Western corn rootworm (Diabrotica virgifera virgifera LeConte) is a major pest of corn in both North America and Europe and as such presents significant challenges for farmers. IPD079Ea protein is encoded by the ipd079Ea gene from Ophioglossum pendulum (a species of fern) and was found to have activity against western corn rootworm in multiple corn events transformed to express the IPD079Ea protein. In chronic laboratory hazard studies, IPD079Ea protein was fed to eleven species in the order Coleoptera and four species in the order Lepidoptera to assess the spectrum of activity. Activity was observed on certain species of the Chrysomelidae and Coccinellidae families, with western corn rootworm as the most sensitive insect tested. No adverse effects on mortality or other sublethal endpoints were observed on any species within Lepidoptera. Overall, IPD079Ea protein appears not to have broad insecticidal properties and has potential value as an effective trait to control western corn rootworm in agricultural systems.
The western corn rootworm, (WCR) (Diabrotica virgifera virgifera LeConte), is one of the most significant and influential pests of corn in both North America and Europe. Economic losses associated with WCR in North America alone have been estimated between $1 and $2 billion annually (Gray et al. 2009, Wechsler and Smith 2018). As such, management of WCR can significantly influence decision making by farmers such as choice of which crop to plant, when to plant it, and what pest control technologies to use to maintain yield and return on investment. Historically, crop rotations along with deploying soil, seed, and/or foliar applied chemicals, along with the use of genetically modified (GM) corn hybrids, have all been utilized to manage WCR pressure (Prasifka et al. 2013, Johnson et al. 2017, Pereira et al. 2019). Despite the variety of management options, WCR have evolved resistance to crop rotations (Levine et al. 2002, Crowder et al. 2005) and numerous chemistries and traits (Gassmann et al. 2014, 2019; Pereira et al. 2015) particularly in areas where a diversified management strategy was not employed (Fishilevich et al. 2016).
More comprehensive and integrated management strategies have been promoted to delay the evolution of resistance in WCR and extend the durability of individual management options when they are deployed collectively (Onstad et al. 2003, Gassmann et al. 2019). One component of more fully integrated management plans is new modes of action to diversify the toolbox available to growers facing high WCR pressure. Historically, GM corn events have utilized several classes of proteins from Bacillus thuringiensis (Bt), to protect plants from WCR damage (Moellenbeck et al. 2001, Carriere et al. 2020). More recently, other actives targeting WCR have been developed including several dsRNA events (Baum et al. 2007, Bachman et al. 2013, Hu et al. 2016, 2019) and one protein from Pseudomonas chlororaphis, IPD072Aa (Schellenberger et al. 2016, Boeckman et al. 2019), and these traits are currently working through global regulatory approval before they can be cultivated commercially. Further complementing these new actives, IPD079Ea protein, encoded by the ipd079Ea gene from Ophioglossum pendulum (a species of fern), was found to have activity against WCR in multiple corn events transformed to express the IPD079Ea protein (Allen et al. 2018).
IPD079Ea protein will diversify the toolbox available to growers in regions impacted by WCR; however, prior to commercial release, an extensive characterization of the potential environmental and safety risks will need to be conducted. The process used to evaluate the potential environmental risks is well established with numerous classes of chemistries and traits having been evaluated under this framework (USEPA 1998, Romeis et al. 2013, Layton et al. 2015). Further, given the relatively recent advancement of different modes of action noted above, this process has again been evaluated for its robustness to evaluate traits from non-Bt sources and found to be sufficiently flexible and informative for the risk assessment (USEPA 2016). As such, one of the steps in the risk assessment process is to understand the spectrum of activity (Tabashnik 2016, Boeckman and Layton 2017). This information is helpful to better inform the necessity and direction of safety testing with nontarget organisms that provide a beneficial ecosystem service in agroecosystems. Traits with narrow activity profiles practically require fewer hazard studies, whereas other traits with either uncertain modes of action or broader activity profiles may require more hazard studies to reduce uncertainty and better inform the risk assessment (Romeis et al. 2013).
Following this framework, the purpose of the current study was to characterize the spectrum of activity of IPD079Ea protein. Given the observed activity against WCR, the target pest of IPD079Ea protein, significant focus was applied to other organisms in the order Coleoptera, as these species are more closely taxonomically related to WCR. Additional testing with surrogate species from four families in the order Lepidoptera were also included as an outgroup and to understand if cross-order activity within Lepidoptera was observed. These data will further inform additional hazard studies with organisms that provide a beneficial ecosystem service in agroecosystems and have a rational exposure pathway identified.
Methods
Test Substance
In order to generate IPD079Ea protein for the studies, the protein was expressed in an Escherichia coli protein expression system as a fusion protein with an N-terminal His tag. The tagged protein was purified using Ni-NTA affinity chromatography. The fusion tag was cleaved with thrombin and the thrombin was removed using heparin Sepharose column chromatography. Prior to lyophilization, tangential flow filtration was used to exchange the buffer to 50 mM ammonium bicarbonate. The protein was then lyophilized and stored in a –80°C freezer until use.
The concentration of IPD079Ea protein in the lyophilized powder was determined by amino acid composition analysis. Briefly, three lyophilized protein samples were solubilized in 70% formic acid. Sub-samples were hydrolyzed in 6N hydrochloric acid for 24 h under argon in a 110°C oven. Hydrolyzed samples were diluted and then isotopically labeled amino acid internal standards were added to a final concentration of 0.5 µg/ml. Isotopically labeled amino acid internal standards were added to amino acid calibration solutions to a final concentration of 0.5 µg/ml. Amino acids in diluted hydrolysate samples and calibration solutions were separated with a Waters Acuity UPLC column (2.1 × 150 mm). The eluent from the column was directed into an electrospray source and analyzed using a Waters Xevo TQ mass spectrometer through multiple reaction monitoring (MRM) for both the non-labeled amino acids and isotopically labeled internal standards. The quantification of the individual amino acids was performed using Waters QuanLynx software. The individual amino acid concentration was used to determine the protein concentration based on the frequency of the specific amino acid in the protein sequence. The concentration of the protein was determined to be 0.63 milligrams of protein per mg of lyophilized powder. The purity of the protein was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which showed > 95% purity on a total protein basis ( Supp Fig. 1 [online only] (toac079_suppl_supplementary_material.docx)). Additional characterization of IPD079Ea was conducted using western blotting, N-terminal sequencing, intact mass determination and peptide mapping by mass spectrometry, glycosylation staining, and insecticidal activity using WCR. Prior to use in insect bioassays, the lyophilized protein was solubilized in ultrapure water. The protein concentration of the resulting stock solution was determined by amino acid composition analysis.
Bioassay Design and Species Selection
Species were selected for testing based on phylogenetic relatedness to WCR, amenability to laboratory bioassays, availability of specific life stages from commercial vendors, and reproducibility and robustness of each observation endpoint (Table 1). In some cases, established standardized bioassay methods were not available; thus, bioassays were developed in-house and validated prior to study. Due to the known activity of IPD079Ea protein against WCR within the order Coleoptera, primary focus was applied to this order to further characterize the spectrum of activity across different families within Coleoptera. Additionally, four families from Lepidoptera were also tested as an outgroup and to assess for cross order activity.
As the IPD079Ea protein is only active via oral ingestion, the protein was incorporated into artificial diets specific for each insect. IPD079Ea protein stock solution was diluted in ultrapure water and mixed with dry diet ingredients, as described below, to support the growth and development of organisms for the duration of the bioassays. Each insect was provided diet incorporated with multiple concentrations of IPD079Ea as shown in Table 2. All studies were conducted under good laboratory practices 40 CFR part 160 with concentration, stability, and homogeneity verification of the test substance characterized in each bioassay using ELISA. Some diets were prepared in bulk and stored frozen for up to 30 d. If applicable, protein stability in the stored diets was also assessed under frozen storage using both a specific antibody-based detection method such as Western blot or enzyme-linked immunoassay (ELISA), and a bioactivity study with a known sensitive insect to demonstrate that the IPD079Ea protein was present and active at the expected potency after the full duration of storage.
Acceptability criteria for all bioassays were determined prior to initiating each study and required less than or equal to 20% mortality in the bioassay control with the exception of WCR and southern corn rootworm (Diabrotica undecimpunctata howardi Barber, Coleoptera: chrysomelidae, SCR), which required less than or equal to 30% mortality due to the greater variation in laboratory studies with these organisms (Ludwick et al. 2018). To demonstrate exposure to the test substance through the diet and the sensitivity of each organism tested, a positive control was added to each bioassay. Either boric acid or cryolite served as a positive control as described in Supp Table 1 (online only) (toac079_suppl_supplementary_material.docx). Generally, at least 80% mortality associated with the positive control was considered acceptable. Further, to demonstrate the biological activity of the IPD079Ea protein after incorporation into artificial diets, a sensitive insect bioassay was conducted alongside each bioassay. A portion of each diet fed to the test organisms either concurrently with or at the conclusion of each bioassay was combined with WCR diet and fed to WCR. Bioactivity of the IPD079Ea protein in each of the insect diets was confirmed by greater than 80% mortality of WCR in these sensitive insect bioassays.
Table 1.
Species tested with vendor, bioassay duration, and endpoints observed
All bioassays were conducted in small environmental chambers (Percival Scientific, Perry, IA). The specific environmental conditions varied for each organism as noted below. Studies with yellow meal-worm (Tenebrio molitor Linnaeus, Coleoptera: Tenebrionidae, MWM), Zophobas morio Fabricius, Coleoptera: Tenebrionidae (SWM), Mexican bean beetle (Epilachna varivestis Mulsant, Coleoptera: Coccinellidae, MBB), and each of the lepidopterans were conducted at 25°C, 65% relative humidity, and no light for 7 d except MWM and SWM which used a 14-d duration to allow time for the positive control to show effectiveness. Coleomegilla maculata De Geer, Coleoptera: Coccinellidae (CMAC), and convergent lady beetle (Hippodamia convergens Guerin-Meneville, Coleoptera: Coccinellidae, CNV) bioassays were both conducted at 27°C, 65% relative humidity, and 16:8 light:dark h photoperiod. The Dalotia coriaria Kraatz, Coleoptera: Staphylinidae (RVB) bioassay was conducted at 25°C, 65% relative humidity, and 16:8 light:dark h photoperiod. WCR and SCR were conditions were 21°C, 65% relative humidity, and no light and the red flour beetle (Tribolium castaneum Herbst, Coleoptera: Tenebrionidae, RFB) bioassay was conducted at 30°C, 70% relative humidity, and no light.
Organisms were provided with fresh diets on a frequency based on the stability of the IPD079Ea protein under bioassay conditions and the needs of the insect. Generally, fresh diet was provided every 3 to 4 d with the exception of Colorado potato beetle (Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae, CPB) and MBB which were provided fresh diet every other day. For WCR, SCR, MWM, SWM, MBB, and each of the lepidopterans, diets were prepared fresh using freshly prepared dosing solutions. Diet for CPB was prepared using a dosing solution that was stored for the duration of the bioassay at 4°C with appropriate IPD079Ea protein stability characterization.
Stonefly heliothis diet (SHD, Ward's Science, Rochester, NY) served as a base diet for all insects with the exception of CMAC, CNV, RVB, and RFB. A proprietary mixture of additional ingredients was added to SHD for some insects to promote growth and development of each species. The SHD based diets were mixed with water or a dosing solution containing the IPD079Ea protein prior to feeding. CMAC were fed lyophilized brine shrimp eggs following Li et al. (2011), and both RVB and CNV were fed lyophilized Esphestia kuehniella Zeller, Lepidoptera: Pyralidae eggs (Beneficial Insectary, Redding, CA). RFB were fed a lyophilized flour-based diet. For those diets that were lyophilized, the IPD079Ea protein was applied to the diet and mixed by hand to a uniform consistency. Then the diet was aliquoted and lyophilized, and after lyophilization, the aliquots were pooled and again mixed by hand and subsequently placed in a –80°C freezer until used in the bioassay.
MWM, SWM, CPB, MBB, and all lepidopteran bioassays were conducted in 12-well Falcon tissue culture plates (Fisher Scientific, Hampton, NH) arranged in a generalized randomized design. A single neonate was placed in each well and the plates were sealed with a heat sealing film with a small hole over each well providing ventilation. The WCR and SCR bioassays were conducted as above except in 24-well tissue culture plates. The CMAC and CNV bioassays were conducted in small Petri dishes (60 × 15 mm, Fisher Scientific) containing a single neonate on day 0 and water source. The dishes were stacked in a randomized complete block design. The RVB and RFB bioassays were conducted in 30-ml plastic cups with lids (Cater Supply Direct, Denton, MD) and provided with an agar moisture source in a randomized complete block design.
Four separate concentration-dependent bioassays were conducted with WCR to characterize the median lethal concentration (LC50) and associated 95% confidence intervals for the target insect. Each bioassay was conducted on different days with separate WCR neonates to better characterize the variability in response of the target insect to IPD079Ea protein. In each bioassay, a target of 30 individual WCR were used per concentration with five IPD079Ea protein concentrations and a negative control (six total treatments). The LC50 also provided context for concentrations to use in subsequent testing with the other organisms noted in this study and provided context for concentrations necessary to achieve in the sensitive insect bioassays to confirm activity of IPD079Ea protein.
Statistical Analysis
Statistical analyses were conducted using SAS software, Version 9.4 (SAS Institute Inc., Cary, NC) separately for each bioassay for the response variables shown in Table 1. For the WCR median lethal concentration, a log-logistic regression model was utilized to analyze the dose-response curve data. SAS PROC NLMIXED was used to fit the log-logistic regression model for each independent bioassay and the model parameter estimates and associated standard errors were obtained using maximum likelihood methods. A random effects meta-analysis approach (Normand 1999) was used in SAS PROC MIXED to derive the estimate of the overall mean LC50 and associated 95% confidence interval across the independent bioassays. For all other studies, statistical comparisons were made between test diets and the bioassay control diet for each response variable with significance declared if the P-value was < 0.05.
For all insects other than WCR, statistical analysis of survival data was conducted using Fisher's exact test (SAS PROC MULTTEST) to determine if the survival observed for each test diet was less than the survival observed with the bioassay control diet.
The statistical analysis methods used for weights of surviving insects were dependent upon validity of statistical assumptions for each data set. For most experiments, a linear model analysis was conducted to test if exposure to the test diet caused growth inhibition as compared to the control diet. Assumptions of independent errors that were normally and identically distributed were confirmed by inspection of residuals from the fitted model. SAS PROC GLIMMIX was used for linear mixed model analysis and to generate estimated treatment means, 95% confidence intervals, and the statistical comparisons between means.
Assessment of the residual plots indicated that the assumptions of the linear model were not satisfied for MBB and SCR weight data; therefore, non-parametric Wilcoxon two-sample tests were conducted to test if exposure to each test diet caused growth inhibition as compared to the control diet. The Wilcoxon two-sample tests were conducted with SAS PROC NPAR1WAY.
Data for days to adult emergence were not normally distributed; therefore, non-parametric Wilcoxon two-sample tests were conducted to examine if exposure to the test diet caused developmental delay.
Results
IPD079Ea protein concentrations, homogeneity in diet, and stability under bioassay conditions were all confirmed for each diet and bioassay. Confirmation of the bioactivity of IPD079Ea protein in diet was verified by greater than 80% mortality of WCR observed with each study ( Supp Table 2 [online only] (toac079_suppl_supplementary_material.docx)). Mortality associated with the negative control met the acceptability criterion of ≤ 20% (≤ 30% for WCR and SCR) in each study, (Table 2), and mortality associated with the positive controls also exceeded the 80% acceptability criterion, ( Supp Table 1 [online only] (toac079_suppl_supplementary_material.docx)), confirming exposure to the test substance via the diet.
Table 2 provides detailed observations on each endpoint collected for each organism; however, a high-level summary is provided below. WCR, the target pest, showed median lethal concentrations ranging from 3.4 ng IPD079Ea/mg diet to 7.9 ng IPD079Ea/mg diet (Table 3) over the course of four separate and independent bioassays, culminating in a summary LC50 of 5.6 ng IPD079Ea/mg diet (95% CIs of 2.9–8.3 ng IPD079Ea/mg diet). WCR was the most sensitive insect tested followed by MBB with a survival No Observed Effect Concentration (NOEC) of 10 ng IPD079Ea/mg diet, though weight was significantly lower in the 10 ng IPD079Ea/mg diet treatment compared with the control (P < 0.001, Table 2). SCR also showed sensitivity to IPD079Ea protein with greater than 50% mortality observed at a concentration of 100 ng/mg (Table 2). These three species, showing sensitivity to IPD079Ea protein, are pests of agricultural crops, primarily corn for WCR and SCR, and MBB is a pest of various legumes (Dobrin and Hammond 1983).
For all other species tested, there were no effects on mortality or weight associated with IPD079Ea protein at concentrations up to 800 ng/mg except for MWM (Table 2). Observed mortality for MWM exposed to 400 ng IPD079Ea/mg diet reached 16.7% compared with 0% for the negative control which was statistically significantly different (P = 0.026). Given there was no dose-dependent response for mortality of MWM exposed to the other IPD079Ea protein concentrations, the observed mortality was below the 20% acceptability criteria for the negative control, and there was no apparent effect on weight of MWM, the result was deemed non-biologically relevant, and no further studies were performed.
Days to adult emergence were observed for CNV and CMAC, both of which are potential predators in agricultural fields. There was no effect of IPD079Ea protein on CNV days to adult emergence; however, CMAC exposed to both 200 and 800 ng IPD079Ea/ mg diet were statistically significantly different from the control (P = 0.031 and P = 0.011, respectively). Given mean adult emergence in the control was 13.6 d compared with 14 and 14.2 d in the 200 and 800 ng IPD079Ea/mg diet treatments, respectively, (Table 2), this represents roughly a 14-h difference between the treatments and control. As there was no dose-dependent response, no concomitant increase in mortality or decrease in weight, and given the relatively small 14-h difference in adult emergence between the negative control and these treatments, this result was considered non-biologically relevant, and no further studies were performed.
Table 3.
Western corn rootworm median lethal concentration and slope with associated standard error and 95% confidence intervals
Overall, of the coleopteran families tested, only certain organisms in the family Chrysomelidae and Coccinellidae showed sensitivity to IPD079Ea protein at concentrations up to 800 ng/mg, but not every species tested in those families were affected. Further, there was no indication that IPD079Ea protein had any effect on mortality or weight of the four lepidopteran families tested.
Discussion
The gene encoding for the IPD079Ea protein originated from the fern Ophioglossum pendulum. The protein was found to have activity against WCR in multiple corn events transformed to express the IPD079Ea protein (Allen et al. 2018). As current commercial GM corn events that provide protection from WCR damage exclusively rely on Cry proteins from the Bacillus thuringiensis genome, and as WCR continue to evolve resistance to these proteins, IPD079Ea protein represents an important new tool to maintain corn yields for global grain trade and extend the durability of traits currently on the market. Prior to commercial release; however, an extensive characterization is required of the IPD079Ea protein focused in part on the environmental safety.
Part of that environmental safety characterization includes understanding the activity spectrum of the trait of interest. Over time and with broader use, traits develop a particular familiarity and empirical studies add little new information to the risk assessment for these traits. Such is the case today with traditional Cry proteins mentioned above. However, as technology developers delve into other sources for new actives, laboratory studies focused on characterizing activity spectra are an important part of the risk assessment process (Boeckman and Layton 2017, Roberts et al. 2020).
Given that IPD079Ea protein was shown to be active against WCR, it was logical to focus a large part of the spectrum of activity effort on other species in the order Coleoptera, under the hypothesis that related species are more likely to share sensitivity to the IPD079Ea protein than more distantly related species (Romeis et al. 2011, Carstens et al. 2014). Additional considerations for selection of species used in laboratory studies included: organism (and specific life stage) availability from commercial vendors, established bioassay protocols and methods, an artificial diet into which IPD079Ea protein could be incorporated for oral exposure, and reproducibility and robustness of the endpoints observed with each species meeting generally standard performance criteria for survival over the duration of the study. At this stage in the risk assessment process, protection of valued ecosystem services is not necessarily considered. As such, many of the species used to understand spectrum of activity are typically considered pest species. For instance, of the species reported here, only CMAC, CNV, and RVB would be considered beneficial species in agroecosystems, with each providing a predatory ecosystem service that would be important to evaluate and preserve in later stages of the full environmental risk assessment. All other species used to evaluate IPD079Ea protein and reported here are either stored grain or agricultural pests and thus, not necessarily a desired species to conserve from an environmental risk assessment context.
Overall, results of the studies detailed here demonstrate that the IPD079Ea protein does not appear to have broad activity against surrogate species from four families within the orders Coleoptera and Lepidoptera. Activity was observed within certain species of the Chrysomelidae family, with WCR showing the greatest sensitivity to the IPD079Ea protein, though not every Chrysomelid was sensitive. CPB showed no adverse effects when exposed to IPD079Ea protein concentrations up to 800 ng/mg. IPD079Ea protein was also active within certain species from the Coccinellidae family. MBB was the second most sensitive species tested with a survival NOEC of 10 ng/ mg, yet again not every Coccinellid was sensitive. CNV showed no effect on survival, time to adult emergence, or adult weight at concentration up to 800 ng/mg. CMAC did show modest increases in time to adult emergence at greater concentrations of IPD079Ea protein; however, as the delay was marginal at 14-h difference between the control and IPD079Ea treatment, this observation is not likely to be biologically meaningful particularly under more realistic exposure scenarios resulting from the potential cultivation of corn events expressing IPD079Ea to control WCR. Further information characterizing the concentration of IPD079Ea protein in various tissues from corn events would be helpful to better characterize the potential risk to predatory Coccinellids.
There were no adverse effects observed for any of the surrogate species from four families of Lepidoptera. These lepidopteran surrogates were specifically selected to diversify at the family level the spectra of species tested. Additional testing from a broader array of insect orders will be conducted as part of the full environmental risk assessment; however, from the studies described herein, IPD079Ea protein appears not to have broad spectrum activity across the orders Coleoptera and Lepidoptera. Based on the testing conducted to date, IPD079Ea protein appears to be most potent against its intended target pest, WCR and future characterization with a greater diversity of valued non-target organisms will complement these data to fully evaluate any potential risk associated with cultivation of corn events expressing IPD079Ea protein. As the evolution of resistance to current technologies and practices used to control WCR continues, additional traits such as IPD079Ea protein become ever more important, not only as a new mode of action but also to help extend the durability of those currently commercial technologies.
Acknowledgments
We wish to acknowledge Ian Lamb for helpful review and comments on early drafts of this manuscript. All authors are currently employed by Corteva Agriscience. C.J.B., C.W., and J.Z. authored this manuscript. C.L., T.O., K.L., and R.W. conducted each bioassay. S.B., T.G., and B.S. provided analytical support for each bioassay. Y.W., E.H., and C.W. provided statistical analysis.
