(Coleoptera: Chrysomelidae) Larvae - PubAg - USDA

3 downloads 0 Views 816KB Size Report
increasing as a result of recent changes in the insect's distribution (Kiss et al. 2005) and biology (resistance to crop rotation) (Krysan and Miller 1986, Levine et ...
ECOLOGY AND BEHAVIOR

Localized Search Cues in Corn Roots for Western Corn Rootworm (Coleoptera: Chrysomelidae) Larvae E. J. BERNKLAU,1,2 L. B. BJOSTAD,2 L. N. MEIHLS,3 T. A. COUDRON,4 E. LIM,5 1,3 AND B. E. HIBBARD

J. Econ. Entomol. 102(2): 558Ð562 (2009)

ABSTRACT Cues that elicit a characteristic localized search behavior by neonate larvae of the western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), were extracted from living corn, Zea mays L., roots with acetone. Larvae were exposed to corn roots or to an acetone extract of corn roots and then transferred into a bioassay arena where their movements were tracked and recorded. After a 5-min exposure to live corn roots, larvae produced highly convoluted tracks that were indicative of a localized search behavior, and these distinctive tracks were also produced by larvae exposed to an acetone extract of corn roots. Larvae exposed to a Þlter paper control moved in relatively straight paths that were indicative of ranging behavior. Larval tracks were recorded by means of a videocamera and tracking software, and four parameters of movement were used to quantify the tracks: mean turn angle, mean meander, total distance, and maximum distance from origin. For every parameter measured, tracks resulting from exposure to the control were signiÞcantly different from tracks resulting from exposure to live corn roots and to all doses of the corn root extract. In a separate experiment, larvae exposed to corn root pieces or corn root juice exhibited the localized search behavior, but larvae exposed to oat root pieces and oat root juice (nonhost) exhibited ranging behavior. KEY WORDS Diabrotica virgifera virgifera, Zea mays, host search behavior, bioassay

Strnad and Dunn (1990) reported that the behavior of neonate western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), larvae immediately after removal from the roots of corn is distinctly different from the behavior of larvae removed from the roots of a nonhost plant. After exposure to host plant roots, a larva exhibits a localized search movement pattern, whereby it travels relatively slowly, with increased turns and path crossings and little overall area covered. In contrast, a larva exposed to the roots of a nonhost plant exhibits a broader ranging behavior characterized by an increased rate of travel, with fewer turns and path crossings and more area covered. Oyediran et al. (2004) used this difference in behavior to evaluate responses of northern corn rootworm, Diabrotica barberi Smith This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or recommendation for its use by the USDA, Colorado State University, University of Missouri, or Gyeongsang National University. 1 USDAÐARSÑMWA, BSPM Dept., Colorado State University, Ft. Collins, CO 80523. 2 Department of Bioagricultural Sciences & Pest Management, Colorado State University, Fort Collins, CO 80523. 3 205 Curtis Hall, University of Missouri, Columbia, MO 65211. 4 Biological Control of Insects Research Laboratory, 1503 S. Providence, RES. PK., Columbia, MO 65203. 5 Division of Applied Life Science (BK21 Program), Graduate School of Gyeongsang National University, 900 Gajwa-dong, Jinju 660-701 Korea.

& Lawrence, to alternate hosts. The Strnad and Dunn (1990) bioassay has recently been used to assess larval responses to Bacillus thuringiensis (Bt) corn as well as to native sources of resistance to western corn rootworm feeding damage (B.E.H., unpublished data). The goals of the current study were to use behavioral bioassays to determine whether the localized search cues were chemical versus physical in nature and to recover the chemical cues from corn roots. Materials and Methods Insects. Western corn rootworm eggs (nondiapausing strain) were obtained from the USDAÐARS Plant Genetics Research Unit in Columbia, MO, and from the USDAÐARS Laboratory, Brookings, SD. Survivorship of these laboratory rootworm colonies has been compared with that of wild-type insects in Þeld studies (Hibbard et al. 1999). The source insects were reared on corn plants grown in soil using methods described by Jackson (1985) and modiÞed by Hibbard and Bjostad (1988). The eggs were incubated in soil at 27⬚C, and larvae were used for bioassays within 16 h of hatching. Plant Tissues and Juices for Bioassays. For all test materials, seeds were washed in soapy water, rinsed well, and then germinated on moist blotter paper (Steel Blue, Anchor Paper Company, St. Paul, MN) at 26⬚C in a closed polyethylene tub (4 Ð5 d for corn and

April 2009

BERNKLAU ET AL.: ROOTWORM SEARCH CUES

5Ð 8 d for oat). Nine test materials were prepared: moist Þlter paper (negative control), corn root (positive control), oat root (negative control), corn root juice, oat root juice, corn root slices, oat root slices, corn root residue, and oat root residue. For corn juice, the roots were cut off the germinated seedlings and processed through a juicer (model 67800, Hamilton Beach/Proctor-Silex Inc., Southern Pines, NC). The resultant homogenate was frozen, then thawed and applied to Þlter paper (wetting the paper to saturation) just before bioassays. For the oat root juice, roots were crushed onto a piece of Þlter paper until the paper was saturated with the liquid. For the corn slice and oat slice, pieces of fresh roots were cut (1 cm, cross section) from germinated plants. The corn residue and oat residue test materials consisted of root pieces that had been crushed between two pieces of Þlter paper, rinsed thoroughly in distilled water and allowed to air dry. For bioassays, germinated seedlings, root slices or root residues were placed on moistened Þlter paper in a large (9-cm-diameter) petri dish. A single larva was placed directly on the test material, then removed after 5 min and transferred to the bioassay arena for recording of its tracks by procedures described below under Larval Bioassays. For the corn root juice and oat root juice (described previously), the Þlter paper was placed treatment-side down in the petri dish so the larva would not be exposed to any root particles remaining in the juice. A single larva was placed on the treated Þlter paper and then removed after 5 min and transferred to the bioassay arena. Extract of Corn Roots for Bioassays. Preliminary tests showed that the corn root compounds responsible for the localized search behavior are soluble in acetone. In contrast, extracts made with hexane or ethyl acetate were not active, and acetone was therefore used for all subsequent work. For this experiment, six materials were tested: corn root (positive control), Þlter paper (negative control), and four concentrations of corn root extract (0.02, 0.04, 0.08, and 0.16 g equivalents (GE). Dry, untreated seed corn (Pioneer 3730) was washed and germinated as described previously. Roots were cut from 50 germinated corn seeds, placed in a 100-ml Erlenmeyer ßask and acetone (100 ml) was added. After 3 d, the liquid was strained through a stainless steel kitchen strainer. The eluent was evaporated under a nitrogen stream to a concentration of 2 GE of root tissue (dry weight). Three more concentrations of the corn root extract were prepared by transferring 4 ml of the concentrated extract to each of three glass vials and concentrating the extract twofold (0.04 GE), four-fold (0.08 GE), or eight-fold (0.16 GE) under a gentle nitrogen stream. Filter paper (Whatman no. 4, catalog no. 1004-090, SpringÞeld Mill, Maidstone, Kent, England) was cut with a gasket punch (Blue Point 11 piece gasket punch set, Snap-On Tools Corp., Kenosha, WI) to make small disks (1.5 cm in diameter). The disks were washed by agitating them in distilled water for 8 min and then air-dried. An aliquot (30 ␮l) of corn extract was ap-

559

plied to each disk, and the disks were air-dried for 24 h to ensure removal of any solvent. Larval Bioassays. The bioassay arena consisted of a circle of Þlter paper (Whatman no. 2, 18.5 cm) that was thoroughly moistened with water. The Þlter paper was blotted brießy between two paper towels and laid ßat on a clean glass plate (30 by 30 cm, 2 mm in thickness). After being exposed to a test material (described above) for 5 min, a neonate larva was transferred with a soft, camelÕs-hair brush to the center of the bioassay arena. The movements of the larva were tracked and recorded (for 5 min) using a videocamera and the EthoVision software system (see below). Track Recording and Data Collection. Behavioral bioassays were recorded and analyzed using the Noldus EthoVision camera and software system (Pro Version 3.1, Noldus, Wageningen, Netherlands). Parameters of movement measured were maximum distance from the origin (the farthest distance traveled by the center of gravity of the larvae from the point of origin), mean turn angle (change in direction of movement between two samples), mean meander/tortuosity (change in direction of movement of an object relative to the distance [amount of turning per unit distance]), and total distance traveled. Total distance and maximum distance from origin indicate distance moved, and mean turn angle and mean meander describe the shape of the path traveled by the larvae. The following settings and Þlters were used in the EthoVision analysis program for the speciÞc parameter measurements: maximum distance from origin, downsize Þlter (1/25); mean turn angle, absolute setting, downsize Þlter (1/25); mean meander, absolute setting, downsize Þlter (1/25); total distance, downsize Þlter (1/25), and minimum distance Þlter (0.2 cm). Statistical Analysis. In total, 26 replicates were completed for each test material in the experiment with root pieces and root liquids and 30 replicates were completed for the experiment with the acetone extract of corn roots and a randomized complete block design was used for both experiments. Analysis of variance (ANOVA) was conducted for each parameter (maximum distance from origin, total distance, mean meander, and mean turn angle) with MINITAB (Addison-Wesley Publishing Co. Inc., Reading, MA). Fisher least signiÞcant difference (LSD) test was used for all a posteriori comparisons, with ␣ ⫽ 0.05. The combination of limited larval movement and the program Þlters occasionally resulted in no value being calculated for a particular parameter. These omissions are indicated by the unequal degrees of freedom reported in the statistical details for each analysis. Results Plant Tissues and Juices. In the behavioral bioassays, tracks of larvae exposed to corn root slices, corn root juice, and corn root residue were not signiÞcantly different from tracks of larvae exposed to germinating corn seedlings (Fig. 1). The mean turn angle for all corn root test materials was signiÞcantly greater (F ⫽

560

JOURNAL OF ECONOMIC ENTOMOLOGY

Vol. 102, no. 2

Fig. 1. Response parameters of movement tracks of neonate western corn rootworm larvae after 5-min exposure to the test plant parts and juices. (A) Mean turn angle. (B) Mean meander. (C) Maximum distance from origin. (D) Total distance.

8, 24.01; df ⫽ 8, 232; P ⬍ 0.0001) than the mean turn angle for the control and for all oat root test materials (Fig. 1A). The mean meander values for all corn root test materials were signiÞcantly greater (F ⫽ 8, 17.84; df ⫽ 8, 232; P ⬍ 0.0001) than the mean meander values for the control and for all oat root test materials (Fig. 1B). The maximum distance from origin and total distance for all corn root test materials, except the corn residue, were signiÞcantly lower (maximum distance from origin, F ⫽ 13.45; df ⫽ 8, 232; P ⬍ 0.0001; total distance, F ⫽ 15.12; df ⫽ 8, 230; P ⬍ 0.0001) than the maximum distance from origin and total distance for the control and for all oat root test materials (Fig. 1C and D). Bioassays with Acetone Extract of Corn Roots. Larvae exposed to corn roots and to an acetone extract of corn roots produced the distinctive localized search tracks, but larvae exposed to the control produced ranging tracks. For every parameter measured, the response to the control was signiÞcantly different than the response to corn roots and to all concentrations of corn root extract (mean turn angle, F ⫽ 12.35; df ⫽ 5, 173; P ⬍ 0.0001; mean meander, F ⫽ 11.58; df ⫽ 5, 173; P ⬍ 0.0001; maximum distance from origin, F ⫽ 14.72; df ⫽ 5, 173; P ⬍ 0.0001; total distance, F ⫽ 12.84; df ⫽ 5, 162; P ⬍ 0.0001). For each parameter, the concentration of extract that resulted in larval tracks most similar to the tracks of larvae exposed to corn roots was 0.08 GE (Fig. 2). Discussion In behavioral bioassays, larvae exposed for 5 min to an acetone extract of corn roots exhibited the localized search behavior Þrst described by Strnad and Dunn (1990). Quantitative analysis of four parameters of movement conÞrmed that tracks produced after exposure to the corn root extract were not signiÞ-

cantly different from tracks produced after exposure to corn roots, indicating that the compounds responsible for the localized search behavior were present in the extract. The localized search tracks were characterized by slow crawling, sharp and frequent turning, and a minimal distance moved away from the point of origin. When exposed to Þlter paper (control), larvae produced tracks that were indicative of a ranging search behavior (Strnad and Dunn 1990), characterized by fast crawling, few but wide turns, and a greater distance moved from the point of origin. The same differences in larval tracks were obtained in an experiment with root parts and root juices. Larvae exposed to corn root pieces or corn root juice created the localized search tracks, but larvae exposed to oat root parts and oat root juice (nonhost) did not. Host recognition by insects involves speciÞc behaviors of palpation (Harrison 1987, Chapman and Sword 1993), biting (Bernays and Chapman 1994, Eigenbrode and Espelie 1995), oviposition (Sta¨dler 1978, Eigenbrode and Espelie 1995, Schoonhoven et al. 1998, Mu¨ ller and Riederer 2005), or arrestant behavior (Heisswolf et al. 2007). To our knowledge, a speciÞc search behavior, after exposure to a substrate, has not been reported as a determinant of host recognition. In our experiments, as in previous studies (Strnad and Dunn 1990, Oyediran et al. 2004), rootworm larvae separated from a known host plant (or substrate treated with host plant materials) consistently exhibited localized search behavior that is distinctly different from ranging behavior exhibited by larvae separated from a known nonhost plant. In the soil environment, localized search behavior would likely result in a larva locating a resource in its immediate vicinity. Conversely, ranging behavior of larvae exposed to a nonhost plant (or substrate treated with non host plant materials) in the soil environment would result in the larvae eventually locating a more

April 2009

BERNKLAU ET AL.: ROOTWORM SEARCH CUES

561

Fig. 2. Response parameters of movement tracks of neonate western corn rootworm larvae after 5-min exposure to an acetone extract of corn roots. (A) Mean turn angle. (B) Mean meander. (C) Maximum distance from origin. (D) Total distance.

distant resource. It could be argued that a larva exhibits localized search behavior because it has recognized the plant (or substrate) as a host and a larva exhibits ranging behavior because it did not recognize the plant (or substrate) as a host. We previously identiÞed a speciÞc blend of compounds from germinating corn roots that elicits strong feeding by neonate western corn rootworm larvae (Bernklau and Bjostad 2008). We tested this feeding stimulant blend, as well as the individual compounds in the blend, for localized search activity, but preliminary experiments indicated that the chemical cues involved in feeding stimulation are not responsible for the localized search behavior. Our results suggest that the localized search behavior of western corn rootworm larvae is elicited by compounds of little or low volatility. In the current study, treated disks were allowed to dry overnight and, therefore, highly volatile compounds in the corn root extract would have evaporated and would not have been available to the larvae. Heisswolf et al. (2007) reported that host plant selection by C. canaliculata is based on the presence of speciÞc compounds rather than on the exact quantities or ratios of the compounds. In the current study, a range of corn root extract concentrations from 0.02 to 0.16 GE elicited robust responses, which suggests a qualitative, rather than quantitative, basis for the localized search behavior by western corn rootworm larvae. Strnad and Dunn (1990), who Þrst reported the localized search behavior, conducted their experiments by placing a glass plate over the arena and after the movement of neonate western corn rootworm larvae with a

felt pen on an acetate sheet. The EthoVision system has facilitated documentation and standardization of measurements from the Strnad and Dunn (1990) assay and allows for additional analysis of larval movements. Larval tracks can now be quantitatively described by speciÞc movement parameters, including meander (tortuosity), turn angle, distance traveled, and maximum distance from the point of origin. In our study, the parameter data, as collected and calculated using the EthoVision system, correlated to the shape of the track produced by a neonate rootworm larva (Fig. 3). Using this system, larval responses to host and nonhost materials can readily be differentiated using any one of the movement parameters, but the combination of all four parameters provides a more comprehensive evaluation of the larval tracks and provides an accurate correlation with the track proÞle. The economic impact of the western corn rootworm is in excess of $1 billion each year (Metcalf 1986) and is increasing as a result of recent changes in the insectÕs distribution (Kiss et al. 2005) and biology (resistance to crop rotation) (Krysan and Miller 1986, Levine et al. 2002). Although a variety of tools are still available for rootworm management, many of the current options, including crop rotation, controlling adults to prevent egg laying, granular and liquid soil insecticides, insecticidal seed treatments, and perhaps Bt corn, have issues that reduce their effectiveness. A better understanding of rootworm-host interactions could improve current control strategies or lead to the development of new practical applications. We demonstrated previously that the efÞcacy of insecticides can be increased when applied with corn infochemicals (Hibbard and Bjostad 1989, Hibbard et al. 1995, Bernklau and Bjostad 2005) and that

562

JOURNAL OF ECONOMIC ENTOMOLOGY

Fig. 3. Tracks of neonate western corn rootworm larvae after 5-min exposure to the test materials. (A) Filter paper. (B) Corn root. (C) Oat root. (D) Corn root extract (0.08 GE).

this concept may apply to localized search factors as well. Alternatively, it may be possible to reduce or eliminate key behaviorally-active compounds from corn roots through plant breeding and thereby protect corn roots from rootworm damage. Acknowledgments We thank Wilant Vangiessen of Noldus Information Technology, Inc., for assistance with the EthoVision data analysis. Elements of the discussion include very helpful suggestions provided by the anonymous reviewers of the manuscript. Funding for this project was provided by USDA Biotechnology Risk Assessment Award 2006-33522-17716 and Colorado Agricultural Experiment Station project number 622. E.L. was supported by a grant from the BK21 Program, the Ministry of Education and Human Resources Development, Korea.

References Cited Bernays, E. A., and R. F. Chapman. 1994. Host-plant selection by phytophagous insects. Chapman & Hall, New York. Bernklau, E. J., and L. B. Bjostad. 2005. Insecticide enhancement with feeding stimulants in corn for western corn rootworm larvae (Coleoptera: Chrysomelidae). J. Econ. Entomol. 98: 1150 Ð1156. Bernklau, E. J., and L. B. Bjostad. 2008. IdentiÞcation of feeding stimulants in corn roots for western corn rootworm larvae (Coleoptera: Chrysomelidae). J. Econ. Entomol. 101: 341Ð351. Chapman, R. F., and G. Sword. 1993. The importance of palpation in food selection by a polyphagous grasshopper (Orthoptera: Acrididae). J. Insect Behav. 6: 79 Ð91. Eigenbrode, S. D., and K. E. Espelie. 1995. Effects of plant epicuticular lipids on insect herbivores. Annu. Rev. Entomol. 40: 171Ð194.

Vol. 102, no. 2

Harrison, G. D. 1987. Host-plant discrimination and evolution of feeding preference in the Colorado potato beetle Leptinotarsa decemlineata. Physiol. Entomol. 12: 407Ð 415. Heisswolf, A., D. Gabler, E. Obermaier, and C. Mu¨ ller. 2007. Olfactory versus contact cues in host plant recognition of a monophagous chrysomelid beetle. J. Insect Behav. 20: 247Ð 266. Hibbard, B. E., and L. B. Bjostad. 1988. Behavioral responses of western corn rootworm larvae to volatile semiochemicals from corn seedlings. J. Chem. Ecol. 14: 1523Ð1539. Hibbard, B. E., and L. B. Bjostad. 1989. Corn semiochemicals and their effects on insecticide efÞcacy and insecticide repellency toward western corn rootworm larvae (Coleoptera: Chrysomelidae). J. Econ. Entomol. 82: 773Ð 781. Hibbard, B. E., L. L. Darrah, and B. D. Barry. 1999. Combining ability of resistance leads and identiÞcation of a new resistance source for western corn rootworm (Coleoptera: Chrysomelidae) larvae in corn. Maydica 44: 133Ð139. Hibbard, B. E., F. B. Peairs, S. D. Pilcher, M. E. Schroeder, D. K. Jewett, and L. B. Bjostad. 1995. Germinating corn extracts and 6-methoxy-2-benzoxazolinone: western corn rootworm (Coleoptera: Chrysomelidae) larval attractants evaluated with soil insecticides. J. Econ. Entomol. 88: 716Ð724. Jackson, J. J. 1985. Diabrotica spp., pp. 237Ð254. In P. Singh and R. F. Moore [eds.], Handbook of insect rearing. Elsevier, New York. Kiss, J., J. Komaromi, K. Bayar, C. R. Edwards, and I. HatalaZseller. 2005. Western corn rootworm (Diabrotica virgifera virgifera LeConte) amid crop rotation systems in Europe., pp. 189 Ð220. In S. Vidal, U. Kuhlmann, and R. Edwards [eds.], Western corn rootworm: ecology and management. CABI, Wallingford, United Kingdom. Krysan, J. L., and T. A. Miller. 1986. Methods for the study of pest Diabrotica. Springer, New York. Levine, E., J. L. Spencer, S. A. Isard, D. W. Onstad, and M. E. Gray. 2002. Adaptation of the western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), to crop rotation: evolution of a new strain in response to a cultural management practice. Am. Entomol. 48: 94 Ð107. Metcalf, E. R. 1986. Forward. In J. L. Krysan and T. A. Miller [eds.], Methods for the study of pest Diabrotica. Springer, New York. Mu¨ ller, C., and M. Riederer. 2005. Plant surface properties in chemical ecology. J. Chem. Ecol. 31: 2621Ð2651. Oyediran, I. O., B. E. Hibbard, and T. L. Clark. 2004. Selected grassy weeds as alternate hosts of the northern corn rootworm (Coleoptera: Chrysomelidae). Environ. Entomol. 33: 1497Ð1504. Schoonhoven, L. M., T. Jermy, and A. van Loon. 1998. Hostplant selection: how to Þnd a host plant., pp. 121Ð153. In L. M. Schoonhoven, T. Jermy, and A. van Loon [eds.], Insect-plant biology; from physiology to evolution. Chapman & Hall, London, United Kingdom. Sta¨ dler, E. 1978. Chemoreception of host plant chemicals by ovipositing females of Delia (Hylemya) brassicae. Entomol. Exp. Appl. 24: 711Ð720. Strnad, S. P., and P. E. Dunn. 1990. Host search behavior of neonate western corn rootworm (Diabrotica virgifera virgifera). J. Insect Physiol. 36: 201Ð205. Received 27 May 2008; accepted 30 October 2008.