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Survivorship NOTE of Field-Collected European Corn Borer (Lepidoptera: Crambidae) Larvae and Its Impact on Estimates of Resistance to Bacillus thuringiensis ~erliner' R. C. Venette, J. C. Luhman2 and W. D. Hutchison3 USDA-APHIS, Department of Entomology, and Midwest Ecological Risk Assessment Center, 1980 Folwell Ave., University of Minnesota, St. Paul, MN 55108 USA J. Entomol. Sci. 35(2): 20E212 (April 2000) Key Words European corn borer, Bacillus thuringiensis, Bt corn, resistance management, resistance monitoring, parasitism, pathogens Widespread production of transgenic corn, Zea mays (L.), engineered to express insecticidal crystal proteins (e.g., crylAb) from the bacterium Bacillus thuringiensis subsp, kurstaki Berliner (Bt), has generated concerns over targeted insects developing resistance (Gould, 1998, Annu. Rev. Entomol. 43: 701-726). The primary target for Bt-corn is Ostrinia nubilalis (Hiibner), the European corn borer. Resistance management plans for 0. nubilalis (Ostlie et al., 1997, Bt corn and European corn borer: long term success through resistance management, University of Minnesota, St. Paul; Vlachos et al., 1999, http://www.ncga.com/02profits/insectMgmtPlan/main.htm) assume that resistant individuals are rare (e.g., <1 in 1000 insects or To evaluate this assumption, sampling strategies must provide a high probability of detection and correct classification of larvae with resistance to Bt. In a previous study, we used an in-field screen with Bt-sweet corn to detect resistant larvae; live 0 . nubilalis larvae (==thirdinstar) that had been feeding on Bt expressing tissue (independently confirmed with immunoassay) were operationally considered resistant (Venette et al., 2000, J. Econ. Entomol., in press). However, the National Corn Growers Association recently suggested more restrictive criteria to characterize resistance (Vlachos et al. 1999). Specifically, a larva collected from Bt expressing tissue is "resistant" if it also survives a discriminating concentration of an appropriate crylA protein in a laboratory assay. In the event that a discriminating concentration is not available, a larva shall be considered resistant if its offspring satisfy the following criteria: (a) the concentration of crylA protein required to kill 50% L, is statistically greater than the historical L , for the popuC of the population ( C) 'Received 06 July 1999; accepted for publication 04 September 1999. 'Minnesota Department of Agriculture, 90 West Plato Boulevard, St. Paul, MN 55107. 'Department of Entomology & Midwest Ecological Risk Assessment Center, 1980 Folwell Ave., University of Minnesota, St. Paul, MN 55108. VENETTE et al.: Su~ivorship European Corn Borer of 209 lation; and (b) 230% of progeny survive and >25% of the leaf area is damaged in a laboratory bioassay using leaf tissue expressing the appropriate crylA protein. Presumably field-collected larvae that do not survive the bioassay or produce offspring will not be considered resistant. During a discriminating dose bioassay with larvae collected from Bt fields, mortality may be due to susceptibility to Bt toxins, mechanical damage incurred during larval collections, inappropriate environmental conditions, pathogens, parasitoids, or poor genetic fitness. Because Bt-resistant larvae are rare, controls to account for non-Bt related mortality are not practical. If we assume that larvae in transgenic and isogenic corn are equally likely to be infected, parasitized, or damaged during collection, the degree of non-Bt related mortality could be measured from larvae sampled from non-Bt corn. However, the incorporation of such information into an estimate of the frequency of resistance remains problematic. In this study, we report annual larval mortality for fifth-instar 0. nubilalis collected from non-Bt field corn throughout Minnesota. Given estimates of larval mortality, we then provide probabilities for the number of larvae that are likely to survive to adulthood and explore how non-Bt related mortality may affect estimates of the frequency of resistance. Between 1984 and 1997, annual surveys for 0. nubilalis larvae were conducted in corn-producing counties throughout Minnesota. From each country, -30 apparently healthy fifth-instar larvae were collected from 5 to 6 arbitrarily selected fields (-6 larvaelfield). Stalks were selected randomly within 45 m from the edge of a field. Larvae from the 5 fields were pooled to form a single sample for the county. Larvae were placed in doubled paper bags inside a plastic bag to prevent escape and given pieces of corn stalk to allow boring. The sample was kept cool and returned to the lab for processing. Larvae were removed from corn stalks using a knife and forceps. Any larva with emergent hyphae of Beauveria bassiana (Balsamo) Vuillemin or damaged during the extraction process was discarded. Larvae obviously infected with B. bassiana were eliminated to prevent contamination of other larvae. Discarded larvae were not included in statistical analyses. Remaining larvae were dipped in a 5% bleach solution (volume bleach: volume water) and placed individually in cups containing an agar-based medium (1.88% agar, 0.16% methyl paraben, 0.06% sorbic acid, and 98% water wlw). Larvae were stored for -8 wks at 4"C, 80% relative humidity, and complete darkness to allow larvae to complete diapause. After completing diapause, larvae were inspected for disease, parasitism, or inviability. Status of larvae was recorded, and dead larvae were removed. Larvae were placed at 2g C, 80% relative humidity, and full light to break diaspause. Mortality due to diseases, parasites, or unknown causes was noted weekly. Infection by B. bassiana was confirmed by light microscopy. Emerging parasitoids were identified as Macrocentrus grandii Goidanich, Eriborus terebrans Gravenhorst, Eumeae caesar (Aldrich), Sympiesis viridula (Thornson), or other. The number of surviving adult 0. nubilalis was recorded. Data were transformed using ARSIN(SQRT(x)) and analyzed by one-way analysis of variance using PROC GLM with mean separation by Ryan-Einot-Gabriel-Welsch multiple range test (SAS Institute, 1995, SASISTAT user's guide, Cary, NC). For probability analyses, we assume each larva represents an independent Bernoulli trial and allow the average probability of success (i.e., surviving to adulthood) to be rep- 210 J. Entornol. Sci. Vol. 35, No. 2 (2000) resented by p 1-p is average mortality. If N larvae are collected from the field, the ; probability that x larvae will survive to adulthood is given by: (Equation For 1). these analyses, we assume a maximum N of 4, which represents the maximum number of 0. nubilalis larvae collected to date from large plots of Bt sweet corn (Andow and Hutchison, 1998, pp. 19-66, In M. Mellon and J. Rissler [eds.], Now or never: serious new plans to save a natural pest control, Union of Concerned Scientists, Cambridge, MA). Mortality of field-collected, fifth-instar 0. nubilalis was greatest in 1984 and 1991 when -75% of all larvae died (Table 1). In these years, -50% of larvae died due to unknown causes. Low rates of mortality were observed between 1987-89 and 199293 (Table 1). Across these periods, mortality averaged 27% which was still predominantly due to the effects of unknown causes. Infection by Nosema pyrausta (Paillot) may have caused some of the unknown deaths. Assays in 1994, 1995, and 1997, indicated that 27%, 39%, and 63%, respectively, of all 0. nubilalis collected in this survey, including individuals that pupated or emerged as adults, were infected with N. pyrausta (D. Ragsdale, Univ. Minnesota, unpubl. data). Over the 11 years of this study, ~ 1 0 % all larvae collected died due to parasitoids. On average, M. grand;; of accounted for 51.2 + 7.6% (X + SEM) of the deaths due to parasitism; Eriborus terebrans, 37.6 & 7.4%; Eumeae caesar, 2.9 + 1.2%; and S. viridula, 1.2 ~t 0.5%. As mortality due to parasitoids, pathogens, or other non-Bt related factors increases, the probability that all larvae from a small subsample (i.e., $4 individuals) will survive is reduced (Table 2). If mortality is -30% (i.e., 70% survivorship) and 2 2 larvae are collected, we expect 2 1 deaths. If mortality increases to -70% we expect 2 2 deaths. As more larvae are collected, the probability that at least one larva will survive to an adult becomes greater (Table 2). These calculations rely on the assumption that larvae from Btand non-Btcorn experience the same likelihood of dying due to infection, parasitism, mechanical damage, or other non-Bt related factors. If a field-collected larva must survive to undergo a laboratory bioassay before it can be considered resistant, non-Bt related mortality will lower the estimated frequency of resistant individuals. Venette et al. (2000) estimated the frequency of resistance, E[f], using the equation E[f] = (S + l)/(ML), where S is the number of resistant larvae found, M is the number of Bt plants examined, and L is the average density of resistant and susceptible larvae per non-Bt plant. If 4 resistant larvae (S) were observed in a sample of 1200 Bt plants (M) and an average of 3 larvae were observed per non-Bt plant (L), we expect the frequency of resistance to be 1.4 If larval survivorship from non-Bt corn averages 60% (Table I ) , we expect only 2 to 3 of the resistant larvae will survive to adults for further laboratory bioassays (Table 2). Therefore, S is now 2, and if all other variables are the same, the frequency of resistance becomes 8.4 x Appropriate doses of Bt to distinguish resistant from susceptible individuals have been established for neonates of certain lepidopteran pests (e.g., Sims et al., 1996, p. 229-242, In T. M. Brown [ed], Molecular genetics and evolution of pesticide resistance, American Chemical Society, Washington, DC; Marcon et al., 1999, J. Econ. Entomol. 92: 279-285). These doses do not apply to later-instars because larger susceptible insects exhibit greater tolerance to Bt (Davis and Coleman, 1997, J. Table 1. Percent mortality (&EM) of Minnesota field-collected, iifth-instar Ostrinia nubilalis caused b y parasitoids or pathogens under standardized laboratory conditions % Mortality due to: c rn 6 2 Year 1984 M. grandii 1.6 + 0.4ab E. terebrans 0.6 E. caesar 0.0 + O.Oc S. viridula 0.0 + O.Ob B. bassiana 6.0 + 1.9bc Unknown* 64.1 + 3.8a Total 72.3 r 3.4a Counties (n) 34 Total Larvae 1424 3 rn El .- 0.3de 15, Mean 4.0 + 0.8 2.3 r 0.5 0.2 + 0.1 0.1 + " 6.4 + 3.1 27.4 + 4.6 40.5 + 5.9 683 20520 Percentages wlthin a column followed by the same letter are not stat~sticallydifferent (60.05) as determined by Ryan-Einot-Gabriel-Welsch multiple range test. ' Includes larvae that apparently dled due to unidentified bacterial or fungal pathogens or dead larvae with normal shape and coloration. " SEM<O.l . L? - 212 J. Entomol. Sci. Vol. 35, No. 2 (2000) Table 2. Effect of average survivorship of fifth-instar Ostrinia nubilalis larvae on the probability that field-collected individuals will survive t o adults Average survivorship Collected larvae ( W ) Probability of having 3 0 surviving larvae - IVumber of survivors ( x ) 0.70 1 0.30 0 1 0.70 2 - 3 - 4 - 3 (All years) 2 0.16 0.48 0.36 ' Variables N and x relate to Equation 1 , described in the text. " Example: When average survivorship is 70% and 2 larvae are collected from a f~eld, there is a 9% probability that no fifth instar larvae will survive to adulthood. Kansas Entornol. Soc. 70: 31-38; VanFrankenhuyzen et al., 1997, J. Econ. Entornol. 90: 560-565). In-field identification currently requires that a larva be 2 third instar and actively feeding on Bt-expressing tissue (confirmed by immunoassay) to be considered resistant (Venette et al. 2000). It is improbable that all larvae identified as resistant in the field will survive to adulthood for additional laboratory analysis (Table 2). Furthermore, not all survivors will successfully mate and produce viable offspring (R. C. Venette, unpubl. data). A larva is definitively resistant if it is collected from Bttissue and it, or its offspring, can survive an appropriate bioassay. However, a larva collected from Bt tissue that does not survive to undergo bioassay is not necessarily susceptible. Accounting for the effects of non-Bt related mortality on resistance frequency estimates is difficult because resistant larvae are likely to be scarce. If additional laboratory confirmation is required before a larva can be considered resistant, the levels and risks of Bt resistance in targeted pest populations will be underestimated. The opinions expressed by individuals in this report do not necessarily represent the policies of the U.S. Department of Agriculture (USDA). Mention of companies or commercial products does not imply recommendation or endorsement by the USDA over others not mentioned. USDA neither guarantees nor warrants the standard of any product mentioned. Product names are mentioned solely to report factually on available data and to provide specific information. This is manuscript number 991 170026 of the Minnesota Agricultural Experiment Station. 1 I

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