J. Appl. Entomol.

Assessing larval rootworm behaviour after contacting maize roots: impact of germplasm, rootworm and diapause status D. A. Prischmann1, K. E. Dashiell1 & B. E. Hibbard2

1 United States Department of Agriculture, Agricultural Research Service, North Central Agricultural Research Laboratory, Brookings, SD, USA 2 United States Department of Agriculture, Agricultural Research Service, Plant Genetics Research Unit, University of Missouri, Columbia, MO, USA

Keywords Abstract v. virgifera, D. barberi, Chrysomelidae, behaviour, plant Current methods of screening maize (Zea mays L.) germplasm for suscep- resistance tibility or resistance to corn rootworms (Coleoptera: Chrysomelidae) rely primarily on information from large-scale field experiments. Due to Correspondence labour and cost constraints associated with field trials, alternative evalu- Deirdre A. Prischmann (corresponding author), ation methods are desirable. We used a previously developed behaviour- United States Department of Agriculture, al bioassay to: (1) investigate the host search behaviour of rootworm Northern Plains Area, North Central Agricultural Research Lab, 2923 Medary larvae after contact with 14 maize genotypes, (2) compare the behav- Avenue, Brookings, SD 57006-9401, USA. iour of non-diapausing Diabrotica virgifera virgifera LeConte, diapausing E-mail: [email protected] D. v. virgifera, and diapausing D. barberi Smith & Lawrence and (3) deter- mine if this technique can be used to separate susceptible vs. resistant Received: March 24, 2008; accepted: June 17, maize genotypes. The majority of rootworm larvae engaged in intensive 2008. (local search) behaviour after exposure to maize roots, whereas larvae continued to exhibit extensive (ranging) behaviour after contact with doi: 10.1111/j.1439-0418.2008.01313.x negative controls. Even though a transgenic hybrid with resistance to D. v. virgifera was included in analyses, quantitative path measurements were similar among genotypes and only differed between specific maize lines and controls. Notably, there were differences in host search behav- iour among rootworm groups, with non-diapausing D. v. virgifera having more convoluted paths and engaging in intensive search more fre- quently than diapausing rootworms. Correlations between larval path measurements and historic root damage ratings were not significant, although there were weak positive correlations between historic adult emergence densities and measures of path linearity. However, due to the lack of significant behavioural differences among maize lines with a range of susceptibility levels, we concluded that this bioassay is not useful in screening maize germplasm for rootworm resistance.

subterranean larval stages, which can also utilize Introduction roots of several alternative plant species to support Corn rootworms (Chrysomelidae: Diabrotica spp.) are their development (Branson and Ortman 1967, widespread maize (Zea mays L.) pests that can cause 1970; Moeser and Vidal 2004; Oyediran et al. 2004a; economic damage by consuming plant tissues, thus b). Soil insecticides and crop rotation are commonly negatively affecting plant physiology and function used to manage rootworms (Wilson et al. 2005), (Riedell 1990; Riedell and Reese 1999), and grain although insecticide resistance has been documented yield (Sutter et al. 1990; Spike and Tollefson 1991). (Ball and Weekman 1962; Wright et al. 2000). In The majority of plant damage is caused by the addition, western corn rootworms (WCR, Diabrotica

J. Appl. Entomol. 133 (2009) 21–32 ª 2008 The Authors Journal compilation ª 2008 Blackwell Verlag, Berlin 21 Assessing rootworm behaviour after exposure to maize D. A. Prischmann, K. E. Dashiell and B. E. Hibbard virgifera virgifera LeConte) have adapted to crop rota- distinct, and have been studied for several insect tion in some areas by ovipositing in other crops taxa (Bell 1985, 1990; Visser 1988; De´jean et al. (Rondon and Gray 2004), while in some regions 1993; Ettifouri and Ferran 1993; Guershon and Ger- northern corn rootworms (NCR, Diabrotica barberi ling 2006). Smith & Lawrence) have extended their diapause Analysis of larval corn rootworm behaviour has from one to two years (Krysan et al. 1984; Levine been used to determine their response to carbon et al. 1992). Transgenic maize hybrids effective dioxide (Bernklau and Bjostad 1998a), host plant against corn rootworms are commercially available; volatiles (Hibbard and Bjostad 1988), toxic plant however, non-genetically modified hybrids are still compounds (Branson et al. 1969; Xie et al. 1992a), desirable for use as refuge partners or for organic and transgenic maize hybrids (Clark et al. 2006). and European markets. Due to negative aspects of Rootworm larvae use carbon dioxide as a general broad-spectrum pesticide use, the decline of the orientation cue to locate potential host plants effectiveness of crop rotation in many areas and the (Bernklau and Bjostad 1998b). Larvae can detect establishment of the western corn rootworms in feeding stimulants using their maxillary palpi (Bran- Europe, there is an increasing need for alternate son and Ortman 1969), and rapidly begin feeding on rootworm management strategies, including using acceptable food sources (Clark et al. 2006). Recently resistant and/or tolerant corn hybrids. feeding stimulants from corn have been chemically Scientists have been trying to identify maize lines identified as a blend of short-chain sugars and long- with rootworm resistance in the hopes of lessening chain free fatty acids (Bernklau and Bjostad 2008). reliance on insecticides and developing viable alter- Strnad and Dunn (1990) developed a bioassay to natives to transgenic hybrids (Branson et al. 1983; investigate larval rootworm host search behaviour Gray and Steffey 1998; Hibbard et al. 1999b). Inves- by studying their movement after contact with tigations have focused on screening germplasm of potential food sources. Larval rootworms engaged in maize and its relatives for use in breeding programs local search patterns after contact with suitable host (Branson 1971; Moellenbeck et al. 1995; Hibbard plants, but exhibited ranging behaviour after contact et al. 1999b; Eubanks 2002), refining evaluation with non-hosts (Strnad and Dunn 1990). This bioas- methods regarding plant susceptibility (Moellenbeck say has been used to test responses of non-diapaus- et al. 1994; Knutson et al. 1999), and exploring ing WCR (Strnad and Dunn 1990) and diapausing mechanisms of corn resistance (Ajani and Lonnquist northern corn rootworms (Oyediran et al. 2004a) to 1979; Xie et al. 1992b; Assabgui et al. 1995). One maize, grassy weeds, and other plant species and mechanism of plant resistance to herbivorous pests is investigate WCR behaviour after contact with differ- non-preference (or antixenosis), with non-preferred ent maize cultivars (Knutson et al. 1999). plants not eaten or used for oviposition as frequently Our objectives were to: (1) use the larval corn as preferred ones (Painter 1951). Another resistance rootworm behavioural bioassay developed by Strnad mechanism is antibiosis, where plants have negative and Dunn (1990) to compare the host search behav- effects on pest life history parameters or performance iour of neonate rootworm larvae after contact with (Painter 1951). Effects of non-preference and anti- 14 different maize genotypes, (2) compare the host biosis on rootworms can be difficult to distinguish, search behaviour of three rootworm groups: WCR and root damage ratings and number of adult root- from a non-diapausing colony, WCR from a diapaus- worms that emerge from the soil have been used to ing colony and NCR from a diapausing colony and measure both traits (Branson et al. 1981b, 1983). (3) determine if this technique can be used to sepa- Insect behaviour is an important indicator of rate susceptible vs. resistant maize genotypes. resource quality, with exposure to acceptable resources stimulating local search behaviour, or Materials and Methods intensive movement (Jander 1975; Bell 1985, 1990; Visser 1988). Local search behaviour is characterized Lab experiments were performed from January to by highly convoluted paths, and helps keep February 2007. We assessed 15 treatments, which within profitable patches (Jander 1975; Bell 1985, included 14 maize genotypes and a negative control 1990; Visser 1988). This is in contrast to ranging consisting of filter paper (9 cm diameter; Schleicher behaviour (extensive movement), which is when & Schuell Inc., Keene, NH) moistened with insects move in relatively linear paths between deionized water. Maize genotypes (table 1) included patches (Jander 1975; Bell 1985, 1990; Visser 1988). a transgenic commercial hybrid expressing the Local search patterns vs. ranging behaviour are quite Cry3Bb1 endotoxin with rootworm resistance

J. Appl. Entomol. 133 (2009) 21–32 ª 2008 The Authors 22 Journal compilation ª 2008 Blackwell Verlag, Berlin D. A. Prischmann, K. E. Dashiell and B. E. Hibbard Assessing rootworm behaviour after exposure to maize

Table 1 Background information on maize hybrids and experimental synthetic maize populations grown in field trials near Brookings, SD during 2005 and 2006

Pedigree Background1 Source2 References3

DeKalb46-23 H Transgenic resistant hybrid Monsanto CRW DeKalb46-26 H Non-transgenic isoline of DeKalb46-23 CRW Monsanto Monsanto company 2006 B73 · MO17 H Susceptible hybrid, some tolerance Public line GRIN NPGS 2006; B.E.H. B37 · H84 H Highly susceptible hybrid Public line GRIN NPGS 2006 NGSDCRW1 SP Moderate resistance in MO NCRPI, USDA-ARS, SDSU-AES Kahler et al. (S2)C4 (1985); Hibbard et al. (1999b) CRW8-1 SP High resistance in MO, derived from BS19/20 USDA-ARS, Columbia, MO Russell et al. (1976) CRW8-2 SP High resistance in MO, derived from BS19/20 USDA-ARS, Columbia, MO Russell et al. (1976) CRW8-3 SP High resistance in MO, derived from BS19/20 USDA-ARS, Columbia, MO Russell et al. (1976) CRW2(C5) SP High resistance, derived from CIMMYT in USDA-ARS, Columbia, MO Russell et al. (1976); Hibbard et al. 1999 Hibbard et al. (1999b) CRW3(C6) SP High resistance, derived from CIMMYT in USDA-ARS, Columbia, MO Russell et al. (1976); Hibbard et al. 1999 Hibbard et al. (2007) BS29-18-01 SP Susceptible in MO, derivative of BS29 Iowa State University GRIN NPGS 2006; Hallauer 1994; B.E.H. BS29-07-01 SP Susceptible in MO, derivative of BS29 Iowa State University GRIN NPGS 2006; Hallauer 1994; B.E.H. BS29-11-01 SP Susceptible in MO, derivative of BS29 Iowa State University GRIN NPGS 2006; Hallauer 1994; B.E.H. SD10 SP Moderate resistance in MO, tolerant to SDSU Chiang 1973; B.E.H. rootworms

1SP – experimental synthetic maize population, H – maize hybrid; MO – Missouri; resistance/susceptibility assessed in MO trials using root damage ratings. 2NCRPI – North Central Regional Plant Introduction Station; SDSU-AES – South Dakota State University, Agricultural Experiment Station; USDA-ARS – United States Department of Agriculture, Agricultural Research Service. 3B.E.H. – B.E. Hibbard (personal communication); GRIN NPGS – Germplasm Resources Information Network, National Plant Germplasm System.

(DeKalb46-23 CRW; Monsanto, St Louis, MO), its 6; Ritchie et al. 2005) were used in larval bioassays non-transgenic isoline (DeKalb46-26), two public or discarded. hybrids (B73 · MO17 and B37 · H84) and 10 Three rootworm groups were tested: non-diapaus- experimental synthetic lines [NGSDCRW1(S2)C4, ing WCR (nd-WCR), diapausing WCR (d-WCR) and CRW8-1, CRW8-2, CRW8-3, CRW2(C5), CRW3(C6), diapausing NCR (d-NCR). Rootworm eggs were BS29-18-01, BS29-07-01, BS29-11-01, SD10]. Maize obtained from colonies maintained at the North lines had a range of rootworm resistance levels Central Agricultural Research Laboratory (NCARL) (table 1), as demonstrated by varying root damage in Brookings, SD. Background information regarding ratings, compensatory root growth ratings and the establishment of the non-diapausing and diapa- density of adult rootworm emergence from field using WCR colonies can be found in Kim et al. experiments (Prischmann et al. 2007). (2007). The diapausing NCR colony was started in Maize seeds were produced in either 2005 or 1995 from 125 000 adult field-collected in 2006, and were kept in cold storage (4C) to Brookings county, SD, and has been reared for maintain their viability. DeKalb46-23 CRW and approximately 11 generations. Both diapausing colo- DeKalb46-26 seeds came pretreated with two fun- nies are maintained in a similar manner, although gicides (metalaxyl and mefenoxam) and the insecti- the NCR colony is smaller. Petri dishes containing cide clothianidin. All seeds were separated according rootworm eggs in 80 mesh soil were placed in a to genotype, washed in tepid soapy water to remove growth chamber (25C, 12 : 12 light:dark) and any seed treatments, and thoroughly rinsed in de- allowed to hatch for eight to 23 days. There were no ionized water. Seeds were then imbibed with water significant differences in egg hatch times between for 20–28 h, laid on moist germination paper in Petri rootworm groups (anova,df2,21, F = 0.09, P = 0.92). dishes, and placed in a dark room at approximately Fifteen hours prior to testing, dishes were checked 25C. After four days, germinated seedlings (stage 4– and any larvae removed, thus ensuring that only

J. Appl. Entomol. 133 (2009) 21–32 ª 2008 The Authors Journal compilation ª 2008 Blackwell Verlag, Berlin 23 Assessing rootworm behaviour after exposure to maize D. A. Prischmann, K. E. Dashiell and B. E. Hibbard starved 1st instars < 15 h old (neonates) were used observed frequencies of occurrence for each maize in experiments. genotype compared to those from the negative filter There were eight replicates with one larva per paper control (expected negative response) using the maize treatment per rootworm group, with each rep- Pearson chi-square crosstabulation test for two-way licate performed on a different day. Bioassays were tables (Systat, SPSS Inc. 1998). Due to small sample performed on a lab benchtop under fluorescent light- sizes, P-values from the Fisher exact test (two-tail) ing at approximately 22C. Using a fine camel-hair were reported rather than Pearson chi-square statis- paintbrush, one active larva was transferred to a Petri tics for tests comparing individual maize genotypes dish containing one germinated maize seed or a neg- to the control. One-way crosstabulation tests were ative filter paper control. For each maize bioassay, used to compare the total frequency of positive and one larva was gently placed on the central region of negative responses within each rootworm group the radicle. For negative controls, one larva was (TOTALS without controls; table 3), and Pearson placed in the centre of a moist filter paper. The neo- chi-square statistics were reported. nate was left undisturbed for 5 min, although during For quantitative analysis of path measurements, this period if the larva left the maize seedling or filter path area, length, sinuosity and crossings data were paper it was moved back to its original starting posi- log(X + 1) transformed to normalize the data, tion. Arenas were not covered during this time to although only path crossing data had zeroes. Data prevent a build-up of carbon dioxide. After 5 min, were analysed using the GLIMMIX procedure (SAS the larva was transferred to the centre of a moist- 2004, 2005) followed by Tukey’s Honest Significant ened, circular filter paper arena (24 cm diameter; Difference multiple comparison test. #6310-2400, Ahlstrom, Mt Holly Springs, PA) resting In order to determine if this larval bioassay could on a clear plexiglass square (30.5 cm2). An identical be used to separate susceptible vs. resistant maize plexiglass square with an affixed clear acetate sheet genotypes, we investigated correlations between lar- was then placed above the filter paper on four sup- val path measurements and historic root damage and ports (4 mm high). A fine-tipped marker was used to adult rootworm emergence data (Prischmann et al. trace the neonate’s movement (i.e. larval path) on 2007) for all maize lines, with the exception of SD10. the suspended acetate sheet for the next 5 min. If a Mean root damage ratings (Iowa 1–6 scale; Hills and larva was visibly damaged during the transfer pro- Peters 1971) and adult emergence densities (mal- cess, the bioassay was terminated and repeated with es + females) from a 2-year field experiment investi- another larva. New larvae, filter paper arenas, and gating the susceptibility of these maize genotypes to acetate sheets were used for each bioassay. d-WCR infestations (Prischmann et al. 2007) were Using definitions and examples of local search and used in analyses. Correlations between parameters ranging movement behaviours (Jander 1975; Bell were tested using zero correlation analysis (Systat, 1985, 1990; Visser 1988; Strnad and Dunn 1990), SPSS Inc. 1998), with the null hypothesis being that larval paths were qualitatively scored as demonstrat- there was no correlation between the variables. ing positive (local search) or negative responses (ranging behaviour). Several quantitative aspects of Results each larval path were also assessed, including path area, path length, path sinuosity and number of Qualitative path analysis times a larva crossed its own path (path crossings). Larval path area was measured by placing a clear Regardless of rootworm group, all larvae had nega- grid (10 squares/25 mm) over the traced path, tive responses (ranging behaviour) after exposure to counting all intersected squares, and converting to filter paper controls, while exposure to maize roots mm2, while path length was measured in mm using generally elicited positive behavioural responses (i.e. a ruler. Sinuosity is a measurement of path linearity, local search; table 2). After contact with maize roots, and was calculated by dividing total path length by nd-WCR engaged in local search behaviour more the distance between path beginning and end points frequently than after exposure to filter paper (De´jean et al. 1993). controls, regardless of maize genotype. Exposure to 11 of the 14 maize lines invoked more frequent posi- tive reactions in d-WCR larvae compared to controls. Statistics However, 50% of the time, d-WCR had negative Qualitative positive/negative rootworm response responses after contact with B37 · H84, DeKalb data were converted to binary (1, 0) data and 46–23 CRW, and DeKalb46-26 roots, which was

J. Appl. Entomol. 133 (2009) 21–32 ª 2008 The Authors 24 Journal compilation ª 2008 Blackwell Verlag, Berlin D. A. Prischmann, K. E. Dashiell and B. E. Hibbard Assessing rootworm behaviour after exposure to maize

Table 2 Results from qualitative analysis of larval host-search paths

nd-WCR1,2 d-WCR1,2 d-NCR1,2

Maize line ) +P ) + P ) + P

Control 8 0 n/a 8 0 n/a 8 0 n/a DeKalb46–23 CRW 2 6 0.007 4 4 0.08 5 3 0.20 DeKalb46–26 1 7 0.001 4 4 0.08 6 2 0.47 B73 · MO17 3 5 0.03 1 7 0.001 3 5 0.03 B37 · H84 2 6 0.007 4 4 0.08 3 5 0.03 NGSDCRW1(S2) 1 7 0.001 3 5 0.03 1 7 0.001 CRW8-1 1 7 0.001 2 6 0.007 4 4 0.08 CRW8-2 2 6 0.007 3 5 0.03 2 6 0.007 CRW8-3 1 7 0.001 3 5 0.03 3 5 0.03 CRW2(C5) 3 5 0.03 3 5 0.03 4 4 0.08 CRW3(C6) 3 5 0.03 2 6 0.007 3 5 0.03 BS29-18-01 0 8 < 0.001 3 5 0.03 4 4 0.08 BS29-07-01 0 8 < 0.001 3 5 0.03 4 4 0.08 BS29-11-01 0 8 < 0.001 0 8 < 0.001 2 6 0.007 SD10 0 8 < 0.001 1 7 0.001 6 2 0.47 TOTALS (without controls) 19 93 < 0.001 36 76 < 0.001 50 62 0.26

1nd-WCR – non-diapausing western corn rootworms, d-WCR – diapausing western corn rootworms, d-NCR – diapausing northern corn rootworms; ) negative responses indicated extensive search patterns or ranging behavior, + positive responses indicated intensive search patterns or local search behaviour. 2P-values are from Pearson chi-square crosstabulation tests. not significantly different from controls, although CRW8-2, SD10, NGSDCRW1(S2)C4 and BS29-18-01 the P-value was marginal. Only half the lines elicited having shorter path lengths than filter paper controls local search behaviour in d-NCR larvae significantly (fig. 1). There was a significant main effect of more often than controls, with negative responses rootworm identity on path length, which was due to occurring in ‡ 50% of trials with DeKalb46–23 nd-WCR moving further than d-NCR (fig. 2). CRW, DeKalb46-26, CRW8-1, CRW2(C5), BS29- All three main effects impacted path sinuosity 18-01, BS29-07-01 and SD10 roots. When looking at (table 3), with larvae exposed to CRW8-1, the overall frequency of behavioural responses after CRW3(C6) and BS29-11-01 having higher path contact with maize roots regardless of genotype, sinuosity than rootworms exposed to filter paper nd-WCR and d-WCR exhibited positive responses controls (figs 1 and 2). Non-diapausing WCR had more often than negative responses (P < 0.001), while more convoluted paths than other rootworm groups d-NCR displayed negative search patterns as frequ- (df2,357, F = 9.83, P < 0.0001; d-WCR, P = 0.009; ently as positive behavioural movements (P = 0.26). d-NCR, P < 0.0001). Maize treatment and rootworm identity affected the number of path crossings, although replicate did Quantitative path analysis not affect this parameter (fig. 2). Larvae exposed to There were no interactions between maize treatment, maize roots had significantly more path crossings rootworm identity and replicate for any measured than larvae exposed to filter paper controls, with larval path parameters (table 3). Maize treatment the exception being DeKalb46-23 CRW and affected path area, with larvae exposed to maize roots DeKalb46-26 (fig. 1). Paths of nd-WCR had signifi- having a smaller path area than those exposed to fil- cantly higher numbers of crossings than diapausing ter paper controls (fig. 1). However, there were no rootworms (df2,357, F = 2.68 P < 0.0001; d-WCR, significant differences in path area among maize P = 0.003; d-NCR, P < 0.0001; fig. 2). genotypes (P > 0.05). Corn rootworm identity did not impact the area covered by neonate larvae (fig. 2), Correlations although replicate did have a significant main effect. Maize treatment and replicate also affected path Correlations between historic root damage ratings length, with DeKalb46-23 CRW, DeKalb46-26, and larval path area, length and sinuosity were not

J. Appl. Entomol. 133 (2009) 21–32 ª 2008 The Authors Journal compilation ª 2008 Blackwell Verlag, Berlin 25 Assessing rootworm behaviour after exposure to maize D. A. Prischmann, K. E. Dashiell and B. E. Hibbard

Table 3 ANOVA results from analysis of quantitative path measure- significant (P > 0.05, fig. 3). There was a significant ments positive correlation between historic root damage ratings and the number of path crossings (P = 0.02), Parameter1 df F P although this relationship was driven by one maize genotype (DeKalb46-23 CRW, mean root damage Path area Maize 14 196 4.77 < 0.001 rating = 1.0). When this data point was removed Rootworm 2196 0.78 0.46 from the analysis, the correlation was not significant Replicate 7196 9.15 < 0.001 (P = 0.07). There were no correlations between his- Maize · rootworm 28 196 1.06 0.39 toric densities of emerged adult (male + female) Maize · replicate 98 196 1.09 0.30 rootworms and larval path area or length (P > 0.05). Rootworm · replicate 14 196 1.27 0.23 However, there were significant positive correlations Path length between historic adult emergence and path sinuosity Maize 14 196 2.97 < 0.001 Rootworm 2196 3.18 0.04 (P = 0.01) and number of path crossings (P = 0.04). Replicate 7196 6.82 < 0.001 If log, and not log(X + 1) transformations, were used Maize · rootworm 28 196 0.93 0.57 for path sinuosity data, the correlation between his- Maize · replicate 98 196 0.94 0.64 toric adult emergence and path sinuosity was weak- Rootworm · replicate 14 196 1.52 0.11 ened, with an R2 value of 0.38 and P = 0.02. Path sinuosity Maize 14 196 2.30 0.006 Rootworm 2196 10.21 < 0.001 Discussion Replicate 7196 2.83 0.008 Maize · rootworm 28 196 0.70 0.87 Evaluating maize germplasm in the field is the pri- Maize · replicate 98 196 0.93 0.66 mary method used to determine its susceptibility to Rootworm · replicate 14 196 0.88 0.59 corn rootworms (Riedell and Evanson 1993; Knut- Path crossings son et al. 1999). Because this process can be time Maize 14 196 3.22 < 0.001 consuming and costly, alternative screening methods Rootworm 2196 13.88 < 0.001 are desirable. The rootworm larval host-search bioas- Replicate 7196 0.40 0.90 Maize · rootworm 28 196 0.99 0.49 say has been used to separate acceptable and unac- Maize · replicate 98 196 0.66 0.99 ceptable species of host plants (Strnad and Dunn Rootworm · replicate 14 196 1.63 0.07 1990; Oyediran et al. 2004a). With regard to intra- specific host plant acceptability, Knutson et al. 1Maize – maize treatment (14 maize genotypes and filter paper con- (1999) concluded that this bioassay was not a useful trol), rootworm – rootworm group (nd-WCR, d-WCR or d-NCR).

2.5 Area Length a a 2.0 b b b b b b b b b b b b b ab b abab ab abab ab b b ab b b Fig. 1 Effect of maize treatment 1.5 b b on larval rootworm path parame- ters. White bars – filter paper con- trols, black bars – maize hybrids, 1.0 grey bars – experimental synthetic 1.5 Sinuosity Crossings maize populations. Maize treat- ment abbreviations are as follows: Cntl – filter paper control, DKC b b b 1.0 ab ab b ab b b b CRW – DeKalb 46-23 CRW, DKC46- ab ab abab ab b b b Lg (Path measurement + 1) ab ab b b b b b 26 – DeKalb 46-26 and NGSDCRW1 ab abab a – NGSDCRW(S2)C4. Area measured in mm2 and length measured in 0.5 mm. Data are means Æ SEMs. Col- a umn means with the same letter are not significantly different SD10 BS29-07-01 BS29-11-01 BS29-18-01 DKC CRW SD10 BS29-11-01 DKC CRW NGSDCRW1 CRW8-1 B73xMO17 B37xH84 CRW3(C6) CRW2(C5) Cntl Cntl B37xH84 NGSDCRW1 CRW8-1 DKC 46-26 B73xMO17 CRW3(C6) BS29-18-01 DKC 46-26 CRW8-2 BS29-07-01 CRW8-2 CRW8-3 CRW8-3 CRW2(C5) 0.0 according to Tukey’s HSD test Maize treatment (P > 0.05).

J. Appl. Entomol. 133 (2009) 21–32 ª 2008 The Authors 26 Journal compilation ª 2008 Blackwell Verlag, Berlin D. A. Prischmann, K. E. Dashiell and B. E. Hibbard Assessing rootworm behaviour after exposure to maize

2.5 Area Length

2.0

a a a 1.5 a ab b 1.0

0.5

0.0 1.0 Sinuosity Crossings

0.8 Fig. 2 Effect of rootworm group a a on larval path parameters. nd-WCR b Lg (Path measurement + 1) 0.6 – non-diapausing western corn b b rootworms, d-WCR – diapausing 0.4 b western corn rootworms, d-NCR – diapausing northern corn root- 0.2 worms. Data are means Æ SEMs. Column means with the same let- 0.0 ter are not significantly different nd-WCR d-WCR d-NCR nd-WCR d-WCR d-NCR according to Tukey’s HSD test (P > 0.05). Rootworm group tool for separating resistant vs. susceptible maize decreased path length, is one way stay lines, but they did not evaluate NCR and Strnad and within favourable patches (Bell 1990). Alternatively, Dunn (1990) and Oyediran et al. (2004a) did not decreased path length could be due to toxic effects evaluate different maize varieties. In addition, we of the transgenic hybrid on neonates. Clark et al. felt additional exploration of this bioassay’s utility (2006) found that when WCR larvae fed on trans- was needed because qualitative path characteristics genic maize they either wandered around sampling and path sinuosity were not assessed in the previous different root regions or stopped moving, with the study. Furthermore, only non-diapausing WCR were latter situation therefore resulting in a shorter path tested in behavioural bioassays and only diapausing length. However, in our study, larvae exhibited simi- larvae were tested in field work. lar behaviour after contact with the transgenic In our study, the majority of rootworm larvae hybrid’s isoline, DeKalb46-26, so movement pat- engaged in intensive search patterns after exposure terns after exposure to DeKalb46-23 CRW may not to maize roots, whereas larvae continued to exhibit be related to insecticidal proteins derived from Bacil- ranging behaviour after contact with filter paper lus thuringiensis Berliner. controls. This parallels results from studies investi- Given that decreased path length, which is directly gating larval rootworm behaviour after contact with related to path velocity (velocity = length/time), can maize, alternative host, and non-host roots (Strnad indicate either a positive or negative movement and Dunn 1990; Oyediran et al. 2004a). There were response, this parameter may not be useful in assess- few instances when contact with maize roots of a ing host-search behaviour. In addition, larval search particular genotype resulted in intensive search in area can be similar between two paths that portray all replicates, in contrast to filter paper controls, extensive vs. intensive search (fig. 4). In this figure, which resulted in consistent negative responses. larval path area and length are the same, although Overall, there was significant variability among repli- sinuosity and number of path crossings are clearly cates, which is not uncommon in behavioural stud- different. In previous studies where the larval ies (Bell 1985). behavioural bioassay was used (Strnad and Dunn Frequency of local search and number of path 1990; Oyediran et al. 2004a), exposure to maize and crossings after contact with the transgenic hybrid alternative host plants that can support adult root- DeKalb46-23 CRW did not differ from controls for worm development (Echinochloa crus-galli L. Scop., d-WCR and d-NCR, although path length was signif- Triticum aestivum L.; Branson and Ortman 1967; icantly shorter. Decreased movement, leading to Clark and Hibbard 2004; Oyediran et al. 2004a)

J. Appl. Entomol. 133 (2009) 21–32 ª 2008 The Authors Journal compilation ª 2008 Blackwell Verlag, Berlin 27 Assessing rootworm behaviour after exposure to maize D. A. Prischmann, K. E. Dashiell and B. E. Hibbard

Area, R 2 = 0.04 consistently triggered intensive search patterns, 2.0 Root damage Length, R 2 = 0.12 which were characterized by reduced search area, Sinuosity, R 2 = 0.16 slower velocity, and increased turns and/or path Crossings, R 2 = 0.38 crossings. However, rootworms exposed to plants that supported larval, but not adult development, 1.5 also had reduced search area and velocity compared to filter paper controls, but most did not exhibit increased turns and/or path crossings. Similar results

1.0 occurred after contact with specific non-host plants that provided no or extremely limited support for larval development (Festuca arundinacea Schreb. Sor- ghum bicolor L.; Wilson and Hibbard 2004; Oyediran 0.5 et al. 2004a), possessed feeding deterrents, or had 0.51.0 1.5 2.0 2.5 3.0 3.5 4.0 toxic effects on larvae (Avena sativa L.; Branson and Mean root damage rating Ortman 1967, 1969, 1970; Branson et al. 1969; Str- nad and Dunn 1990; Clark and Hibbard 2004). 2 2.0 Emergence Area, R = 0.01 Therefore, sinuosity and number of turns and/or 2 Length, R = 0.05 path crossings may be more useful parameters to 2

Lg (Path measurement + 1) Sinuosity, R = 0.45 measure when exploring host-search behaviour Crossings, R 2 = 0.34 using the larval bioassay, especially when investigat- 1.5 ing different cultivars of the same plant species. Another parameter that could provide useful information is the duration of local search. Unre- warded larvae will eventually shift from local search 1.0 back to ranging behaviour (Bell 1990), and thus the length of time larvae persist in intensive search may indicate the strength of their preference for a specific

0.5 food source. However, in our study larval behaviour 0 1020304050was only recorded for 5 min, and duration of local Mean adult rootworm emergence search behaviour was not measured.

Fig. 3 Correlations between larval path parameters for all rootworm There were no significant differences in quantita- groups and historic mean root damage ratings and adult rootworm tive path measurements among maize genotypes. emergence densities (Prischmann et al. 2007). Maize roots were rated Analysis of path characteristics indicated that specific using the Iowa 1–6 scale (Hills and Peters 1971). maize lines were more preferred than controls, although favourable genotypes varied depending on the path parameter being assessed. According to search area and number of path crossings, all maize lines were more preferred than controls, with the exception of DeKalb46-23 CRW and DeKalb46-26 for the latter parameter. In contrast, only seven maize genotypes were more preferred than controls based on decreased path length [NGSDCRW1(S2)C4, CRW8-2, BS29-18-01, SD10] and increased path sin- uosity [CRW8-1, CRW3(C6), BS29-11-01]. However, in field trials these maize lines had a range of root damage ratings and adult emergence densities (table 1; Prischmann et al. 2007). In gen- eral, NGSDCRW1(S2)C4 (Kahler et al. 1985; Prisch- mann et al. 2007) and SD10 (Branson et al. 1982) Fig. 4 Comparison between ranging behaviour (extensive search, on are tolerant to rootworm damage, CRW lines left) vs. local search behaviour (intensive search, on right). For both paths, area = 75 mm2 and length = 33 mm. The left path has a sinuos- [CRW8-1, CRW8-2, CRW8-3, CRW2(C5), CRW3(C6)] ity of 1.1 and no path crossings, while the right path has a sinuosity have some resistance, and BS lines (BS29-18-01, of 6.6 and four path crossings. BS29-07-01, BS29-11-01) are susceptible (table 1;

J. Appl. Entomol. 133 (2009) 21–32 ª 2008 The Authors 28 Journal compilation ª 2008 Blackwell Verlag, Berlin D. A. Prischmann, K. E. Dashiell and B. E. Hibbard Assessing rootworm behaviour after exposure to maize

Hallauer 1994; Hibbard et al. 2007; Prischmann trials, or any correlations between historic root dam- et al. 2007). age ratings and measured path parameters. This par- Although 5 min of exposure to maize roots stimu- allels results from Knutson et al. (1999), who lated the majority of larval rootworms to engage in concluded that this bioassay is not useful in identify- local search behaviour, this initial response does not ing suitable host plants within the same species or take into account other factors, such as feeding separating susceptible vs. resistant maize cultivars. deterrents, toxins, or nutritional qualities (Moeser Modifying the experimental methods or assess- and Vidal 2004), that can impact root damage rat- ment of path parameters may improve the utility of ings, larval development, and adult emergence lev- this bioassay as a tool for investigating intraspecific els. Roots of numerous plant species can stimulate host plant acceptability. The chemicals responsible intensive host search behaviour in neonate root- for this change in behaviour have recently been worms, regardless of their ability to support larval extracted in organic solvents (B.E. Hibbard, unpub- and adult development (Branson and Ortman 1967, lished data). Once these chemicals are identified, 1970; Strnad and Dunn 1990; Clark and Hibbard analyzing host roots directly for feeding stimulants 2004; Oyediran et al. 2004a,b). Thus, it was not sur- (Bernklau and Bjostad 2008) and/or host recognition prising that correlations between larval path mea- factors may provide a less variable approach. It may surements and historic root damage ratings were not also be possible to reduce the frequency of variable significant. However, there were positive correlations responses in the bioassay by prescreening larvae for between historic adult emergence and measures of activity. Other potential modifications and/or consid- path linearity (sinuosity and number of crossings), erations include: (1) chemical differences between although the R2 values were relatively low, and his- root regions (e.g. root tip, hairs) and initial place- toric data were from trials infested with d-WCR. ment of larvae, (2) larval movement and feeding When investigating differences in host search behaviour while on the root, including time from behaviour among rootworm groups, nd-WCR were contact to feeding, ranging behaviour along the root, more likely to engage in local search after contact and if larvae crawl off the root, (3) varying the with maize roots. In contrast to diapausing root- length of time larvae are in contact with the root worms, especially d-NCR, nd-WCR exhibited inten- prior to transfer, and (4) specifying if this time sive search more frequently than controls regardless period is defined solely by contact or if active feeding of maize genotype and had more convoluted larval is necessary for the entire duration. In addition, paths. It is unclear why there were differences in because rootworm larvae are small and subterra- host search behaviour between rootworm groups. nean, behavioural tests may be more accurate if per- However, reared insects can have altered behaviour formed under low-light conditions with the aid of and food preferences compared to their wild types video tracking systems (Noldus et al. 2002). Video counterparts (Boller 1972; Guthrie et al. 1974), and recording would also reduce potential effects of car- the nd-WCR colony at NCARL is genetically different bon dioxide produced by people conducting the than the d-WCR colony, with the latter being similar assays on larval behaviour. to wild populations (Kim et al. 2007). In field exper- Rootworms from a non-diapausing WCR colony iments, nd-WCR and d-WCR from NCARL colonies responded differently than WCR and NCR from a caused similar amounts of root damage (Hibbard diapausing colony, indicating that this trait should be et al. 1999a), although in other field trials reared considered when comparing feeding behaviour nd-WCR larvae did not cause as much root damage among groups. Although in its current form this lab as those originating from field-collected WCR (Bran- bioassay is not an appropriate tool for screening maize son et al. 1981a). In feeding bioassays, nd-WCR from germplasm, a modified behavioural bioassay or other another rearing facility (French Agricultural lab tests may have promise for identifying resistant Research, Inc., Lamberton, MN) consumed more genotypes, including those that focus on behavioural pollen than field-collected WCR, although the statis- and physiological responses of rootworms to host tical significance of this comparison was not deter- plant chemicals and/or nutritional properties. mined (Hollister and Mullin 1999). In our study, contact with maize roots generally Acknowledgements stimulated local search behaviour regardless of geno- type, and we did not find significant differences in We would like to thank B. Wade French and Chad host search behaviour among maize lines previously N. Nielson for providing rootworm eggs, and the determined to be susceptible or resistant in field anonymous reviewers. Mention of trade names or

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J. Appl. Entomol. 133 (2009) 21–32 ª 2008 The Authors 32 Journal compilation ª 2008 Blackwell Verlag, Berlin