an extensive amount of research has identified the physiology, behavior, ecology, and life history of pest insects and how their numbers affect yield, but more needs to be determined on how the plant host responds to insect injury (Peterson and Higley, 2001). Stress is the reduction of optimal physiological conditions and “injury is a stimulus producing an abnormal change in physiological processes” (Peterson and Higley, 2001). Because gas exchange processes (such as photosynthesis, stomatal conductance, and transpiration) are fundamental physiological processes that ultimately determine plant growth, development, and fitness, understanding how insect herbivory affects these processes would provide valuable perspectives on how a plant responds to different stresses and allow for integration of responses across different levels of biological organization. In addition to alterations in primary metabolism, many plants respond biochemically to insect injury by producing secondary metabolites. Many secondary metabolites function as natural pesticides against insect herbivores. Secondary metabolites are highly diverse among taxa and their presence has led to extensive research on the origin, diversity, and role that these chemicals play in plant-insect 2 interactions, as well as their specificity to insects (Theis and Lerdau, 2003). Because naturally occurring pesticides may be synthesized when plants are under stress, it is expected that injury to plant tissue would instigate synthesis of higher concentrations of the naturally occurring pesticides in the injured and uninjured plant tissue. Because approximately 99% of all toxins consumed by humans are naturally occurring in plant tissue (Ames et al., 1990), insect injury to crops may increase concentrations of toxins, altering dietary risk to humans. Photosynthesis Photosynthesis “encompasses both a complex series of reactions that involve light absorption, energy conversion, electron transfer, and a multipstep enzymatic pathway that converts [carbon dioxide] CO2 and water into carbohydrates” (Malkin and Niyogi, 2000). Photosynthesis involves two different phases: light reactions and “dark” reactions. The light reactions produce O2, ATP, and NADPH. The “dark” reactions (Calvin Cycle) “reduce CO2 to carbohydrates and consume the ATP and NADPH produced in the light reactions” (Malkin and Niyogi, 2000). Although the second phase is called the Calvin Cycle, it does not require light nor does it need to take place in the dark. All the reactions required for photosynthesis take place in particular locations within the chloroplast. Chloroplasts have an outer and inner envelope and all thylakoid membranes are interconnected and enclosed, creating an inner space called the lumen (Malkin and Niyogi, 2000). The internal membrane is known as the thylakoid membrane and is comprised of granal thylakoids, which are organized into stacks called grana and 3 stromal thylakoids, which are unstacked and exposed to the stroma. The stroma is the surrounding fluid medium. Located in the thylakoid membranes are Photosystems I and II, which contain reaction centers that convert light energy and transfer the energy into chemical bond energy. Photosystem I lies in the stromal membrane and absorbs wavelengths at 700 nm. Photosystem II lies in the granal membrane and absorbs wavelengths at 680nm (Malkin and Niyogi, 2000). The electron transfer chain not only involves Photosystems I and II, but cytochrome b6f, plastocyanin, and plastoquinone. Photosystem II has two quinones bound to it. Light is absorbed in Photosystem II, and because Photosystem II has two quinones bound to it: QA and QB, two consecutive electrons are released from the light absorbing pigments (P680), transferred to QA, which are then transferred to QB, eventually yielding a fully reduced quinone (QB 2-). The QB 2- then takes on two protons (QBH2) from the stromal side of the membrane, dissociates from Photosystem II, diffuses into the lipid bilayer to become a mobile electron carrier, and a free quinone refills the QB site on the Photosystem II complex (Buchanan et. al., 2000; Malkin and Niyogi, 2000). In the cytochrome b6f complex, QBH2 (plastoquinol) transfers its electrons to plastocyanin in the lumen. The cytocrome b6f complex allows the transfer of two protons across the lumen for every electron transferred to plastocyanin. In addition to the oxidation of water, this establishes a proton gradient and on the surface of the ATP synthase, located in the thylakoid membrane, ADP is phosphorylated to produce ATP (Buchanan et. al., 2000; Malkin and Niyogi, 2000). Plastocyanin transfers its electrons to Photosystem I, where the electrons are transferred to a few electron carriers before 4 reaching Ferrodoxin. Through electrostatic interactions, Ferrodoxin binds to Ferredoxin- NADP+ reductase and transfers its electrons to NADP+ to produce NADPH in the stroma (Buchanan et. al., 2000; Malkin and Niyogi, 2000). Using the light energy absorbed, the light reactions generate the energy required by the Calvin Cycle. In the Calvin Cycle, 13 steps fix CO2 into carbohydrates in three phases: carboxylation, reduction, and regeneration. Carboxylation is the enzymatic combination of CO2 and water from the environment with ribulose 1,5-bisphosphate (RuBP) using ribulose bisphosphate carboxylase/oxygenase (Rubisco) to produce two 3- phosphoglycerate (3-PGA) molecules (Buchanan et. al., 2000; Malkin and Niyogi, 2000). The reductive phase involves two steps: the conversion of 3-PGA into triose phosphate and triose phosphate to glyceraldehyde 3-phosphate (GAP) using ATP and NADPH synthesized during the light reactions. The GAP molecules generated can then be used to build carbohydrates such as sucrose and starch or other cellular constituents necessary to maintain the growth, development, and fitness of the plant. Sources are leaf or leaf-like structures that carry out the majority of photosynthesis and transfer photoassimilates, sucrose and/or starch, to sinks. Sinks are young leaves, stems, roots, fruits, or seeds and the photoassimilates within sinks may be utilized for respiration and for the synthesis of other molecules required for their growth. Sucrose is synthesized in the cytosol of mesophyll cells in source leaves. Most of the sucrose synthesized during photosynthesis is directly transferred into the phloem and exported into sink leaves where it is then used for respiration. The last phase of the Calvin Cycle is the regeneration phase and involves the regeneration of RuBP, which is required to continue the process of carbon fixation and carbohydrate synthesis. 5 Stomatal Conductance For photosynthesis to occur, atmospheric CO2 must diffuse into the leaf through the stomata and reach the carboxylation site of Rubisco. The appropriate gradients are required for the diffusion of CO2 to maintain carbon fixation and variations in these gradients highly influence the diffusion rates (Taiz and Zeiger, 1998). Carbon dioxide is first absorbed through the stomatal pore into the substomatal cavity and intercellular air spaces between the mesophyll cells, eventually diffusing into the mesophyll cells, through the cytoplasm, and across the chloroplast membrane before reaching the carboxylation site of Rubisco (Taiz and Zeiger, 1998). Higher stomatal conductance increases the diffusion of CO2 into the leaf via the stomata. The increased diffusion of CO2 into the leaf favors higher photosynthetic rates due to the continuous influx of carbon available for fixation in the Calvin Cycle as described above. In C3 plants, which are most crops including potatoes (Solanum tuberosum L.), increasing the CO2 levels beyond the level fixed by photosynthesis and produced by respiration causes an increase in photosynthesis and is limited by the ability of the enzymes in the Calvin Cycle to regenerate RuBP. If CO2 concentrations are low, however, photosynthesis is limited by the carboxylation capacity of Rubisco (Taiz and Zeiger, 1998). Transpiration Water transpired by a leaf evaporates from the wet mesophyll cells into the intercellular air spaces to exit out of the stomatal pore. Because water, a liquid, and CO2, 6 a gas, share the same pathway of release and absorption, and because CO2 diffuses through the air 1.6 times more slowly than water, plants have to overcome substantial water loss to maintain adequate influx of CO2. At high relative humidity, “the diffusion gradient that drives water loss is about 50 times greater than the gradient that drives CO2 uptake” (Taiz and Zeiger, 1998). Therefore, a plant reduces its stomatal resistance allowing for a greater concentration of CO2 to diffuse into the leaf. Substantial water loss may still occur even when stomatal resistance is lowered. In sunny conditions where there is sufficient water available to the plant, it is advantageous for the plant to decrease stomatal resistance to allow for the increased diffusion of CO2 to support photosynthetic demands (Taiz and Zeiger, 1998). In sunny conditions where there is lack of water, it is advantageous for the plant to open the stomatal pore only slightly or keep it closed entirely (Taiz and Zeiger, 1998); slight and complete closure of the stomata would inhibit photosynthesis due to the reduction in carbon available for fixation. In addition to a plant reducing its stomatal resistance, at night when photosynthesis is limited and CO2 demand is limited, the stomatal pores are kept as small as possible (Taiz and Zeiger, 1998). Insect Injury Guilds There are different types of injury to a plant that insects may impose and they may be grouped into injury guilds based upon the appearance of the injury alone. Examples of these injury guilds include leaf skeletonizing, seed feeding, leaf mining, stem boring, and fruit scarring. Because different species may impose similar injury types and because these injury types elicit a similar response in plants, those species may be placed in injury guilds based upon physiological response. The injury guilds 7 aforementioned were later augmented to incorporate plant physiological responses from each injury type. These injury guilds include fruit feeders, stand reducers, assimilate sappers, turgor reducers, plant architectural modifiers, and leaf-mass consumers (Boote, 1981; Pedigo et. al., 1986). Incorporating the injury guilds described above, Higley et al. (1993) reclassified the insect injury guilds to include leaf senescence alterators, leaf-mass reducers, leaf photosynthetic-rate reducers, light reducers, assimilate removers, water- balance disruptors, population or stand reducers, seed or fruit destructors, architecture modifiers, and phenological disruptors. Insect Injury Guilds and Photosynthetic Rates Photosynthetic rate is the “net amount of carbon fixed per unit leaf area per unit time” (Taiz and Zeiger, 1998) and therefore determines the amount of carbon available to the source leaf. Physiological factors that could affect photosynthetic rate include “increased assimilate demand after defoliation, reduced competition between leaves for mineral nutrients necessary for cytokinin production, and delayed leaf senescence” (Peterson, 2001). Variable photosynthetic responses to insect herbivory have been observed among different combinations of plant taxa and types of injury insects impose (Welter 1989). A large amount of research has been conducted on the photosynthetic and other gas exchange responses of soybean (Glycine max L.) injured by different species of insects. Most of the research has involved the leaf-mass consumption injury guild (Peterson et al., 2004). The Japanese beetle (Popillia japonica Newman), the corn earworm (Helicoverpa zea Bodie) (Aldea et al., 2005), the cabbage looper (Trichoplusia ni Hübner), the green 8 cloverworm (Plathypena scabra F.), and the Mexican bean beetle (Epilachna varivestis Mulsant) would all be classified as leaf-mass consumers and each can remove soybean (Glycine max L.) leaf tissue. Although each species is a leaf-mass consumer, all except the Mexican bean beetle elicit a similar photosynthetic response (Peterson et al., 1998). The Japanese beetle, the corn earworm (Aldea et al., 2005), the cabbage looper, and the green cloverworm caused no change in photosynthetic rates of remaining uninjured tissue on an injured leaf (Hammond and Pedigo, 1981; Ostlie and Pedigo, 1984). The only change observed was a transient increase in water loss, suggesting that the photosynthetic pathway is not affected by defoliation of insects. Reductions in photosynthetic rates that have been observed after leaf-mass consumption injury (Delaney and Higley, 2006; Peterson et al., 1998) may also be due to the disruption of water and nutrient transport, changes in the performance of photosynthetic enzymes, and diversion of resources to defenses. At the canopy level, reductions in photosynthetic rate have also been observed, but many of these reductions were temporary and were caused by a reduction in leaf-area indices, smaller leaf size, and decreased light inception (Peterson, 2001). Many studies have indicated that removal of either partial or entire leaves by insects increases photosynthetic rates (Welter, 1989). Other studies have observed no changes in photosynthetic rates on the remaining leaf tissue of leaves injured by insects (Poston et al., 1976; Welter, 1989, 1991; Higley, 1992; Peterson et al. 1992; Peterson and Higley, 1996; Burkness et al., 1999) suggesting that photosynthesis is unaffected by leaf- mass consumers. Changes in photosynthetic rates observed are caused from the reduction in leaf photosynthesizing tissue and not from an increase or decrease in the 9 photosynthetic capacity of the remaining leaf tissue. In alfalfa and soybean defoliator systems, defoliation of whole plants or plant canopies resulted in a delay of normal, progressive leaf senescence (Higley, 1992; Peterson et al., 1992). Sources, Sinks, and Photosynthetic Rate To further explain plant response to insect injury, it is best to divide the plant into two tissue types based on physiological function: sources and sinks. Sources are leaf or leaf-like structures that carry out the majority of photosynthesis and transfer photoassimilates, sucrose and/or starch, to sinks. Sinks are young leaves, stems, roots, fruits, or seeds and the photoassimilates within sinks may be utilized for respiration and for the synthesis of other molecules required for their growth. Sink size and activity affects the mobility of photoassimilates. Sink activity is “the rate of uptake of assimilates per unit weight of sink tissue” and sink size is “the total weight of the sink tissue” (Taiz and Zeiger, 1998). By altering the size and activity of a sink, the rate of transport of photoassimilates may be altered. The larger the sink, the greater the photoassimilates that may be stored and the greater the activity of a sink, the greater the number of photoassimilates processed. The quicker sinks metabolize photoassimilates, the greater the amount of photoassimilates can be transported (Taiz and Zeiger, 1998). If the source to sink ratio is altered, so that the number of sources per sink is reduced, an increase in the photosynthetic rate of that single source leaf would be observed. The photosynthetic rate increases when the sink demand increases, but when the sink demand decreases, the photosynthetic rate often decreases (Taiz and Zeiger, 1998). 10 When all but one source leaf of a soybean plant was shaded for approximately eight days, a decrease in starch concentration of source leaves and increases in photosynthetic rate, activity of Rubisco, sucrose concentration and transport to sink, and orthophosphate concentration all were observed in the source leaf (Thorne and Koller, 1974). For plants that mainly store starch, there is a drastic reduction in the photosynthetic rate when sink demand decreases, and this may be caused by the accumulation of assimilates such as starch, sucrose, or hexoses in the source leaf (Taiz and Zeiger, 1998). There are three mechanisms which may explain decreases in photosynthetic rates when sink demands decrease. When sink demands are low, starch accumulation in the source leaves could disrupt the chloroplasts by either interfering with CO2 diffusion or by blocking the absorption of light (Taiz and Zeiger, 1998). Synthesis of sucrose is often reduced with low sink demands, causing less phosphate to be available for exchange in the chloroplasts. If starch synthesis, which releases orthophosphates in the chloroplast, could not recycle phosphate fast enough, ATP and CO2 fixation would decline, and the lack of free orthophosphates within the chloroplasts would cause a reduction in the rate of photosynthesis (Taiz and Zeiger, 1998). High sugar levels in source leaves as a result of inhibition of export to sink leaves may also decrease the rate of transcription of mRNA encoding photosynthetic enzymes through a feedback loop (Taiz and Zeiger, 1998). Signals between source and sink leaves may be physical, chemical, hormonal, or through carbohydrate concentrations. Physical signals between source and sink leaves may involve turgor pressure. The signal using turgor pressure may pass through the sieve elements from sink to source leaves. Photoassimilates are transferred from source leaves 11 into the phloem. From the phloem, sinks unload the photoassimilates and if the unloading was rapid, there would then be a reduction in turgor pressure in the sieve elements of sinks. This reduction in turgor pressure would than be transmitted to source leaves. If the phloem unloading was not rapid, there would be an increase in turgor pressure, and this would also be transmitted to the source leaves. Thus source leaves would reduce photosynthetic rates. Turgor pressure across plasma membranes has been shown to affect the activity of the proton pumping ATP-ase in the membranes. Using the taproot of a sugar beet incubated in a mannitol solution, the tugor pressure was reduced, and there was an increase in proton release (Taiz and Zeiger, 1998). Hormonally, shoots can produce growth regulators such as auxin and roots can produce growth regulators such as cytokinin. These regulators are easily transferred either via the phloem into the roots or via the xylem up into the shoots (Taiz and Zeiger, 1998). Gibberellic acid and abscisic acid also are transported via the plants vascular system. Although plant hormones may not have a direct effect on photosynthetic rate, they do control growth and related developmental processes such as sink growth and leaf senescence (Taiz and Zeiger, 1998). Carbohydrates such as sucrose have been shown to have inhibitory and stimulatory effects on gene expression (Taiz and Zeiger, 1998), potentially affecting the transcription of molecules needed to either stimulate or inhibit photosynthesis. Potato The potato (Solanum tuberosum L.) is in the Solanaceae family and was first cultivated by people in the high Andes of western South America at least 7,000 years ago 12 (Petroff, 2002). From Peru, Chile, and Colombia, Spanish and English explorers introduced potatoes to Europe in the mid 1500’s (Petroff, 2002). Today, the world produces approximately 350 million tons of potatoes per year with a U.S. annual consumption per capita of 61 kg fresh weight (Lachman et al., 2001). Potatoes are the fourth largest crop grown in the world and are exceeded in total hectares only by rice (Oryza sativa L.), wheat (Triticum aestivum L.), and corn (Zea mays L.) (Petroff, 2002). They are grown primarily for human consumption, providing a major source of energy and protein (Lachman et al., 2001) Potato plants are perennial in nature and survive year-to-year as a tuber. However, in commercial production potatoes are grown as an annual crop by planting certified, disease free, seed potatoes each year (IPMPWUS, 1986). There are four stages of growth and development for potato plants: vegetative growth, tuber initiation, tuber growth, and maturation (IPMPWUS, 1986). The vegetative growth stage begins when approximately three sprouts emerge with eight to 12 leaves to form the main stems (IPMPWUS, 1986). When the plant begins to flower, tuber growth is initiated. As tubers begin to develop at the tips of the stolons, more vegetative growth is observed (IPMPWUS, 1986). More carbohydrates are produced during photosynthesis than are required to sustain growth of the plant which allows for tuber enlargement. Maturation is the final growth stage and is characterized by the thickening of the tuber skin and senescing of potato plant tissue (IPMPWUS, 1986). Late season cultivars are indeterminate and have a vine type of growth with three stages of flowering (IPMPWUS, 1986). The primary flowering typically coincides with the tuber growth phase and after the plant has grown eight to 12 leaves, a branch 13 develops from the axillary bud producing eight to 12 more leaves to initiate secondary flowering. The process continues until after tertiary flowering when growth of the plant stops. Indeterminate varieties continue to produce tubers until disease, weather conditions, or lack of nutrients and water terminates their growth (IPMPWUS, 1986). Early season cultivars are determinate and have a bush type growth (IPMPWUS, 1986). Branches may develop after primary flowering, but always they develop from the nodes on the main stem. Determinate varieties tend to initiate tuber growth earlier and stop the growth of new foliage (IPMPWUS, 1986). Unlike indeterminate varieties, which typically complete their growth cycle in 100 days, determinate varieties complete their growth cycle in 60 to 80 days. Colorado Potato Beetle The Colorado potato beetle (Leptinotarsa decemlineata Say) was discovered in the Rocky Mountain region of North America in 1824, defoliating weeds such as buffalo bur (Solanum rostratum Ramur) and nightshades (Solanum dulcamara L.) within the Solanaceae family (Muka and Semel, 1983). Within 30 years, as settlers moved westward and started planting the cultivated potato, Colorado potato beetles shifted their preference of Solanaceae weeds to potato, a Solanaceae crop. In 1859, larvae and adult Colorado potato beetles were first recognized as a pest by settlers in the Eastern Rocky Mountain range (Ragsdale and Radcliffe, 2002). By 1874, Colorado potato beetles had migrated eastward an average of 85 miles per year, following potatoes grown on farms and in gardens in the Great Plains and Ohio River Valley (Ragsdale and Radcliffe, 2002). 14 Since reaching the East Coast, Colorado potato beetles have been introduced to Europe and now can be found as far east as the Ural Mountains. Adult Colorado potato beetles have black spotted orange heads and their stout, oval bodies have 10 black-and-yellow alternating longitudinal lines on their elytra. The insect overwinters in the adult stage approximately 5-20 cm below the soil surface (Dwyer et al., 2001). In the spring, adult Colorado potato beetles emerge from the soil and search for suitable hosts. Once the beetles find a suitable host, they feed, mate, and lay eggs. Females, over a four week period, can lay 400-600 yellowish-orange eggs in rows of 10-30 clusters on the underside of leaves (Dwyer et al., 2001), and over a lifetime, the female may lay more than 3500 eggs (Stetter Neel, 1992). Eggs hatch within 4-10 days, and the neonates immediately start feeding. The dark orange larvae are humpbacked with two lateral rows of black spots. Within a 2-3 week period, the dark orange larvae undergo four molts, getting larger with each molt (Dwyer et al., 2001). On the final molt, the larvae construct a small chamber in the ground to pupate and emerge as adults in 5-10 days. Depending on the season, as many as three different generations of Colorado potato beetles may occur. Colorado Potato Beetle Injury and Potato Response Colorado potato beetle adults and larvae defoliate potato leaves. Defoliation often affects the production, transport, or allocation of resources within the plant, reducing the final yield of the crop due to decreased growth. Dripps and Smilowitz (1989) found that injury from Colorado potato beetles during the tuber growth or full-bloom phase caused a greater reduction of leaf, stem, and tuber growth than injury during the vegetative phase 15 or before bloom. Decreases in vegetative and tuber growth at the flowering stage may be caused by a decrease in leaf area and thus photosynthesis and carbohydrate production in plants with high levels of defoliation. A decrease in the production of carbohydrates at a growth stage where carbohydrate production is critical to maintain growth, decreases total yield. Potato Biochemical Response to Colorado Potato Beetle Injury Plants have developed both physical and chemical defense mechanisms to reduce injury by insects and/or the impact of the injury by insects. Plant compensation is for every unit of injury, there is no plant damage. In some plant species, insect injury will affect the level of plant compensation, so that if the growth rate is high, the demand for photoassimilates will be high, increasing the overall photosynthetic rate (Delaney and Macedo, 2001). Plants can also induce resistance through the production of allelochemicals that may act as attractants and/or deterrents to beneficial and non- beneficial insects and/or use degrees of tolerance where they can endure or compensate for varying levels of injury (Delaney and Macedo, 2001). Primary metabolites are compounds involved in photosynthesis, respiration, solute transport, translocation, nutrient assimilation, or differentiation (Taiz and Zeiger, 1998). Unlike primary metabolites, secondary metabolites have no known direct function in growth and development, but function as biochemical defenses against injury and stress (Taiz and Zeiger, 1998). Secondary metabolites are highly diverse among taxa and their presence has lead to extensive research on the origin, diversity, and role that these chemicals play in plant-insect interactions, as well as their specificity to insects (Theis 16 and Lerdau, 2003). Through natural selection, secondary metabolites are thought to have evolved with little metabolic costs and toxicity to the plants themselves (Theis and Lerdau, 2003). Many of these secondary compounds function as natural pesticides within plants, and there is increasing interest to enhance these natural pesticides for commercial use (Fenwick et al., 1990). Plants are being bred to contain not only a greater diversity of natural compounds, but also greater quantities (Hlywka et al., 1994). At certain concentrations, these compounds are toxic to humans and livestock (Theis and Lerdau, 2003). There are three types of secondary compounds: terpenes, phenolics, and nitrogen- containing alkaloids. Although terpenes are the largest class of secondary metabolites (Thies and Lerdau, 2003), glycoalkaloids, in North America are thought to be the most highly consumed natural toxin (Hall, 1992). However, little is known about the human dietary risks associated with consumption of these chemicals and how those risks are altered by insect injury. In potatoes, glycoalkaloids serve as natural defense mechanisms against pathogens and insects (Lachman et al., 2001). Because naturally occurring pesticides may be synthesized when plants are under stress, it is expected that injury to plant tissue would instigate synthesis of higher concentrations of the naturally occurring pesticides in both the injured and uninjured plant tissue. Hlywka et al. (1994) found tubers from plants subjected to Colorado potato beetle defoliation contained higher glycolalkaloid concentrations than tubers from undefoliated plants. In field experiments, Hlywka et al. (1994) observed a 37.53% increase in glycoalkaloid concentrations of tubers from 17 ‘Superior’ plants defoliated at high levels by Colorado potato beetles in comparison to control plants. In tubers from ‘Superior’ plants manually defoliated, there was a 13.33% increase in glycoalkaloid concentration in comparison to tubers from control plants. Hlywka et al. (1994) argued that potato plants protected by pesticides may result in a higher degree of safety by reducing the natural production of glycoalkaloids in tubers when plants are stressed. Glycoalkaloids In the potato plant, glycoalkaloids are found in high concentrations in the leaves, stems, and sprouts. Lower concentrations of glycoalkaloids can be found in the skin of tubers and areas where sprouts emerge (Lachman et al., 2001). Friedman and Dao (1992) found that leaves of red, white, and Russet Burbank potatoes grown in a greenhouse had a concentration of glycoalkaloids 10 times greater than the tubers and a sprout glycoalkaloid concentration nearly 68 times greater than the tubers. Phillips et al. (1996) observed a greater concentration of glycoalkaloids in the leaves compared to tubers from the same plants; however, there was a great deal of variability among leaf glycoalkaloid concentrations within the same variety of plants. In tubers, the greatest concentration of glycoalkaloids was found in the skin (Bejarano et al., 2000), and the greater the concentration of glycoalkaloids present in tubers, the more bitter the taste (Lachman et al., 2001). Although there are many glycoalkaloids, α-chaconine and α-solanine account for up to 95% of the total glycoalkaloids present (Friedman and McDonald, 1997); α- 18 solanine is found in greater concentrations than α-chaconine, and α-solanine has only half as much specific activity as α-chaconine (Lachman et. al., 2001). Other glycoalkaloids that are present, but in much lower concentration, are β- and γ- solanines and chaconines, α- and β-solamarines, and aglycones demissidine and 5-β-solanidan-3-a- ol (Friedman and McDonald, 1997). In wild potato, leptines, commersonine, demissine, and tomatine are also present (Friedman and McDonald, 1997). Roddick and Rijnenberg (1986) observed a degree of synergism between α- solanine and α-chaconine on the lysis of phospholipids/sterol liposomes, which was dependent upon the concentrations of α-solanine and α-chaconine with the greatest effect observed when α-chaconine comprised over 40% of the glycoalkaloids present. In addition, Roddick and Rijnenberg (1986) tested the synergism between α-solanine and α- chaconine, α-tomatine, digitonin, and β2-chaconine, and observed synergistic effects only between α-solanine and α-chaconine. Factors that increase glycoalkaloid levels in tubers include tuber exposure to light, bruising, cutting, rotting by fungi or bacteria, and other forms of mechanical damage (Lachman et al., 2001). Lachman et al. (2001) found that between damaged tubers and undamaged tubers, damaged tubers had 89% higher glycoalkaloid content and in 1994, when weather conditions were unfavorable and dry, the glycoalkaloid content was 71% higher than the content in 1995. In a greenhouse study, tubers harvested from a “hot” chamber versus a “cool” chamber contained a greater concentration of glycoalkaloids (Lachman et al., 2001). In addition to growing conditions, handling, and storage, tubers 19 exposed to light often turn green and can have especially high glycoalkaloid concentrations (Friedman and McDonald, 1997). Health Risks Tablestock tubers typically have about 75 mg/kg fresh weight (FW) or 500 mg/kg dry weight (DW) total of α-chaconine and α-solanine (Zeiger, 1998). Neither α-solanine nor α-chaconine are regulated in the US. The U.S. Department of Agriculture recommended food-safety level for glycoalkaloids is 200 mg/kg FW or 1000 mg/kg DW (Bejarano et al., 2000; Zeiger, 1998). Most commercial tablestock tubers contain between 20 and 130 mg/kg FW (Zeiger, 1998) or 133 and 867 mg/kg DW. Levels of α- solanine greater than 140 mg/kg FW or 933 mg/kg DW taste bitter, and levels greater than 200 mg/kg FW or 1333 mg/kg DW cause a burning sensation in the throat and mouth (Lachman et al., 2001). Dry weights of potatoes typically are calculated by assuming a fresh weight reduction of 85% (Eborn, 2000). In humans, the toxic dose for glycoalkaloids is 2 to 5 mg/kg of body weight (BW) and the fatal dose is 3 to 6 mg/kg BW (Morris and Lee, 1984). According to Friedman and McDonald (1997), the toxic effect level seems to be closer to 1.0 mg/kg BW, but there are few human toxicity studies. Hellanäs et al. (1992) used seven volunteers who abstained from eating potatoes for two days and were then given potatoes containing glycoalkaloids at a dose of 1 mg/kg BW per individual. Six out of the seven subjects experienced burning sensation of the mouth and light to severe nausea and one of the six experienced diarrhea; the first initial symptoms were observed 30 minutes after 20 consumption and lasted for approximately four hours. Animal studies have shown that in mouse, rat, and hamster tissue, α-chaconine and α-solanine reached their highest concentrations within 6 to 14 hours of ingestion and in less than 35 hours, peak concentrations in the blood were reached (Zeiger, 1998). The symptoms of “solanine” poisoning include nausea, vomiting, diarrhea, stomach and abdominal cramps, headache, fever, rapid and weak pulse, rapid breathing, hallucinations, delirium, and coma (Friedman and McDonald, 1997). Effects on the nervous system include increased heart, pulse, and respiratory rates, sedation, and coma (Zeiger, 1998). Effects from cell membrane disruption include internal hemorrhaging, edema, diarrhea, constriction of the abdominal muscles, and lesions in the stomach and duodenum of the large intestines. Teratogenic effects have been observed mainly in the central nervous system and include exencephaly, cranial bleb, encephalocele, and anophthalmia (Zeiger, 1998). Alpha-chaconine seems to exert teratogenic effects at lower concentrations than α-solanine (Zeiger, 1998). Tissues that were observed to accumulate α-chaconine and α-solanine were abdominal fat, adrenals, blood, brain, heart, kidney, liver, lungs, muscle, pancreas, spleen, testis, thymus, and thyroid. Alpha-chaconine and α-solanine remained unchanged or as solanidine when excreted in urine and feces (Zeiger, 1998). Peeling reduces the quantity of glycoalkaloids in tubers 30 to 80% (Zeiger, 1998). Alpha-chaconine and α-solanine are not broken down from cooking or frying because they are heat stable and only begin to break down between 230 to 280°C (Bejarano et al., 2000). 21 Although the potential hazard associated with human consumption of plants injured by insects has been recognized (Hlwyka et al., 1994), there has not been an analytical consideration of the human dietary risks associated with increased production of glycoalkaloids as a result of Colorado potato beetle injury. Risk Assessment Risk assessment is a science-based framework for the objective evaluation of risk (NRC 1983). It involves three major steps: problem formulation, data analysis, and risk characterization (USOSTP, 1999). Problem formulation establishes the objective and scope of the risk assessment, whereas the data analysis phase incorporates two steps: hazard identification and exposure assessment. In terms of human dietary risk assessment, hazard identification identifies the inherent ability for a substance to cause harm to a human subgroup and may either be an estimate or based on a direct measurement. Hazard identification often utilizes dose-response relationships to determine the dose required to elicit a harmful response. Exposure assessment identifies the human subgroups at greatest risk for adverse health effects, the route of exposure (dermal, oral, or inhalation), the type of exposure (acute, subchronic, or chronic), and the duration of exposure; it also is the phase where exposure levels are determined based on consumption data and potential changes in the exposure level due to food preparation and storage practices. Risk characterization is the phase where the estimate of risk to the human subgroups is determined by integrating the information gathered in the data analysis phase; this involves the identification of areas where assumptions were made, 22 areas of uncertainties, and areas where more information is required to refine the estimates of risk. 23 GAS EXCHANGE RESPONSES OF POTATO TO COLORADO POTATO BEETLE DEFOLIATION Abstract Gas exchange was measured on potato leaves (Solanum tuberosum L.) on two varieties in response to varying levels of simulated and actual Colorado potato beetle (Leptinotarsa decemlineata Say) defoliation. For larval and adult Colorado potato beetle single-leaf defoliation experiments, no alteration in photosynthesis was observed on the remaining tissue of an injured leaf for Cal Red and Russet Burbank leaves defoliated by larval Colorado potato beetles nor for Russet Burbank leaves defoliated by adult Colorado potato beetles. No significant differences were observed between actual Colorado potato beetle and manual defoliation for both varieties. This indicates that artificial defoliation techniques may be appropriate to measure certain responses of potato to Colorado potato beetle injury and adult and larvae Colorado potato beetles may be assigned to the same injury guild. There was a significant alteration in photosynthetic rate for Cal Red leaves defoliated by adult Colorado potato beetles, but there were fewer replications of defoliation levels with that variety. In both of the whole-plant defoliation studies, defoliation type or level consistently did not result in increased or decreased gas exchange parameters compared to undefoliated controls. Although there were significant differences between gas exchange variables in upper and lower leaves in this study, there was no evidence of delayed senescence in defoliated treatments. Although, there was a significant difference among defoliation levels in both whole plant studies, defoliation type or level consistently did not result in increased or decreased gas exchange 24 parameters compared to undefoliated controls. Therefore, Colorado potato beetle injury on Cal Red and Russet Burbank potato plants most likely can be classified as leaf-mass consumption. Introduction Understanding the physiological responses of plants to insect injury is important for understanding plant population dynamics, life histories of both plants and insects, and for determining yield loss relationships and economic injury levels in agroecosystems. Plant gas exchange incorporates photosynthesis, stomatal conductance, respiration, and transpiration; each is a critical process for plant growth, development, and fitness. Variable photosynthetic responses to insect herbivory have been observed among different combinations of plant taxa and types of insect injury. Photosynthetic rates are often dependent upon the type of insect injury imposed (Welter, 1989; Higley et al., 1993; Haile, 2001; Peterson, 2001). Injury Guilds Most studies have focused on the effects of plant gas exchange from insects classified as leaf-mass consumers (defoliators), which consume large amounts of leaf tissue in a relatively short time (Welter, 1989; Peterson and Higley, 1993; Peterson and Higley, 1996). Leaf-mass consumption is just one type of insect injury guild. Currently, several insect injury guilds have been identified based on differences in physiological responses of the host plant (Boote, 1981; Pedigo et al., 1986; Higley et al., 1993). These injury guilds include: leaf senescence alteration, leaf-mass consumption, leaf 25 photosynthetic-rate reduction, light reduction, assimilate removal, water-balance disruption, population or stand reduction, seed or fruit destruction, architecture modification, and phenological disruption. Effects of Insect Injury on Photosynthetic Rates A large amount of research has been conducted on the photosynthetic and other gas exchange responses of soybean (Glycine max L.) injured by different species of insects. Most of the research has involved the leaf-mass consumption injury guild (Peterson et al., 2004). In many of these studies, the only change observed after defoliation was a transient increase in water loss, suggesting that the photosynthetic pathway is not affected by defoliation. In two greenhouse experiments, soybean leaves at the V5 and R2 growth stages were defoliated by the green cloverworm (Plathypena scabra (F.)), painted lady butterfly (Vanessa cardui (L.)) larvae, and hole punches from a paper hole puncher and cork-borer punch. Each experienced reductions in photosynthetic rate 12 hours post injury, but no differences 24, 48, 72, or 96 hours post injury (Poston et al., 1976). In a study by Hammond and Pedigo (1981), soybean leaves defoliated by green cloverworm larvae, hole punches, and excision of whole leaves resulted in a significantly greater water loss in leaves defoliated by green cloverworm larvae and by a hole punch than excision of an entire leaf. Although leaf-mass consumption typically does not alter photosynthetic rates of the remaining leaf tissue, reductions in photosynthetic rates after leaf-mass consumption that have been observed may be due to interactions between intrinsic and extrinsic factors. Intrinsic factors that may alter photosynthetic rates include increased assimilate 26 demand after defoliation (Neales and Incoll, 1968), reduced competition between leaves for mineral nutrients necessary for cytokinin production (Wareing et al., 1968), and a diversion of resources to defenses. Extrinsic factors that may alter photosynthetic rates include alterations in sunlight penetration, water and nutrient availability (Welter, 1989; Peterson et al., 1992), alteration of source-to-sink ratios, temperature, and atmospheric carbon dioxide concentrations. At the canopy level, reductions in photosynthetic rate have also been observed, but many of these reductions were temporary and were caused by a reduction in leaf-area indices, smaller leaf size, and decreased light inception (Peterson, 2001). Secondary Compounds and Photosynthetic Rates Plants have evolutionarily developed both physical and chemical defense mechanisms to reduce injury by insects. Plants can induce resistance through the production of chemicals that may act as attractants and/or deterrents to beneficial and non-beneficial insects and/or use degrees of tolerance where they can endure or compensate for varying levels of injury (Delaney and Macedo, 2001). Primary metabolites are compounds involved in photosynthesis, respiration, solute transport, translocation, nutrient assimilation, or differentiation (Taiz and Zeiger, 1998). Unlike primary metabolites, secondary metabolites have no known direct function in growth and development, but have been observed to function as biochemical defenses against injury and stress (Taiz and Zeiger, 1998). Secondary metabolites are highly diverse among taxa and their presence has led to extensive research on the origin, diversity, and role that these chemicals play in plant-insect interactions, as well as their 27 specificity to insects. Through natural selection, secondary metabolites are thought to have evolved with little metabolic costs and toxicity to the plants themselves (Theis and Lerdau, 2003). Many of these secondary compounds function as natural pesticides within plants. Milkweed (Asclepiadaceeae) species have two well-known defense mechanisms: (1) leaf tissue containing one to several mild to severely toxic cardenolides, and (2) leaf tissue containing latex that dries on insect mouths to disrupt feeding. Defoliation using a cork- borer resulted in an eight-fold increase in cardenolides (Malcolm and Zalucki, 1996), and although latex and cardenolides do not always occur together, in species in which they do occur together, the cardenolides may be found in latex fluid. Delaney and Higley (2006) observed reductions in photosynthetic rates of common milkweed (Asclepias syriaca L.) on remaining leaf tissue of individual leaves injured by leaf-mass consumers. Cutting all of the lateral veins on milkweed leaves did not result in as significant of a decrease in photosynthetic rate compared to midrib vein disruption (Delaney and Higley, 2006). The midrib vein contains the xylem and phloem, which are involved in the import and export of water, and disruption of the midrib vein in milkweed resulted in localized decreases in photosynthetic rate in the tissue distal from the wound site as a result of disrupting the xylem and/or phloem (Delaney and Higley, 2006). Although water loss played a role in the decreases in photosynthetic rate on the remaining leaf tissue, because the disruption of the lateral veins did not result in as severe of a decrease in photosynthetic rate as did disruption to the midrib vein, there may be a tradeoff between a plant’s natural defense (i.e., high cardenolide concentrations) and the ability for the leaf to compensate for insect injury. 28 Colorado Potato Beetle Injury and Potato Response In potatoes (Solanum tuberosum L.), glycoalkaloids are well known and highly toxic natural defense compounds against pathogens and insects (Lachman et al., 2001). Because naturally occurring pesticides are synthesized when plants are under stress, it is expected that injury to plant tissue would instigate synthesis of higher concentrations of the naturally occurring pesticides in the injured versus uninjured plant tissue. Hlywka et al. (1994) found tubers from plants subjected to Colorado potato beetle (Leptinotarsa decemlineata Say) defoliation contained higher glycolalkaloid concentrations than tubers from undefoliated plants. Colorado potato beetle adults and larvae consume leaf-mass of potato. Dripps and Smilowitz (1989) found that injury from Colorado potato beetles during the tuber growth or full-bloom phase, caused a greater reduction of leaf, stem, and tuber growth than injury during the vegetative phase or before bloom. Decreases in vegetative and tuber growth at the flowering stage may be caused by a decrease in leaf area and thus photosynthesis and carbohydrate production in plants with high levels of defoliation. A decrease in the production of carbohydrates at a growth stage where carbohydrate production is critical to maintain growth, decreases total yield. Despite the potato being the fourth most important food crop in the world and Colorado potato beetles being a worldwide potato pest, there is very little information on how potato responds to injury from this pest. This is particularly true for physiological responses such as photosynthesis. Both Colorado potato beetle adults and larvae consume whole-leaf tissue and therefore might be considered leaf-mass consumers. However, to date, the primary physiological responses have not been characterized to test this 29 hypothesis. Alternatively, because potato contains high levels of toxic defensive compounds (glycoalkaloids) it may respond to defoliation similar to milkweed. Objective The current understanding of plant responses to biotic stressors such as insects is limited, and understanding a plants physiological response to injury provides a common language and insight into managing such pests in order to optimize development, fitness, and growth. This research was conducted to characterize the primary physiological responses of potato by measuring photosynthetic rate, stomatal conductance, and transpiration at varying levels of simulated and actual Colorado potato beetle defoliation. Materials and Methods Plant Material All plants were grown in a greenhouse (Montana State University, Bozeman, MT) and planted in 50:50 MSU:Sunshine #1 soil mix, and fertilized weekly with Scott’s Peter Professional Peat-Lite Special 20-20-20. The Sunshine Mix #1 consisted of Canadian Sphagnum Peat Moss, perlite, vermiculite, starter nutrient charge, wetting agent, and Dolomitic lime (Sun Gro Horticulture, Inc., Bellevue, WA). The MSU soil mix consisted of equal parts of Bozeman Silt Loam Soil, washed concrete sand, and Canadian Sphagnum Peat Moss in addition to AquaGro 2000 G wetting agent blended in at 593 g/m3 of soil mix. The MSU soil mix was aerated-steam pasteurized to 80ºC for 45 min. 30 Plants were grown at 21 ± 2°C with a photoperiod of 14:10 (Light:Dark). Two cultivars were used, Cal Red and Russet Burbank, and were obtained from VenHuizen Seed Potatoes, Inc., Belgrade, MT. Each seed tuber was cut, ensuring at least one eye per cut, and placed cut-side down in a 20-L pot filled approximately with 12 to13 cm of pre- moistened 50:50 MSU:Sunshine soil mix and covered with 5 to 9 cm of pre-moistened 50:50 MSU:Sunshine soil mix. Plants were watered 4 to 5 times/week and fertilized with Scott’s Peter Professional Peat-Lite Special 20-20-20 bi-weekly. Once plants reached approximately 9 cm tall, in a process called “hilling,” soil was added to the pots weekly until pots were full. Upon senescence, stems were cut and potatoes were harvested two weeks later. The tubers were washed, placed in brown paper bags, and stored in a dark cold room held at 4°C. Colorado Potato Beetles At the early vegetative stage, Colorado potato beetle eggs were obtained from the Phillip Alampi Beneficial Insect Lab, New Jersey Department of Agriculture, Trenton, New Jersey. Approximately 120 egg masses were placed on approximately 10 to 15 extra Cal Red and Russet Burbank potato plants and allowed to hatch and feed until approximately the third instar. At approximately the third instar, the Colorado potato beetles were placed on the treatment plants and allowed to defoliate. 31 Larval Leaf Defoliation Study – 2005 Responses of individual potato leaves to defoliation injury by Colorado potato beetle larvae were. The greenhouse bay (32 m2) was set at 21 ± 2°C and a photoperiod of 14:10 (Light:Dark). A randomized complete block design (RCBD), repeated measures two-by-five factorial with five replications was used, with five metal halide lamps (1000 W) serving as the blocking factors. The treatment factors were the two cultivars, Cal Red and Russet Burbank, and the five defoliation factors were control (no defoliation), low and high simulated Colorado potato beetle defoliation, and low and high actual larval Colorado potato beetle defoliation. At the early vegetative stage, a leaf in the top one-third of the plant was randomly chosen and covered with a net approximately 12 cm x 12 cm (w x h) made of green or white tulle. At the mid-vegetative stage, three and six third instars were applied to the low and high defoliation treatment leaves, respectively, and allowed to defoliate overnight to allow for approximately 30% (low) and 90% (high) of the leaf area to be removed. Simulated Colorado potato beetle injury treatments were imposed with scissors so that approximately 30% (low) and approximately 90% (high) of the leaf tissue was removed in a pattern consistent with actual larval injury immediately after the larvae were removed from the other treatment plants. Adult Leaf Defoliation Study – 2005 Responses of individual potato leaves to defoliation injury by Colorado potato beetle adults were determined. The greenhouse bay (32 m2) was set at 21 ± 2°C and a photoperiod of 14:10 (Light:Dark). A RCBD, repeated measures two-by-five factorial 32 with three replications was used, with five metal halide lamps (1000 W) serving as the blocking factors. The treatment factors were the two cultivars, Cal Red and Russet Burbank, and the five defoliation factors were control (no defoliation), low and high simulated defoliation, and low and high adult Colorado potato beetle defoliation. At the early vegetative stage, a leaf in the top one-third of the plant was randomly chosen and covered with a net approximately 12 cm x 12 cm (w x h) made of green or white tulle. At the mid-vegetative stage, one and three adult Colorado potato beetles were applied to the low and high defoliation treatment plants, respectively, and allowed to defoliate until approximately 30% (low) and 90% (high) of the plant leaf area was removed. Target defoliation levels were reached after approximately 9.5 hours. Simulated Colorado potato beetle injury treatments were imposed the same as described above. For both larval and adult Colorado potato beetle experiments, a portable photosynthesis system (Model LI-6400, LI-COR, Inc., Lincoln, NE) with CO2 injector and light source (to allow for stable CO2 and light concentrations) was used to measure plant-gas exchange (photosynthesis, stomatal conductance, and transpiration). These variables were measured 24 and 48 hours after injury. All measurements were taken on a 6-cm2 leaf area with the following settings: light source at 1200 µmol/m2/s photosynthetic photon flux density, 400 µmol of CO2/m2/s, and chamber humidity between 40 and 60%, from mid-morning to mid-afternoon. After the final measurements were recorded, the leaves were removed, labeled, and scanned using an HP Scanjet 7400c scanner (Hewlett-Packard Company, Palo Alto, CA) with the HP Precision Scan Pro 3.02 software (Hewlett-Packard Company, Palo 33 Alto, CA). Percentage of actual leaf area removed was measured using the image analysis software Sigma Scan Pro 5.0 (SPSS Inc., Chicago, IL) and Adobe Photoshop CS2 9.0 (Adobe Systems Inc., San Jose, CA). Manual Defoliation Study – 2004 Responses of individual potato leaves over time to whole-plant defoliation injury were determined in two experiments conducted in 2004 and 2005. In a greenhouse (32 m2) set at 21 ± 2°C and a photoperiod of 14:10 (Light:Dark), five replicates of Russet Burbank cultivar of the following treatments—control (no defoliation), low, medium, and high manual defoliation—were arranged in a two-by-four repeated measures factorial within an RCBD; low defoliation corresponded to approximately 30% leaf-mass removal, medium defoliation corresponded to approximately 60% leaf-mass removal, and high defoliation corresponded to approximately 90% leaf-mass removal. Metal halide lamps (1000 W) served as the blocking factors. Plants were defoliated over a 10-day period at the mid-vegetative stage, removing the following percentages of leaf-mass corresponding to their treatment levels (day 1 to 10): 1%, 1%, 5%, 5%, 10%, 10%, 30%, 30%, 4%, and 4%. Leaf-mass removal was followed according to these percentages to mimic larval Colorado potato beetle defoliation from the first to fourth instars just prior to pupation. The LI-6400 portable photosynthesis system was used to measure photosynthetic rate, as described above, on a leaf in the bottom third and a leaf on the top third of the plant. These gas exchange variables were measured one day after the cessation of 34 defoliation and thereafter weekly until the initiation of senescence (i.e., 10 weeks of measurements). Manual and Colorado Potato Beetle Defoliation Study – 2005 A greenhouse bay (32 m2) was set at 21 ± 2°C with a photoperiod of 14:10 (Light:Dark). A RCBD, repeated measures two-by-two-by-five factorial with five replications was used, with five metal halide lamps (1000 W) serving as the blocking factors. The treatment factors were the two cultivars, Cal Red and Russet Burbank, the two leaf factors were upper and lower leaves, and the five defoliation factors were control (no defoliation), low, medium, and high Colorado potato beetle and manual defoliation. At the early vegetative stage, all plants were individually covered with nets approximately 91 cm x 40 cm x 40 cm (h x w x d) made of white tulle. Approximately 120 egg masses were placed on 10 host Cal Red and Russet Burbank potato plants and allowed to hatch and feed until the larvae reached the third instar. At the mid-vegetative stage, just before flowering, approximately 15, 20, and 25 third instars were applied to the low, medium and high treatment plants, respectively, and allowed to defoliate. Once the low, medium, and high treatment plant leaf area was reduced by approximately 30% (low), 60% (medium), and 90% (high) the Colorado potato beetles were removed. The manually defoliated plants were defoliated with scissors throughout the same period to simulate the percentage and patterns of leaf-mass removed by the Colorado potato beetles. The LI-6400 portable photosynthesis system was used to measure gas exchange rate on a 2-cm2 leaf area using the procedure described above. A leaf in the bottom third 35 and a leaf in the top third of the plant was measured each time. Gas exchange parameters were measured one week following the application of Colorado potato beetles and weekly thereafter until the initiation of senescence (i.e., six weeks of measurements). Statistical Analyses Control and treated groups were compared (α = 0.05) using an analysis of variance (ANOVA) and interaction plots fitted with standard error. Data were analyzed using R 2.0.1 (R: A Language and Environment for Statistical Computing; The R Foundation for Statistical Computing, 2004, Vienna, Austria). To meet normality, the necessary data were log transformed. Linear and non-linear regression analyses were conducted to determine the relationship between percent defoliation and gas exchange parameters using SAS system for Windows V8 (SAS Institute, Inc. ©1999-2001, Cary, NC). Results Larval Colorado Potato Beetle Leaf Defoliation Study – 2005 The mean (± SE) for high Colorado potato beetle defoliation, low Colorado potato beetle defoliation, high manual defoliation, and low manual defoliation were 51.4 ± 6.1%, 26.8 ± 1.4%, 47.8 ± 2.4%, 14.3 ± 1.8%. There was no significant block, variety, defoliation, or time effect on photosynthetic rate (Table 1, Figures 1, 2). There was a significant interaction between variety, defoliation, and time (F = 2.592, df = 4, P = 0.044). Because there were no other interaction effects and because there were no variety or defoliation main effects, it is unlikely the three-way interaction has biological 36 Table 1. ANOVA for Larval Colorado Potato Beetle Leaf Defoliation Experiment (2005). Photosynthesis Stomatal Conductance Transpiration df p-value p-value p-value Block 4 0.5063 0.344 0.146 Variety 1 0.6467 0.0006 0.0013 Defoliation 4 0.5511 0.715 0.7804 Time 1 0.0566 0.0001 < 0.0001 Variety x Defoliation 4 0.6291 0.2661 0.319 Variety x Time 1 0.4971 0.0483 0.0734 Defoliation x Time 4 0.4405 0.2473 0.2841 Variety x Defoliation x Time 4 0.0439 0.2977 0.2407 37 0 2 4 6 8 10 12 14 16 1 2 Day C al R ed P ho to sy nt he tic R at e Control Low Manual Defoliation High Manual Defoliation Low Colorado Potato Beetle Defoliation High Colorado Potato Beetle Defoliation Figure 1. Photosynthetic Rates of the Larval Colorado Potato Beetle Leaf Defoliation Study (2005) on Cal Red plants. P ho to sy nt he tic R at e (μ m ol C O 2/m 2 /s ) 38 Figure 2. Photosynthetic Rates of the Larval Colorado Potato Beetle Leaf Defoliation Study (2005) on Russet Burbank Plants. 0 2 4 6 8 10 12 14 16 18 1 2 Day R us se t B ur ba nk P ho to sy nt he tic R at e Control Low Manual Defoliation High Manual Defoliation Low Colorado Potato Beetle Defoliation High Colorado Potato Beetle Defoliation P ho to sy nt he tic R at e (μ m ol C O 2/m 2 /s ) 39 relevance. Linear and quadratic relationships between percent defoliation and photosynthesis were not significant. Stomatal conductance was log transformed and variety (F = 13.077, df = 1, P = 0.0006), time (F = 18.133, df = 1, P = 0.0001), and the interaction between variety and time (F = 4.04, df = 1, P = 0.048) had a significant effect on stomatal conductance (Table 1). Linear and quadratic relationships between percent defoliation and conductance were not significant. Transpiration was log transformed and variety (F = 11.27, df = 1, P = 0.001) and time (F = 57.905, df = 1, P < 0.0001) had a significant effect on transpiration (Table 1). Linear and quadratic relationships between percent defoliation and transpiration rate were not significant. Adult Colorado Potato Beetle Leaf Defoliation Study – 2005 The mean (± SE) for high Colorado potato beetle defoliation, low Colorado potato beetle defoliation, high manual defoliation, and low manual defoliation were 55.7 ± 12.0%, 32.2 ± 11.4%, 47.5 ± 2.5%, 16.9 ± 1.9%. There was no significant block, variety, defoliation, or time effect on photosynthetic rate (Table 2, Figures 3, 4) The interaction between variety and defoliation had a significant effect on photosynthetic rate (F = 4.621, df = 4, P = 0.005) (Table 2). As defoliation level and type increased (i.e., more severe), Cal Red plants’ photosynthetic rate decreased (Figure 3); this same trend was not observed in Russet Burbank plants (Figure 4). Linear and quadratic relationships between percent defoliation and photosynthesis were not significant. 40 Table 2. ANOVA for Adult Colorado Potato Beetle Leaf Defoliation Experiment (2005). Photosynthesis Stomatal Conductance Transpiration df p-value p-value p-value Block 2 0.2326 0.385 0.3117 Variety 1 0.1417 0.0121 0.0277 Defoliation 4 0.2651 0.4824 0.5152 Time 1 0.5953 0.0395 0.0345 Variety x Defoliation 4 0.0054 0.1676 0.1095 Variety x Time 1 0.4523 0.8275 0.7971 Defoliation x Time 4 0.3659 0.1227 0.1094 Variety x Defoliation x Time 4 0.1597 0.5789 0.4954 41 0 5 10 15 20 25 1 2 Day C al R ed P ho to sy nt he tic R at e Control Low Manual Defoliation High Manual Defoliation Low Colorado Potato Beetle Defoliation High Colorado Potato Beetle Defoliation Figure 3. Photosynthetic Rates of the Adult Colorado Potato Beetle Leaf Defoliation Study (2005) Cal Red Plants. P ho to sy nt he tic R at e (μ m ol C O 2/m 2 /s ) 42 0 3 6 9 12 15 18 1 2 Day R us se t B ur ba nk P ho to sy nt he tic R at e Control Low Manual Defoliation High Manual Defoliation Low Colorado Potato Beetle Defoliation High Colorado Potato Beetle Defoliation Figure 4. Photosynthetic Rates of the Adult Colorado Potato Beetle Leaf Defoliation Study (2005) on Russet Burbank Plants. P ho to sy nt he tic R at e (μ m ol C O 2/m 2 /s ) 43 Stomatal conductance was log transformed and variety (F = 7.32, df = 1, P = 0.012) and time (F = 4.72, df = 1, P = 0.04) had a significant effect on conductance (Table 2). The linear relationship between percent defoliation and transpiration rate was not significant. A significant quadratic relationship was measured between percent defoliation and conductance 48 hours post injury (F = 5.62, df = 1, P = 0.0279, βo = 0.7501, β1 2 = 0.00001, r2 = 0.21932). Transpiration was log transformed and variety (F = 5.464, df = 1, P = 0.028) and time (F = 5.001, df = 1, P = 0.035) had a significant effect on transpiration (Table 2). The linear relationship between percent defoliation and transpiration rate was not significant. A significant quadratic relationship was measured between percent defoliation and transpiration 48 hours post injury (F = 6.24, df = 1, P = 0.0214, βo = 1.1226, β1 2 = 0.00003, r2 = 0.23771). Manual Defoliation Study – 2004 Photosynthesis was log transformed and block (F = 18.36, df = 4, P < 0.0001), defoliation (F = 4.218, df = 1, P = 0.006), leaf location (F = 4.742, df = 1, P = 0.03), and time (F = 134.87, df = 9, P < 0.0001) had a significant effect on photosynthetic rate (Table 3). The interaction between leaf and time also had a significant effect on photosynthetic rate (F =1.914, df = 9, P = 0.05) (Table 3). Photosynthetic rates for upper and lower leaves across all defoliation levels varied considerably throughout the 10 weeks of measurements (Figures 5, 6). Linear and quadratic relationships between percent defoliation and photosynthesis were not significant. 44 Table 3. ANOVA for Manual Defoliation Experiment (2004). Photosynthesis Stomatal Conductance Transpiration df p-value p-value p-value Block 4 < 0.0001 0.1905 0.5593 Leaf 1 0.0302 0.0092 0.0697 Defoliation 3 0.0061 0.0097 0.0186 Time 9 < 0.0001 < 0.0001 < 0.0001 Leaf x Defoliation 3 0.5196 0.2337 0.0977 Leaf x Time 9 0.0496 0.2738 0.103 Defoliation x Time 27 0.6196 0.0002 < 0.0001 Leaf x Defoliation x Time 27 0.9919 0.9088 0.7127 45 Figure 5. Photosynthetic Rates of the Manual Defoliation Study (2004) on Upper Leaves of Russet Burbank Plants. 0 5 10 15 20 25 30 35 40 45 50 1 2 3 4 5 6 7 8 9 10 Week U pp er L ea f P ho to sy nt he tic R at e Control Low Manual Defoliation Medium Manual Defoliation High Manual Defoliation P ho to sy nt he tic R at e (μ m ol C O 2/m 2 /s ) 46 Figure 6. Photosynthetic Rates of the Manual Defoliation Study (2004) on Lower Leaves of Russet Burbank Plants. 0 5 10 15 20 25 30 35 40 45 1 2 3 4 5 6 7 8 9 10 Week Lo w er L ea f P ho to sy nt he tic R at e Control Low Manual Defoliation Medium Manual Defoliation High Manual Defoliation P ho to sy nt he tic R at e (μ m ol C O 2/m 2 /s ) 47 Stomatal conductance was log transformed and treatment (F = 3.878, df = 3, P = 0.01), time (F = 55.749, df = 9, P < 0.0001), and leaf location (F = 6.877, df = 1, P = 0.0092) had a significant effect on stomatal conductance (Table 3). The interaction between defoliation level and time (F = 2.444, df = 27, P = 0.0002) also had a significant effect on stomatal conductance. Transpiration was log transformed and defoliation level (F = 3.392, df = 3, P = 0.019) and time (F = 27.773, df = 9, P < 0.0001) had a significant effect on mean transpiration (Table 3). The interaction between defoliation and level and time had a significant effect on mean transpiration (F = 2.635, df = 27, P < 0.0001) (Table 3). Manual and Colorado Potato Beetle Defoliation Study – 2005 Block (F = 6.077, df = 4, P = 0.0001), defoliation level (F = 2.843, df = 4, P = 0.024), time (F = 54.208, df = 5, P < 0.0001), and leaf location (F = 22.483, df = 1, P < 0.0001) had a significant effect on photosynthetic rate (Table 4). The interaction between variety and leaf location (F = 5.453, df = 1, P = 0.020) and defoliation level and leaf location (F =3.199, df = 4, P = 0.013) also were significant (Table 4). Although defoliation treatments had a significant effect on photosynthetic rates, no consistent pattern was evident (Figures 7 to 10). However, there was a trend for upper leaves on defoliated plants to have higher photosynthetic rates than lower leaves (Figures 7 to 10). Linear and quadratic relationships between percent defoliation and photosynthesis were not significant. Stomatal conductance was log transformed and block (F = 6.700, df = 4, P = 0.0001), defoliation level (F = 9.190, df = 4, P = 0.0001), time (F = 51.300, df = 5, P = 48 Table 4. ANOVA for Manual and Colorado Potato Beetle Defoliation Experiment (2005). Photosynthesis Stomatal Conductance Transpiration df p-value p-value p-value Block 4 0.0001 < 0.0001 0.071 Leaf 1 < 0.0001 < 0.0001 < 0.0001 Defoliation 4 0.0238 < 0.0001 0.002 Time 5 < 0.0001 < 0.0001 < 0.0001 Variety 1 0.4574 0.0962 0.4371 Leaf x Defoliation 4 0.0131 0.0001 0.0008 Leaf x Time 5 0.4322 0.8214 0.2014 Defoliation x Time 20 0.981 0.9596 0.9803 Variety x Time 5 0.917 0.2095 0.3491 Variety x Leaf 1 0.02 0.0001 0.0021 Variety x Defoliation 4 0.417 0.2447 0.7925 Defoliation x Time x Leaf 20 0.7981 0.785 0.6947 Variety x Defoliation x Time 20 0.9048 0.9869 0.9722 Variety x Defoliation x Leaf 4 0.3856 0.013 0.1266 Variety x Time x Leaf 5 0.8377 0.7867 0.8033 Variety x Defoliation x Time x Leaf 20 0.611 0.5272 0.6114 49 0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 Week C al R ed U pp er L ea f Ph ot os yn th et ic R at e Control Low Manual Defoliation High Manual Defoliation Low Colorado Potato Beetle Defoliation High Colorado Potato Beetle Defoliation Figure 7. Photosynthetic Rates of the Manual and Colorado Potato Beetle Defoliation Study (2005) on Upper Leaves of Cal Red Plants. P ho to sy nt he tic R at e (μ m ol C O 2/m 2 /s ) 50 0 1 2 3 4 5 6 7 1 2 3 4 5 6 Week C al R ed L ow er L ea f Ph ot os yn th et ic R at e Control Low Manual Defoliation High Manual Defoliation Low Colorado Potato Beetle Defoliation High Colorado Potato Beetle Defoliation Figure 8. Photosynthetic Rates of the Manual and Colorado Potato Beetle Defoliation Study (2005) on Lower Leaves of Cal Red Plants. P ho to sy nt he tic R at e (μ m ol C O 2/m 2 /s ) 51 0 1 2 3 4 5 6 7 8 1 2 3 4 5 6 Week U pp er L ea f R us se t B ur ba nk Ph ot os yn th et ic R at e Control Low Manual Defoliation High Manual Defoliation Low Colorado Potato Beetle Defoliation High CPB Defoliation Figure 9. Photosynthetic Rates of the Manual and Colorado Potato Beetle Defoliation Study (2005) on Upper Leaves of Russet Burbank Plants. P ho to sy nt he tic R at e (μ m ol C O 2/m 2 /s ) 52 0 1 2 3 4 5 6 7 1 2 3 4 5 6 Week Lo w er L ea f R us se t B ur ba nk Ph ot os yn th et ic R at e Control Low Manual Defoliation High Manual Defoliation Low Colorado Potato Beetle Defoliation High Colorado Potato Beetle Defoliation Figure 10. Photosynthetic Rates of the Manual and Colorado Potato Beetle Defoliation Study (2005) on Lower Leaves of Russet Burbank Plants. P ho to sy nt he tic R at e (μ m ol C O 2/m 2 /s ) 53 0.0001), leaf location (F = 63.420, df = 1, P < 0.0001) and the interaction between defoliation level and leaf location (F = 5.800, df = 4, p = 0.0001) and variety and leaf location (F = 15.630, df = 1, p = 0.0001) had a significant effect on conductance (Table 4). Linear and quadratic relationships between percent defoliation and conductance were not significant. Transpiration was log transformed and defoliation levels (F = 4.303, df = 4, P = 0.002), time (F = 26.6176, df = 5, P < 0.0001), and leaf location (F = 43.964, df = 1, P < 0.0001) had a significant effect on transpiration (Table 4). The interaction between variety and leaf (F = 9.607, df = 1, P = 0.0021) and defoliation level and leaf (F = 4.828, df = 4, P = 0.008) also had a significant effect on transpiration (Table 4). Linear and quadratic relationships between percent defoliation and transpiration rate were not significant. Discussion In both the larval and adult Colorado potato beetle single-leaf defoliation experiments, there were generally no significant differences in photosynthetic rates among defoliation type or levels. This indicates that there was no alteration in photosynthesis of remaining tissue on an injured leaf. This response has been observed in numerous studies with the leaf-mass consumption injury type (Welter, 1991; Higley, 1992; Peterson et al., 1992, 1996; Burkness et al., 1999; Peterson, 2001). There was a significant relationship between Cal Red leaves and defoliation level, but there were fewer replications of defoliation levels with that variety. Additionally, there were no significant differences between actual Colorado potato beetle and manual defoliation, 54 indicating that artificial defoliation techniques may be appropriate to measure certain responses of potato to Colorado potato beetle injury. Although the photosynthetic responses of larval and adult Colorado potato beetles were not compared in the same experiment, it seems likely that both life stages elicit similar responses and therefore belong to the same injury guild. For both larval and adult Colorado potato beetle single-leaf defoliation experiments, both variety and time had significantly altered stomatal conductance and transpiration. Although previous studies on other plant species have noted significant, yet transient effects of insect and manual defoliation on those physiological parameters (i.e., typically higher transpiration and lower stomatal conductance until the cut edges of tissues healed) (Poston et al., 1976; Hammond and Pedigo, 1981; Ostlie and Pedigo, 1984; Peterson and Higley, 1996; Aldea et al., 2005), those effects were not observed in these studies 24- or 48-hours after injury. Because Hlywka et al. (1994) and this research (Chapter 2) found that tubers from plants subjected to Colorado potato beetle defoliation contained higher glycolalkaloid concentrations than tubers from undefoliated plants, but photosynthetic rates among treatments were unaffected, there does not seem to be a tradeoff between a potato plant’s natural defense and photosynthesis. This is counter to the observations with common milkweed where defoliation caused a decrease in photosynthetic rate, indicating there may be a tradeoff between milkweed’s natural defense (i.e., high cardenolide concentrations) and the ability for the leaf to compensate for insect injury. In both of the whole-plant defoliation experiments, there were significant defoliation effects on photosynthesis, stomatal conductance, and transpiration when 55 measured on individual leaves. However, there were no consistent trends to establish a generalized treatment effect. That is, defoliation type or level did not result consistently in increased or decreased gas exchange parameters compared to undefoliated controls. Although not consistent, the undefoliated controls seemed to have lower photosynthetic rates than defoliated treatments over several dates (Figures 7 to 10). This was not unexpected because defoliated plants have altered source-sink relationships, resulting in increased photosynthesis of remaining leaves through increased assimilate demand from sinks. Photosynthetic rates also could have increased from improved water and nutrient availability. According to Taiz and Zeiger (1998), if the source-to-sink ratio is altered so that the number of sources per sink is reduced, an increase in the photosynthetic rate of that single source leaf would be observed. In addition, when sink demand increases, the photosynthetic rate often increases, but when the sink demand decreases, the photosynthetic rate often decreases. Because source leaves transfer their photoassimilates to sink tissue, and sinks store the photoassimilates for use in respiration and to make other critical compounds, it is likely that the source-to-sink ratio was not severely alt