Effects of Cucurbitacin on the Activity of Nucleopolyhedroviruses Against Pickleworm Larvae 1,2

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1 Effects of Cucurbitacin on the Activity of Nucleopolyhedroviruses Against Pickleworm Larvae 1,2 D. Michael Jackson, 3 B. Merle Shepard, 4 Martin Shapiro, 4 and S. El Salamouny 5 J. Agric. Urban Entomol. 26(2): (April 2009) ABSTRACT The cucurbitacin-containing feeding stimulant (InviteH EC) was attractive to second instar pickleworm larvae in the absence of a food source. Second instar pickleworm larvae also fed preferentially on cucumber fruits that were treated with a 1% (0.013) solution of Invite EC compared with untreated fruit. However, 1% Invite did not stimulate feeding on artificial diet, squash leaves, or squash fruits. Concentrations of Invite as low as 0.001% were effective in increasing the efficacy of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, multiply-embedded nucleopolyhedrovirus (AgMNPV) against second instar pickleworms. A 1% Invite solution also increased the efficacy of the celery looper, Anagrapha falcifera (Kirby), multiply-embedded NPV (AnfaMNPV) against second instar pickleworms. KEY WORDS cucurbitacin Baculovirus, Diaphania nitidalis, Pyralidae, Invite EC, The pickleworm, Diaphania nitidalis (Stoll) (Lepidoptera: Pyralidae), is a major pest of Cucurbitaceae (York 1992, Capinera 2001, 2004). Larvae typically feed within the fruits of various cucurbits, such as cucumber (Cucumis sativus L.), cantaloupe (Cucumis melo L.), pumpkin (Cucurbita pepo L.), and squash (Cucurbita spp.) (Dupree et al. 1955, Reid & Cuthbert 1956, Elsey 1981). Pickleworm larvae are especially destructive to pickling cucumbers because of the near zero tolerance for infestations (York 1992). Damaged fruits are unmarketable and even a single infested fruit may lead to rejection of an entire shipment of pickling cucumbers (Hughes et al. 1983, Zehnder et al. 1996). This pest can be one of the most important production constraints for cucurbit growers in the eastern United States (Anonymous 1952, Canerday & Dilbeck 1968, Gianessi 2009). Because of the low threshold for pickleworm damage to many cucurbits (Hughes et al. 1983), crops are often sprayed on a schedule whether or not the insects are actually present (Kaplan 1989). Insecticide applications for the 1 Accepted for publication 25 October The use of trade, firm, or corporation names in this paper is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable. 3 Corresponding Author, USDA, ARS, U.S. Vegetable Laboratory, 2700 Savannah Highway, Charleston, SC, USA; mike.jackson@ars.usda.gov, , Coastal Research and Education Center, Clemson University, Charleston, SC, USA. 5 Department of Plant Protection, College of Food and Agricultural Sciences, King Saud University, Riyadh, P.O. Box 2460, Kingdom of Saudi Arabia. 95

2 96 J. Agric. Urban Entomol. Vol. 26, No. 2 (2009) control of pickleworm larvae may give unsatisfactory results, especially when the pest is abundant (Reid & Cuthbert 1953). Female pickleworm moths typically lay their eggs singly or in small clusters on leaf buds, flowers, small leaves, and fruits (Anonymous 1952, Sorensen et al. 1994, Capinera 2004). On squash and cucumber plants, newly hatched larvae feed on leaves, stem tips, or flowers before late-stage larvae migrate to the fruits (Van Balen 1976, Elsey 1980, 1981). Some larvae may even complete development on large squash flowers (Elsey 1981). Because early instars feed near where the eggs are laid (Reid & Cuthbert 1956), there is an opportunity to control these pests before they enter cucurbit fruits. Control of the early instars is important because once larvae penetrate the fruits, they are very difficult to kill with insecticides (Fulton 1947, Reid & Cuthbert 1953, Dupree et al. 1955). Larvae may spin a silken web over the entrance hole, which may make them even less susceptible to natural enemies or insecticide sprays (Reid & Cuthbert 1956). Thus, frequent applications of insecticides are used to protect cucurbits, especially in the late season after these pests have built up (Sorensen 1993). This approach is not only expensive, but it also destroys natural enemies, negatively affects the environment, and could lead to the development of insecticide resistance. Of the approximately 15 families of viruses that infect invertebrates, the baculoviruses (Baculoviridae) are by far the most important for the development of pest management tools (Evans 2000). Baculoviruses (Nucleopolyhedroviruses [NPV] and Granuloviruses) are generally regarded as safe and effective bioinsecticides against a wide range of lepidopteran pests (Moscardi 1999, Lacey et al. 2001). Baculoviruses are especially attractive biological control agents against lepidopterous pests because of their specificity and safety (Burges & Jones 1986). Recently, we (Jackson et al. 2008) demonstrated that several nucleopolyhedroviruses were active against second instar pickleworms. A multiply-embedded nucleopolyhedrovirus (AgMNPV) from velvetbean caterpillar, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) (Carner & Turnipseed 1977), and a multiply-embedded nucleopolyhedrovirus (AcMNPV) from alfalfa looper, Autographa californica (Speyer) (Lepidoptera: Noctuidae) (Vail et al. 1972), were the most efficacious against second-instar pickleworms. Despite the advantages of using baculoviruses as microbial control agents, their widespread use has been limited by their relatively high cost, slow activity, and susceptibility to UV degradation (Moscardi 1999). However, we found that five of six stilbene fluorescent brighteners significantly increased efficacy of AgMNPV against pickleworm larvae (Jackson et al. 2008). Also, we recently have investigated the use of plant extracts as virus enhancers (Shapiro et al. 2007a, b) and sunlight protectants (Shapiro et al. 2008, 2009a, b, El Salamouny et al. 2009) to increase efficacy and persistence of NPV formulations against lepidopteran pests. Cucurbitacins are a diverse group of oxgenated triterpenoid substances characterized by a tetracyclic cucurbitane nucleus skeleton (Chen et al. 2005). Cucurbitacins, isolated from Cucurbitaceae and several other plant families, have been placed into 12 categories (Chen et al. 2005). These distasteful (bitter) compounds are highly toxic to vertebrates, and they can be detected by humans at extremely low concentrations (1 ppb) (Metcalf et al. 1980). Therefore, cucurbitacin has been bred out of commercial cucurbit varieties (Andeweg &

3 JACKSON et al.: Pickleworm Baculoviruses and Cucurbitacin 97 De Bruyn 1959, Chambliss et al. 1968, Bisognin 2002). Cucurbitacins also serve as oviposition or feeding deterrents for many insects (Tallamy et al. 1997). On the other hand, cucurbitacins are strong phagostimulants to diabroticite beetles of the tribe Lupernini, including species in the genera Diabrotica and Acalymma (Chambliss & Jones 1966, Metcalf et al. 1980, 1987, Metcalf 1986). A water extract of a bitter mutant of the Hawkesbury watermelon, Citrullus lanatus (Thunb.) Matsum & Nakai (Syn. Citrullus vulgaris Schrad), elicits a positive feeding response in banded cucumber beetles, Diabrotica balteata LeConte (Coleoptera: Chrysomelidae) (Chambliss & Jones 1966, Peterson & Schalk 1985). DeMilo et al. (1998) identified this water-soluble compound as cucurbitacin E-glycoside. The bitter Hawkesbury watermelon contains % cucurbitacin E-glycoside by fresh weight (Matsuo et al. 1999). This cucurbitacin arrests Diabrotica beetles, and acts as a gustatory stimulant (Peterson & Schalk 1985, Schroder et al. 1998). A commercial corn rootworm (Coleoptera: Chrysomelidae) bait derived from bitter Hawkesbury watermelon (Invite ECH, Florida Food Products, Eustis, FL) (Anonymous 2002) in combination with a variety of insecticides, has been tested for corn rootworm control throughout the corn belt of the United States (Parimi et al. 2003, Siegfried et al. 2004, French et al. 2007). The use of cucurbitacin baits for control of corn rootworm adults can reduce insecticide application for this adult insect by 95 98% (Tallamy et al. 2005, Van Rozen & Ester 2010). Commercial cucurbitacin products derived from the juice of the bitter Hawkesbury melon received an exemption from the requirement of tolerance for residues when applied as an inert ingredient in pesticide formulations (Anonymous 2000). In addition, cucurbitacins do not attract non-target insects, so they are safe for bees and other beneficial insects. In this paper we report on the effects of this cucurbitacin product (Invite) on the efficacy of entomopathogenic viruses against pickleworm larvae. Materials and Methods Pickleworm colony. Pickleworms used for bioassay studies were reared at the USDA, ARS, U.S. Vegetable Laboratory (USVL), Charleston, SC by methods modified from those of Elsey et al. (1984). Modifications included using a multipurpose Lepidoptera diet (BioServe Product# F9772; Wheat Germ, Soy Flour Base) (BioServe, Frenchtown, NJ), the elimination of plastic louvers in the rearing crispers, and using layered instead of crumpled paper towels for pupation sites (Jackson et al. 1998, 2008). Larvae for bioassays were fed on the multipurpose diet until the second instar. In 2008, our pickleworm colony became infected with a disease that we suspected was an NPV. Because of this infection, we were forced to re-establish our colony using a remnant virus-free group of larvae. Samples of infected larvae were sent to Johannes Jehle and colleagues (Laboratory for Biotechnological Crop Protection, Department of Phytopathology, Agricultural Service Center Palatinate [DLR Rheinpfalz], Neustadt, Germany) for identification through DNA amplification and sequencing procedures. Virus isolates. Six nucleopolyhedroviruses were evaluated: AgMNPV; AcMNPV; the celery looper, Anagrapha falcifera (Kirby) (Lepidoptera: Noctuidae) multiply-embedded NPV (AnfaMNPV) (Hostetter & Puttler 1991); the old-

4 98 J. Agric. Urban Entomol. Vol. 26, No. 2 (2009) world bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), singly-embedded NPV (HearSNPV) (McCarthy et al. 1978, Hamm 1982); the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), multiplyembedded NPV (PlxyMNPV) (Kariuki & McIntosh 1999); and Rachiplusia ou (Guenée) (Lepidoptera: Noctuidae) multiply-embedded NPV (RoMNPV) (Paschke & Hamm 1961). The AcMNPV (strain 6-R) was obtained from E. M. Dougherty (USDA-ARS, Beltsville, MD). The AnfaMNPV was obtained from D. L. Hostetter (USDA-ARS, Kimberly, ID). We originally obtained the PlxyMNPV from A. H. McIntosh (USDA-ARS, Columbia, MO). This isolate was produced in larvae of P. xylostella and Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) (Farrar et al. 2007). We obtained a Brazilian isolate of AgMNPV from J. R. Fuxa (Louisiana State University, Baton Rouge, LA). This isolate originally came from diseased larvae collected in southern Brazil (Carner & Turnipseed 1977). The AgMNPV was produced in laboratory-reared A. gemmatalis by standard techniques (Fuxa & Richter 1999). The RoMNPV was obtained from the virus collection at the Insect Biocontrol Laboratory (USDA-ARS, Beltsville, MD). The HearSNPV (V-83 strain from China) was obtained from J. J. Hamm (USDA-ARS, Tifton, GA). Invite ECH (lot number 8319C17AP+T) was received directly from the manufacturer (Florida Food Products, Inc., Eustis, FL). This product is a water-soluble emulsifiable concentrate (EC) containing juice from the bitter Hawkesbury watermelon, and an 80% principal functioning agent (Anonymous 2002). The primary active ingredient of Invite EC is cucurbitacin, and it is labeled as a gustatory, arrestant/feeding stimulant (Anonymous 2002). Invite was developed under USDA license (van Rosen & Ester 2010), and it is registered for attract-and-kill control of corn rootworm/cucumber beetles (Diabrotica spp.) (Anonymous 2002). Behavioral response of pickleworm larvae to Invite. The behavioral response of pickleworm larvae to 1% Invite (0.013) was examined in choice experiments during 2009 and 2010 at the USVL. All behavioral experiments were done under ambient laboratory conditions (23 26uC). For the first experiment, filter paper disks (4.2 cm diam.) were treated by soaking them in a 1% solution of Invite or distilled water (control disks) and letting them air-dry. The disks were placed into 14-cm diam. glass Petri dishes each containing two treated and two untreated disks arranged in an alternating pattern at the outer edge of the dish. A single drop of distilled water was placed on each disk just before placing 10 larvae (2 nd instar) at the center of each dish. The location of each larva was recorded after 15, 30, 60, and 120 min. In a similar design, Invite-treated and untreated artificial diet (multi-purpose Lepidoptera diet), cucumber fruits ( Poinsett ; Cucumis sativus L), squash fruits ( Early Prolific Straightneck ; Curcurbita pepo L.), and squash leaves ( Early Prolific Straightneck ) were assayed. Plugs (2 cm diam) of fruit with skin intact, leaves, or artificial diet were taken with a cork-hole borer. They were then immersed in 1% Invite or distilled water (control) for one minute and allowed to air dry. Twelve replications were run for each of these experiments (120 larvae per treatment). In a separate observational experiment, pickleworm larvae were observed to determine their orientation behavior to Invite-treated and untreated filter paper disks. For this test, individual second instars were placed in the center of 14-cm

5 JACKSON et al.: Pickleworm Baculoviruses and Cucurbitacin 99 diam. glass Petri dishes containing two treated and two untreated disks (4.2 cm diam) arranged in an alternating pattern at the outer edge of the dish. Twelve larvae (12 replications) were observed for 15 min, and details of their behaviors and locations were recorded. The laboratory had overhead fluorescent lighting, and the Petri dishes were oriented randomly under these lights. Susceptibility of pickleworm larvae to viruses with and without Invite. Bioassays of pickleworm larvae were modified from published techniques (Shapiro & Farrar 2003). Experiments were done in new 9-cm diam plastic Petri dishes. The dishes contained 0.6 cm of the same multipurpose artificial diet used for colony rearing. One milliliter of the virus suspensions were pipetted onto the surface of the diet and allowed to air-dry. Sterile distilled water was pipetted onto the surface of the diet in the control treatments. Second-instar pickleworm larvae from the laboratory colony were gently placed on the surface of the diet using soft forceps (Bioquip Products, Rancho Dominguez, CA). Dishes were monitored several times over a two-week period, and the numbers of dead larvae, live larvae, prepupae, and pupae were recorded. In the first NPV-efficacy experiment, five rates of Invite were evaluated alone and in combination with AgMNPV. The 12 treatments consisted of the untreated control, five rates of Invite alone (0.1, 0.01, 0.001, , and ), the same five rates of Invite in combination with AgMNPV applied at 10 7 viral occlusion bodies (OB) per dish, and AgMNPV alone (10 7 OB per dish). One milliliter of the treatments was pipetted onto the surface of the diet. This experiment was replicated twice. For each replication, there were five Petri dishes with 10 larvae in each dish. Thus, a total of 100 larvae were bioassayed for each treatment. These experiments were done under ambient laboratory conditions (23 26uC). In the second NPV-efficacy experiment, the six NPVs with and without the addition of 1% Invite (0.013) to artificial diet were evaluated. The 14 treatments were AcMNPV, AnfaMNPV, AgMNPV, HearSNPV, PlxyMNPV, and RoMNPV with and without Invite, Invite alone (0.013), and an untreated control. The NPVs were applied at 10 7 OB per dish. For each replication, there were five Petri dishes with 10 larvae in each dish. An equal number of controls (untreated diet) were set up. This experiment was replicated four times. Thus, a total of 200 larvae were bioassayed for each treatment. Data analysis. Data from larval bioassays were subjected to analysis of variance (ANOVA) using the General Linear Model (GLM) procedure of the SAS system, version 8.02 (SAS 1999). When treatment effects were significant at the 5% level, means were separated by Fisher s least significant difference (LSD) at the 5% probability level (SAS 1999). Results Behavioral response of pickleworm larvae to Invite. Within 15 min, second instar pickleworms preferentially chose the Invite-treated filter papers over untreated ones (Table 1). Because there was no food source, some larvae left the Invite-treated filter papers after one hour, and after two hours nearly onethird of the larvae were found wandering on the Petri dishes not on either the treated or the untreated filter papers. This contrasted with the artificial diet experiment, where 90% of the larvae were on one of the diet pieces after two

6 100 J. Agric. Urban Entomol. Vol. 26, No. 2 (2009) Table 1. Preference of pickleworm larvae for filter-paper disks, artificial diet, cucumber fruits, squash fruits, or squash leaves treated with or without Invite (0.013) in choice bioassays. Treatment Time (minutes) Percent on treatment (1% Invite) Percent on control (no Invite) Percent on dish (not on treatment) Filter paper a a 11.6 c 26.7 b Filter paper a 10.0 c 28.3 b Filter paper a 15.0 c 31.7 b Filter paper a 14.2 c 30.8 b Artificial diet ns b Artificial diet a 45.8 a 14.2 b Artificial diet a 46.7 a 11.6 b Artificial diet a 47.5 a 10.0 b Cucumber fruit a 27.5 b 20.8 b Cucumber fruit a 27.5 b 15.8 b Cucumber fruit a 27.5 b 14.2 b Cucumber fruit a 28.3 b 6.7 c Squash leaves ab 40.8 a 21.7 b Squash leaves ab 44.2 a 19.2 b Squash leaves a 45.0 a 17.5 b Squash leaves a 45.8 a 10.0 b Squash fruit a 40.0 a 19.2 b Squash fruit a 40.0 a 17.5 b Squash fruit a 35.8 a 16.7 b Squash fruit a 37.5 a 13.3 b a Means in the same row followed by the same number are not significantly different according to Fisher s least significant difference (LSD) at the 5% probability level (SAS 1999). b ns 5 nonsignificant F value for treatment means in ANOVA. hours (Table 1). However, there was no significant difference in the number of larvae choosing the Invite-treated over the untreated pieces of artificial diet. Within 15 min, second instar pickleworms preferentially chose the Invitetreated cucumber fruits over untreated ones (Table 1). However, unlike the treated filter papers, no larvae left the Invite-treated pieces of cucumber. After two hours, most larvae that had not been on either cucumber treatment moved to the Invite-treated cucumber pieces. For squash leaves and squash fruits, there were no significant differences in the percentage of larvae choosing Invite-treated over untreated pieces. However, there was a trend for more larvae that were not on either treatment to move to the Invite-treated pieces by the end of the experiments (2 h) (Table 1). For the observational experiment, larvae showed typical orientation behavior for lepidopterous larvae (Jones 1977, Jackson 1982, Becher & Guerin 2009). They normally moved along the surface of the Petri dish for a few seconds before raising the anterior portion of their bodies and waving it back and forth. For this experiment, eight of the twelve larvae were on the Invite-treated disks, three were wandering on the dish, and one was on the water control after 15 min. It took an average of 127 s for the eight larvae to first find the Invite-treated disks.

7 JACKSON et al.: Pickleworm Baculoviruses and Cucurbitacin 101 Table 2. Effect of five rates of Invite in combination with AgNPV (10 7 OB per plate) on mortality of second instar pickleworms in laboratory bioassays. Treatment AgMNPV plus Invite (0.0013) AgMNPV plus Invite (0.13) AgMNPV plus Invite (0.013) AgMNPV plus Invite ( ) AgMNPV plus Invite ( ) AgMNPV Invite (0.13) Invite (0.013) Invite (0.0013) Invite ( ) Invite ( ) Control Percent Mortality 95.0 a a 94.0 a 94.0 a 93.0 a 91.0 a 23.0 b 0.0 c 0.0 c 0.5 c 0.5 c 0.0 c 0.0 c a Means followed by the same number are not significantly different according to Fisher s least significant difference (LSD) at the 5% probability level (SAS 1999). Effects of Invite on efficacy of AgMNPV on pickleworm larvae. In the first NPV-efficacy experiment, ANOVA indicated that treatment effects were highly significant (F , df 5 11, 1, P, ). Invite alone had no effect on pickleworm mortality at any of the rates applied to the artificial diet (Table 2). However, all rates of Invite caused a significant increase in mortality in the AgMNPV treatments compared with AgMNPV alone. Larval mortality for the AgMNPV treatment without Invite was 20.0%, but mortality was increased to over 90% for each of the AgMNPV plus Invite treatments (Table 2). Susceptibility of pickleworm larvae to viruses with and without Invite. In the second NPV-efficacy experiment, ANOVA indicated that treatment effects were highly significant (F , df 5 13, 3, P, ). For the NPV treatments without Invite, only AgMNPV and AnfaMNPV produced mortalities that were significantly higher than the untreated control or Invite control (Table 3). However the addition of 1% Invite increased the efficacy of AcMNPV to a level that was significantly higher than the controls. The addition of Invite also significantly increased the efficacy of AgMNPV and AnfaMNPV in this experiment (Table 3). This increase was especially dramatic for AgMNPV, which had a 3.5-fold increase in efficacy with the addition of 1% Invite. Discussion Pickleworm larvae travelled quickly to Invite-treated filter papers and many remained there for the duration of the bioassays (120 min) (Table 1). However, some larvae continuously left the treated as well as the untreated filter papers and began searching for a food source. Therefore, there were several larvae that were not on either the treated or the untreated filter papers throughout this bioassay. However, when a food source was added to the bioassay, most larvae did

8 102 J. Agric. Urban Entomol. Vol. 26, No. 2 (2009) Table 3. Effects of Invite on six baculoviruses tested on pickleworm, Diaphania nitidalis, larvae. Treatment AgMNPV plus Invite (0.013) AnfaMNPV plus Invite (0.013) AcMNPV plus Invite (0.013) AgMNPV AnfaMNPV HearSNPV plus Invite (0.013) RoMNPV plus Invite (0.013) PlxyMNPV plus Invite (0.013) AcMNPV RoMNPV PlxyMNPV HearSNPV Invite (0.013) Control Percent Mortality 86.5 a a 57.5 b 28.5 c 24.0 c 21.5 c 19.0 cd 16.0 cd 16.0 cd 15.5 cd 15.0 cd 13.0 cd 13.0 cd 0.0 d 0.0 d a Means followed by the same number are not significantly different according to Fisher s least significant difference (LSD) at the 5% probability level (SAS 1999). not leave either the treated or untreated artificial diet, cucumber fruits, squash fruits, or squash leaves. It is interesting to note the different response between cucumber fruit and squash fruit (Table 1). Invite stimulated the aggregation of larvae on cucumber fruit, but this was not the case for squash fruit or leaves. Perhaps squash fruit and leaves have compounds (including cucurbitacins) that masked the effects of Invite. Peterson & Elsey (1995) reported that gravid female pickleworm moths are stimulated to oviposit by non-volatile and highly polar amphoteric compounds found on the leaves of yellow squash. However, this study is the first report of pickleworm larvae being affected behaviorally by a naturally occurring cucurbitacin. Even the lowest concentration of Invite ( ) increased efficacy of AgMNPV for pickleworm larvae (Table 2), indicating that cucurbitacin E-glycoside is active even at low levels. This is similar to the response for Diabrotica undecimpunctata, where it has been reported that these insects can detect cucurbitacin E at very low levels ( mg) (Metcalf & Lampman 1989). We (Jackson et al. 2008) previously reported that AgMNPV, AcMNPV, and AnfaMNPV were the most efficacious NPVs against pickleworm larvae in laboratory bioassays. We also demonstrated that the addition of stilbene fluorescent brighteners (virus enhancers and UV protectants) (Shapiro et al. 1992, Dougherty et al. 1996) can significantly increase efficacy of AgMNPV against this pest (Jackson et al. 2008). The present study also demonstrated that AgMNPV was the most efficacious nucleopolyhedrovirus against pickleworm larvae, and that the addition of Invite could further increase its efficacy. Through DNA amplification, sequencing procedures, and a BLAST (Basic Local Alignment Search Tool) search (Altschul et al. 1990), we concluded that the pathogen from the pickleworm colony was not a novel NPV for D. nitidalis, but

9 JACKSON et al.: Pickleworm Baculoviruses and Cucurbitacin 103 was AnfaMNPV. Despite standard hygiene practices, AnfaMNPV had become established in our pickleworm colony with devastating results, providing additional evidence that this nucleopolyhedrovirus could be a promising candidate for biological control of this pest. Therefore, these promising NPVs should be tested in combination with Invite and UV-protectants in replicated field experiments on cucurbit crops (small and large plot studies) (Shapiro & Farrar 2003, Shapiro et al. 2002). These promising viruses also should be passed through pickleworm larvae to determine if their potencies can be increased further, as has been shown with other baculoviruses (Pavan et al. 1981). Acknowledgments We thank Jeremy Greene for serving as both subject editor and editor of this paper to avoid a conflict of interest by the senior author who serves as editor of this journal. We thank Louise Cauthen, Jennifer Cook, and Nan Lu Ling for technical assistance, and A. M. Simmons and K. S. Ling for critical reviews of this paper. We thank Regina Kleespies (Julius Kuhn-Institute, Federal Research Centre for Cultivated Plants, Institute for Biological Control, Darmstadt, Germany), and Youngjie Wang and Johannes Jehle (Laboratory for Biotechnological Crop Protection, Department of Phytopathology, Agricultural Service Center Palatinate [DLR Rheinpfalz], Neustadt, Germany) for identification of AnfaMNPV that contaminated the pickleworm colony. This research was funded in part by the Pickle Packers International, Inc. Technical contribution No of the Clemson University Experiment Station. References Cited Andeweg, J. M. & J. W. De Bruyn Breeding of non-bitter cucumbers. Euphytica 8: Anonymous The pickleworm. Plate 51. In Insects. The Yearbook of Agriculture. USDA, Washington, D.C. Anonymous Rule- Cucurbitacins; Exemption from the requirement of a tolerance. 65 FR 15248, Federal Register, 22 March 2000, pages 15,248 15,252., federalregister.gov/articles/2000/03/22/ /cucurbitacins-exemption-from-therequirement-of-a-tolerance#p-3.. Anonymous Invite EC, Specimen Label. Page 949, CPR Crop Protection Reference, C&P Press, New York, NY. Altschul, S. F., W. Gish, W. Miller, E.W. Myers & D. J. Lipman Basic local alignment search tool. J. Mol. Biol. 215: Becher, P. G. & P. M. Guerin Oriented responses of grapevine moth larvae Lobesia botrana to volatiles from host plants and an artificial diet on a locomotion compensator. J. Insect Physiol. 55: Bisognin, D. A Origin and evolution of cultivated cucurbits. Ciência Rural, Santa Maria 32: Burges, D. H. & K. Jones Formulations of bacteria, viruses, and protozoa to control insects, pp In H. D. Burges [Ed.], Formulation of microbial pesticides. Kluwer Academic Publishers, Dordrecht, The Netherlands. Canerday, T. D. & J. D. Dilbeck The pickleworm: its control on cucurbits in Alabama. Auburn Univ., Agric. Exp. Stn. Bull. 381: Capinera, J. L Handbook of vegetable pests. Academic Press, New York. Capinera, J. L Pickleworm, Diaphania nitidalis (Stoll) (Insecta: Lepidoptera: Pyralidae). Univ. Florida, Fla. Coop. Exten. Serv., Institute Food Agric. Sci. (IFAS) EENY 164. Carner, G. R. & S. G. Turnipseed Potential of a nuclear polyhedrosis virus for control of the velvetbean caterpillar in soybean. J. Econ. Entomol. 70:

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