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DATE DUE DATE DUE DATE DUE 5/08 K;IPrq/Acc&Pres/ClRC/DateDue indd CHARACTERIZING THE SUB-LETHAL EFFECTS OF Two INSECT GROWTH REGULATORS ON PLUM CURCULIO, CONOTRA CHELUS NENUPHAR (HERB ST), IN APPLES AND CHERRIES By KiDukKim A THESIS Submitted to Michigan State University In partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Entomology 2009 ABSTRACT CHARACTERIZING THE SUB-LETHAL EFFECTS OF TWO INSECT GROWTH REGULATORS ON PLUM CURCULIO, CONOTRACHELUS NENUPHAR (HERBST), IN APPLES AND CHERRIES By Ki Duk Kim With the implementation of Food Quality Protection Act 1996, fruit growers are forced to use pesticides that are becoming available in the form of reduced-risk and organophosphate-altemative compounds. Two of newly registered compounds, novaluron and pyriproxyfen, from a class of insecticides categorized as insect growth regulators were investigated in laboratory and field for their potential sub-lethal performance against plum curculio. Adult exposure to novaluron reduced the number of viable eggs in laboratory bioassays. Field trials have also Shown this sub-lethal activity when adults were exposed to sprayed shoots containing either fruits or leaves. In case of sprayed fruit exposure, novaluron sub-lethal activity lasted up to 14 d post spray. Pyriproxyfen reduced the survival rate of overwintering northern strain plum curculio. Both novaluron and pyriproxyfen have Shown unique sub-lethal activity that can be utilized in apple and cherry IPM, but more investigation is needed to determine the applicability of these compounds. To my parents, Gyoung Hwan Kim and Mi Sook Kim, for their love and support ACKNOWLEDGMENTS I would like to thank my major professor, Dr. Mark Whalon for everything he has done to help me get through my maters. It was an honor and great privilege working with him. I also would like to thank Dr. John Wise. I could have never finished my thesis without his help and continuous support. I cannot forget to mention a visiting professor from Turkey, Dr. Ayhan Gokce, who had Shown me how an enthusiastic scientist should do his research. I thank Dr. Rufus Isaacs and Dr. Kevin Walker for their input at the defense. It really opened my eyes to see what it means to be a scientist. I thank everyone in the Whalon lab for their assistance; Willye Bryan, Dan Nortman, Soo-hoon Kim, Peter Nelson, Renee Pereault, Alex Johnson, Jeannette Wilson, Abbra Puvalowski, Andrew Skwiercz, Lisa Losievsky, Christopher Archangeli, Saunte Sutton, and Zach Koan. A special thanks for my friend Soo-hoon Kim who had been there with me at every Step of my thesis research. It had been a pleasure working with him. Thanks to everyone at the MSU Agricultural Experiment Stations in Clarksville and Fennville who helped me carry out my field trials. Funding contributions were provided by Michigan Apple Research Committee, Cherry Marketing Institute, and USDA-CAR. iv TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii INTRODUCTION ............................................................................................................... 1 CHAPTER 1 Novaluron reduces larval survival in plum curculio (Conotrachelus nenuphar Herbst) after adult exposure .............................................................................................................. 7 Abstract .......................................................................................................................... 7 Introduction .................................................................................................................... 7 Materials and methods ................................................................................................... 8 Insect material ......................................................................................................... 8 Chemical materials and application methodology .................................................. 9 Plum curculio larval survival and emergence ....................................................... 10 Dissection methods ................................................................................................ I I Results and discussion ................................................................................................. 11 CHAPTER 2 Influence of reproductive maturity, exposure, and residue field age on novaluron’s sub- lethal activity on plum curculio ......................................................................................... 14 Abstract ........................................................................................................................ 14 Introduction .................................................................................................................. 15 Materials and methods ................................................................................................. 18 General methods .................................................................................................... 18 Insect materials ...................................................................................................... I 8 Chemical materials ................................................................................................ 19 Laboratory studies .................................................................................................. 19 Exposure methods .................................................................................................. 19 Maturation level of the ovary ................................................................................. 21 Field studies ........................................................................................................... 22 Field plots and treatment applications .................................................................. 22 Residual activity bioassay ...................................................................................... 23 Novaluron residue profile analysis ........................................................................ 25 Plant growth. .......................................................................................................... 26 Results .......................................................................................................................... 27 Laboratory studies .................................................................................................. 27 Exposure methods .................................................................................................. 27 Maturation level of the ovary ................................................................................. 28 Field studies ........................................................................................................... 29 Residual activity bioassay ...................................................................................... 29 Novaluron residue profile analysis ........................................................................ 32 Plant growth. .......................................................................................................... 33 Discussion .................................................................................................................... 34 Laboratory studies .................................................................................................. 34 Field studies ........................................................................................................... 35 Novaluron residue profile analysis and plant growth ............................................ 37 CHAPTER 3 Efficacy of single pyriproxyfen treatment in comparison to multiple treatments ............. 38 Introduction .................................................................................................................. 38 Materials and methods ................................................................................................. 39 Results and discussion ................................................................................................. 39 CHAPTER 4 Use of pyiproxyfen’s diapause breaking activity on plum curculio, Conotrachelus nenuphar (Herbst), as a population control strategy .......................................................... 41 Abstract ........................................................................................................................ 41 Introduction .................................................................................................................. 42 Materials and methods ................................................................................................. 44 General methods .................................................................................................... 44 Insect materials ...................................................................................................... 44 Field efficacy ......................................................................................................... 45 Chemical materials and application methods ........................................................ 45 Dissection and larval emergence ........................................................................... 46 Overwintering survival .......................................................................................... 47 Chemical materials and application methods ........................................................ 47 Deployment site and methods ................................................................................ 48 Statistical analysis ................................................................................................. 49 Results .......................................................................................................................... 50 Field efficacy ......................................................................................................... 50 Overwintering survival .......................................................................................... 52 Discussion .................................................................................................................... 54 CONCLUSION .................................................................................................................. 55 APPENDICES ................................................................................................................... 59 REFERENCES .................................................................................................................. 61 vi LIST OF TABLES Table l. l. Plum curculio larval emergence after adult exposure to treated fruit. Total sum of larvae over 35 day emergence period .................................................................... 11 Table 1.2. Plum curculio ovary development and mating status following treatments with tebufenozide and novaluron ............................................................................................... 12 vii LIST OF FIGURES Figure 2.1. Schematic drawing of novaluron force-feeding method. ............................... 21 Figure 2.2. Schematic drawing of novaluron spray application apparatus. ...................... 22 Figure 2.3. Experimental setup for plum curculio bioassay after exposure to field-aged novaluron residue. Treatment groups were categorized according to applied pesticide (novaluron / control), plum curculio Strain exposed (northern / southern), exposure media (fruits I leaves), and collection time (4 M d/14 d). ........................................................... 24 Figure 2.4. Average number (:l: SE) of plum curculio larvae emerged after adult exposure to novaluron, treated through ingestion and contact methods. The larval emergence with * is significantly different from control (paired T-test, P<0.05). ...................................... 28 Figure 2.5. Average number (:I: SE) of plum curculio larval emergence after adult exposure to novaluron. The larval emergence with * is Significantly different from control (paired T-test, P<0.05) ........................................................................................... 29 Figure 2.6. Plum curculio, Conotrachelus nenuphar (Herbst), larval emergence following adult exposure to novaluron-treated fruit or leaves harvested at different intervals (4 h, 7 d, and 14 (I) post field application. The larval emergence with * is Significantly different from control (paired t-test, P<0.05) ................................................ 31 Figure 2.7. Mean residue profiles (6 samples) of novaluron surface and sub-surface residues on apple fruits and leaves. Residues measured are in micrograms active ingredient per gram fruit and leaf tissue taken at 4 h, 7 d, and 14rd post field application using a conventional air-blast sprayer ................................................................................ 33 Figure 2.8. Novaluron surface residues per cm2 on apple and leaves. Residues calculated from mean residue profile (figure 2.7), surface area, and weight data. ............................. 34 Figure 3.1. Conotrachelus nenuphar F1 mean larval emergence (:SE) after parent pyriproxyfen treatment. One time treatment (NSl), treatment every 30d (NS llm), treatment every 2wk (NS 1/2w), and untreated control (NS - control) ............................. 40 Figure 4.1. Schematic drawing of plum curuclio exposure chamber, 32 oz deli container (Fabri-Kal, Kalamazoo, MI) with wet florofoam, paraffin wax, and ventilated top ......... 46 Figure 4.2. Schematic drawing of plum curculio incubation chamber, 4 oz soufflé cup (SOLO cup company, Urbana, IL) with fitted wire mesh (30 mm x 30 mm) ................... 47 Figure 4.3. Schematic drawing of plum curculio overwintering Site, rooftop and drainage setup. .................................................................................................................................. 49 viii Figure 4.4. Plum curculio overwintering deployment setup. Group 1 + fruit = provisioned with a thinning apple, Group 2 -— fruit = not provisioned with a thinning apple, UTC = untreated control. ........................................................................................ 50 Figure 4.5. Average number (:t SE) of plum curculio larval emergence from pyriproxyfen treated (direct Spray in field and 3d residue exposure) adults after 2 wk incubation. * indicates Significant difference. ................................................................... 51 Figure 4.6. Percent (:1: SE) female plum curculios with egg development at dissection after exposure to direct spray of pyriproxyfen in field and 3d residue exposure. * indicates Significant difference. ......................................................................................... 51 Figure 4.7. Survival rate (:I: SE) of diapause bound northern strain plum curculio adults. Untreated Control (UT C), Pyriproxyfen (Pyri), Deployment with fruit provision (+ fruit), without (- fruit). * indicates Significant difference. ........................................................... 53 Introduction The plum curculio, Conotrachelus nenuphar (Herbst), is an important native weevil pest of cultivated pome and stone fruits found in eastern and central North America (Chapman 1938). The host range of plum curculio includes a variety of rosaceous fruits in the Amygdaloideae (Prunoideae) and Maloideae (Pomoideae) subfamilies. Varying degrees of plum curculio feeding may occur on a range of tropical and subtropical fi'uits, but oviposition and complete development occurs mostly on rosaceous fruits such as apple, cherry, peach, and plum (Chapman 1938, Armstrong 1958, Maier 1990, Racette et al. 1992, Hallman and Gould 2004, Brown 2005, and Jenkins et al. 2006). Plum curculio has also been found in wild blueberries and is known to infest cultivated blueberries causing significant loss in yield (Beckwith 1943, Mampe and Neunzig 1967, Polavarapu et al. 2004). There are two strains of plum curculio, northern and southern, of which the northern is univoltine and southern multivoltine (McGiffen et al. 1987, Smith and Salkeld 1964, Smith and Flessel 1968). Northern strain plum curculios are characterized by an obligate adult reproductive winter diapause, which is broken during the course of overwintering (Smith and Flessel 1968). In the spring, female plum curculios oviposit (300 — 800 DDlooc) in developing fruits leaving a distinctive crescent-shaped scar. Larvae eclose and feed (500 — 1200 DDlooC) internally on the fruit pulp, then drop down to the orchard ground, burrow into the soil, pupate and eclose as adults (900 — 1600 DDlooc) (Armstrong 1958, Chapman 1938, Quaintance and Jenne 1912, Whalon and Korson 2008). Through the summer, adult plum curculios puncture a small round hole about one-tenth of an inch (0.254 cm) in diameter, then consume the pulp underneath the skin as far as their snout can reach (Stedman 1904). Plum curculio larval presence and/or feeding damage in the fruit at harvest as well as the scars resulting from oviposition and adult feeding can cause serious economic losses in tree fruit crops and blueberries. Furthermore, developing larvae release pectic enzymes and cellulase as they feed internally and these enzymes can cause a premature dropping of infested fruit by inducing fruit abscission (Levine and Hall 1977, 1978a and 1978b); however, it is difficult to determine the effects on yield because this phenomenon overlaps with natural fruit abscission (Racette et al. 1992). At any rate, plum curculio can cause serious damage in both pome and stone fruits thus is a key pest in terms of its potential economic damage if left unchecked (Hoyt et al. 1983). In unsprayed orchards, plum curculio fruit damage at harvest reach up to 85 percent (Vincent and Roy 1992) and it only takes about three years after discontinuation of pesticides for a plum curculio population to return to primary economic importance (Glass and Lienk 1971, Hagley et al. 1977, Hall 1974). Increasing plum curculio population pressure is a growing concern because of the tightly set statutory and consumer tolerances for damaged or infested fruit both in the US domestic but especially in export markets. US grades standards for apples and cherries require low in fresh apples, but a zero-tolerance of worms for processed apples and especially cherries (USDA 1941, 1961, 1971, 2002). In processed cherries, a zero tolerance standard, meaning that no live larvae may be present in fruit at harvest, has been set by the US. Department of Agriculture (USDA 1941). This standard creates severe economic pressure on growers Since failure to comply can result in a rejection of the grower’s entire delivery load to processor. Over the past 50 years, plum curculio control has been based on organophosphate insecticides, primarily azinphos-methyl (Guthion®, Makhteshim A gan of North America Corp.) and to a much lesser extent phosmet (Imidan®, Gowan Company LLC), aimed at killing adult plum curculios before they oviposit into fruit. Historically two to three applications of organophosphate insecticides have effectively prevented plum curculio injury and allowed fruit growers to meet the US Department of Agriculture grade standards for fruit quality and condition (Howitt 1993). However, as a result of the implementation of Food Quality Protection Act 1996 (FQPA) (US EPA 1996), US Environmental Protection Agency (US EPA) after a protracted, volumous and heated debate with tree fruit industries in the upper Midwest and northeast, has made a decision to phaseout azinphos-methyl uses by September 30, 2012 (US EPA 2008). Even though azinphos-methyl provides important pest control benefits for growers of cherries and apples as well as other crops, US EPA determined that it poses health risks to children, farm workers, pesticide applicators, and aquatic ecosystems (Eskenazi 1999, Coggon 2002, Alavanja et al. 2004, Anderson et al. 2006); thus with the azinphos-methyl phaseout decision, growers are forced to transition to reduced-risk and organophosphate alternatives or other non-chemical methods of pest control. Therefore to facilitate the tree fruit industries’ transition, US EPA has established a Reduced Risk Pesticide Program to expedite the process of registering commercially viable alternative management methods that pose less risk to human health and the environment while ensuring the availability of these reduced risk pesticides to growers (US EPA 1997). A series of new reduced-risk and organophosphate-altemative insecticides have become registered for use in fruit production, several of which have demonstrated promise for plum curculio control. The neonicotinoid compounds thiarnethoxam (Actara®, Syngenta, Greensboro, NC) and thiacloprid (Calypso®, Bayer Cropscience, Research Triangle Park, NC) and the oxidiazine indoxacarb (Avaunt®, DuPont, Wilmington, DE) have all exhibited Significant levels of damage reduction in field efficacy trials on apple (Wise and Gut 2004). The insect growth regulators, many of which fit the US. Environmental Protection Agency’s Reduced—Risk category (US EPA 2008) are also being registered for use in fruits but some may not be labeled by pesticide companies for plum curculio control because of their lack of acute lethal toxicity to this pest and the risk of legal consequences for failed control where “zero thresholds” are in place. The term insect growth regulator describes a new class of bio-rational compounds introduced in late 19005 as a result of research on insect juvenile hormones (Staal 1975). US EPA classify insect growth regulators as pesticides that disrupt the molting, maturity from pupal stage to adult, or other life processes of insects (US EPA 2009). Insect growth regulators act by disrupting normal developmental processes of the pest unlike direct nerve toxins like many of the FQPA targeted conventional insecticides (i.e. organophosphates, carbamates, synthetic pyrethroids). Insect growth regulators are generally absent of direct effects on mammals, birds and reptiles although they may greatly impact invertebrates (Staal 1975). There are three basic types of insect growth regulators registered for US. fruit producers; juvenile hormone analogs (Group 7), ecdysone agonists (Group 18), and chitin biosynthesis inhibitors (Group 15) (IRAC 2008). These modes of action are thought to make insect growth regulators safer for humans, since molting and chitin synthesis are Specific to invertebrates. Novel insect growth regulator insecticides can provide a wide range of strategic approaches to pest managers and thereby support resistance management programs as well (Ishaaya et a1. 2001, Ishaaya 2007, Wise et al. 2007, Whalon et al. 2008). Among registered insect growth regulator pesticides, juvenile hormone analogue pyriproxyfen (Esteem®, Valent, Walnut Creek, CA) and chitin synthesis inhibitor novaluron (Rimon®, Chemtura, Middlebury, CT) have shown indirect sub-lethal effects against plum curculio. Pyriproxyfen was found to activate reproduction in the diapause bound northern strain plum curculio (Hoffmann et al. 2007). AS mentioned above, northern strain plum curculios have obligate adult winter diapause where reproductive maturation and oviposition occur in the spring only after undergoing the hibernation process. However, topical application of pyriproxyfen in the laboratory condition induced oocyte development and reproductive maturation in all treated females (Hoffmann et al. 2007). Novaluron, however, in my initial laboratory bioassay (Chapter 1), was found to reduce larval survival following adult exposure (Wise et al. 2007). A female plum curculio lays an average of 73 eggs yet is capable of laying anywhere from 250 to 400 eggs, and egg stage survival is about 15 percent (Stedman 1904). This high rate facultative fecundity is the key issue making plum curculio a key pest in tree fruit and blueberries by oviposition yielding damage well above the economic injury levels if unchecked by producers. However, when adult plum curculios were exposed to a novaluron treated substrate then incubated on an untreated substrate for oviposition, egg development or larval emergence; Significantly fewer plum curculio larvae emerged compared to the control (Wise et al. 2007). To date, the plum curculio insecticide control has focused on killing the adult life stage of this pest by the means of acute lethal toxicity through timing of conventional insecticides such as azinphos-methyl before they oviposit in fruit. With unparalleled performance, azinpho-methyl was widely used essentially setting the industry standard on the quality and condition of fruit produce for nearly half a century. However increased regulation of conventional pesticide tolerance through implementation of Food Quality Protection Act resulted in a restricted use and eventual phase out decision of azinphos- methyl; therefore for successful transition from conventional to reduced risk, it will be critical that fruit producers learn the effects of these new reduced risk compounds such as insect growth regulators against key pest of fruit like plum curculio, and incorporate them into current integrated pest management programs. The goal of the research herein is to determine the effects and field efficacy of the insect growth regulator insecticides, novaluron and pyriproxyfen, against plum curculio; and to propose strategies to incorporate them into current apple and cherry integrated pest management. Chapter 1 Novaluron reduces larval survival in plum curculio, Conotrachelus nenuphar (Herbst), after adult exposure Abstract Laboratory treated apple bioassays were used to determine if the insect growth regulator insecticides novaluron and tebufenozide have physiological effects on plum curculio larvae following adult exposure. The results revealed that adult exposure to novaluron results in reduced larval survival. Introduction Plum curculio, Conotrachelus nenuphar (Herbst), is a native beetle that occurs east of the Rocky Mountains and is a key pest of cultivated pome and stone fruits in eastern and central North America (Chapman 1938, Racette et al. 1992, Yonce et al. 1995). The plum curculio is a direct pest of apples, causing damage to fruit in a variety of ways. In the spring, female plum curculios make crescent-shaped oviposition scars in the developing fruit, and larvae feed internally on the fruit flesh. Adult plum curculios also feed on fruit by making a small puncture hole in the skin and eating the flesh underneath. Internal feeding damage can render the fruit unmarketable, as does the scar tissue resulting from adult feeding and oviposition. Developing larvae tunnel in the fruit and, if left unchecked, can cause Significant yield loss in commercial apple orchards (Levine and Hall 1977). Over the past 50 years, plum curculio control has been based primarily on organophosphate insecticides, such as azinphos-methyl, aimed at killing adult plum curculios before they oviposit in fruit. More recently a series of new reduced-risk and organophosphate alternative insecticides have become registered for use in US apple production, several of which Show promise for plum curculio control. The neonicotinoid compounds thiamethoxam and thiacloprid, and the oxidiazine indoxacarb have all shown significant levels of control in field efficacy trials on apple (Wise and Gut 2004). Three insect growth regulators, the chitin synthesis inhibitor benzoylurea novaluron and the two closely related ecdysone agonists tebufenozide and methoxyfenozide, have also been recently registered for use in apples, but are not labeled for plum curculio control because of their lack of acute lethal toxicity to this pest. To date, research on plum curculio insecticide control has focused on the adult life stage of this pest, with the assumption that once eggs are laid the immature larvae inside the fruit are out of reach of a toxicant. Also, little to no work has been done to determine if exposure to insect growth regulator compounds have sub-lethal effects that impact consecutive generational life-stages and pest population dynamics (Calkins et al. 1977). The objective of this study was to determine if insect growth regulator insecticides have physiological effects on plum curculio larvae following adult exposure. Materials and methods Insect material Southern-strain (non-diapausing) plum curculios were used from a continuous colony at the Michigan State University Center for Integrated Plant Systems. This colony has been in production since 1998, with occasional additions from collaborators from the southeastern USA. Adults were reared on thinning apples for food/oviposition substrate (Smith 1957). Southern-strain weevils from this colony maintained at 23 :t 1 °C with a 16:8 h lightzdark photoperiod were collected, sexed at emergence before mating and held separately in plastic cages provisioned with green thinning apples for oviposition (Thomson 1932). The cages (10 cm x 10 cm x 8 cm) (Tupperware, Wooster, OH) were fitted with a 3 :l: 1 cm high wire mesh (4 x 4 m, 0.5 cm diameter mesh) platform, which allowed emerging larvae to drop into the bottom of the cage for monitoring and collection. Replicated cages were used as exposure arenas, rearing cages, and emergence cages throughout the experiment. After emergence, adults were collected over a period of 10 days to obtain a sufficient number of newly emerged, unmated plum curculios for experimentation. Chemical materials and application methodology Two commercial insect growth regulator formulations, novaluron 100 g / L EC (Rimon 0.83EC; Chemtura USA Corporation, Middlebury, CT) and tebufenozide 240 g / L SC (Confirm 2F; Dow AgroSciences LLC, Indianapolis, IN), were diluted to label rates, 0.24 g a.i. / L (equivalent to 224 g a.i. / ha at 935 L / ha) for novaluron, and 0.37 g a.i. / L (equivalent to 346 g a.i. / ha at 935 L / ha) for tebufenozide. Latron B-1956 (a spreader and sticker by Dow AgroSciences LLC, Indianapolis, IN) was added at 38mL / L to both treatments and a Latron-water control, but not to the water-only control. Pesticide applications were made using a hand Sprayer (Lansing Sanitary Supply Inc.), which delivered 100 mL of finished spray to 55 apples per treatment, carried out in a glasshouse (10 m x 30 m x 3 m) facility at CIPS. To ensure complete coverage, treatment apples (3.5 :r: 1 cm diameter) were suspended from 1mm diameter string attached to a 2 cm x 9 cm x 180 cm wooden slat suspended from the ceiling over a bench. After the apples had been sprayed and air dried (30 min), five were placed in a 1.2 L plastic container (Rubbermaid®, Wooster, OH) on 1.6 mm woven wire hardware cloth. Plum curculio larval survival and emergence Fifteen male and 15 female plum curculios were placed inside each container and sealed with a vented and fitted top. Each container of treated apples (holding 30 weevils) was considered an experimental replicate. There were three replicates per treatment. After exposure for 4 days, plum curculios were transferred to holding containers with three untreated apples for feeding and oviposition. Apples were replaced every 5 days, and apples with oviposition scars were held in plastic emergence cages, with woven wire hardware cloth. Emergence cages were lined with moistened paper towel (Kimberly- Clark, Neenah, WI) to prevent larval desiccation. Treatment containers were monitored 2—3 times per wk for larval emergence, survival, malformations, ablations or other anomalies, and held until all potential larvae had emerged (35 days). At every apple change, two males and two females were removed from each replicate for dissection. This design was replicated 11 times from 9 January to 30 January 2006. Each starting day (design replication) was considered a block owing to the possible variation in the colony cohort over the 3 wk period. Statistical comparisons were made on the number of emerged larvae per adult female using analysis of variance. Mean separations were done using Tukey’s HSD (a = 0.05). 10 Dissection methods For dissection, females were placed in 70 percent ethanol and their egg and spermatheca developmental status was assessed in Ringer’s solution under a Nikon SMZlOOO (Mager Scientific, Inc., Dexter, MI) stereo dissecting microscope. A reproductive progress assignment was made according to four stages of ovary development: (1) no oocytes; (2) one to several small oocytes in the vitellarium; (3) numerous unchorionated oocytes in the vitellarium; (4) chorionated eggs in the median oviduct (calyx). Mating status was complemented with a binary code: 1 = mated (translucent white), 0 = unmated (clear). Data were submitted to chi-square analysis (PROC User’s Manual 2002). Results and Discussion Significantly fewer plum curculio larvae emerged from fruit following adult exposure to novaluron than when adults were exposed to any other treatments (F = 13.37, df= 88, a < 0.0001) (Table 1.1). Treatment Insecticide concentration Mean number 0f larvae a (g 3-1- / L) emerged per adult PC (:SE) Control 0.95 (:2.26)A Controlb 0.94 (12.29)». Tebufenozideb 0.37 1.08 (12.17» Novaluronb 0.41 0.07 (:tO.25)B Table 1. 1. Plum curculio larval emergence after adult exposure to treated fruit. Total sum of larvae over 35 d emergence period. a Raw data were log transformed [log(x + 1)] prior to ANOVA. Untransformed means are shown for comparison. Means followed by the same letter are not Significantly different (a < 0.05 LSD). b Latron B-1956 was added at 3mL / L to these treatments. 11 When plum curculio adults were exposed to water + Latron or tebufenozide + Latron there was no reduction in larval emergence. No differences were detected in mating status or ovary development between novaluron, tebufenozide and the water control (mating: x2 = 2.08, df= 3, a = 0.55; ovary: fl = 3.18, df= 9, a = 0.95) (Table 1.2). Treatment Ovary development (:I:SE)a’b Mating status (:tSE)“ Control 3.25 (10.35) a 0.91 (10.08) a Control“ 3.00 (10.30) a 1.00 (10.00) a Tebufenozided 3.00 (10.34) a 1.00 (10.00) a Novaluron“ 3.00 (10.34) a 0.91 (10.08) a Table 1.2. Plum curculio ovary development and mating status following treatments with tebufenozide and novaluron. a Chi-square analysis was performed on the raw data. Means followed by the same letter are not Significantly different (a < 0.05). b Ovary development score (1 = no oocytes, 2 = one to several small oocytes in the vitellarium, 3 = numerous unchorionated oocytes in the vitellarium, 4 = chorionated eggs in the median oviduct). c Mating status score (0 = not mated, l = mated). d Latron B-l956 was added at 3.75mL / L to these treatments. The fact that novaluron did not affect mating and egg development and yet significantly fewer larvae emerged suggests that there was chemical toxicity on the developing larvae. The objective of this study was to determine if insect growth regulator insecticides have physiological effects on plum curculio larvae following adult exposure. Novaluron exhibited a reduction in larval survival following adult exposure to treated substrate. Similar results were recorded by Kostyukovsky and Trostanesky (2006) with novaluron on T ribolium castaneum (Herbst), and by Calkins et al. with diflubenzuron on plum curculio. This apparent sub-lethal action from treated adult to egg/larval viability 12 presents a new mode of insecticide activity for plum curculio control in tree fruits. It cannot be concluded from these data that larvae successfully hatched from oviposited eggs, so further research is needed to determine if this compound acts specifically on the embryonic or the post-hatch larval stage of plum curculio. 13 Chapter 2 Influence of reproductive maturity, exposure, and residue field age on novaluron’s sub-lethal activity against plum curculio Abstract In this study, we investigated novaluron’s sub-lethal activity against plum curculio Conotrachelus nenuphar Herbst (Coleoptera: Curculionidae) as field residues age over time. Previous laboratory study showed reduction in subsequent plum curculio egg viability after adult exposure to novaluron. Laboratory bioassays were conducted to compare efficacy of different modes of exposure and different life stage exposure. Field trials were conducted on both northern and southern strains with concurrent residue analysis to determine novaluron field efficacy as amount of field residue decreases over time. In-field novaluron surface and sub-surface fruit and leaf residues were analyzed and plant growth was measured. Laboratory results showed that novaluron’s sub-lethal activity is active through both coming in contact with surface residue, and direct ingestion; and effective even after female egg development. These results indicate possible treatment synchrony for plum curculio management with existing novaluron Spray timing for codling moth Cydia pomonella (Linnaeus). Field trial results showed initial efficacy through Sprayed fruit and leaf Shoots collected 4 h after the application on both northern and southern strains, but only fruit exposure on southern strain Showed continued residual efficacy lasting up to 14 d after the application. The impact of different exposure substrates, progression of plum curculio phenology, and age of field residue on novaluron’s sub-lethal activity against plum curculio is discussed. l4 Introduction Over the past 50 years, plum curculio control has relied heavily on the use of organophosphate insecticides, primarily azinphos—methyl (Guthion®, Makhteshim Agan of North America Corp.) and to a lesser extent phosmet (Imidan®, Gowan Company LLC). However with the implementation of Food Quality Protection Act, organophosphate insecticides collectively are under strict reevaluation by US Environmental Protection Agency for their human and environmental risk and final decision is already made for azinphos-methyl to phase out its uses by 2012 (U SEPA 2008). As a result of this phase out, growers are losing their best means of insecticide control against plum curculio and forced to use other viable alternatives in the form of reduced-risk and organophosphate-alternative compounds. Many of these replacement chemicals, such as the neonicotinoids and oxidiazines, have Shown good performance on pests yet they have generally exhibited a lower efficacy against plum curculio compared to the organophosphates (Wise and Gut 2004, Whalon and Korson 2008). Many of these new chemistries do not exhibit solely the acute lethal mode of action seen from conventional insecticides, but rather they appear to encompass a collection of lethal and sub-lethal mechanisms working in unison to achieve prescribed crop protection (Nauen 1995, Hu and Prokopy 1998, Biddinger and Hull 1999, Kunkle et al. 2001, Isaacs et al. 1999, Wise et al 2006). The insect growth regulators, many of which fit the US. Environmental Protection Agency’s reduced risk category (US EPA 1997) are also being registered for use in fruits but not labeled for plum curculio control because of their lack of acute lethal toxicity to this pest. Insect growth regulators act by disrupting normal developmental 15 processes of the pest thus direct lethal activity is generally absent and effects are often indirect and sub-lethal (Staal 1975). Among insect growth regulator pesticides, the chitin synthesis inhibitor novaluron (Rimon®, Chemtura Corp.) is registered in the US. for pome fruit use, targeting primarily Lepidopteran pests such as the codling moth, Cydia pomonella (Linnaeus). Novaluron belongs to a group of chemicals named benzoylphenyl ureaS that is known to disrupt cuticle formation and deposition that occur during an insect’s molting process (Ishaaya 1990). Novaluron may also exhibit various sub-lethal effects which may provide novel compatibilities with integrated pest management systems, including developmental delays and sterilization (Malinowski 1992, Kostyukovsky and Trostanetsky 1992, Cutler et al. 2005). The potential use of novaluron as a plum curculio management strategy was first suggested from laboratory bioassays in which adults were reared on novaluron sprayed apples. Though novaluron did not Show acute toxicity to treated females, it reduced subsequent larval emergence (W ise et al. 2007). This reduction in offspring survival has been demonstrated in other coleopterans such as Colorado potato beetles, Leptinotarsa decemlineata, (Malinowski 1992, Cutler et al. 2005) and the rust—red flower beetle, Tribolium castaneum, (Kostyukovsky and Trostanetsky 2006). In this study, we investigated how novaluron’s sub-lethal activity could be incorporated into current plum curculio management programs in Upper Midwest US. apple orchards. In addition to conducting a field experiment, the impact of different application methods and different maturation level of ovary were compared in the laboratory. Exposure to novaluron residues may occur in oviposition through a treated substrate or by ingesting treated leaf or fruit substrates. A previous laboratory study that 16 suggested the potential use of novaluron as a plum curculio management tool involved newly emerged adults prior to egg developments (Wise et al. 2007). The purpose of the laboratory experiment involving a different stage of ovary maturation was to investigate the efficacy of novaluron exposure after egg development. Manipulation of application tinting to target several different susceptible pest species is an integrated pest management conservation practice that reduces pesticide inputs, improves the economics of management and reduces non-target exposure to critical beneficial Species. Since novaluron is already registered and recommended for codling moth management at petal fall, especially where organophosphates resistance has been demonstrated or is suspected, capacity to adjust novaluron application timing to also intercept plum curculio exposure could afford economic, ecological and environmental benefits. These two aspects, different modes of exposure and level of ovary maturation, were also taken into account in field experiment by means of differentiating exposure substrates fruit and leaf, and conducting experiment with both northern and southern strains. Through field-based bioassays, this research investigated temporal progression of the sub-lethal effects of novaluron as the residues aged in the field. The objectives of this research were to: 1. Determine the impact of contact and ingestion exposures and ovary maturity on plum curculio egg viability. 2. Determine the impact of field-aged residue exposure on plum curculio egg viability as plant and adult plum curculio phonology progress in synchrony. 17 3. Determine the impact of field-aged residue exposure on plum curculio egg viability when adult plum curculio maturity is controlled. 4. Determine the influence of different exposure substrates on the sub-lethal effects as residues age under field condition. Materials and methods General methods Insect materials Two different strains of plum curculio (PC), southern and northern, were used in the experiments. Southem—strain (non-diapausing) plum curculios were from a continuous colony at the Michigan State University (MSU) Center for Integrated Plant Systems (CIPS) (East Lansing, MI) and the MSU Trevor Nichols Research Complex (TNRC) (Fennville, MI). Adults were reared at 23: 1°C with a 16:8 h lightzdark photoperiod on thinning apples for feeding and oviposition (Smith 1957). Plum curculio adults from this colony were collected, sexed at emergence before mating (Thomson 1932), and held separately in plastic cages provisioned with thinning apples for oviposition. The cages (100 mm x 100 mm x 80 mm) (Tupperware, Wooster, OH) were fitted with a 30 mm high wire mesh (5 mm diameter mesh) platform, which allowed emerging larvae to drop into the bottom of the cage lined with wet paper towel for monitoring and collection. Apples were renewed twice a week and removed apples were held in separate cages for larval emergence. Larvae that emerged were allowed to pupate in glass jars (Alltrista Corporation, Muncie, Indiana) with moistened soil, and fitted boll 18 weevil trap tops (Great Lakes IPM, Vestaburg, MI) allowing the collection of eclosed adults. Northern strain plum curculios were only used in part of field experiments. Northern strain plum curculio adults were collected from organic cherry farms in Manistee County, Michigan concurrent to the experiment. Whalon modified Tedders traps (Great Lakes IPM, Vestaburg, MI) and screen traps equipped with plum essence and benzaldehyde lures were used to attract and capture the plum curculio adults from the field (T edders and Wood 1994, Coombs 2001). Northern strain plum curculios were held separately from the southern strain and maintained as above in the rearing facility at MSU CIPS. Chemicals materials The commercial formulation of novaluron (Rimon® 0.83 EC, Chemtura Corp., Middlebury, CT) was diluted to 20 oz in 200 gallons of water per acre; 0.78 ml Rimon in l L water, 77.53 mg a.i. / L. This is the lower end of the current US. pome fruit codling moth label rate. Latron Bl956 (Dow AgroSciences LLC, Indianapolis, IN) was added (0.125% by volume) as a spreader and sticker to the novaluron and the control treatment. Laboratory studies Exposure methods For contact exposure, novaluron and control treatments were pipetted (2 ml) to tops and bottoms of 100 mm diam. Petri dishes, and then allowed to dry at room temperature. After drying, 4 southern strain plum curculio adults (2 males and 2 females) 19 were placed inside the Petri dish for 1 d of exposure and thereafter reared on a thinning apple in 4 oz souffle cup (SOLO cup company, Urbana, IL) with fitted wire mesh (30 mm x 30 mm). Apples were renewed every 7 d for 28 d and apples with oviposition scars were kept to observe larval emergence. There were 6 replications per treatment (24 PC). To determine effects of ingestion exposure, 4 southern strain plum curculio adults (2 males and 2 females) were force-fed with novaluron or control using a micro applicator (Hamilton Company, Reno, Nevada) (Figure 2.1). Two droplets of novaluron (0.4 ul, 0.062 p. g a.i. per plum curculio) or control were applied to the end of the proboscis and drops were observed to be immediately ingested without any smudging on facial area of the insect. After ingestion, plum curculios were reared on a thinning apple for 28 (1 inside a 4 oz souffle cup fitted with wire mesh as described above. There were 7 replications per treatment (28 PC). For both contact and ingestion exposure methods, statistical comparisons were made between novaluron treated and untreated using paired T test (a = 0.05) on number of emerged larvae collected per adult female. 20 Plum Curculio Q J Petri dish with sticky tape on top Figure 2.1. Schematic drawing of novaluron force-feeding method. Maturation level of the ovary Newly emerged male and female southern strain plum curculio adults were held together for at least 16 d to assure mating, egg production, and oviposition. Egg development was confirmed by dissecting cohorts (Smith 1964). Pesticide applications were made on thinning apples (Empire, collected in summer 2007 from MSU TNRC). Apples were suspended from a pole horizontally positioned in a rectangular box (609 mm length x 330 m depth x 660 mm height) using cotton strings (Phoenix Rope and Cordage Co., Illinois) 254 mm long. One end of the string was tied to the Stem of the apple, while the other to the pole. The apples were then Sprayed to drip using a handheld Sprayer (Lansing Sanitary Supply Inc, MI) and dried at room temperature (Figure 2.2). After drying, 4 southern strain plum curculio adults (2 males and 2 females) were exposed to sprayed apples for 3 d inside previously described 4 oz souffle cup fitted with 21 wire mesh. After 3 d of exposure, plum curculios were transferred onto untreated apples. Apples were renewed at every 7 d for 21 d and apples with oviposition scars were kept to observe larval emergence. There were 10 replications per treatment (40 PC). Statistical comparisons were made between novaluron treated and control using paired T test (a = 0.05) on number of emerged larvae collected per adult female. 6° V 61cm ‘ 67cm G 6 O O v " Figure 2.2. Schematic drawing of novaluron spray application apparatus. Field Studies Field plots and treatment applications Plots consisted of 9-yr-old apple red delicious trees at the MSU TNRC in Fennville, MI. Tree spacing was 5.5 m by 6.1 m, with one buffer row. Regular maintenance applications of fungicides were applied to all treatment blocks. Novaluron and control treatments were applied with a FMC 1029 airblast sprayer calibrated to 22 deliver 935.3 liters of water per ha (100 gal / acre). Applications were made on 18 May 2007 and these plots served as the source of apple fruit or leaves for use in plum curculio bioassays and residue analysis. Residual activity bioassay The field trial was carried out from 18 May 2007 to 1 June 2007. Fruit or leaves were randomly collected from Sprayed apple trees 4 h after the application (0 d) and then again at 7 and 14 (I later. Each shoot (2 150 mm) was pruned to contain only 5 of the preferred exposure media (only 5 fruits or 5 leaves). In order to preserve the integrity of the plant, each shoot was placed in water-soaked Oasis floral foam (Smithers—Oasis Company, Kent, OH) in clear plastic 950-ml containers (Fabri-Kal, Kalamazoo, MI) and the floral foam was covered with a layer of unscented candle wax (Yaley Enterprises, Redding, CA). The container lid was ventilated by cutting a 50 mm x 50 mm square Space and covering it with fabric mesh. This ventilation was used to reduce condensation of water vapor inside the container and minimize potential fumigation effects. Each of these containers was considered an experimental unit in the bioassay. At each post-application time interval, collected and pruned fruit or leaf shoots were placed onto the prepared floral foam container, and these containers were used as an exposure arena. Four plum curculio adults (2 males and 2 females) were placed in the bottom of each exposure arena, and held in the laboratory at =5 21 °C and a photoperiod of 16:8 (LzD) h. After 6-7 (I of exposure, plum curculio adults were transferred and reared on untreated apples in previously described 4 oz souffle cup fitted with wire mesh. Apples were renewed every 7 d for 28 d and apples with oviposition scars were kept for 23 larval emergence. Treatment groups were categorized according to applied pesticide (novaluron vs. control), plum curculio strain exposed (northern vs. southern), and exposure media (fruits vs. leaves) (Figure 2.3). There were 6 replicates for each of 8 different treatment groups at each of the post-application time intervals (576 PCS). Novaluron Treated Untreated Control Northern Strain Northern Strain Southern Strain Southern Strain Figure 2.3. Experimental setup for plum curculio bioassay after exposure to field—aged novaluron residue. Treatment groups were categorized according to applied pesticide (novaluron / control), plum curculio strain exposed (northern / southern), exposure media (fruits / leaves), and collection time (4 M d/14 d). Statistical comparisons were made at each collection period for a pair of novaluron treated and untreated control from each treatment group (4 N7 d/l4 d, northern/southem, fruit/leaf) using paired T test (a = 0.05) on number of emerged larvae collected per adult female. When entire data set of all three collection periods was collected, it was differentiated according to the strain (northern and southern) then 24 submitted to analysis of variance PROC MDIED procedure to analyze the significance of overall novaluron treatment effects. Novaluron residue profile analysis A parallel series of fruit and leaf samples were taken from field plots at each of three post application timings (4 h and 7 and 14 (1). Compared to the initial 4 h post application collection, fewer fruit and leaves were collected per sample because of the plant growth. However the sample volume was always standardized to fit 4 oz glass bottle (The Glass Group Inc., Park Hills, Missouri) and contained a minimum of 10 g of plant substrate. The samples were immediately transported to the MSU Pesticide Analytical Laboratory in East Lansing, Michigan. A novel pesticide extraction procedure was used to separate dislodgeable residues on the surface of the fruit and leaf samples from the proportions in and below the plant cuticle, to provide a Spatial profile of each compound over time (Wise et al. 2006). To determine the amount of residue on the fruit and leaf surfaces, replicate samples of apple fruit and leaves were placed in 150 ml of acetonitrile and sonicated for 10-15 s. The acetonitrile was decanted through 5 g of anhydrous sodium sulfate to remove all water. The sample was dried via rotary evaporation and brought up in acetonitrile for high-performance liquid chromatography (HPLC) or gas chromatography / mass spectrometry (GC/MS) analysis. To determine the residue from the fruit and leaf sub-surfaces, the remaining solid fruit and leaf samples were ground with 200 ml of dichloromethane. The extracts were then vacuum-filtered, and the filtrate was passed through 5 g of anhydrous sodium sulfate. 25 The samples were dried via rotary evaporation and brought up in acetonitrile. Any remaining particulates were removed by passing the sample through a 0.45 pm filter. Novaluron samples were analyzed using GC/MSD (Agilent 6890 gas chromatograph with a 5973N MSD) equipped with a Zebron ZB-Sms 30 m, 0.25 mm ID. column and a 0. 25 um film thickness. The GC/MSD settings for analysis were as follows: the oven was held at 115 °C for 5 min with a ramp of 9 °C per min to 280 °C, followed by 30 °C per min to 310 °C. The inlet was held at 200 °C in a pulsed splitless mode with 78324 Pa and a pulse pressure of 103421 Pa, with a purge flow of 50.0mL per min of helium gas. The MSD transfer line was held at 285 °C. The MSD was set to scan 28—535 Da. The injector was rinsed 3 times with acetone and 3 times with dichloromethane before each injection to eliminate contamination between injections. All compounds were quantitated against a standard curve, and recovery data were recorded. Level of detection (LOD) and level of quantitation (LOQ) recoveries ranged from 50 to 150 percent. Plant Growth At each collection period, 20 apple fruits and leaves were randomly collected from experimental plot from which average weight and surface area was calculated. Sphere surface area calculated from measured diameter of fruit represented fruit surface area and leaf surface area was measured through digital image analysis (O’Neal et al. 2002). Leaf surface area represented by scanned image was calculated using computer program Scion Image (Scion, Frederick, MD). The plant growth data allowed for conversion of residue data from a u g a.i. per g basis to a u g a.i. per cm2 surface area basis. 26 Weight per surface area (g/cmz) x surface residue profile (pg/g) = surface residue per cm2 (ug/ cmz). Rme Laboratory studies Exposure methods For both contact and ingestion application methods, significantly fewer plum curculio larvae emerged from fruit following adult exposure to novaluron compared to the control (contact T=7.924, df=5, P=0.001; ingestion T=8.407, df=6, P=0.000) (Figure 2.4). Both Petri dish residue and force fed ingestion exposure methods delivered sufficient novaluron to plum curculio adults to elicit the sub-lethal effects of reduced larval emergence. 27 ET (I) 3.". 360 is. .5. 040 “a“ £30 “620 3'1 210 :0 2 Figure 2.4. Average number (:1: SE) of plum curculio larvae emerged after adult exposure to novaluron, treated through ingestion and contact methods. The larval emergence with * is significantly different from control (paired T-test, P<0.05). 70- * * UTC * l Novaluron Ingestion Maturation level of PC ovaries Significantly fewer plum curculio larvae emerged from fruit following adult (>16 UTC Contact Novaluron d old since eclosion, with egg development) exposure to novaluron compared to the control (T=4.926, df=9, P=0.001) (Figure 2.5). This result is similar to the chapter 1 data with newly eclosed adults (Table 1.1). 28 E ii 35 i I 8 30 - 8° . l E 25 . 0) O m 20 J E J.“ u- 15 i o g 10 . g 5 4 * 0 I E ..-'f.'.‘ Control Novaluron Figure 2.5. Average number (:1: SE) of plum curculio larval emergence after adult exposure to novaluron. The larval emergence with * is significantly different from control (paired T-test, P<0.05) Field Studies Residual activity bioassay Results Showed that field-sprayed novaluron residues initially elicited the same sub-lethal effects for southern and northern strain plum curculio as were seen in laboratory studies (Fig 2.6). For northern strain plum curculio, only exposure to leaves and fruits collected at 4 h after novaluron field-Spray significantly lowered subsequent larval emergence (leaf, T=6.303, df=5, P=0.001; fruit, T=4.150 , df=5 , P=0.009). Compared to the exposure at 4 h post-spray, there was a dramatic decline of larval emergence in untreated control at 7 d and 14 d. This suggests that there could have been a different factor such as the ovipositional behavior that might have had impact on plum curculio egg viability. For southern strain plum curculio, novaluron lowered subsequent larval emergence up to 7 d post-spray in case of leaf exposure, and up to 14 d post-spray 29 for fruit exposure (Figure 2.6). This suggests that exposure to fruit residues may be more important for causing long term sub-lethal effects. The emergence of southern strain plum curculio larvae from the untreated leaf exposure at 14 d post-spray was unexpectedly low, which may have limited any potential treatment effect in that test. 30 Northern Strain DControl 40 ~ INovaluron I: 35 ~ .11 3 30 — 5 25 . E’ g 20 — |.l.l - 15. * E * 3 10 « H- o “ " i ES 0 aLiiii o . ._1 i1 . 4h 7d 14d 4h 7d 14d Leaf Fruit Southern Strain EIControI 40 , INovaluron 35 ‘13 g 30 . 'U a, 25 ~ 2 20 « g * * a 15 “ L“ n- 10 ~ ° * a: 5 ~ * i * o -_... m, _ ..... __- ._.. 1...- Time post 4h 7d 14d 4h 7d 14d treatment Leaf Fruit Figure 2.6. Plum curculio, Conotrachelus nenuphar (Herbst), larval emergence following adult exposure to novaluron-treated fruit or leaves harvested at different intervals (4 h, 7 d, and 14 (1) post field application. The larval emergence with * is significantly different from control (paired t-test, P<0.05). 31 Novaluron residue profile analysis Residue profile results showed a pattern of gradual decline of surface and sub- surface novaluron residues on both apple fruit and leaves under the 14 d field conditions (Fig 2.7). Residue samples at 21 d were also collected, and showed dramatic declines in surface and sub-surface residues for both fruit and leaves down to ng a.i. per g level (data not shown in figure). The residues recovered from the plant surface were consistently greater than sub-surface, and sub—surface residues on leaves were proportionally more than what was recovered in fruit. This suggests that novaluron penetrate into leaf tissue more readily than into fruit. Amount of rainfall during the study period was 7.36 mm on 25 May (7 d post), 13.46 mm on 1 June (14 (I post), and 33.52 mm on 8 June (21 a post) (Michigan Automated Weather Network). 32 60 - I Apple Suface Residue DApple Sub-surface Residue 504 304 20a Novaluron Residue (us/g) 10~ Figure 2.7. Mean residue profiles (6 samples) of novaluron surface and sub-surface residues on apple fruits and leaves. Residues measured are in micrograms active ingredient per gram fruit and leaf tissue taken at 4 h, 7 d, and 14 (1 post field application using a conventional air-blast sprayer. Plant Growth Converting residue data to a u g AI per cm2 is likely more representative of the surface dislodgeable residues that a plum curculio adult encountered in the field trial bioassays. For both apple fruit and leaf the residue levels were similarly highest at 4 h and lower at 7 and 14 d post-application. These data show that there are very little differences between fruit and leaf in the contribution of surface dislodgeable residues. Thus the greater extent of sub-lethal effects per fruit exposure seen (Fig 2.6) is not likely related to contact exposure. 33 5 o, 4.5 IApple Fruit g 5 i DApple Leaf % 4 i 3.6 r: 4 i 1 . i c 3 2 1 2 . 3 2 11 1.2 1.3 N g 1 ‘1 0,6 0.8 1 I ll i 0 l ' ' . 4h 7d 14d Figure 2.8. Novaluron surface residues per cm2 on apple and leaves. Residues calculated from mean residue profile (figure 2.7), surface area, and weight data. More non-quantified feeding of PC was observed on fruit than leaves in this study, suggesting that the ingestion exposure is responsible for the extended activity of novaluron on plum curculio at 14 d post. Discussion Laboratory Studies The objective of the laboratory studies was to determine the importance of exposure method and ovary maturity on novaluron’s sub-lethal activity against plum curculio. Both Petri dish—contact and force-feeding methods resulted in reduced number of viable larvae after adult exposure. Novaluron’s sub-lethal activity against plum curculio acts by both ingestion and contact exposure. However, issues with plum curculio grooming behavior pose questions if Petri dish method used in this experiment represents solely contact exposure. Novaluron treatment of adult plum curculios with matured 34 ovaries, holding developed eggs, resulted in reduced number of viable larvae. This result Showed that novaluron sub-lethal activity can be expected for both on newly eclosed and mature plum curculios. Thus, novaluron application timing will not be limited based on egg development but can be manipulated within the boundaries of time when plum curculio oviposition starts to occur. Field Studies The second objective of this research was to determine the impact of field-aged residue exposure on plum curculio egg viability as plant and plum curculio phenology progress in synchrony. Northern strain plum curculios collected concurrent to the experiment represented insect phenology progression over the course of this experiment. Such setup would best describe the actual field performance of novaluron spray under natural condition. Though larval emergence at 4 h suggests significance of novaluron sub-lethal effect, there was a sudden decline in overall number of larval emergence observed at 7 d and 14 d. The probable explanation for this phenomenon is the natural ovipositional pattern of northern strain plum curculios. According to the plum curculio phenology model from Whalon and Korson (2008), oviposition starts to decline from 500 DDmoc and ends shortly after 650 DDmoc. After the exposures at 4 h, 7 d, and 14 d post- spray, there was a 28 d incubation period. The degree days with base 10°C at the end of 28 d incubation period were calculated using Michigan Automated Weather Network (MAWN) station at Bear Lake, MI resulting 675 DDlooC (4 h), 803 DDlooc (7 d) and 925 (14 d). This suggests that compared to 4 h, plum curculios exposed at 7 d and 14 (1 post novaluron application had been incubated well beyond the period when oviposition ends. 35 Such information provides convincing explanation for the overall decline of northern strain plum curculio larval emergence after 7 d, and provides valuable description of how novaluron would perform under actual orchard conditions. The third objective of this research was to determine the effect of residue age on sub-lethal activity when plum curculio maturity is controlled. As seen in case of northern strain plum curculios, natural progression of plum curculio phenology could become a factor when determining performance of novaluron sub-lethal activity against plum curculio. Therefore, colony reared southern strain plum curculios with controlled maturity were used to accurately determine the effect of field-aged novaluron residue. Significant novaluron sub-lethal activity was observed for southern strain plum curculio at 4 h, 7 d for leaf exposure, and 7 d and 14 d in fruit exposure clusters. The result that continued novaluron sub-lethal activity was observed in only fruit exposure brings in focus objective 4 of this research, to determine the impact of different exposure substrates on the efficacy of novaluron sub-lethal activity against plum curculio. Ingestion exposure seems to be the important exposure methods for continued novaluron sub-lethal activity against plum curculio. Novaluron is found to be selective sometimes in regards to exposure methods acting by ingestion on lepidopteran larvae and by contact on white flies (Ishaaya et al 2003). Plum curculio feeding and grooming behavior on fruit and leaves were observed during all post-application timings of this experiment, confirming the importance of ingestion as a delivery mechanism for novaluron on both substrates. Contact exposure was relevant for both forms of exposure, but the dislodgeable residues from fruit and leaves may have been different, thus inducing different levels of novaluron sub-lethal activity against plum curculio. The results of this 36 research suggest the importance of both apple fruit and leaf residues in novaluron field applicability against plum curculio. Novaluron residue profile analysis and plant growth Novaluron residue profile analysis shows gradual decline of novaluron residue under field conditions up to 14 d then drastic decline. This result corresponds to the pre- harvest interval of 14 (1 suggested by the label. The weight-based residue analysis data (p. g a.i. per g of plant substrate) alone suggests that apple leaves harbor and maintain more novaluron residue, hence Should play a more important role in field activity of novaluron. This was not the case as field bioassay results Showed importance of apple fruit exposure. Representing the surface residue data on a p. g a.i. per cm2 surface area basis showed largely equivalent values for the surface dislodgeable residues that would likely represent the novaluron contact activity on plum curculio. These data suggest that optimal timing for spraying novaluron for sub-lethal activity on plum curculio is petal fall, such that both fruit and leaves are covered, adult contact and ingestion is maximized, and so that the egg development is still underway. The results of this study have several positive applications for tree fruit 1PM. Novaluron’s sub-lethal activity against plum curculio works through both contact and ingestion exposure; reproductive maturity does not have a significant influence on the sub-lethal activity thus allows manipulation of timing of novaluron application to target other pest species; and the sub-lethal activity continues up to 14 d post-spray. This suggests that this insect growth regulator, thought of as a selective insecticide, has a broader Spectrum of activity than what was previously understood. 37 Chapter 3 Efficacy of single pyriproxyfen treatment in comparison to multiple treatments Introduction Plum curculio, Conotrachelus nenuphar (Herbst), is an important native pest of cultivated pome and stone fruits in eastern and central North America. There are two strains of plum curculio, northern and southern, of which the univoltine northern strain has an obligate adult reproductive winter diapause that requires hibernation to overcome (Smith and Salkeld 1964, Smith and Flessel 1968). Previous laboratory studies have demonstrated that pyriproxyfen activates reproduction in prediapause northern strain plum curculios (Hoffmann et al. 2007). Watanabe and Tanaka (1999) have shown that such sub-lethal effects of pyriproxyfen reduced chill tolerance in Chrysomelid and caused Significantly fewer days of survival in cold condition. Induction of plum curculio reproductive development prior to overwintering could inhibit necessary physiological preparations for the colder months, and a post-harvest pyriproxyfen application in cherries can reduce the number of plum curculio adults that successfully overwinter. Even though pyriproxyfen was found to exhibit sub-lethal activity against plum curculio that can potentially be utilized in field, the dose and duration of field-applied- exposure is not well understood. Therefore, the objective of this research was to compare the efficacy of a single pyriproxyfen treatment to multiple treatments in regards to their ability to activate the reproductive process in female prediapause northern Strain plum curculio. 38 Materials and methods Newly eclosed northern strain plum curculios collected from our rearing facility were exposed to pyriproxyfen (262 mg a.i. / 1L) dipped apples for 3d and thereafter incubated with untreated apples in a 4 oz souffle cup (SOLO cup company, Urbana, IL) with fitted wire mesh (30 mm x 30 mm). There were a total of three pyriproxyfen treated groups and one control. While one treatment group was incubated with untreated apples throughout the 2 mo incubation period, other two treatment groups were re-exposed to pyriproxyfen dipped apples either every 2 wk or every 1 mo. The apples that plum curculios were incubated on were held for larval emergence. A replicate was represented by a 4 oz souffle cup housing 2 plum curculios (1 male + 1 female) and there were four replicates per treatment. After 2 mo of incubation, the total number of larval emergence from each group was square root (x+1) transformed then submitted to analysis of variance. Comparison was done using ANOVA Tukey HSD (a = 0.05) and data were square root (x+l) transformed before ANOVA analysis. Results and discussion While the control Showed no reproduction activity, all three pyriproxyfen treated groups Showed statistically same level of reproduction (F =18.75, df=3, P=0.000). 39 7o — A E, A b 50 « E 0 g 50 ~ A .L' A 40 - “621 ‘6 ~— 30 ~ .1: S c 20 - Si. 10 - g B < o 1 NS 1 NS 1/m NS 1/2w NS - Control Treatments Figure 3.1. Conotrachelus nenuphar F1 mean larval emergence (:tSE) after parent pyriproxyfen treatment. One time treatment (NS 1), treatment every 30d (NS 1/m), treatment every 2wk (NS 1/2w), and untreated control (NS - control) Hoffmann et al. (2007) discussed that induction of reproduction appears to be dose dependent in terms of the distribution of plum curculio ovary development stages after pyriproxyfen treatment, according to the result that females treated with the highest topical dose had a greater proportion of fully mature eggs after 10 d. However in terms of diapause breaking performance and the fecundity, all tested doses appeared sufficient. It appears that the diapause in plum curculio works like an on and off switch and that even at relatively low rates, pyriproxyfen successfully breaks the diapause and switches on the reproduction. Chapter 4 Use of pyriproxyfen’s diapause breaking activity on plum curculio, Conotrachelus nenuphar (Herbst), as a population control strategy Abstract Northern strain plum curculio, Conotrachelus nenuphar (Herbst), has an obligate winter reproductive diapause where reproduction does not take place until the following year’s spring emergence. Laboratory assessment of the effects of pyriproxyfen treatment showed activation of reproduction in prediapause adults. The applicability of such sub- lethal activity of pyriproxyfen, disrupting diapause and inducing egg development and oviposition in prediapause plum curculio, was examined in field trials. Field treatment of prediapause adults with direct spray and exposure to residue resulted in successful induction of oviposition and F1 emergence in 85 percent of the adults tested. Only 5 percent of the controls were observed to have oviposition and F l emergence. Also, laboratory treated prediapause plum curculios were taken out to the overwintering Site and assessed for survival after overwintering. Results showed Significantly reduced survival rate in pyriproxyfen treated adults compared to the controls. These results indicate that pyriproxyfen could be used in overall population management of plum curculio, via mortality to overwintering adults and reducing number of adults in spring emergence. 41 Introduction Plum curculio, Conotrachelus nenuphar (Herbst), is an important native pest of cultivated pome and stone fruits in eastern and central North America. There are two strains of plum curculio, northern and southern, of which the univoltine northern strain has an obligate adult reproductive winter diapause that requires hibernation to overcome (Smith and Salkeld 1964, Smith and Flessel 1968). Southern strain is multivoltine and does not have obligatory reproductive diapause. This beetle has a broad host range and is a key pest of apples and cherries, as well as other stone fruits such as plums and peaches (Chapman 1938, Armstrong 1958, Maier 1990, Racette et al. 1992, Hallman and Gould 2004, Brown 2005, and Jenkins et al. 2006). Although blueberries are not among the primary host of plum curculio, the beetle can cause Significant loss in yield to them as well (Mampe and Neunzig 1967, Polavarapu et al. 2004). Increasing plum curculio population pressure is a growing concern because of the tightly set statutory and consumer tolerances for damaged or infested fruit. A zero tolerance standard for processed cherries, meaning that no larvae may be present in fruit at harvest, has been set by the US. Department of Agriculture (USDA 1941). This standard creates severe economic pressure on growers since failure to comply results in a rejection of the grower’s entire delivery to the processor. The use of conventional broad- spectrum insecticides over the past 50 yrs, particularly organophosphates, has helped growers comply with zero tolerance standards in tart cherries. However, through the implementation of the 1996 US. Food Quality Protection Act (FQPA) (US EPA 1996), some organophosphates are being phased out in favor of compounds with reduced human and environmental toxicity (US EPA 2008). Many of these replacement chemicals, such 42 as the neonicotinoids and oxidiazines, have Shown good performance on pests yet they have generally exhibited a lower efficacy against plum curculio compared to the organophosphates (Wise and Out 2004, Whalon and Korson 2008). Therefore, it is imperative for the survival of domestic fruit industries to investigate innovative management strategies with novel insecticides or biopesticides for appropriate management of plum curculio. Pyriproxyfen is one of the insect growth regulators that exhibit juvenoid activity suppressing embryogenesis, metamorphosis and formation of adults (Dhadialla et al. 1998). Its potential pesticidal activity has been studied for the management of various arthropods, including beetles, fleas, whiteflies, flies, thrips, and mosquitoes (Koopmanschap et al. 1989, Hargrove and Lagley 1990, Ishaaya et al. 1994, Meola et al. 1996, Liu and Chen 2001, Paul et al. 2006). Application of pyriproxyfen to the Chrysomelid beetle, Aulacophora nigripennis (Motschulsky), terminated diapause and inhibited the accumulation of the cryoprotectant myo-inositol, which is considered a necessary energy source for the overwintering process (W atanabe and Tanaka 1998). Watanabe and Tanaka (1999) also demonstrated that such sublethal effects of pyriproxyfen reduced chill tolerance and caused significantly fewer days of survival in cold condition. In northern strain plum curculios that have obligate winter reproductive diapause, pyriproxyfen application is found to activate reproduction (Hoffmann et al. 2007). The above studies suggest that induction of plum curculio reproductive development prior to overwintering may inhibit necessary physiological preparations for the colder months, and that an after-harvest application in cherries, even at relatively low rates, could reduce the number of plum curculio adults that successfully overwinter. 43 In this study, the potential use of pyriproxyfen treatment for management of plum curculio in tart cherry production in Michigan was examined. Our objectives were to: a) verify efficacy of a pyriproxyfen field spray to induce reproduction (egg development, oviposition, and subsequent larval emergence) in female plum curculios, and b) demonstrate that pyriproxyfen treated female exhibits reduced survival rates through overwintering. Materials and methods General Methods Insect Materials Northern strain plum curculio adults were captured using pyramid (Whalon modified tedder’s trap) and screen traps (circle trunk trap) equipped with plum essence and benzaldehyde lures to attract the plum curculio adults from the field (Great Lakes IPM). For a 2006 field efficacy study, plum curculio adults were collected from a private production cherry orchard located in Traverse City, Michigan (Grand Traverse County). The collection was made in August 2006, when adult emergence mostly consists of pre- diapause generation plum curculios. Adult Northern strain plum curculios were held separately from the southern strain and maintained at 23:1: 1°C with a 16:8 h lightzdark photoperiod on thinning apples for feeding (Smith 1957) in the rearing facility at the Michigan State University (MSU), Department of Entomology, Center for Integrated Plant Systems (CIPS). For the 2007 overwintering study, northern strain plum curculio adults were collected from organic farms in Manistee County, Michigan. The collection was made in July 2007, and maintained as above until experimental use. Field Efficacy Chemical Materials and Application Methods Commercial formulation of pyriproxyfen Esteem® 35WP (V alent U.S.A. Corporation Walnut Creek, CA) was diluted to 350.8 g in 936.9 L of water per 1 ha (131.0 mg a.i. / L). This was the most concentrated level of dilution suggested by the current label (5 oz in 100 gal of water per acre). The experimental plot consisted of ‘Montmorency’ cherry trees planted in 1994 with tree spacing of 18 m by 14 m at the MSU Clarksville Horticultural Experimental Station located in Clarksville, Michigan (Ionia County). Regular maintenance applications of fungicides were made to all treatment blocks. Treatments were applied with an FMC 350 air blast sprayer calibrated to deliver 935.3 L of water per ha (100 gall acre) at a pressure of 180 psi. Pyriproxyfen was applied on August 18th 2006. A row of 20 trees was sprayed with pyriproxyfen, and with a buffer row in between another row of 20 trees was sprayed with water only as the control. During the Spraying, 20 cylindrical metal mesh cages, each containing 10 plum curculios, were randomly hung on the trees; coupled in opposite directions (North - South and/or East - West) leaving no cages bundled in one direction. After the spray was dried (5h), plum curculios were taken out of the metal cages and transferred to exposure chambers (32 oz deli container, Fabri-Kal, Kalamazoo, MI) containing shoots (branch terminals with attached fruits and leaves collected from sprayed trees) set in wet floral foam (Smithers-Oasis North America, Kent, OH) with paraffin coating; so as to maintain the integrity of the plant tissue (Figure 4.1). Each 45 exposure chamber containing 10 plum curculio adults was considered a replicate and there were a total of 20 replicates per treatment in the experiment. Ventilated Top /‘ Paraffin Wax Layer a \‘ ,4 Figure 4.1. Schematic drawing of plum curuclio exposure chamber, 32 oz deli container (Fabri-Kal, Kalamazoo, MI) with wet florofoam, paraffin wax, and ventilated top Dissection and Larval Emergence The plum curculios were left for 3 d inside the exposure chambers described above, and then were transferred to a 4 oz soufflé cup (SOLO cup company, Urbana, IL) with fitted wire mesh (30 mm x 30 mm) and incubated on untreated thinning apples for 2 wk, providing sufficient time for egg laying and development (Figure 4.2). The thinning apples that had been used in the 2 wk incubation period of the plum curculios were held for larval emergence. Because larval emergence for the control group was zero, one sample t—test was conducted to determine whether the mean was significantly different from zero for the pyriproxyfen treatment. Figure 4.2. Schematic drawing of plum curculio incubation chamber, 4 oz souffle cup (SOLO cup company, Urbana, IL) with fitted wire mesh (30 mm x 30 m) At the end of the 2 wk period, a female plum curculio from each replicate was dissected for observation of egg development in Ringer’s solution (Online Medical Dictionary) under a Nikon SMZ1000 (Mager Scientific, Inc., Dexter, MI) stereo dissecting microscope (Hoffmann et al. 2004). The presence of egg development inside female ovaries was recorded in binary code (1=yes; 0=no). This egg development data was submitted to a two proportions test and Fisher’s exact test to determine statistical difference between treated and control groups. Overwintering Survival Chemical Materials and Application Methods Adult Northern strain plum curculios were reared on thinning apples, as described in the insect materials section. Pyriproxyfen treatment with Esteem® 35WP (Valent 47 U.S.A. Corporation Walnut Creek, CA) took place 1 month before deployment (October 2007) by incubating plum curculios on apples dipped in a solution of 262 mg a.i. / 1L of water. The control group of plum curculios remained untreated. Deployment Site and Methods An apple orchard located at MSU, East Lansing, Michigan (Ingham County) was selected for the plum curculio overwintering site. This orchard has not been managed for commercial production, and thus no pesticides used in the past 2 or more years. Six northern strain plum curculios (3 males and 3 females) were placed inside 4 oz sterile polypropylene container (Medegen Medical Products, Gallaway, TN) filled with apple leaves collected from the orchard ground concurrent to the deployment. Deployment site was setup with drainage system and rooftop providing protection against flooding, predators, snow, and other natural adversity (Figure 4.3). 48 Ground level rooftop cover Ground Level I \ PVC Pipe Figure 4.3. Schematic drawing of plum curculio overwintering site, rooftop and drainage setup. Each container housing 6 plum curculios was considered a replicate. A total of 4 sets (2 groups of 4 replicates of 2 treatments) were deployed with each set brought back to the laboratory every month for 4 months (from October 2007 until February 2008) to assess plum curculio survival rate over the winter (Figure 4.4). Statistical Analysis At each collection, survival data was submitted to ANOVA Tukey HSD test for Statistical comparison of different treatments. Entire data set was also submitted to the zero-inflated count model for analyzing the significance of pyriproxyfen treatment, presence of fruit, and duration of overwintering process on plum curculio survival over the entire course of 4 month overwintering process. The main motivation for zero- 49 inflated count models is that real-life data frequently display overdispersion and excess zeros (Lamber 1992, Greene 1994). Replicate SCI (2 groups, 4 replicates of 2 treatments) Pyriproxyfen Treated UTC MEEEE X 4 Pyriproxy_fen Treated 6 Plum fl . UTC mm EEEE Figure 4.4. Plum curculio overwintering deployment setup. Group 1 + fruit = provisioned with a thinning apple, Group 2 - fruit: not provisioned with a thinning apple, UTC = untreated control. Results Field Efficacy While no larvae emerged from apples on which untreated plum curculios were incubated, a significantly higher average number of 9.65 larvae emerged from pyriproxyfen treated (one sample t. test, T=9.024, P=0.000) (Figure 4.5). Also at dissections, Significantly more pyriproxyfen treated females had egg development than untreated controls (two sample proportions test, Z=-8.55, P=0.000). Only 1 out of 20 (5%) untreated females was observed with egg development, while 17 out of 20 (85%) pyriproxyfen treated females were observed with egg development (Figure 4.6). 50 12~ * Number of larvae emerged (:1: SE) Control Pyriproxyfen Figure 4.5. Average number (t SE) of plum curculio larval emergence from pyriproxyfen treated (direct spray in field and 3d residue exposure) adults after 2 wk incubation. * indicates Significant difference. 100— * 90— g 80- 53 70— a: 60— g; 50— _l-l-’ _ as. :13- 92 20- 81:10- E 0 'l' . Control Pyriproxyfen Figure 4.6. Percent (i SE) female plum curculios with egg development at dissection after exposure to direct spray of pyriproxyfen in field and 3d residue exposure. * indicates Significant difference. 51 Overwintering Survival Mortality was first observed from the second collection of plum curculios (December 2007) although there were no significant differences. It was the last month’s collection (February 2008), significant differences were found in survival rate from different treatments (ANOVA Tukey HSD, F=8.415, P=0.003). According to the last month’s collection of plum curculio, 70 percent (UTC 4» Fruit) and 83 percent (UT C - Fruit) of untreated controls survived while only 29 percent (both Pyri + Fruit, and Pyri - Fruit) of pyriproxyfen treated plum curculios survived (Figure 4.7). According to the zero-inflated Poisson regression model pyriproxyfen treatment (T=3.69, df=64, P=0.0005) and duration of overwintering process (T =-2.01, df=64, P=0.0483) showed significant effects on the survival of plum curculio when there is a 4 month duration of overwintering. 52 Group 1 D urc + Frult I Pyri + Fruit a g; i i', 80 1 75 60 .2 ‘ * S 40 J W . ‘E 20 4‘ m 1 L) o .1“... -1“ 1-... 1‘ __ g r 7 r 1 2 3 4 GroupZ DUTC-Frult IPyri-Fruit A 100 l a i :1 8°? 71’ 6° ~§ a j >1: .2 4° . 15 l (u 20 l 2 i an: o WIT—fl ’7’—T_‘—“ ’ 'T‘ -F‘l 1 2 3 4 Months after deployment Figure 4.7. Survival rate (:t SE) of diapause bound northern Strain plum curculio adults. Untreated Control (UTC), Pyriproxyfen (Pyri), Deployment with fruit provision (+ fruit), without (- fruit). * indicates significant difference. 53 Discussion In this research, a single application of pyriproxyfen field spray successfully induced reproduction in diapause bound northern strain plum curculios and more importantly significantly reduced their survival over the winter. With reduced toxicity compared to that of organophosphates, pyriproxyfen would be a good candidate to incorporate into current integrated pest management strategies in cherry orchards. With increased evidence of pest resistance to conventional pesticides, not only the discovery of new ‘reduced-risk’ chemistries, but also investigation of how they can be utilized and incorporated into current integrated pest management is necessary to continue the progress towards sustainable agricultural. Though pyriproxyfen may well reduce the physiological fitness of plum curculio, factors regarding resident weather conditions and duration of overwintering also play an important role in reducing plum curculio survival over the winter. The results indicated that reduced survival was achieved only after 4 months of overwintering suggesting that the benefit is likely to be seen in long winter states such as Michigan, but may not in Short winter states like Georgia. Inducing reproduction and oviposition behavior is not an issue in early—harvested crops such as cherries, because diapause-bound plum curculio adults emerge after harvest. However, in late-harvest crops such as apples, inducing reproduction while fruits are still vulnerable to oviposition could result in severe economic damage to growers if plum curculio oviposit on mature fruit. To evaluate the applicability of this strategy, future laboratory. and field-based studies need to be conducted on the acceptable age of fruit for oviposition and population migration monitoring after pyriproxyfen spray. 54 Conclusion An organophosphate insecticide Azinphos—methyl was widely used by US fruit growers essentially setting the standards on quality and condition of fruit commodities for its remarkable insecticidal performance, short residues, and proven curative abilities (Wise et al. 2006, Whalon and Korson 2008). However with implementation of Food Quality Protection Act 1996 enforced by US. Environmental Protection Agency, conventional pesticides that pose high risk of human and environment such as azinphos- methyl are under strict reevaluation. A phase out decision is already made on azinphos- methyl to end its uses by 2012 causing loss of the best means of pest control in fruit production. As a result of this, pesticides with comparably less human and environment risk are being registered and becoming available for growers in the form of reduced-risk and organophosphate-altemative compounds. However performance of these replacement chemicals requires Specialized timing or increased number of sprays to maintain current industry standards for grade of fruits. Such aspects of reduced risk compounds will cause economic pressure with increased cost for pesticides and reduced risk on humans may not be reduced risk for the environment or the ecosystem. Therefore further studies are needed to understand such indirect adverse effects of these new insect growth regulators with novel pesticidal activities found in this thesis research. Two novel plum curculio insecticide control strategies were suggested using currently registered bio-rational insecticides; insect growth regulators novaluron and pyriproxyfen. Novaluron acts on plum curculio by reducing number of viable larvae after adult exposure. This sub-lethal activity was effective in field with residual activity up to 55 14d through apple fruit exposure. Application of novaluron in the Spring when plum curculios oviposit in fruit may reduce resident population of the orchard hence may reduce overall pest population over the years. This application timeline overlaps with current recommendations of novaluron for codling moth control suggesting potential for dual control action with one spray. Such aspects of novaluron make this compound a good candidate for incorporation into current integrated pest management of plum curculio as growers need wide variety of control strategies after azinphos-methyl phase out decision by US EPA. Novaluron cannot be a standalone compound against plum curculio because it does not prevent adult oviposition, and its adverse effects on beneficial and non-target organisms Should be taken into consideration. No adverse effects of novaluron exposure are found on phytoseiid mite field populations (Ishaaya et al. 2001) mortality and development of the soil-dewelling predatory mite Stratiolaelaps scimitus (W omersley) (Cabrera et al. 2005), or greenhouse populations and percent parasitism of the parasitoid Encarsia Formosa Gahan (Ishaaya et al. 2002). Novaluron exposure to natural enemy Trochogramma pretiosum moderately reduced the percentage of parasitized hosts and also the parasitoid emergence (Bastos et al. 2006). While these studies suggest moderate to no adverse effects of novaluron on beneficial and non-target organisms, novaluron exposure to adult Podisus maculiventris (Say), a natural enemy of Colorado potato beetle Leptinotarsa decemlineata (Say), showed adverse effects significantly reducing the oviposition and hatch of eggs from exposed adults (Cutler et al. 2006). The ideal application timing for utilizing novaluron sub-lehtal activity against plum curculio is spring time when adult oviposition occurs. This is also when flowering 56 of tree fruit crops and the activity of important pollinators such as bumblebees occur. Effects of novaluron exposure to such beneficial organisms like bumblebees would be important aspect on compatibility of novaluron use in fruit integrated pest management programs. Recent study involving risk assessment of novaluron towards bumblebees Bombus terrestris resulted in reduced reproduction, egg hating, and larval growth (Mommaerts et al. 2006). Though chitin synthesis inhibitors such as novaluron are considered to be relatively benign towards beneficial and non target insects, recent studies showed novaluron sub-lethal activity not only on targeted pest but also on other beneficial and non target insects. Such results suggest that novaluron should be used with caution and that species specific risk assessment for major beneficial and non target insects should be conducted before safe use of this compound in fruit integrated pest management. Pyriproxyfen acts on northern strain plum curculios by activating reproduction in prediapause female adults. Instead of converting summer feed to accumulated energy in the form of fat body as energy reserves through overwintering process, females seem to exploit such energy by this early activation of reproduction in prediapuse state. Such commitment of energy to reproduction may have caused reduced survival over the winter observed in chapter 4 of this thesis research. These findings suggested plum curculio population control strategy by reducing spring emergence population through late season pyriproxyfen application on previous year. However in case of orchards growing multiple crop variety with different harvest time such as cherry and apple, inducing reproduction according to early harvest crop (cherry) could bring detrimental effects on the late harvest 57 crop (apple) if the insect utilizes both crops as satisfactory hosts. Future studies in such areas would be needed to effectively employ this strategy into plum curculio management. As mentioned in case of novaluron, adverse effects of pyriproxyfen on beneficial and non target insects Should also be taken into consideration. Like novaluron, pyriproxyfen also seem to exhibit contradicting results on beneficial and non target insects according to differences in Species, life stages, and exposure methods (Medina et al. 2003, Cloyd and Dickinson 2006, Mahdian et al. 2007). Again, Species Specific risk assessment for major beneficial and non target insects should be conducted before safe use of this compound in fruit integrated pest management. 58 Appendix 1 Record of Deposition of Voucher Specimens* The specimens listed on the following sheet(s) have been deposited in the named museum(s) as samples of those species or other taxa, which were used in this research. Voucher recognition labels bearing the Voucher No. have been attached or included in fluid-preserved specimens. Voucher No.: 2008-09 Title of thesis or dissertation (or other research projects): Characterizing the sub-lethal effects of two insect growth regulators on plum curculio, Conotrachelus nenuphar (Herbst), in apples and cherries Museum(s) where deposited and abbreviations for table on following sheets: Entomology Museum, Michigan State University (MSU) Other Museums: Investigator’s Name(s) (typed) Ki Duk Kim Date 5/5/09 *Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in North America. Bull. Entomol. Soc. Amer. 24: 141-42. Deposit as follows: Original: Include as Appendix 1 in ribbon copy of thesis or dissertation. Copies: Include as Appendix 1 in copies of thesis or dissertation. Museum(s) files. Research project files. This form is available from and the Voucher No. is assigned by the Curator, Michigan State University Entomology Museum. 59 £292: saw 18222 o5 5 188,5 .2 £586on new: o>onm 65 nozooom 8-88 .2 Loco=o> moowBB memo Ev. x30 2 6098 vaoEmz wLBmmzmoE. Emmmoooc : 33% 55:68 no.3 of 1 Pages Appendix 1.1 Voucher Specimen Data Page 1 or or 23.30 b06533 am: 38.2.: $325: esocogozoo ._u m 1w 10 0+ 3 w m .m a m m m m. m s notwoaou new new: :98“ $50 Lo 8825 w e ”r h u u D m N 9 Lo 860:8 mcoEeonw Le Emu .023 h H t d d U y a g M w .n O A A P N L E co LonEzz References Alavanja, M.C.R., Hoppin, J .A. and Kamel, F. (2004). Health effects of chronic pesticide exposure: cancer and neurotoxicity. Annual Review of Public Health 25: 155-197. Anderson, B.S., Phillips, B.M., Hunt, J .W., Worcester, K., Adams, M., Kapellas, N. and Tjeerdema, RS. (2006). Evidence of pesticide impacts in the Santa Maria River watershed, California, USA. Environmental Toxicology and Chemistry 25(4): 1 160-1 170. Armstrong, T. (1958). Life-history and ecology of the plum curculio, Conotrachelus nenuphar (Hbst.) (Coleoptera: Curculionidae), in the Niagara Peninsula, Ontario. Canadian Entomologist 90: 8-17. Bastos, C.S., de Ahneida, RP. and Suinaga, EA. (2006). Selectivity of pesticides used on cotton (Gossypium hirsutum) to Trichogramma pretiosum reared on two laboratory-reared hosts. Pesticide Management Science 62(1): 91-98. Beckwith, CS. (1943). Insects attacking blueberry fruit. New Jersey Agricultural Experimental Station Circular 472. Biddinger, D. and Hull, L. (1999). Sublethal effects of selected insecticides on growth and reproduction fo a laboratory susceptible strain of tufted apple bud moth (Lepidoptera: Tortricidae). Journal of Economic Entomology 92: 314-323. Brown, M.W. (2005). Host utilization and phenology of injury by plum curculio (Coleoptera: Curculionidae) in West Virginia. Journal of Entomological Science 40: 149-157. Cabrera, A.R., Cloyd, RA. and Zaborski, ER. (2005). Lethal and sub-lethal effects of novaluron (Pedestal) on the soild-dwelling predatory mites, Stratiolaelaps scimitus (Womersely) (Acari: Mesostigrnata: Laelapidae), under laboratory conditions. Journal of Entomological Science 40: 47-53. Calkins, C.O., Hill, A.J., Huettel, MD. and Mitchell, ER. (1977). Effect of diflubenzuron on plum curculio populations in laboratory and field tests. Journal of Economic Entomology 70: 463-466. Chapman, RI. (1938). The plum curculio as an apple pest. New York State Agricultural Experiment Station, Bulletin 684. Cloyd, RA. and Dickinson, A. (2006). Effect of insecticides on mealybug destroyer (Coleoptera: Coccinellidae) and parasitoid Leptomastix dactylopii (Hymenoptera: Encyrtidae), natural enemies of citrus mealybug (Homoptera: Pseudococcidae). Journal of Economic Entomology 99(5): 1596-1604. 61 Coggon, D. (2002). Work with pesticides and organophosphate sheep dips. Occupational Medicine 52: 467-470. Cutler, G.C., Scott-Dupree, C.D., Tolman, J .H. and Harris C.R.(2005). Acute and sublethal toxicity of novaluron, a novel chitin synthesis inhibitor, to Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). Pesticide Management Science 61: 1060-1068. Cutler, G.C., Scott-Dupree, C.D., Tolman, J.H. and Harris, CR. (2006). Toxicity of the insect growth regulator novaluron to the non-target predatory bug Podisus Maculiventris (Heteroptera: Pentatomidae). Biological Control 38: 196-204. Dhadialla, T.S., Carlson, GR. and Le DP. (1998). New insecticides with ecdysteroidal and juvenile hormone activity. Annual Review of Entomology 43: 545-569. Eskenazi, B., Bradman, A. and Castorina, R. (1999). Exposures of children to organophosphate pesticides and their potential adverse health effects. Environmental Health Perspectives 107(3): 409-419. Glass, E.H. and Leink, SE. (1971). Apple insect and mite populations developing after discontinuance of insecticides: 10-year record. Journal of Economic Entomology 64(10): 23-26. Greene, W.H. (1994). Accounting for excess zeros and sample selection in poisson and negative binomial regression models. New York, University Department of Economics Working Paper No. 94-10. Hagley, E.A.C., Moteith, L.G., Herne, D.H.C. and Trottier, R. (1977). Pest population buildup in apple orchards following omission of insecticide and acaricide sprays. Proceedings of the Entomological Society of Ontario 108: 7-11. Hall, ER. (1974). Bioeconomics of apple pests: cost appraisal of crop injury data. Journal of Economic Entomology 67: 517-521. Hallman, G. J. and Gould, W. P. (2004). Evaluation of subtropical and tropical fruits as potential hosts for the southern strain of plum curculio (Coleoptera: Curculionidae). Florida Entomologist 87: 241-243. Hallman, G.J. and Gould, WP. (2004). Evaluation of subtropical and tropical fruits as potential hosts for the southem strain of plum curculio (Coleoptera: Curculionidae). Florida Entomologist 87: 241-243. Hargrove, J .W. and Langley, RA. (1990). Sterilizing Tsetse (Diptera: Glossinidae) in the field: a successful trial. Bulletin of Entomological Research 80: 397-403. 62 Hoffmann, E.J., Coombs, AB. and Whalon, ME. (2004). Reproductive development of northern and southern strains of Plum Curculio (Coleoptera: Curculionidae). Journal of Economic Entomology 97: 27—32. Hoffmann, E.J., Vander Jagt, J. and Whalon, ME. (2007). Pyriproxyfen activates reproduction in prediapuase northern strain plum curculio (Conotrachelus nenuphar Herbst). Pest Management Science 63: 835-840. Howitt, AJ. (1993). Common tree fruit pests, NCR 63 ed. Michigan State University Extension, East Lansing, MI. Hoyt, S.C., Leeper, J .R., Brown, G.C. and Croft, B.A. (1983). Basic biology and management for insect IPM. Pp. 93-151 in B.A. Croft and SC. Hoyt (eds.) Integrated Management of Insect Pests of Pome and Stone Fruits. New York. Hu, X.P. and Prokopy, R.J. (1998). Lethal and sublethal effects of irnidacloprid on apple maggot fly, Rhagoletis pomonella Walsh (Dipt., Tephritidae). Journal of Applied Entomology 122: 37-42. IRAC (2008). Insecticide Resistance Action Committee Mode of Action Classification. Version: 5.3 Revised and re-issued, July 2007. Isaacs, R., Cahill, M. and Byme, D.N. (1999). Host plant evaluation behavior of Bemisia tabaci and its modification by external or internal uptake of irnidacloprid. Physiological Entomology 24: 1-8. Ishaaya, I. (1990). Benzoylphenyl ureas and other selective control agents: mechanism and application. In Casida, J .E. (ed.), Pesticides and alternatives. Elsevier Science Publisher BV 363-376. Ishaaya, I. and Horowitz AR. (2007). In focus: [PM using novel insecticides and other approaches. 63(8): 729. Ishaaya, 1., De Cock, A. and Degheele, D. (1994). Pyriproxyfen, a potent suppressor of egg hatch and adult formation of the Greenhouse Whitefly (Homoptera: Aleyrodidae). Journal of Economic Entomology 87: 1185-1189. Ishaaya, I., Horowitz, A.R., Tirry, L. and Barazani, A. (2002). Novaluron (Rimon), a novel IGR: mechanism, selectivity and importance in 1PM programs. Proc Int Symp Crop Proect Med Fae Landbouww Univ Gent 67: 617-626. Ishaaya, I., Kontsedalov, S. and Horowitz, AR. (2003). Novaluron (Rimon), a novel IGR: potency and cross-resistance. Archives of insect biochemistry and physiology 54: 157-164. 63 Ishaaya, 1., Kontsedalov, S., Mazirov, D. and Horowitz, AR. (2001). Biorational agents — mechanism and importance in IPM and IRM programs for controlling agricultural pests. Proc Int symp Crop Protect Med Fac Landbouww Univ Gent 66: 363-374. Jenkins, D., Cottrell, T., Horton, D., Hodges, A. and Hodges, G. (2006). Host of plum curculio, Conotrachelus nenuphar (Coleoptera: Curculionidae), in central Georgia. Environmental Entomology 35: 48-55. Koopmanschap, A.B., Oouchi, H. and de Kort C.A.D. (1989). Effects of a juvenile hormone analogue on the eggs, post-embryonic development, metamorphosis and diapause induction of the Colorado potato beetle, Leptinotarsa decemlineata. Entomologia Experimentalis et Applicata 50: 255-263. Kostyukovsky, M. and Trostanetsky, A. (2006). The effect of a new chitin synthesis inhibitor, novaluron, on various developmental stages of Tribolium castaneum (Herbst). Journal of Stored Products Research 42: 136-148. Kunkle, B., Held, D. and Potter, D. (2001). Lethal and sublethal effects of bendiocarb, halofenocide and imidacloprid on Harpalus pennsylvanicus (Coleoptera: Carabidae) following different modes of exposure in turfgrass. Journal of Economic Entomology 94: 62-67. Lambert, D. (1992). Zero-inflated poisson regression, with an application to defects in manufacturing. Technometrics 34(1): 1-14. Levine, E. and Hall, RR. (1977). Effect of feeding and oviposition by the plum curculio on apple and plum fruit abscission. Journal of Economic Entomology 70(5): 603- 607. Levine, E. and Hall, F.R. (1978a). Pectinases and cellulases from plum curculio larvae: possible causes of apple and plum fruit abscission. Entomologia Exerimentalis et Applicata 23: 259-268. Levine, E. and Hall, F.R. (1978b). Physiology of plum fruit abscission induced by larvae of the plum curculio. HorScience 13(2): 161. Liu, T.-X. and Chen T.-Y. (2001). Effects of a juvenile hormone analog, pyriproxyfen, on the apterous form of Lipaphis erysimi. Entomologia Experimentalis et Applicata 98: 295-301. Mahdian, K., Van Leeuwen, T., Tirry, L. and De Clercq, P. (2007). Susceptibility of the predatory stinkbug Picromerus bidens to selected insecticides. BioControl 52(6): 765-774. Maier, CT. (1990). Native and exotic rosaceous hosts of apple, plum, and quince curculio larvae (Coleoptera: Curculionidae) in the Northeastern United States. Journal of Economic Entomology 83: 1326-1332. Malinowski, H. (1992). Comparative evaluation of some chitin synthesis inhibitors as insecticides against Colorado beetle Leptinotarsa decemlineata Say. Pesticide Management Science 35: 349-353. Mampe, CD. and Neunzig, H.H. (1967). The biology, parasitism, and population sampling of the plum curculio on blueberry in North Carolina. Journal of Economic Entomology 60: 807-812. McGiffen, Jr., M.E., Meyer, J .R. and Boyne, J .V. (1987). Physiological parameters of diapauses and reproduction in the plum curculio, Conotrachelus nenuphar (Coleoptera: Curculionidae). Annals of the Entomological Society of America 80: 284-287. Medina, P., Smagghe, G., Budia, F., Tirry, L. and Vinuela E. (2003). Toxicity and absorption of azadirachtin, diflubenzuron, pyriproxyfen, and tebufenozide after topical application in predatory larvae of Chrysoperla cornea (N europtera: Chrysopidae). Environmental Entomology 32(1): 196-203. Meola, R.W., Pullen, S. and Meola, S. (1996). Toxicology and histopathology of the growth regulator pyriproxyfen to adults and eggs of the cat flea (Siphonaptera: Pulicidae). Journal of Medical Entomology 33: 670-679. Mommaerts, V., Stork, G. and Smagghe, G. (2006). Hazards and uptake of chitin synthesis inhibitors in bumblebees Bombus terrestris. Pesticide Management Science. 62: 752-758. Nauen, R. (1995). Behaviour modifying effects of low systemic concentrations of irrridacloprid on Myzus persicae with special reference to an antifeeding response. Pesticide Science 44: 145-153. O’Neal, M.E., Landis, DA. and Isaacs, R. (2002). An inexpensive, accurate method for measuring leaf area and defoliation through digital image analysis. Journal of Economic Entomology 95(6): 1190-1194. Paul, A., Harrington, LC. and Scott, J .G. (2006). Evaluation of novel insecticides for control of dengue vector aedes aegypti (Diptera: Culicidae). Journal of Medical Entomology 43(1): 55-60. Polavarapu, S., Kyryczenko-Roth, V. and Barry, J .D. (2004). Phenology and infestation patterns of plum curculio (Coleoptera: Curculionidae) on four hi ghbush blueberry cultivars. Journal of Economic Entomology 97: 1899-1905. 65 Quaintance, AL. and Jenne, EL. (1912). The plum curculio. Bulletin. US Department of Agriculture. Bureau of Entomology 103: 1-250. Racette, G., Chouinard, G., Vincent, C. and Hill, SB. (1992). Ecology and management of the plum curculio, Conotrachelus nenuphar (Coleoptera: Curculionidae), in apple orchards. Phytoprotection 73: 85-100. Smith, EH. (1957). A method of rearing the plum curculio under laboratory conditions including some biological observations. Journal of Economic Entomology 50: 187-190. Smith, E.H. and Flessel, J .K. (1968). Hibernation of the plum curculio and its spring migration to host trees. Journal of Economic Entomology 61: 193-203. Smith, E.H. and Salkeld, EH. (1964). Ovary development and oviposition rates in the plum curculio, Conotrachelus nenuphar (Coleoptera: Curculionidae). Annals of the Entomological Society of America 57: 781-787. Staal, GB. (1975). Insect growth regulators with juvenile hormone activity. Annual Review of Entomology 20: 417-460. Stedrnan, J .M. (1904). The “sting” in the apple - The work of the plum curculio in the apple. University of Missouri Agricultural Experiment Station Bulletin 64: 1-24. Thomson, J .R. (1932). Sex differentiation of adults of Conotrachelus nenuphar. Journal of Economic Entomology 25: 807-810. US EPA (1996). Food Quality Protection Act. Public Law 104-170, Aug. 3, 1996. US EPA (1997). Guidelines for expedited review of conventional pesticides under the reduced-risk initiative and for biological pesticides. Pesticide Registration (PR) Notice 97-3. US EPA (2008). Azinphos-methyl; Product cancellation order and amendments to terminate uses. EPA-HQ-OPP-2005-0061; FRL-8349-8. US EPA (2008). Reduced risk/organophosphate alternative decisions for conventional pesticides. Office of prevention, pesticides and toxic substances. Updated February 12, 2008. US EPA (2009). Types of pesticides. http://www.epa.gov/pesticideS/about/tvpes.htm Last updated on January 29, 2009. USDA Agricultural Marketing Service. (1941). US Standards for grades of red sour cherries for manufacture. 51 .4340-51.4348. 66 USDA Agricultural Marketing Service. (1961). US Standards for grades of apples for processing. 51340-51349. USDA Agricultural Marketing Service. (1971). US Standards for grades of sweet cherries. 51.2646-51.2660. USDA Agricultural Marketing Service. (2002). US Standards for grades of apples. 51.300-51.322. Vincent, C. and Roy, M. (1992). Entomological limits to biological control programs in Quebec apple orchards. Acta Phytopathologica et Entomologica Hungarica 27: 649-657. ' Watanabe, M. and Tanaka, K. (1998). Effect of juvenile hormone analogs on diapause termination and myo-inositol content in Aulacophora nigripennis adults (Coleoptera: Chrysomelidae). Applied Entomology and Zoology 33(2): 259-262. Watanabe, M. and Tanaka, K. (1999). Cold tolerance strategy of the freeze-intolerant Chrysomelid, Aulacophora nigripennis (Coleoptera: Chrysomelidae), in warrn- temperate regions. European Journal of Entomology 96(2): 175-181. Whalon, M. and Korson, P. (2008). Tart cherry azinphos-methyl transition strategy. Pesticide Program Dialogue Committee Azinphos-Methyl (AZM) Transition Issues Workgroup. Whalon, M.E., Mota-Sanchez, D. and Hollingworth, RM. (2008). Ananlysis of global pesticide resistance in arthropods. In Whalon, ME. (ed.), Global pesticide resistance in arthropods. Cabi Publishing 5-31. Wise, J .C. and Gut, L. (2004). Control of Plum curculio, 2003. Arthropod Management Tests 29 (B2). Wise, J .C., Coombs, A.B., Vandervoort, C., Gut, L.J., Hoffmann, J. and Whalon ME. (2006). Use of residue profile analysis to identify modes of insecticide activity contributing to control of plum curculio in apples. Journal of Economic Entomology 99: 2055-2064. Wise, J .C., Kim, K., Hoffmann, E.J., Vandervoort, C., Gokce, A. and Whalon, ME. (2007). Novel life state targets against plum curculio, Conotrachelus nenuphar (Herbst) in apple integrated pest management. Pest management Science 63: 737- 742. Yonce, C.E., Horton, D.L. and Okie, W.R. (1995). Spring migration, reproductive behavior, monitoring procedures and host preference of plum curculio (Coleoptera: Curculionidae) on prunus species in central Georgia. Journal of Entomological Science 30: 82-92. 67 I“ "ll Illll Ill “ u “an Nil U“ 03062 9459 3 1293 lllllllllllllilllll