Synthetic Feeding Stimulants Enhance Insecticide Activity Against Western Corn Rootworm Larvae, Diabrotica Virgifera Virgifera (Coleoptera: Chrysomelidae) E

J. Appl. Entomol.

Synthetic feeding stimulants enhance insecticide activity against western corn rootworm larvae, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) E. J. Bernklau1, L. B. Bjostad1 & B. E. Hibbard2

1 Department of Bioagricultural Sciences & Pest Management, Colorado State University, Fort Collins, CO, USA 2 United States Department of Agriculture, Agricultural Research Service, Plant Genetics Research Unit, University of Missouri, Columbia, MO, USA

Keywords Abstract Zea mays, insect behaviour, tefluthrin, thiamethoxam, western corn rootworm In bioassays, the addition of a synthetic feeding stimulant blend improved the efficacy of the insecticide thiamethoxam against neonate Correspondence western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae. In Elisa J. Bernklau (corresponding author), 4-h bioassays, the concentration of thiamethoxam required for 50% Department of Bioagricultural Sciences & Pest mortality (LC50) was 2800 pg/ml for the insecticide alone, but was Management, Colorado State University, Fort reduced to 0.0075 pg/ml when the synthetic feeding stimulant blend Collins, CO 80523, USA. E-mail: [email protected] was added (more than a 100,000-fold difference). Dilutions of thiamethoxam and tefluthrin (ranging from 0 pg/ml to 10 lg/ml) were tested in This article reports the results of research behavioural bioassays alone and in conjunction with a feeding stimulant only. Mention of a proprietary product does blend containing 30 : 4 : 4 : 1 mg/ml glucose : fructose : sucrose : lino- not constitute an endorsement or leic acid. Larvae were placed on insecticide-treated filter paper disks in recommendation for its use by the USDA or an arena that allowed them to leave the treated area at will. In 30-min Colorado State University. bioassays, and in 4-h bioassays, significantly more larvae fed on thia- Received: August 17, 2009; accepted: methoxam-treated disks when the feeding stimulant blend was present December 20, 2009. for every concentration of insecticide tested. Larvae fed readily on disks treated with tefluthrin when the feeding stimulants were added, but the doi: 10.1111/j.1439-0418.2009.01502.x feeding stimulant blend did not affect the activity of tefluthrin insecticide at any concentration tested.

work in certain areas. For this reason, it is worth- Introduction while to look towards the development of new strat- The western corn rootworm, Diabrotica virgifera virgif- egies that are specifically aimed at improving the era LeConte, continues to be a significant pest of efficacy of current products. corn (Zea mays L.) in the United States. Because of We previously demonstrated the presence of phag- recent changes in their distribution (now in Europe; ostimulation cues for western corn rootworm larvae Kiss et al. 2005) the impact of this pest is growing. in the roots of corn plants. In behavioural bioassays, While a variety of management tools are still avail- neonate larvae fed vigorously on paper disks treated able for growers, some of the current options includ- with liquid pressed from corn roots, or with a ing crop rotation (Gray et al. 2009), baits to control solvent extract of corn roots, but not on disks treated adults (Tollefson 1998), insecticidal seed treatments with distilled water (Bernklau and Bjostad 2005). In (Furlan et al. 2006), Bt corn (Lefko et al. 2008; Mei- the same study, the efficacy of a neonicotinoid insec- hls et al. 2008), adult sprays (Meinke et al. 1998) ticide (thiamethoxam; Syngenta, Inc., Basel, Switzer- and granular soil insecticides (Wright et al. 2000) land) was significantly increased when crude have issues reducing their effectiveness or do not corn root extract was added. The concentration of

J. Appl. Entomol. 135 (2011) 47–54 ª 2010 Blackwell Verlag, GmbH 47 Feeding stimulants enhance insecticide activity E. J. Bernklau, L. B. Bjostad and B. E. Hibbard thiamethoxam required for 50% larval mortality ticide alone and insecticide plus feeding stimulants) (after 24 h) was reduced from 1 ppm without corn were prepared in 50 : 50 acetone : water. extract to 0.01 ppm when the corn extract was Filter paper (Whatman No. 4; Springfield Mill, added, a 100-fold decrease. Maidstone, Kent, UK) was cut with a gasket punch We recently identified a specific feeding stimulant (Blue Point 11 piece gasket punch set; Snap-On blend from corn roots as a combination of simple Tools Corp., Kenosha, WI) to make small (1.5 cm in sugars (30 : 4 : 4 mg/ml glucose : fructose : sucrose diameter) or large (4 cm in diameter) disks. The in the corn root) plus either linoleic acid or oleic disks were washed by agitating them in distilled acid (0.3 mg/ml; Bernklau and Bjostad 2008). In this water for 8 min, and then air-dried. An aliquot study, we tested the synthetic blend (glucose : fruc- (30 ll for the small disks and 90 ll for the large tose : sucrose : linoleic acid, 30 : 4 : 4 :1 mg/ml) in disks) of a test blend was applied to a disk and the conjunction with two classes of insecticides to deter- disks were air-dried for 24 h to ensure removal of mine effects of the synthetic feeding stimulant blend any solvent. on the efficacy of the insecticides against western corn rootworm larvae. Larval bioassays Bioassays were conducted in a chamber designed to Materials and Methods maintain a humid and closed environment condu- cive to larval health and optimal feeding (Bernklau Insects and Bjostad 2005). A ring of blotter paper (Steel Western corn rootworm eggs (non-diapausing strain) Blue; Anchor Paper Company, St. Paul, MN, cut were shipped in soil-filled Petri dishes from the 1 cm wide and 5 cm in diameter) was moistened USDA-ARS Plant Genetics Research Unit in Colum- with distilled water, placed inside the inverted lid of bia, Missouri and from the USDA-ARS Laboratory in a Petri dish (5 cm diameter), and the Petri dish bot- Brookings, SD. In field studies, survivorship and tom was placed lightly (with the flat surface down) infestation levels of these colonies were not signifi- on top of the paper. The ring of blotter paper pro- cantly different from wild type insects (Hibbard et al. vided a 1 mm gap between the stacked halves of 1999). To our knowledge, there are no reports of the Petri dish. This gap maintained the humidity resistance in these colonies to either tefluthrin or and provided minimal space for larvae to feed and thiamethoxam. The source insects were reared on move freely. For bioassays, a treated filter paper corn plants grown in soil using methods described disk was placed, treated side down, in the bioassay by Jackson (1985) and modified by Hibbard and arena and re-moistened with distilled water (30 ll Bjostad (1988). The eggs were incubated in soil at for small disks and 90 ll for large disks). Water- 27C and larvae were used for bioassays within 16 h soluble fluorescent powder (D-282, UV-blue optical of hatching. brightener; DayGlo Color Corp., Cleveland, OH) was added to the distilled water to make a concentration of 0.1%. Preparation of insecticide/feeding stimulant blends Using a camel’s hair brush, neonate western corn The following sugars and free fatty acids were rootworm larvae (less than 16 h old, unfed) were purchased from Sigma-Aldrich, Inc., St. Paul, MN: placed in the centre of the moistened disk and the d-(+)-glucose (reagent grade), sucrose (standard), arena cover was replaced. One larva at a time was b-d-fructose (reagent grade), linoleic acid 99%, oleic placed on the small disk and five larvae were placed acid 99%. Technical grade tefluthrin (organophos- on the large disk. Larvae that left the treated disk phate insecticide) and thiamethoxam (neonicotinoid during the bioassay were not replaced onto the disk. insecticide) were obtained from Syngenta Crop After the allotted time, larvae were removed from Protection, Inc., Basel, Switzerland. the chamber and each larva was evaluated individu- In preliminary tests, feeding stimulant blends con- ally. All observations were collected as binomial data taining 1 mg/ml linoleic acid elicited slightly stronger (0 = no feeding, no staying, dead; 1 = feeding, stay- feeding than blends containing 0.3 mg/ml linoleic ing, alive). For staying behaviour, it was noted acid or any blends containing oleic acid and, there- whether or not the larva had stayed on the treated fore, 1 mg/ml linoleic acid was used in all subse- disk. For feeding evaluation, each larva was observed quent tests, in combination with 30 : 4 : 4 mg/ml under ultra-violet light (GE Model 23301-A with glucose : fructose : sucrose. All test solutions (insec- F15T8 Blacklamp bulb; General Electric Co., St.

48 J. Appl. Entomol. 135 (2011) 47–54 ª 2010 Blackwell Verlag, GmbH E. J. Bernklau, L. B. Bjostad and B. E. Hibbard Feeding stimulants enhance insecticide activity

Louis, MO) in a darkened room. The digestive tracts Statistical analysis of larvae that had fed on the filter paper substrate fluoresced (Bernklau and Bjostad 2008) and larvae For each experiment, mortality data were log trans- that had not fed did not fluoresce. formed (log(x + 0.001)) and the LC50 and 95% con- For mortality evaluation, each larva (regardless of fidence intervals were estimated (for the insecticide condition) was transferred onto the root of an indi- alone and the insecticide with feeding stimulants) vidual 3-day-old germinating corn seed. Each seed using the PROBIT procedure of SAS. The untrans- was placed in a small (5 cm in diametre), covered formed LC50 values are reported. Mortality, feeding Petri dish lined with moist blotter paper. After 24 h, and staying data (binomial) from all experiments larvae that survived the feeding bioassay had bur- were analyzed with PROC GENMOD of the SAS sta- rowed into the corn root and were recovered using a tistical package (SAS Institute, 2000) using a logit modified Berle`se technique (Bernklau and Bjostad link function and a binomial distribution. Individual 2005). Larvae that crawled completely out of the comparisons between data with and without feeding arena during the bioassay were immediately trans- stimulants at a particular insecticide dose were deter- ferred to a corn root. mined with the Least Square Means by using the LSMEANS statement within GENMOD (SAS Insti- tute 2000, Cary, NC). For the tefluthrin experiment, 30-Minute bioassays with thiamethoxam where the higher doses resulted in 100% mortality Six concentrations of thiamethoxam were tested and staying, data for one of the 25 replications was with and without feeding stimulants: 0 pg/ml, changed (from a ‘1’ to a ‘0’) for the GENMOD analy- 0.1 pg/ml, 10 pg/ml, 1 ng/ml, 100 ng/ml and 10 lg/ sis because the model requires variation to run. ml. The small filter paper disks (see Preparation of Insecticide/Feeding Stimulant Blends) were used for Results this experiment and larvae were evaluated after 30 min. Twenty replications were conducted (20 lar- 30-Minute bioassays with thiamethoxam vae total) for each insecticide concentration with and without feeding stimulants. In 30-min bioassays, the LC50 of thiamethoxam with feeding stimulants was 206 pg/ml (95% CI: 9.06– 3.06 · 103) and the LC without feeding stimulants 30-Minute bioassays with tefluthrin 50 was 84 500 pg/ml (95% CI: 1.83 · 103–3.57 · 108; Eight concentrations of tefluthrin were tested, with table 1). Significantly more (P < 0.05) western corn and without feeding stimulants: 0 pg/ml, 0.00001 pg/ml, 0.001 pg/ml, 0.1 pg/ml, 10 pg/ml, 1 ng/ml, 100 ng/ml and 10 lg/ml. The small filter paper disks (see Preparation of Insecticide/Feeding Table 1 LC50 of insecticides tested on neonate Diabrotica virgifera Stimulant Blends) were used for this experiment virgifera LeConte in feeding bioassays with and without feeding stimulants present and larvae were evaluated after 30 min. Twenty five replications were conducted (25 larvae total) for Feeding stimulants each insecticide concentration with and without Present (+) ) 1 2 feeding stimulants. Absent ( )LC50 95% CI

30 min bioassays with tefluthrin 4-Hour bioassays with thiamethoxam ) 9.98 1.23–8.71 · 10a )1 Five concentrations of thiamethoxam were tested + 3.38 3.56 · 10 –3.26 · 10a 30 min bioassays with thiamethoxam with and without feeding stimulants: 0 pg/ml, ) 84 500 1.83 · 103–3.57 · 108a 10 pg/ml, 1 ng/ml, 100 ng/ml and 10 lg/ml. The + 206 9.06–3.06 · 103a large filter paper disks (see Preparation of Insecti- 4 h bioassays with thiamethoxam cide/Feeding Stimulant Blends) were used for this ) 2800 3.95 · 102–1.68 · 104a experiment to allow larvae to wander and still main- + 0.00751 0.00–7.82b tain contact with the insecticide. Larvae were evalu- 1Concentration of insecticide expressed in pg/ml. ated after 4 h. Four replications were conducted (20 2For each experiment, confidence intervals followed by the same let- larvae total) for each insecticide concentration with ter indicates no significant difference in LC50 with and without the and without feeding stimulants. feeding stimulants present (PROBIT model, P < 0.05).

J. Appl. Entomol. 135 (2011) 47–54 ª 2010 Blackwell Verlag, GmbH 49 Feeding stimulants enhance insecticide activity E. J. Bernklau, L. B. Bjostad and B. E. Hibbard rootworm larvae fed on the treatment disks with P < 0.0001; 10 lg/ml: v2 = 10.23; d.f. = 1, 39; feeding stimulants than on disks without feeding P = 0.0014) (fig. 1b). For the 100 ng concentration of stimulants for every concentration of thiamethoxam thiamethoxam, larval mortality was significantly tested (0 pg/ml: v2 = 15.15; d.f. = 1, 39; P < 0.0001; higher when feeding stimulants were present (80% 0.1 pg/ml: v2 = 6.39; d.f. = 1, 39; P = 0.0115.; 10 pg/ mortality) than when feeding stimulants were not ml: v2 = 13.54; d.f. = 1, 39; P = 0.0002; 1 ng/ml: v2 = present (40% mortality) (v2 = 6.16; d.f. = 1, 39; 16.31; d.f. = 1, 39; P < 0.0001; 100 ng/ml: v2 = 11.14; P = 0.0130; fig. 1c). d.f. = 1, 39; P = 0.0008; 10 lg/ml: v2 = 7.59; d.f. = 1, 39; P < 0.0059) (fig. 1a). Significantly more larvae 30-Minute bioassays with tefluthrin stayed on the treatment disks with feeding stimulants than on disks without feeding stimulants for every In 30-min bioassays, the LC50 of tefluthrin with concentration of insecticide except 0 pg/ml (0 pg/ml: feeding stimulants was 3.38 pg/ml (95% CI: 2 2 )1 v = 3.52; d.f. = 1, 39; P = 0.0608; 0.1 pg/ml: v = 3.50 · 10 –3.36 · 10) and the LC50 without feeding 2.50; d.f. = 1, 39; P = 0.1142; 10 pg/ml: v2 = 12.18; stimulants was 9.98 pg/ml (95% CI: 1.23–8.71 · 10; d.f. = 1, 39; P = 0.0005; 1 ng/ml: v2 = 15.35; d.f. = 1, table 1). Significantly more larvae fed on the treat- 39; P < 0.0001; 100 ng/ml: v2 = 15.35; d.f. = 1, 39; ment disks with feeding stimulants than on disks without feeding stimulants for every concentration (a) (–) Feeding simulants (+) Feeding stimulants n = 20 of tefluthrin tested, except the 10 lg/ml, for which 100 there was no feeding either with or without feeding b b b 2 80 b stimulants (0 pg/ml: v = 17.46; d.f. = 1, 49; P < 0.0001; 0.00001 pg/ml: v2 = 16.13; d.f. = 1, 49; 60 b b P < 0.0001; 0.001 pg/ml: v2 = 19.93; d.f. = 1, 49; P < 40 0.0001; 0.1 pg/ml: v2 = 10.63; d.f. = 1, 49; P = 2 20 a a a )0.0011; 10 pg/ml: v = 4.80; d.f. = 1, 49; P = % larvae feeding % larvae a a a 2 0 0.0285; 1 ng/ml: v = 9.02; d.f. = 1, 49; P = 0.0027; 0 pg 0.1 pg 10 pg 1 ng 100 ng 10 µg 100 ng/ml: v2 = 4.80; d.f. = 1, 49; P = 0.0285; 10 ug/ ml: v2 = 0.00; d.f. = 1, 49; P = 1.00; fig. 2a). Signifi- (b) (–) Feeding simulants (+) Feeding stimulants n = 20 cantly more larvae stayed on the treatment disks 100 b b with feeding stimulants than on disks without feed- b 80 a a ing stimulants for the 0 and 0.001 pg/ml concentra- b 2 60 tions (0 pg/ml: v = 7.84; d.f. = 1, 49; P = 0.0051; a a 2 40 a 0.001 pg/ml: v = 3.90; d.f. = 1, 49; P = 0.0484; fig. 2b). There were no significant differences in lar- 20 a a % larvae staying % larvae a val mortality for any dose when feeding stimulants 0 0 pg 0.1 pg 10 pg 1 ng 100 ng 10 µg were present compared to when feeding stimulants were not present (fig. 2c). (c) (–) Feeding simulants (+) Feeding stimulants n = 20 100 a 4-Hour bioassays with thiamethoxam b a 80 In 4-h bioassays, the LC50 of thiamethoxam with 60 a feeding stimulants was 0.0075 pg/ml (95% CI: 0.00– a a 40 a 7.82) and the LC50 without feeding stimulants was a a a a 2,800 pg/ml (95% CI: 3.95 · 102–1.68 · 104; % larvae dead % larvae 20 a table 1). Significantly more larvae fed on the treat- 0 0 pg 0.1 pg 10 pg 1 ng 100 ng 10 µg ment disks with feeding stimulants than on disks Thiamethoxam concentration (per ml) without feeding stimulants for every concentration of thiamethoxam tested (0 pg/ml: v2 = 13.74; Fig. 1 Response of neonate western corn rootworm larvae in 30-min d.f. = 1, 39; P = 0.0002; 10 pg/ml: v2 = 16.44; bioassays with thiamethoxam or thiamethoxam plus feeding stimu- d.f. = 1, 39; P < 0.0001; 1 ng/ml: v2 = 11.68; d.f. = 1, lants. (a) Larvae feeding on treated disks. (b) Larvae staying on treated 39; P = 0.0006; 100 ng/ml: v2 = 11.14; d.f. = 1, 39; disks. (c) Larval mortality (evaluated after 24 h). Significant differences 2 (Least Square Means Test, P < 0.05) between an insecticide concentra- P = 0.0008; 10 lg/ml: v = 4.76; d.f. = 1, 39; tion with feeding stimulants and the same concentration of insecticide P = 0.0291; fig. 3a). Significantly more larvae stayed alone are indicated with different lower case letters. on the treatment disks with feeding stimulants than

50 J. Appl. Entomol. 135 (2011) 47–54 ª 2010 Blackwell Verlag, GmbH E. J. Bernklau, L. B. Bjostad and B. E. Hibbard Feeding stimulants enhance insecticide activity

(a) (–) Feeding stimulants (+) Feeding stimulants n = 25 (a) (–) Feeding stimulants (+) Feeding stimulants n=20 100 b 100 b b b b b 80 80 b b b 60 60 b b 40 a b 40 20 a % larvae feeding % larvae a a a a 20 a a a a a a 0 a a 0 0.00001 0.001 0.1 pg 10 pg 1 ng 100 ng 10 µg 0 pg pg 0pg 10pg 1ng 100ng 10 µg (b) (–) Feeding stimulants (+) Feeding stimulants n = 25 (b) a a a aa aa (–) Feeding stimulants (+) Feeding stimulants n=20 100 a a 100 b b aa a b 80 b b 80 60 b a a 60 a a 40 a a 40 a

% larvae staying % larvae 20

0 20 0 0.00001 0.001 0.1 pg 10 pg 1 ng 100 ng 10 µg a pg pg 0 0pg 10pg 1ng 100ng 10 µg (c) (–) Feeding stimulants (+) Feeding stimulants n = 25 a aa 100 a (c) (–) Feeding stimulants (+) Feeding stimulants n=20 a 80 100 a a a b a a 60 80

40 a a a 60 a a a a a aa % larvae dead % larvae 20 a 40 a 0 a a 0 0.00001 0.001 0.1 pg 10 pg 1 ng 100 ng 10 µg a pg pg 20 Tefluthrin concentration (per ml) 0 0pg 10pg 1ng 100ng 10 µg Fig. 2 Response of neonate western corn rootworm larvae in 30-min Thiamethoxam concentration (per ml) bioassays with tefluthrin or tefluthrin plus feeding stimulants. (a) Lar- vae feeding on treated disks. (b) Larvae staying on treated disks. (c) Fig. 3 Response of neonate western corn rootworm larvae in 4-h bio- Larval mortality (evaluated after 24 h). Significant differences (Least assays with thiamethoxam or thiamethoxam plus feeding stimulants. Square Means Test, P < 0.05) between an insecticide concentration (a) Larvae feeding on treated disks. (b) Larvae staying on treated disks. with feeding stimulants and the same concentration of insecticide (c) Larval mortality (evaluated after 24 h). Significant differences (Least alone are indicated with different lower case letters. Square Means Test, P < 0.05) between an insecticide concentration with feeding stimulants and the same concentration of insecticide alone are indicated with different lower case letters. on disks without feeding stimulants for every concentration of thiamethoxam except 1 ng/ml (0 pg/ ml: v2 = 9.98; d.f. = 1, 39; P = 0.0016; 10 pg/ml: of thiamethoxam (0 pg/ml: v2 = 0.47, d.f. = 1, 39, v2 = 6.92; d.f. = 1, 39; P = 0.0085; 1 ng/ml: P = 0.4917; 1 ng/ml: v2 = 3.60, d.f. = 1, 39, P = v2 = 0.00; d.f. = 1, 39; P = 1.0; 100 ng/ml: v2 = 7.55; 0.0577; 100 ng/ml: v2 = 0.35, d.f. = 1, 39, P = d.f. = 1, 39; P = 0.006; 10 lg/ml: v2 = 7.59; d.f. = 1, 0.5557; 10 lg/ml: v2 = 0.35, d.f. = 1, 39, P = 0.5557; 39; P = 0.0059; fig. 3b). fig. 3c). For the 10 pg/ml concentration of thiamethoxam, larval mortality was significantly higher when feed- Discussion ing stimulants were present than when feeding stimulants were not present (10 pg/ml: v2 = 13.82, In this study, the addition of a synthetic feeding stim- d.f. = 1, 39, P = 0.0002). There were no significant ulant blend (containing glucose, fructose, sucrose and differences in mortality for any other concentrations linoleic acid) improved the efficacy of the insecticide

J. Appl. Entomol. 135 (2011) 47–54 ª 2010 Blackwell Verlag, GmbH 51 Feeding stimulants enhance insecticide activity E. J. Bernklau, L. B. Bjostad and B. E. Hibbard thiamethoxam against western corn rootworm larvae. toms commonly seen in insects as a result of nicoti- In 4-h bioassays, the concentration of thiamethoxam noid poisoning (Nauen et al. 2003). However, the required for 50% mortality (LC50) was 2800 pg/ml for fact that most larvae stayed on the insecticide-trea- the insecticide alone, but was reduced to 0.0075 pg/ ted disks when the feeding stimulants were present ml when the feeding stimulant blend was added suggests, instead, that thimethoxam has a mild (more than a 100 000-fold difference). This reduction repellant effect that is overcome by the feeding stim- in the LC50 with the addition of feeding stimulants is ulants. Other studies have shown that thiamethox- consistent with the results of previous experiments in am is not repellent to whiteflies, B. tabaci which larvae fed on a solvent extract of germinating (Gennadius) (Mason et al. 2000) or wireworms, corn (Bernklau and Bjostad 2005). The decrease in Agriotes obscurus (L.) (Van Herk et al. 2008). the LC50 is reflected in the concentration response Although the synthetic blend elicited robust feed- whereby significantly higher mortality was observed ing by neonate larvae on tefluthrin, the feeding when feeding stimulants were present for the lowest stimulants had no effect on the efficacy of this insec- concentration of thiamethoxam tested. For the ticide. Although tefluthrin has been reported to 30-min bioassay with thiamethoxam, we did not cause short-lived feeding inhibition in larvae of the observe a significant difference in the LC50 with or southern corn rootworm (Diabrotica undecimpunctata without the feeding stimulant blend present howardi Barber) at both lethal and sublethal concen- (although there was a single significant difference in trations (Michaelides 1995; Michaelides and Wright mortality at the 100 ng dose). 1997), we observed no evidence of antifeedant Thiamethoxam is a neonicotinoid insecticide that effects of tefluthrin on neonate western corn root- acts by binding to nicotinic acetylcholine receptors. It worm larvae. Larvae fed readily (as high as 90% has been shown to have contact, stomach and feeding) on disks treated with tefluthrin and feeding systemic activity (Maienfisch et al. 2001) and is stimulants, and even larvae that exhibited obvious equally effective whether ingested or applied topi- toxicity (writhing and curling) were observed chew- cally (Harrewign et al. 1998). Mason et al. (2000) ing on the feeding stimulant-treated disks. demonstrated that thiamethoxam does not have an Tefluthrin is a pyrethroid insecticide and its pri- anti-feedant effect on the whitefly, Bemisia tabaci mary mode of action is on the nervous system (Gennadius), and the results of our study are consis- where it interferes with the sodium channel and dis- tent with this previous report. In the 30-min and the turbs the function of neurons. It has a ‘knock down’ 4-h bioassays, 50–90% of larvae fed upon the treat- effect and provides good toxicity upon contact, but ment disks when the feeding stimulants were pres- has been shown to be less toxic when ingested ent, indicating that thiamethoxam did not have an (Michaelides et al. 1997). In this study, rootworm inhibitory effect on rootworm feeding. larvae exhibited ‘knock down’ at a concentration of During the 30-min test with thiamethoxam, 85% 10 pg/ml, but mortality for this dose (after 24 h) was of larvae stayed on the 100 ng/ml concentration of no greater than for the control, because the affected insecticide when feeding stimulants were present. larvae recovered after being removed from the trea- This staying behaviour was likely responsible for the ted disk and were subsequently extracted alive from high mortality (80%) that resulted from this treat- inside the corn roots. A few larvae exhibited mild ment because, for the same concentration of insecti- toxic effects (lethargy) even at the lowest concentra- cide alone, only 15% of larvae stayed, resulting in tion tested (0.00001 pg/ml) and most larvae were only 40% mortality. Most larvae (at least 60%) rendered immobile when placed on disks with stayed on the disks treated with thiamethoxam and 0.1 pg tefluthrin. Sublethal doses of tefluthrin feeding stimulants for every concentration of insecti- have been reported to cause curling, writhing and cide tested. When feeding stimulants were not regurgitation in insect larvae, but these symptoms present, 40–45% of larvae stayed on disks with low commonly diminish when exposure to tefluthrin concentrations of insecticide (0–0.1 pg/ml), but with ends (Michaelides et al. 1997). In preliminary exper- concentrations of 10 pg/ml or higher, most of the iments, moribund larvae that had been feeding on larvae (65–95%) quickly crawled off the disks when tefluthrin-treated disks recovered completely when feeding stimulants were not present. Rapid move- removed from the treated disks and placed on corn ment away from thiamethoxam alone was observed roots. In this study, we transferred all larvae (regard- in a previous study with neonate western corn root- less of condition) to the roots of corn and reported worm larvae (Bernklau and Bjostad 2005). This larvae as dead only if they were not recovered alive activity could be a manifestation of excitatory symp- from the corn roots after 24 h.

52 J. Appl. Entomol. 135 (2011) 47–54 ª 2010 Blackwell Verlag, GmbH E. J. Bernklau, L. B. Bjostad and B. E. Hibbard Feeding stimulants enhance insecticide activity

In this study, 50% or more larval mortality was Bernklau EJ, Bjostad LB, 2008. Identification of feeding achieved with low (10 pg/ml) concentrations of thia- stimulants in corn roots for western corn rootworm methoxam when larvae ingested the insecticide. This larvae (Coleoptera: Chrysomelidae). J. Econ. Entomol. suggests the possibility of combining feeding stimu- 101, 341–351. lants and thiamethoxam to form a toxic bait for Furlan L, Canzi S, Di Bernardo A, Edwards CR, 2006. rootworm larvae similar to adult baits with cucurbi- The ineffectiveness of insecticide seed coatings and tacins incorporated as feeding stimulants that are planting-time soil insecticides as Diabrotica virgifera used in areawide management programs (Tollefson virgifera LeConte population suppressors. J. Appl. 1998; Pingel et al. 2001). In field trials, hand-made Entomol. 130, 485–490. Gray ME, Sappington TW, Miller NG, Moeser J, Bohn granules coated with thiamethoxam (2% ai) and MO, 2009. Adaptation and invasiveness of western feeding stimulants (crude extract of germinating corn rootworm: intensifying research on a worsening corn) were effective in preventing damage to corn pest. Ann. Rev. Entomol. 54, 303–321. roots, but the feeding damage was not significantly Harrewign P, de Kogel WJ, Piron PGM, 1998. CGA different (P < 0.05) from damage to roots treated 293¢343 effects on Myzus persicae: electrical penetration with insecticide granules without feeding stimulants graph studies and effect on non-persistent virus (Bernklau 2003). The concentration of thiamethox- transmission. BCPC Conference Proceedings. Wagenin- am in the granules was relatively high (2% active gen Univ. and Research Center Publications ingredient) compared with the effective concentra- (Netherlands), pp. 813–818. tions in this study (10–100 ng/ml), suggesting that Hibbard BE, Bjostad LB, 1988. Behavioral responses of further soil or field tests should be conducted using western corn rootworm larvae to volatile semiochemi- much lower concentrations of thiamethoxam. The cals from corn seedlings. J. Chem. Ecol. 14, 1523– earlier field experiment was conducted using a crude 1539. (acetone) extract of germinating corn as a source of Hibbard BE, Barry BD, Darrah LL, Jackson JJ, Chandler the feeding stimulants (Bernklau 2003). With the LD, French LK, Mihm JA, 1999. Controlled field infes- recent identification of the active components tations with western corn rootworm (Coleoptera: (Bernklau and Bjostad 2008), it is now possible to Chrysomelidae) eggs in Missouri: effects of egg strains, prepare treatments that contain the proportions of infestation dates, and infestation levels on corn root feeding stimulant components that provide the damage. J. Kan. Entomol. Soc. 72, 214–221. strongest feeding and then add materials to optimize Jackson JJ 1985. Diabrotica spp. In: Handbook of insect longevity in the soil. rearing. Ed. by Singh P, Moore RF, Elsevier, New York, 237–254. Kiss J, Komaromi J, Bayar K, Edwards CR, Hatala-Zseller Acknowledgements I 2005. Western corn rootworm (Diabrotica virgifera virgifera LeConte) amid crop rotation systems in We would like to thank Catherine E. Savinelli, Syn- Europe. In: Western corn rootworm: ecology and genta Crop Protection for providing the technical management. Ed. by Vidal S, Kuhlmann U, Edwards R, thiamethoxam and tefluthrin. We thank Mark CABI, Wallingford, UK, 189–220. Ellersieck, Agricultural Experiment Station Statisti- Lefko SA, Nowatzki TM, Thompson SD, Binning RR, cian, University of Missouri, for assistance in analyz- Pascual MA, Peters ML, Simbro EJ, Stanley BH, 2008. ing the data. Funding for this project was provided Characterizing laboratory colonies of western corn by the Biotechnology Risk Assessment Award No. rootworm (Coleoptera: Chrysomelidae) selected for 2006-33522-17716 and the Colorado Agricultural survival on maize containing event DAS-59122-7. Experiment Station project number 622. J. Appl. Entomol. 132, 189–204. Maienfisch P, Angst M, Brandl F, Fischer W, Hofer D, Kayser H, Kobel W, Rindlisbacher A, Senn R, 2001. References Chemistry and biology of thiamethoxam: a second gen- Bernklau EJ, 2003.Behavioral effects of carbon dioxide eration neonicotinoid. Pest Manag. Sci. 57, 906–913. on western corn rootworm and subterranean termites Mason G, Rancati M, Bosco D, 2000. The effect of thia- with implications for pest management. Ph.D. Disserta- methoxam, a second generation neonicotinoid insecti- tion. Colorado State University, Fort Collins, CO. cide, in preventing transmission of tomato yellow leaf Bernklau EJ, Bjostad LB, 2005. Insecticide enhancement curl geminivirus (TYLCV) by the whitefly Bemisia tabaci with feeding stimulants in corn for western corn root- (Gennadius). Crop Prot 19, 473–479. worm larvae (Coleoptera: Chrysomelidae). J. Econ. Meihls LN, Higdon ML, Siegfried BD, Spencer TA, Miller Entomol. 98, 1150–1156. NJ, Sappington TW, Ellersieck MR, Hibbard BE, 2008.

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Increased survival of western corn rootworm on cursor converted to clothianidin in insects and plants. transgenic corn within three generations of on-plant Pestic. Biochem. Phys. 76, 55–69. greenhouse selection. Proc. Nat. Acad. Sci. 105, 19177– Pingel RL, Behle RW, McGuire MR, Shasha BS, 2001. 19182. Improvement of the residual activity of a cucurbitacin- Meinke LJ, Siegfried BD, Wright RJ, Chandler LD, 1998. based adult corn rootworm (Coleoptera: Chrysomeli- Adult susceptibility of Nebraska western corn dae) insecticide. J. Entomol. Sci. 36, 416–425. rootworm (Coleoptera: Chrysomelidae) populations to SAS Institute, 2000. SAS 9.1.3 Help and Documentation. selected insecticides. J. Econ. Entomol. 91, 594– SAS Institute, Cary, NC. 600. Tollefson JJ, 1998. Rootworm areawide management Michaelides PK, 1995. Toxic effects of soil insecticides on program in Iowa. J. Agric. and Urban Entomol. 15, immature stages of the southern corn rootworm. Ph.D. 351–357. University of London, London. Van Herk WG, Vernon RS, Roitberg BD, 2008. Michaelides PK, Wright DJ, 1997. Insecticide penetra- Repellency of a wireworm, Agriotes obscurus (Coleoptera tion and symptomology studies on larvae of Diabrotica Elateridae), on exposure to synthetic insecticides in a undecimpunctata howardi (Barber). Pestic. Sci. 49, 353– soil-less bioassay. Environ. Entomol. 37, 534–545. 361. Wright DJ, Scharf ME, Meinke LJ, Zhou XG, Siegfried Michaelides PK, Cleverly AL, Wright DJ, 1997. Sub-lethal BD, Chandler LD, 2000. Larval susceptibility of an effects of tefluthrin on Diabrotica undecimpunctata howar- insecticide-resistant western corn rootworm (Cole- di, Barber: plant protection and larval development. optera: Chrysomelidae) population to soil insecticides: Crop Prot. 16, 423–429. Laboratory bioassays, assays of detoxification Nauen R, Ebbinghouse-Kintscher U, Salgado V, Kauss- enzymes, and field performance. J. Econ. Entomol. mann M, 2003. Thiamethoxam is a neonicotinoid pre- 93, 7–13.

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