Host Recognition Responses of Western (Family: Chrysomelidae) Corn Rootworm Larvae to RNA Interference and Bt Corn

Host Recognition Responses of Western (Family: Chrysomelidae) Corn Rootworm Larvae to RNA... Western corn rootworm Diabrotica virgifera virgifera LeConte is an important pest of corn whose larvae exhibit particular quantifiable patterns of locomotion after exposure to, and removal from, host roots and nonhost roots. Using EthoVision software, the behavior and locomotion of the western corn rootworm larvae was ana- lyzed to determine the level of host recognition to germinated roots of differing corn hybrids containing either rootworm targeted Bt genes, RNA interference (RNAi) technology, the stack of both Bt and RNAi, or the isoline of these. The behavior of the rootworm larvae indicated a significant host preference response to all corn hy- brids (with or without insecticidal traits) compared to the filter paper and oat roots. A weaker host response to the RNAi corn roots was observed in the susceptible larvae when compared to the resistant larvae, but not for the Btþ RNAi vector stack. Additionally, the resistant larvae demonstrated a weaker host response to the isoline corn roots when compared to the susceptible larvae. Although weaker, these host responses were significantly different from those observed in the negative controls, indicating that all hybrids tested do contain the contact cues necessary to elicit a host preference response by both Cry3Bb1-resistant and Cry3Bb1-susceptible larvae that would work to hinder resistance development in refuge in a bag fields. Key words: Bacillus thuringiensis, Bt, corn, Diabrotica virgifera virgifera, EthoVision The western corn rootworm (WCR), Diabrotica virgifera virgifera locate potential host plants (Robert et al. 2012). Interestingly, WCR LeConte, is considered to be the most important insect pest of corn larvae are also attracted to (E)-b-caryophyllene, which is an induced (Zea mays L.) in major corn-producing regions (Stamm et al. 1985, plant volatile given off when WCR larvae feed on the roots of certain Krysan et al. 1986) with crop losses and control costs estimated to corn varieties. Robert et al. (2012) discovered that both of these vola- be over $2 billion annually in the United States alone (Mitchell tiles are used by the larvae to evaluate the health of the plant from a 2011). WCR larvae are subterranean and specialize on corn roots. distance. Although older larvae can survive starvation for up to 96 h, Although these larvae will feed on most grass roots (Family neonate larvae need to locate host roots within 12–36 h or risk being Poaceae), they can only complete their development on a select few too weaktoburrow into the root (Strnad and Bergman 1987b). species other than corn (Branson and Ortman 1970, Clark and Once the WCR larvae find potential host roots, contact cues are Hibbard 2004). Root damage caused by intense larval feeding can picked up by the maxillary palps that aid the larvae in making feeding severely limit the ability of the plant to absorb nutrients and water decisions (Branson and Ortman 1969). Feeding stimulants used by and can weaken the plant base, making the plant vulnerable to lodg- the WCR larvae to identify a host have been identified as a combin- ing (Kahler et al. 1985, Sutter et al. 1990). ation of simple sugars, 30:4:4 mg/ml glucose:fructose:sucrose, and WCR eggs are laid in soil near the base of the plant, and after one of the free fatty acids in germinating corn roots, oleic acid, or hatching larvae use CO (a volatile given off by all plants) as a long linoleic acid (Bernklau and Bjostad 2008). Interestingly, individual range attractant as they move through the soil in search of host roots components by themselves did not elicit a major feeding response by (Branson 1982, Strnad et al. 1986, Hibbard and Bjostad 1988, the WCR larvae, but together, they did (Bernklau and Bjostad 2008). Bernklau and Bjostad 1998, Miller et al. 2006). In addition to CO , Larvae of the WCR have a set of behaviors that help the larvae ethylene, a volatile phytohormone in corn, is used by WCR larvae to locate food patches and stay within food patches. When WCR V C The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/59/3739026 journals.permissions@oup.com by Ed 'DeepDyve' Gillespie user on 17 July 2018 2 Journal of Insect Science, 2017, Vol. 17, No. 2 larvae are exposed to a substrate that is not recognized as a host and approved, this product will be the first of its kind for rootworm then are removed, they exhibit a “ranging” behavior, where they control. crawl in a relatively straight direction and move quickly (Strnad and WCR host recognition behavior is unknown for these transgenic Dunn 1990). Until the larvae encounter host volatiles, they will con- genes and the recent discovery of populations of WCR resistant to tinue searching in this manner. In contrast, when WCR larvae are Cry3Bb1 Bt corn in the field (Gassmann et al. 2011) raises concerns exposed to a host root and then are removed, they exhibit a about the rootworm–transgenic corn interactions. If WCR larvae “localized searching” behavior. This behavior involves a restricted were to demonstrate nonhost, ranging behavior to a corn hybrids’ area of search with greater number of turns, path crossings and a de- roots, this could be beneficial for WCR management as the corn crease in speed (Strnad and Dunn 1990, Bell 1991). Throughout plant would not be recognized as a host and thus not be damaged. their development, WCR larvae move to higher quality, younger The larvae would presumably wander away from the plant and root whorls (Apple and Patel 1963, Strnad and Bergman 1987a), starve to death. However, if resistant larvae perceived the isoline as and this localized searching behavior likely helps the larvae stay in nonhosts over Bt hybrids, then in refuge in a bag (RIB) hybrid the root zone while moving around. These behaviors are important planted fields, this could add to the resistant population. Therefore, for larval survival and contribute to the highly successful nature of the objective of this study was to investigate how mCry3A, this pest (Strnad and Dunn 1990). Cry3Bb1, Cry34/35Ab1, and RNAi corn influence the host recogni- In behavioral bioassays, Strnad and Dunn (1990) analyzed the tion behavior of neonate WCR larvae. paths that WCR larvae took after exposure to germinated roots of corn and other grasses. They found that after being exposed to corn Materials and Methods and wheat roots, the rootworms initiated localized search. The WCR larvae exposed to giant foxtail (Setaria faberi Herrm) and oat The study was conducted at the USDA-ARS Plant Genetics Research (Avena sativa L.) seedling roots showed in part localized search by Unit on the University of Missouri-Columbia campus in 2010 and having a somewhat reduced area of search and reduced velocity. 2011. To assess the host recognition behavior of WCR neonates on However, these larvae did not show any differences in the number different varieties of corn roots, we conducted two sets of bioassays. of turns and path crossing as the moist filter paper control. The first set of bioassays consisted of a randomized complete block of Although the rootworm larvae will feed briefly on the oats, they will nine treatments replicated 20 times. In this set, susceptible WCR were abandon them due to a feeding deterrent (Branson and Ortman exposedtoseven typesof germinatedcornroots,MIR604 (mCry3A), 1969). WCR larvae have been shown to survive on giant foxtail DAS59122-7 (Cry34/35Ab1), MON88017 (Cry3Bb1), SmartStax roots (Clark and Hibbard 2004), but Chege et al. (2005) discovered (Cry3Bb1þ Cry34/35Ab1), and their corresponding isolines, as well weed host phenology may affect larval survival. Bernklau et al. as germinated oat roots (nonhost, living plant control) and filter paper (2009) found that WCR larvae will initiate localized search when (control). The second set of bioassays included a full factorial random- exposed to root extracts, corn root pieces, and corn root juice. ized complete block design of 14 treatments with 20 replicates per Transgenic corn lines with genes from Bacillus thuringiensis treatment. These treatments consisted of two sources of eggs (resistant Berliner (Bt) with resistance to WCR feeding are commonly used for and susceptible colonies) on one of five types of corn, MON88017 rootworm management in the United States. These products range (Cry3Bb1), RNAi, the vector stack (RNAiþCry3Bb1), their corres- from single event hybrids to pyramided hybrids that have two or ponding isolines, filter paper (control), or oats (nonhost control). more Bt genes targeting rootworms. Current commercially available Bt hybrids targeting the WCR produce one or more of the following Insects proteins mCry3A, Cry3Bb1, eCry3.1Ab, or Cry34/35Ab1. SmartStax WCR eggs were obtained from nondiapausing (Branson 1976)colo- is a stacked corn hybrid from Monsanto/Dow Agrosciences (St. nies maintained in our laboratory. In the first set of bioassays, the egg Louis, Missouri, USA/Midland, Michigan, USA), which includes two type used was from a susceptible WCR line (Janesville control, see pyramided rootworm genes, Cry3Bb1 and Cry34/35Ab1 as well as Meihls 2010). In the second set of bioassays, eggs from the same sus- three Bt toxins targeted towards above ground pests. Syngenta’s ceptible colony were used and eggs from a line selected for resistance (Basel, Switzerland) next generation product, Agrisure Duracade, to MON88017 (Janesville selected) (Meihls 2010). WCR eggs were which includes mCry3Aþ eCry3.1Ab, is now commercially, but was placed in 15 cm 10 cm oval containers (708 ml, The Glad Products not included in this study. Company, Oakland, CA) and filled approximately 4-cm deep with a RNA interference (RNAi) is a novel way of controlling target moistened growth medium of 2:1 autoclaved soil and ProMix pests by using double-stranded RNA (dsRNA) to silence-specific (Premier Horticulture Inc. Quakertown, Pennsylvania, USA). The mRNA genes by way of interference with the target pests own RNAi eggs were incubated in the soil at 25 C for approx. 2 wk before pathway (Fire et al. 1998, Bolognesi et al. 2012). This pathway hatching. Unfed neonate larvae <24 h old were used in the bioassays. regulates gene expression and is also used for protection against viral infections (Waterhouse et al. 2001, Wang et al. 2006, Plant Material Bolognesi et al. 2012). Artificial diet incorporating dsRNA and gen- All of the corn used was soaked in a 10% bleach solution for etically engineered plants that express dsRNA can be fed to WCR 10 min, rinsed well and allowed to dry completely prior to germina- larvae that cause mortality after entering the midgut cells (Baum tion. The corn was then soaked in water at room temperature for et al. 2007, Whyard et al. 2009, Bolognesi et al. 2012). After inges- tion, these dsRNA travel to other tissues and then are diced into 8 h. After soaking, corn kernels were placed onto a saturated paper short interfering RNA and interfere with gene expression causing towel in closed oval containers and placed in a growth chamber at larval mortality and stunting (Baum et al. 2007, Whyard et al. 2009, 25 C to germinate. Oats were treated with a soapy water solution, Bolognesi et al. 2012). Monsanto’s next-generation rootworm prod- rinsed well, and placed on a saturated paper towel in oval containers uct contains Corn Rootworm III (more information can be found for germination in the growth chamber. Upon germination, all here http://www.monsanto.com/SiteCollectionDocuments/whistle plants were kept moist on clean, saturated filter paper in closed oval stop-corn-pipeline.pdf) which includes RNAiþ Cry3Bb1 genes. If containers. Corn seedlings were used in bioassays when they reached Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/59/3739026 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 3 3–4 d old; oats were used at 4–5 d old. All roots used in the assays larvae were placed in the arena and the door to the arena closed. were approximately 1.5–2 inches in length. The recording continued for 5 min unless the larva left the filter Gene checks were performed on MON88017 and SmartStax roots paper before time expired. To account for any changes in the set- at the end of the study using QuickStix test strips (EnviroLogix, tings due to replacing the filter paper between bioassays, the detec- Portland, ME), and tissue samples were also taken from RNAi and the tion variables were updated before the start of each trial. Vector Stack seedlings and sent to Monsanto for expression studies. Parameters measured by the EthoVision system during bioassays All plants used in the assays tested positive for the appropriate genes. included total distance moved (the distance traveled by the center of gravity of the larva), maximum distance from the origin (the farthest Bioassays distance traveled by the center of gravity of the larva from the point of Assays used in this study were modified from those developed by origin), mean velocity (mm/s), mean turn angle (the change in direction Strnad and Dunn (1990). During the bioassays, a single, clean seed- of movement between two samples), and mean meander (the change in ling was placed on moistened filter paper in a petri dish and one neo- direction of movement of an object relative to the distance it moves). nate larvae was placed on the root (or on the filter paper for the To mitigate image noise and larval body wobbles being recorded as control) using a moistened camel’s-hair paintbrush. After exposure to true movement, the following filters and settings were used when calcu- the root for 5 min, the larvae were immediately transferred to the cen- lating the above parameters: total distance moved, downsize filter ter of a specially designed 12.5 cm arena on lightly moistened filter (1/25) and minimum distance moved (0.2 cm); maximum distance paper where the larvae’s host-searching behaviors were recorded for from origin, downsize filter (1/25); mean velocity, downsize filter (1/ 5 min using the EthoVision system (version 3.1; Noldus Information 25); mean turn angle, absolute setting, and downsize filter (1/25); mean Technology, The Netherlands). The bioassay was terminated early if meander, absolute setting, and downsize filter (1/25). Limited larval any larvae exited the arena during the 5-min trial period. Each bioas- movement coupled with the above filters sometimes resulted in no say resulted in one track file in the EthoVision program. A total of value being calculated for a specific parameter. For trials that did not 460 unique track files were recorded for this study. last the full 5 min as a result of larvae leaving the arena during their search, total distance traveled was adjusted to reflect the distance the EthoVision Protocol larvae would have traveled during the five minute period using their The EthoVision arena comprised a moist 125-mm filter paper circle average velocity as calculated by the EthoVision software. mounted on a clean glass plate and replaced between each larval exposure. The arena was enclosed in a clear acrylic box Statistical Analysis (20 20 18 cm) mounted under the EthoVision system video cam- An analysis of variance was used for these data analyses and was era (Panasonic wv BP334) positioned 0.64 cm above the box with a calculated by using the PROC MIXED of the SAS statistical package 15-W fluorescent light located on top for even lighting. For opti- (SAS Institute 2008; Cary, North Carolina, USA). For the mean mum viewing of larvae with the EthoVision system, the tracking set- meander, total distance moved, mean turn angle, maximum distance tings were set to the following specifications: detection method, subtraction; processing settings, only detect objects that are darker from origin and the velocity, the linear statistical model contained the main plot effect of treatment. Data were transformed by square than background; scan window of 50 pixels set to search the com- plete arena; minimum object size, one pixel; maximum object size, root (xþ 0.5) to meet the assumptions of the analysis. Both of the 20 pixels; sample rate, 5.994 samples/s. Recording began after the experiments were run as a randomized complete block. Table 1. Effect of treatment on each parameter measured of the movement of the western corn rootworm during bioassays from experi- ment one (using susceptible insects) and experiment two (using both susceptible and resistant insects) Assay set Analysis df fP One Distance moved Medium 4, 149 9.80 <0.0001 Mean velocity Medium 8, 149 19.16 <0.0001 Mean turn angle Medium 8, 147 41.56 <0.0001 Mean meander Medium 8, 147 36.91 <0.0001 Maximum distance from origin Medium 8, 147 22.29 <0.0001 Two Distance moved Medium 6,226 56.29 <0.0001 Colony 1,226 0.37 0.5411 Medium  colony 6,226 1.71 0.1195 Mean velocity Medium 6,233 61.71 <0.0001 Colony 1,233 0.07 0.7961 Medium  colony 6,233 1.99 0.0683 Mean turn angle Medium 6,231 277.34 <0.0001 Colony 1,231 1.02 0.3134 Medium  colony 6,231 4.2 0.0005 Mean meander Medium 6,235 100.85 <0.0001 Colony 1,235 0.03 0.8732 Medium  colony 6,235 2.02 0.0643 Maximum distance from origin Medium 6,227 104.4 <0.0001 Colony 1,227 3.84 0.0513 Medium  colony 6,227 3.47 0.0027 Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/59/3739026 by Ed 'DeepDyve' Gillespie user on 17 July 2018 4 Journal of Insect Science, 2017, Vol. 17, No. 2 A B Total Distance Moved Maximum Distance from Origin 180 60 a 120 40 b 30 b b 80 b b b b b b b b 20 b 0 0 C D Velocity Turn Angle a a 0.8 a 140 a a a 0.7 0.6 0.5 b b b 80 0.4 b 0.3 0.2 b b 0.1 0 0 E Meander a a 50 a Fig. 1. The total distance moved (A), maximum distance from origin (B), velocity (C), turn angle (D), and meander (E) of the western corn rootworm larvae in 5 min after exposure to a different plant seedlings or filter paper for experiment one. Letters indicate significant differences between corn types (P 0.05). Analysis was done with square root transformed data; figures represent untransformed data. Results origin, moved faster, turned less often and crossed their own path less than the resistant larvae after RNAi corn exposure (Fig. 2b–e). For all parameters that were measured in the first set of bioassays The resistant larvae moved farther from the origin after exposure to using only susceptible colonies, the two negative controls (moist filter the isoline than the susceptible colonies (Fig. 2b). Despite differences paper and germinated oat seedlings) were significantly different than in every parameter measured between the two colonies on the all corn treatments (Table 1; Fig. 1). The larvae that were exposed to RNAi, there were no differences between the susceptible and the the controls had significantly longer paths and traveled farther from resistant colonies for the vector stack exposure treatment (Fig. 2). the distance from the origin than the larvae exposed to corn plants The magnitude of the difference between the resistant and the sus- including the Bt plants (Fig. 1a and b). The larvae exposed to the neg- ceptible colony on RNAi was small compared to the susceptible col- ative controls traveled significantly faster, turned less, and crossed ony on the controls (Fig. 2). their paths less than the larvae exposed to the corn plants (Fig. 1c–e). In the second set of assays using both susceptible and resistant colonies, the two negative controls were also significantly different Discussion from all of the corn treatments for all parameters measured There were no dramatic differences between the localized search (Table 1; Fig. 2). After exposure to the RNAi roots, susceptible responses of WCR larvae exposed to any of the corn lines tested; larval behavior was significantly different than behavior for resistant larvae for all parameters measured (Fig. 2). The susceptible larvae however, the rootworm larvae consistently demonstrated a ranging had a significantly longer path on the RNAi corn than the resistant behavior after contact with the filter paper and oats, indicating that larvae (Fig. 2a). Also, the susceptible larvae moved farther from the they did not recognize the controls as hosts. This was expected since Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/59/3739026 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Velocity (mm/s) Distance traveled (mm) Meander (degrees/mm) Mean turn angle (degrees) Distance traveled (mm) Journal of Insect Science, 2017, Vol. 17, No. 2 5 AB Total Distance Moved Maximum Distance from Origin Susceptible Susceptible Resistant Resistant a a * * bc bc b 10 b bc bc c bc 2 c c Filter Oat Iso 88017 Smart RNAi Vector Filter Oat Iso 88017 Smart RNAi Vector Stax Stack Stax Stack CD Velocity Turn Angle Susceptible Susceptible 0.09 Resistant Resistant 0.08 120 0.07 a a a a 0.06 b 80 0.05 c c 0.04 0.03 0.02 c c 0.01 Filter Oat Iso 88017 Smart RNAi Vector Filter Oat Iso 88017 Smart RNAi Vector Stax Stack Stax Stack Meander Susceptible Resistant ab ab ab ab ab 1000 b 200 c Filter Oat Iso 88017 Smart RNAi Vector Stax Stack Fig. 2. The total distance moved (A), maximum distance from origin (B), velocity (C), turn angle (D), and meander (E) of the western corn rootworm larvae in 5 min after exposure to different plant seedlings or filter paper for experiment B. Letters indicate significant differences between corn types within colony (P 0.05). *Significant differences between resistant and susceptible colonies with seed type (P 0.05). Analysis was done with square root transformed data; figures represent untransformed data. larvae exposed to germinated oat roots had showed a ranging that Cry34/35Ab1 was perceived as a poor host for WCR larvae. behavior in previous studies (Strnad and Dunn 1990) and oats may However, the factors responsible for host recognition require spe- contain a larval feeding deterrent (Branson and Ortman 1969). cific extraction techniques if they are to be separated from corn Binning et al. (2005) conducted assays that were somewhat simi- (Bernklau et al. 2009), and these factors are likely not present in lar to the current experiment, except that in their experiment they artificial diet. In addition, Cry proteins are tied up in plant cells exposed the insects to artificial diet (modified after Pleau et al. under normal circumstances and not directly available to searching 2002) with and without Cry34/35Ab1 proteins. They concluded larvae as was done by Binning et al.(2005). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/59/3739026 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Mean velocity (cm/s) Distance traveled (cm) Mean meander (degrees/cm) Mean turn angle (degrees) Distance traveled (cm) 6 Journal of Insect Science, 2017, Vol. 17, No. 2 rootworm (Coleoptera: Chrysomelidae) larvae. J. Econ. Entomol. 102: Contact cues associated with the roots are the driving factor of 558–562. host recognition (Branson and Ortman 1969, Strnad and Dunn Binning, R., S. Lefko, F. C. Cheng, and K. Liao. 2005. Searching behavior of 1990), and this study demonstrates that each corn type, Bt, RNAi, western corn rootworm (Diabrotica virgifera virgifera) after exposure to the vector stack, and isoline, contains sufficient contact cues to elicit Cry34Ab1/Cry35Ab1 insecticidal proteins. Annual Meeting of the North a localized search response by both Cry3Bb1 resistant and suscepti- Central Branch, Entomological Society of America, March 20-23, West ble larvae when the larvae are removed from the germinated corn Lafayette, IN. roots. The toxins present in the Bt/RNAi roots did not significantly Bolognesi, R., P. Ramaseshadri, J. Anderson, P. Bachman, W. Clinton, R. affect the host response despite what may have happened in other Bt Flannagan, O. Ilagan, C. Lawrence, S. Levine, and W. Moar. 2012. assays such as in the study by Clark et al. (2006). Phenological dif- Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte). ferences in the corn may play a role in this discrepancy since corn PloS One. 7: e47534. seedlings of a slightly younger age were used in this study. Branson, T. F. 1976. The selection of a non-diapause strain of Diabrotica vir- Overall the data between the two sets of assays were similar with gifera (Coleoptera: Chrysomelidae). Entomol. Exp. Appl. 19: 148–154. the primary difference being the addition of a paired resistant col- Branson, T. F. 1982. Olfactory response of larvae of Diabrotica virgifera virgi- ony. Although the susceptible colony had significantly less host rec- fera to plant roots. Entomol. Exp. Appl. 31: 303–307. ognition patterns than the resistant colony on RNAi, the susceptible Branson, T. F., and E. E. Ortman. 1969. 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Nature. 411: 834–842. corn rootworm (Diabrotica virgifera virgifera). J. Insect Physiol. 36: Whyard, S., A. D. Singh, and S. Wong. 2009. Ingested double-stranded RNAs 201–205. can act as species-specific insecticides. Insect Biochem. Mol. Biol. 39: Sutter, G., J. Fisher, N. Elliott, and T. Branson. 1990. Effect of insecticide 824–832. treatments on root lodging and yields of maize in controlled infestations of Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/59/3739026 by Ed 'DeepDyve' Gillespie user on 17 July 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Insect Science Oxford University Press

Host Recognition Responses of Western (Family: Chrysomelidae) Corn Rootworm Larvae to RNA Interference and Bt Corn

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Abstract

Western corn rootworm Diabrotica virgifera virgifera LeConte is an important pest of corn whose larvae exhibit particular quantifiable patterns of locomotion after exposure to, and removal from, host roots and nonhost roots. Using EthoVision software, the behavior and locomotion of the western corn rootworm larvae was ana- lyzed to determine the level of host recognition to germinated roots of differing corn hybrids containing either rootworm targeted Bt genes, RNA interference (RNAi) technology, the stack of both Bt and RNAi, or the isoline of these. The behavior of the rootworm larvae indicated a significant host preference response to all corn hy- brids (with or without insecticidal traits) compared to the filter paper and oat roots. A weaker host response to the RNAi corn roots was observed in the susceptible larvae when compared to the resistant larvae, but not for the Btþ RNAi vector stack. Additionally, the resistant larvae demonstrated a weaker host response to the isoline corn roots when compared to the susceptible larvae. Although weaker, these host responses were significantly different from those observed in the negative controls, indicating that all hybrids tested do contain the contact cues necessary to elicit a host preference response by both Cry3Bb1-resistant and Cry3Bb1-susceptible larvae that would work to hinder resistance development in refuge in a bag fields. Key words: Bacillus thuringiensis, Bt, corn, Diabrotica virgifera virgifera, EthoVision The western corn rootworm (WCR), Diabrotica virgifera virgifera locate potential host plants (Robert et al. 2012). Interestingly, WCR LeConte, is considered to be the most important insect pest of corn larvae are also attracted to (E)-b-caryophyllene, which is an induced (Zea mays L.) in major corn-producing regions (Stamm et al. 1985, plant volatile given off when WCR larvae feed on the roots of certain Krysan et al. 1986) with crop losses and control costs estimated to corn varieties. Robert et al. (2012) discovered that both of these vola- be over $2 billion annually in the United States alone (Mitchell tiles are used by the larvae to evaluate the health of the plant from a 2011). WCR larvae are subterranean and specialize on corn roots. distance. Although older larvae can survive starvation for up to 96 h, Although these larvae will feed on most grass roots (Family neonate larvae need to locate host roots within 12–36 h or risk being Poaceae), they can only complete their development on a select few too weaktoburrow into the root (Strnad and Bergman 1987b). species other than corn (Branson and Ortman 1970, Clark and Once the WCR larvae find potential host roots, contact cues are Hibbard 2004). Root damage caused by intense larval feeding can picked up by the maxillary palps that aid the larvae in making feeding severely limit the ability of the plant to absorb nutrients and water decisions (Branson and Ortman 1969). Feeding stimulants used by and can weaken the plant base, making the plant vulnerable to lodg- the WCR larvae to identify a host have been identified as a combin- ing (Kahler et al. 1985, Sutter et al. 1990). ation of simple sugars, 30:4:4 mg/ml glucose:fructose:sucrose, and WCR eggs are laid in soil near the base of the plant, and after one of the free fatty acids in germinating corn roots, oleic acid, or hatching larvae use CO (a volatile given off by all plants) as a long linoleic acid (Bernklau and Bjostad 2008). Interestingly, individual range attractant as they move through the soil in search of host roots components by themselves did not elicit a major feeding response by (Branson 1982, Strnad et al. 1986, Hibbard and Bjostad 1988, the WCR larvae, but together, they did (Bernklau and Bjostad 2008). Bernklau and Bjostad 1998, Miller et al. 2006). In addition to CO , Larvae of the WCR have a set of behaviors that help the larvae ethylene, a volatile phytohormone in corn, is used by WCR larvae to locate food patches and stay within food patches. When WCR V C The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/59/3739026 journals.permissions@oup.com by Ed 'DeepDyve' Gillespie user on 17 July 2018 2 Journal of Insect Science, 2017, Vol. 17, No. 2 larvae are exposed to a substrate that is not recognized as a host and approved, this product will be the first of its kind for rootworm then are removed, they exhibit a “ranging” behavior, where they control. crawl in a relatively straight direction and move quickly (Strnad and WCR host recognition behavior is unknown for these transgenic Dunn 1990). Until the larvae encounter host volatiles, they will con- genes and the recent discovery of populations of WCR resistant to tinue searching in this manner. In contrast, when WCR larvae are Cry3Bb1 Bt corn in the field (Gassmann et al. 2011) raises concerns exposed to a host root and then are removed, they exhibit a about the rootworm–transgenic corn interactions. If WCR larvae “localized searching” behavior. This behavior involves a restricted were to demonstrate nonhost, ranging behavior to a corn hybrids’ area of search with greater number of turns, path crossings and a de- roots, this could be beneficial for WCR management as the corn crease in speed (Strnad and Dunn 1990, Bell 1991). Throughout plant would not be recognized as a host and thus not be damaged. their development, WCR larvae move to higher quality, younger The larvae would presumably wander away from the plant and root whorls (Apple and Patel 1963, Strnad and Bergman 1987a), starve to death. However, if resistant larvae perceived the isoline as and this localized searching behavior likely helps the larvae stay in nonhosts over Bt hybrids, then in refuge in a bag (RIB) hybrid the root zone while moving around. These behaviors are important planted fields, this could add to the resistant population. Therefore, for larval survival and contribute to the highly successful nature of the objective of this study was to investigate how mCry3A, this pest (Strnad and Dunn 1990). Cry3Bb1, Cry34/35Ab1, and RNAi corn influence the host recogni- In behavioral bioassays, Strnad and Dunn (1990) analyzed the tion behavior of neonate WCR larvae. paths that WCR larvae took after exposure to germinated roots of corn and other grasses. They found that after being exposed to corn Materials and Methods and wheat roots, the rootworms initiated localized search. The WCR larvae exposed to giant foxtail (Setaria faberi Herrm) and oat The study was conducted at the USDA-ARS Plant Genetics Research (Avena sativa L.) seedling roots showed in part localized search by Unit on the University of Missouri-Columbia campus in 2010 and having a somewhat reduced area of search and reduced velocity. 2011. To assess the host recognition behavior of WCR neonates on However, these larvae did not show any differences in the number different varieties of corn roots, we conducted two sets of bioassays. of turns and path crossing as the moist filter paper control. The first set of bioassays consisted of a randomized complete block of Although the rootworm larvae will feed briefly on the oats, they will nine treatments replicated 20 times. In this set, susceptible WCR were abandon them due to a feeding deterrent (Branson and Ortman exposedtoseven typesof germinatedcornroots,MIR604 (mCry3A), 1969). WCR larvae have been shown to survive on giant foxtail DAS59122-7 (Cry34/35Ab1), MON88017 (Cry3Bb1), SmartStax roots (Clark and Hibbard 2004), but Chege et al. (2005) discovered (Cry3Bb1þ Cry34/35Ab1), and their corresponding isolines, as well weed host phenology may affect larval survival. Bernklau et al. as germinated oat roots (nonhost, living plant control) and filter paper (2009) found that WCR larvae will initiate localized search when (control). The second set of bioassays included a full factorial random- exposed to root extracts, corn root pieces, and corn root juice. ized complete block design of 14 treatments with 20 replicates per Transgenic corn lines with genes from Bacillus thuringiensis treatment. These treatments consisted of two sources of eggs (resistant Berliner (Bt) with resistance to WCR feeding are commonly used for and susceptible colonies) on one of five types of corn, MON88017 rootworm management in the United States. These products range (Cry3Bb1), RNAi, the vector stack (RNAiþCry3Bb1), their corres- from single event hybrids to pyramided hybrids that have two or ponding isolines, filter paper (control), or oats (nonhost control). more Bt genes targeting rootworms. Current commercially available Bt hybrids targeting the WCR produce one or more of the following Insects proteins mCry3A, Cry3Bb1, eCry3.1Ab, or Cry34/35Ab1. SmartStax WCR eggs were obtained from nondiapausing (Branson 1976)colo- is a stacked corn hybrid from Monsanto/Dow Agrosciences (St. nies maintained in our laboratory. In the first set of bioassays, the egg Louis, Missouri, USA/Midland, Michigan, USA), which includes two type used was from a susceptible WCR line (Janesville control, see pyramided rootworm genes, Cry3Bb1 and Cry34/35Ab1 as well as Meihls 2010). In the second set of bioassays, eggs from the same sus- three Bt toxins targeted towards above ground pests. Syngenta’s ceptible colony were used and eggs from a line selected for resistance (Basel, Switzerland) next generation product, Agrisure Duracade, to MON88017 (Janesville selected) (Meihls 2010). WCR eggs were which includes mCry3Aþ eCry3.1Ab, is now commercially, but was placed in 15 cm 10 cm oval containers (708 ml, The Glad Products not included in this study. Company, Oakland, CA) and filled approximately 4-cm deep with a RNA interference (RNAi) is a novel way of controlling target moistened growth medium of 2:1 autoclaved soil and ProMix pests by using double-stranded RNA (dsRNA) to silence-specific (Premier Horticulture Inc. Quakertown, Pennsylvania, USA). The mRNA genes by way of interference with the target pests own RNAi eggs were incubated in the soil at 25 C for approx. 2 wk before pathway (Fire et al. 1998, Bolognesi et al. 2012). This pathway hatching. Unfed neonate larvae <24 h old were used in the bioassays. regulates gene expression and is also used for protection against viral infections (Waterhouse et al. 2001, Wang et al. 2006, Plant Material Bolognesi et al. 2012). Artificial diet incorporating dsRNA and gen- All of the corn used was soaked in a 10% bleach solution for etically engineered plants that express dsRNA can be fed to WCR 10 min, rinsed well and allowed to dry completely prior to germina- larvae that cause mortality after entering the midgut cells (Baum tion. The corn was then soaked in water at room temperature for et al. 2007, Whyard et al. 2009, Bolognesi et al. 2012). After inges- tion, these dsRNA travel to other tissues and then are diced into 8 h. After soaking, corn kernels were placed onto a saturated paper short interfering RNA and interfere with gene expression causing towel in closed oval containers and placed in a growth chamber at larval mortality and stunting (Baum et al. 2007, Whyard et al. 2009, 25 C to germinate. Oats were treated with a soapy water solution, Bolognesi et al. 2012). Monsanto’s next-generation rootworm prod- rinsed well, and placed on a saturated paper towel in oval containers uct contains Corn Rootworm III (more information can be found for germination in the growth chamber. Upon germination, all here http://www.monsanto.com/SiteCollectionDocuments/whistle plants were kept moist on clean, saturated filter paper in closed oval stop-corn-pipeline.pdf) which includes RNAiþ Cry3Bb1 genes. If containers. Corn seedlings were used in bioassays when they reached Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/59/3739026 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 3 3–4 d old; oats were used at 4–5 d old. All roots used in the assays larvae were placed in the arena and the door to the arena closed. were approximately 1.5–2 inches in length. The recording continued for 5 min unless the larva left the filter Gene checks were performed on MON88017 and SmartStax roots paper before time expired. To account for any changes in the set- at the end of the study using QuickStix test strips (EnviroLogix, tings due to replacing the filter paper between bioassays, the detec- Portland, ME), and tissue samples were also taken from RNAi and the tion variables were updated before the start of each trial. Vector Stack seedlings and sent to Monsanto for expression studies. Parameters measured by the EthoVision system during bioassays All plants used in the assays tested positive for the appropriate genes. included total distance moved (the distance traveled by the center of gravity of the larva), maximum distance from the origin (the farthest Bioassays distance traveled by the center of gravity of the larva from the point of Assays used in this study were modified from those developed by origin), mean velocity (mm/s), mean turn angle (the change in direction Strnad and Dunn (1990). During the bioassays, a single, clean seed- of movement between two samples), and mean meander (the change in ling was placed on moistened filter paper in a petri dish and one neo- direction of movement of an object relative to the distance it moves). nate larvae was placed on the root (or on the filter paper for the To mitigate image noise and larval body wobbles being recorded as control) using a moistened camel’s-hair paintbrush. After exposure to true movement, the following filters and settings were used when calcu- the root for 5 min, the larvae were immediately transferred to the cen- lating the above parameters: total distance moved, downsize filter ter of a specially designed 12.5 cm arena on lightly moistened filter (1/25) and minimum distance moved (0.2 cm); maximum distance paper where the larvae’s host-searching behaviors were recorded for from origin, downsize filter (1/25); mean velocity, downsize filter (1/ 5 min using the EthoVision system (version 3.1; Noldus Information 25); mean turn angle, absolute setting, and downsize filter (1/25); mean Technology, The Netherlands). The bioassay was terminated early if meander, absolute setting, and downsize filter (1/25). Limited larval any larvae exited the arena during the 5-min trial period. Each bioas- movement coupled with the above filters sometimes resulted in no say resulted in one track file in the EthoVision program. A total of value being calculated for a specific parameter. For trials that did not 460 unique track files were recorded for this study. last the full 5 min as a result of larvae leaving the arena during their search, total distance traveled was adjusted to reflect the distance the EthoVision Protocol larvae would have traveled during the five minute period using their The EthoVision arena comprised a moist 125-mm filter paper circle average velocity as calculated by the EthoVision software. mounted on a clean glass plate and replaced between each larval exposure. The arena was enclosed in a clear acrylic box Statistical Analysis (20 20 18 cm) mounted under the EthoVision system video cam- An analysis of variance was used for these data analyses and was era (Panasonic wv BP334) positioned 0.64 cm above the box with a calculated by using the PROC MIXED of the SAS statistical package 15-W fluorescent light located on top for even lighting. For opti- (SAS Institute 2008; Cary, North Carolina, USA). For the mean mum viewing of larvae with the EthoVision system, the tracking set- meander, total distance moved, mean turn angle, maximum distance tings were set to the following specifications: detection method, subtraction; processing settings, only detect objects that are darker from origin and the velocity, the linear statistical model contained the main plot effect of treatment. Data were transformed by square than background; scan window of 50 pixels set to search the com- plete arena; minimum object size, one pixel; maximum object size, root (xþ 0.5) to meet the assumptions of the analysis. Both of the 20 pixels; sample rate, 5.994 samples/s. Recording began after the experiments were run as a randomized complete block. Table 1. Effect of treatment on each parameter measured of the movement of the western corn rootworm during bioassays from experi- ment one (using susceptible insects) and experiment two (using both susceptible and resistant insects) Assay set Analysis df fP One Distance moved Medium 4, 149 9.80 <0.0001 Mean velocity Medium 8, 149 19.16 <0.0001 Mean turn angle Medium 8, 147 41.56 <0.0001 Mean meander Medium 8, 147 36.91 <0.0001 Maximum distance from origin Medium 8, 147 22.29 <0.0001 Two Distance moved Medium 6,226 56.29 <0.0001 Colony 1,226 0.37 0.5411 Medium  colony 6,226 1.71 0.1195 Mean velocity Medium 6,233 61.71 <0.0001 Colony 1,233 0.07 0.7961 Medium  colony 6,233 1.99 0.0683 Mean turn angle Medium 6,231 277.34 <0.0001 Colony 1,231 1.02 0.3134 Medium  colony 6,231 4.2 0.0005 Mean meander Medium 6,235 100.85 <0.0001 Colony 1,235 0.03 0.8732 Medium  colony 6,235 2.02 0.0643 Maximum distance from origin Medium 6,227 104.4 <0.0001 Colony 1,227 3.84 0.0513 Medium  colony 6,227 3.47 0.0027 Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/59/3739026 by Ed 'DeepDyve' Gillespie user on 17 July 2018 4 Journal of Insect Science, 2017, Vol. 17, No. 2 A B Total Distance Moved Maximum Distance from Origin 180 60 a 120 40 b 30 b b 80 b b b b b b b b 20 b 0 0 C D Velocity Turn Angle a a 0.8 a 140 a a a 0.7 0.6 0.5 b b b 80 0.4 b 0.3 0.2 b b 0.1 0 0 E Meander a a 50 a Fig. 1. The total distance moved (A), maximum distance from origin (B), velocity (C), turn angle (D), and meander (E) of the western corn rootworm larvae in 5 min after exposure to a different plant seedlings or filter paper for experiment one. Letters indicate significant differences between corn types (P 0.05). Analysis was done with square root transformed data; figures represent untransformed data. Results origin, moved faster, turned less often and crossed their own path less than the resistant larvae after RNAi corn exposure (Fig. 2b–e). For all parameters that were measured in the first set of bioassays The resistant larvae moved farther from the origin after exposure to using only susceptible colonies, the two negative controls (moist filter the isoline than the susceptible colonies (Fig. 2b). Despite differences paper and germinated oat seedlings) were significantly different than in every parameter measured between the two colonies on the all corn treatments (Table 1; Fig. 1). The larvae that were exposed to RNAi, there were no differences between the susceptible and the the controls had significantly longer paths and traveled farther from resistant colonies for the vector stack exposure treatment (Fig. 2). the distance from the origin than the larvae exposed to corn plants The magnitude of the difference between the resistant and the sus- including the Bt plants (Fig. 1a and b). The larvae exposed to the neg- ceptible colony on RNAi was small compared to the susceptible col- ative controls traveled significantly faster, turned less, and crossed ony on the controls (Fig. 2). their paths less than the larvae exposed to the corn plants (Fig. 1c–e). In the second set of assays using both susceptible and resistant colonies, the two negative controls were also significantly different Discussion from all of the corn treatments for all parameters measured There were no dramatic differences between the localized search (Table 1; Fig. 2). After exposure to the RNAi roots, susceptible responses of WCR larvae exposed to any of the corn lines tested; larval behavior was significantly different than behavior for resistant larvae for all parameters measured (Fig. 2). The susceptible larvae however, the rootworm larvae consistently demonstrated a ranging had a significantly longer path on the RNAi corn than the resistant behavior after contact with the filter paper and oats, indicating that larvae (Fig. 2a). Also, the susceptible larvae moved farther from the they did not recognize the controls as hosts. This was expected since Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/59/3739026 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Velocity (mm/s) Distance traveled (mm) Meander (degrees/mm) Mean turn angle (degrees) Distance traveled (mm) Journal of Insect Science, 2017, Vol. 17, No. 2 5 AB Total Distance Moved Maximum Distance from Origin Susceptible Susceptible Resistant Resistant a a * * bc bc b 10 b bc bc c bc 2 c c Filter Oat Iso 88017 Smart RNAi Vector Filter Oat Iso 88017 Smart RNAi Vector Stax Stack Stax Stack CD Velocity Turn Angle Susceptible Susceptible 0.09 Resistant Resistant 0.08 120 0.07 a a a a 0.06 b 80 0.05 c c 0.04 0.03 0.02 c c 0.01 Filter Oat Iso 88017 Smart RNAi Vector Filter Oat Iso 88017 Smart RNAi Vector Stax Stack Stax Stack Meander Susceptible Resistant ab ab ab ab ab 1000 b 200 c Filter Oat Iso 88017 Smart RNAi Vector Stax Stack Fig. 2. The total distance moved (A), maximum distance from origin (B), velocity (C), turn angle (D), and meander (E) of the western corn rootworm larvae in 5 min after exposure to different plant seedlings or filter paper for experiment B. Letters indicate significant differences between corn types within colony (P 0.05). *Significant differences between resistant and susceptible colonies with seed type (P 0.05). Analysis was done with square root transformed data; figures represent untransformed data. larvae exposed to germinated oat roots had showed a ranging that Cry34/35Ab1 was perceived as a poor host for WCR larvae. behavior in previous studies (Strnad and Dunn 1990) and oats may However, the factors responsible for host recognition require spe- contain a larval feeding deterrent (Branson and Ortman 1969). cific extraction techniques if they are to be separated from corn Binning et al. (2005) conducted assays that were somewhat simi- (Bernklau et al. 2009), and these factors are likely not present in lar to the current experiment, except that in their experiment they artificial diet. In addition, Cry proteins are tied up in plant cells exposed the insects to artificial diet (modified after Pleau et al. under normal circumstances and not directly available to searching 2002) with and without Cry34/35Ab1 proteins. They concluded larvae as was done by Binning et al.(2005). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/59/3739026 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Mean velocity (cm/s) Distance traveled (cm) Mean meander (degrees/cm) Mean turn angle (degrees) Distance traveled (cm) 6 Journal of Insect Science, 2017, Vol. 17, No. 2 rootworm (Coleoptera: Chrysomelidae) larvae. J. Econ. Entomol. 102: Contact cues associated with the roots are the driving factor of 558–562. host recognition (Branson and Ortman 1969, Strnad and Dunn Binning, R., S. Lefko, F. C. Cheng, and K. Liao. 2005. Searching behavior of 1990), and this study demonstrates that each corn type, Bt, RNAi, western corn rootworm (Diabrotica virgifera virgifera) after exposure to the vector stack, and isoline, contains sufficient contact cues to elicit Cry34Ab1/Cry35Ab1 insecticidal proteins. Annual Meeting of the North a localized search response by both Cry3Bb1 resistant and suscepti- Central Branch, Entomological Society of America, March 20-23, West ble larvae when the larvae are removed from the germinated corn Lafayette, IN. roots. The toxins present in the Bt/RNAi roots did not significantly Bolognesi, R., P. Ramaseshadri, J. Anderson, P. Bachman, W. Clinton, R. affect the host response despite what may have happened in other Bt Flannagan, O. Ilagan, C. Lawrence, S. Levine, and W. Moar. 2012. assays such as in the study by Clark et al. (2006). Phenological dif- Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte). ferences in the corn may play a role in this discrepancy since corn PloS One. 7: e47534. seedlings of a slightly younger age were used in this study. Branson, T. F. 1976. The selection of a non-diapause strain of Diabrotica vir- Overall the data between the two sets of assays were similar with gifera (Coleoptera: Chrysomelidae). Entomol. Exp. Appl. 19: 148–154. the primary difference being the addition of a paired resistant col- Branson, T. F. 1982. Olfactory response of larvae of Diabrotica virgifera virgi- ony. Although the susceptible colony had significantly less host rec- fera to plant roots. Entomol. Exp. Appl. 31: 303–307. ognition patterns than the resistant colony on RNAi, the susceptible Branson, T. F., and E. E. Ortman. 1969. Feeding behavior of larvae of the colony never reached the level of ranging behaviors that was demon- western corn rootworm: normal larvae and larvae maxillectomized with strated by larvae exposed to the controls. The same is true for the laser radiation. Ann. Entomol. Soc. Am. 62: 808–812. resistant colony on the isoline corn roots compared to the suscepti- Branson, T. F., and E. E. Ortman. 1970. The host range of the western corn rootworm: further studies. J. Econ. Entomol. 63: 800–803. ble. The differences observed in the response levels of resistant and Chege, P. G., L. Clark, and B. E. Hibbard. 2005. Alternate host phenology af- susceptible neonates to RNAi but not RNAiþ Bt suggest that the Bt fects survivorship, growth, and development of western corn rootworm provides more of a contact cue when coupled with RNAi than when (Coleoptera: Chrysomelidae) larvae. Envir. Entomol. 34: 1441–1447. RNAi is used alone. Alternatively, the RNAi may interfere with the Clark, P. L., T. Vaughn, L. J. Meinke, J. Molina-Ochoa, and J. E. Foster. contact cues needed to produce a stronger recognition response by 2006. Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) larval the larvae when used alone. feeding behavior on transgenic maize (MON 863) and its isoline. J. Econ. Contact cues in the isoline root as perceived by the resistant lar- Entomol. 99: 722–727. vae may not be as strongly perceived as the in the susceptible larvae Clark, T. L., and B. E. Hibbard. 2004. Comparison of nonmaize hosts to sup- port western corn rootworm (Coleoptera: Chrysomelidae) larval biology. or perhaps there is a slight difference in contact cue chemical com- Environ. Entomol. 33: 681–689. position. In the current set of assays, although differences were Fire, A., S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, and C. C. detected between colonies, overall all corn lines were recognized as Mello. 1998. Potent and specific genetic interference by double-stranded suitable hosts. 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Agronomy J. 77: 769-744 Acknowledgments Krysan, J., T. Miller, and J. Andersen. 1986. Methods for the study of pest We would like to thank Monsanto, Syngenta and Dow for the seeds used in Diabrotica. Springer series in experimental entomology (USA). this study, and Bill Moar and Tom Clark for their helpful comments. Thanks Meihls, L. N. 2010. Development and characterization of resistance to trans- to Bruce Hibbard for his support in this research. genic corn in western corn rootworm. Ph. D. dissertation, University of Missouri, Columbia. Miller, N. J., K. S. Kim, S. T. Ratcliffe, A. Estoup, D. Bourguet, and T. References Cited Guillemaud. 2006. Absence of genetic divergence between western corn Apple, J. W., and K. K. Patel. 1963. Sequence of attack by northern corn root- rootworms (Coleoptera: Chrysomelidae) resistant and susceptible to control worms on the crown roots of corn. Proc. North Cent. Branch Entomol. Soc. by crop rotation. J. Econ. Entomol. 99: 685–690. Am. 18: 80–81. 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