Journal of Invertebrate Pathology 109 (2012) 34–40

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Journal of Invertebrate Pathology

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Directional movement of entomopathogenic nematodes in response to electricalfield: effects of species, magnitude of voltage, and infective juvenile age

David I. Shapiro-Ilan a,⇑, Edwin E. Lewis b, James F. Campbell c, Daniel B. Kim-Shapiro d

a USDA-ARS, Southeastern Fruit and Tree Nut Research Lab, 21 Dunbar Road, Byron, GA 31008, United Statesb UC Davis, Department of Nematology, Department of Entomology, University of California, Davis, CA 95616, United Statesc USDA-ARS, Center for Grain and Animal Health, Manhattan, KS 66502, United Statesd Wake Forest University, Department of Physics, Winston-Salem, NC 27109, United States

a r t i c l e i n f o a b s t r a c t

Article history:Received 1 July 2011Accepted 6 September 2011Available online 12 September 2011

Keywords:ElectricityEntomopathogenic nematodeHeterorhabditisHost findingSteinernema

0022-2011/$ - see front matter Published by Elsevierdoi:10.1016/j.jip.2011.09.004

⇑ Corresponding author. Fax: +1 478 956 2929.E-mail address: David.Shapiro@ars.usda.gov (D.I. S

Entomopathogenic nematodes respond to a variety of stimuli when foraging. Previously, we reported adirectional response to electrical fields for two entomopathogenic nematode species; specifically, whenelectrical fields were generated on agar plates Steinernema glaseri (a nematode that utilizes a cruiser-typeforaging strategy) moved to a higher electric potential, whereas Steinernema carpocapsae, an ambush-typeforager, moved to a lower potential. Thus, we hypothesized that entomopathogenic nematode directionalresponse to electrical fields varies among species, and may be related to foraging strategy. In this study,we tested the hypothesis by comparing directional response among seven additional nematode species:Heterorhabditis bacteriophora, Heterorhabditis georgiana, Heterorhabditis indica, Heterorhabditis megidis, Ste-inernema feltiae, Steinernema riobrave, and Steinernema siamkayai. S. carpocapsae and S. glaseri were alsoincluded as positive controls. Heterorhabditids tend toward cruiser foraging approaches whereas S. sia-mkayai is an ambusher and S. feltiae and S. riobrave are intermediate. Additionally, we determined thelowest voltage that would elicit a directional response (tested in S. feltiae and S. carpocapsae), and weinvestigated the impact of nematode age on response to electrical field in S. carpocapsae. In the experi-ment measuring diversity of response among species, we did not detect any response to electrical fieldsamong the heterorhabditids except for H. georgiana, which moved to a higher electrical potential; S. gla-seri and S. riobrave also moved to a higher potential, whereas S. carpocapsae, S. feltiae, and S. siamkayaimoved to a lower potential. Overall our hypothesis that foraging strategy can predict directional responsewas supported (in the nematodes that exhibited a response). The lowest electric potential that elicited aresponse was 0.1 V, which is comparable to electrical potential associated with some insects and plantroots. The level of response to electrical potential diminished with nematode age. These results expandour knowledge of electrical fields as cues that may be used by entomopathogenic nematodes for host-finding or other aspects of navigation in the soil.

Published by Elsevier Inc.

1. Introduction

Entomopathogenic nematodes (EPNs) in the genera Heteror-habditis and Steinernema are important regulators of natural insectpopulations and are used as biocontrol agents in a variety of urbanand agricultural systems (Shapiro-Ilan et al., 2002; Grewal et al.,2005; Stuart et al., 2006). These nematodes have a mutualisticsymbiosis with a bacterium (Xenorhabdus spp. and Photorhabdusspp. for steinernematids and heterorhabditids, respectively) (Poin-ar, 1990). Infective juveniles (IJs), the only free-living stage, enterhosts through natural openings (mouth, anus, and spiracles), orin some cases, through the cuticle. After entering the host’s

Inc.

hapiro-Ilan).

hemocoel, nematodes release their bacterial symbionts, whichare primarily responsible for killing the host within 24–48 h,defending against secondary invaders, and providing the nema-todes with nutrition (Dowds and Peters, 2002). The nematodesmolt and, depending on the suitability of host resources, completeup to three generations within the host after which IJs exit thecadaver to find new hosts (Kaya and Gaugler, 1993).

Elucidation of mechanisms in host-finding and foraging strate-gies is needed to enhance our ability to use EPNs in biocontrol pro-grams and understand their role in nature. Foraging strategiesamong EPN species vary along a continuum between ambushers,which generally sit and wait for a passing host, and cruisers thatactively search for hosts. EPNs can exhibit a combination of thesebehaviors to locate hosts and although some species exhibitprimarily ambusher-type behaviors and others are mainly cruisertypes, others are considered intermediate in their foraging

Table 1Statistical results from an experiment investigating the directional response ofvarious entomopathogenic nematode species to electrical fields.

Treatmenta Statistic Value df P

Hb with e- t 0.74 23 0.4679Hb without e- t 0.70 23 0.4895Hb F 1.50 1, 44 0.2269Hg with e- t 3.26 23 0.0034Hg without e- t 0.32 23 0.7494Hg F 5.57 1, 44 0.0228Hi with e- t 0.32 23 0.7492Hi without e- t 2.02 23 0.0556Hi F 0.63 1, 44 0.4307Hm with e- t 0.80 23 0.4293Hm without e- t 1.26 23 0.2212Hm F 2.24 1, 44 0.1416Sc with e- t 5.58 23 0.0001Sc without e- t 0.00 23 1.0000Sc F 83.41 1, 44 0.0001Sf with e- t 5.40 23 0.0001Sf without e- t 0.63 23 0.5336Sf F 82.04 1, 44 0.0001Sg with e- t 6.30 23 0.0001Sg without e- t 1.63 23 0.1165Sg F 104.84 1, 44 0.0001Sr with e- t 3.04 23 0.0058Sr without e- t 0.97 23 0.3410Sr F 6.58 1, 44 0.0138Ss with e- t 6.69 23 0.0001Ss without e- t 0.43 23 0.4293Ss F 47.82 1, 44 0.0001

a e- = electrical field; Hb = Heterorhabditis bacteriophora; Hg = H. georgiana;Hi = H. indica; Hm = H. megidis; Sc = Steinernema carpocapsae; Sf = S. feltiae;Sg = S. glaseri; Ss = S. siamkayai; Sr = S. riobrave.

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behavior (Campbell and Kaya, 1999; Lewis, 2002; Kruitbos et al.,2010). Additionally, IJs respond to a variety of stimuli such asCO2 (Lewis et al., 1993; Lewis, 2002), vibration (Torr et al. 2004),temperature (Burman and Pye, 1980; Byers and Poinar, 1982)and chemical compounds (Pye and Burman, 1981; Shapiro et al.,2000), presumably to increase their chances of finding a host. Re-sponse to stimuli may also be useful in other aspects of fitness suchas sensing and avoiding adverse environmental conditions, e.g.,moisture and temperature extremes (Ishibashi and Kondo, 1990).

A number of animals use electrical signals to find host or prey,e.g., moles and certain fish (Gould et al., 1993; von der Emde andBleckmann, 1998). Additionally, certain plant and animal parasitic,and free living nematodes respond to electrical fields, and in the caseof parasitic nematodes, the response has been linked to foragingbehavior (Sukul et al., 1975; Croll and Matthews, 1977; Viglierchioand Yu, 1983; Riga, 2004). Recently, EPNs were also found to responddirectionally to electrical fields (Shapiro-Ilan et al., 2009a). When anelectric potential was generated across an agar plate Steinernemacarpocapsae (Weiser) (an ambusher) moved toward a lower electricpotential and Steinernema glaseri (a cruiser) moved in the oppositedirection. Given that the two nematodes tested showed differing re-sponses we hypothesized that directional response to electricalfields varies across EPN species and the response may be related toforaging strategy. Thus, the primary objective of this study was totest that hypothesis by investigating the response to electrical fieldin a broader array of EPN species. Specifically, we measured direc-tional response to electrical fields in seven nematode species thathad not been tested previously: Heterorhabditis bacteriophora Poin-ar, Heterorhabditis georgiana Nguyen, Shapiro-Ilan and Mbata, Het-erorhabditis indica Poinar, Karunkar and David, Heterorhabditismegidis Poinar, Jackson and Klein, Steinernema feltiae (Filipjev), Ste-inernema riobrave Cabanillas, Poinar and Raulston, and Steinernemasiamkayai Stock, Somsook and Reid. S. carpocapsae and S. glaseri werealso included as positive controls. Heterorhabditids tend towardcruiser foraging approaches whereas S. siamkayai is an ambusherand S. feltiae and S. riobrave are intermediate (Lewis, 2002;Shapiro-Ilan et al., 2009b). In addition to the characterization of spe-cies-specific response, our objectives were to estimate the lowestpotential that would elicit a directional response, and to determineif response is affected by IJ age.

2. Materials and Methods

2.1. Nematodes and experimental conditions

All nematodes (H. bacteriophora [Hb strain], H. georgiana [Keshastrain], H. indica [Hom-1 strain], H. megidis [UK211 strain], S. carpo-capsae [All strain], S. feltiae [SN strain], S. glaseri [NJ43 strain]),S. riobrave [355 strain], and S. siamkayai) were cultured in commer-cially obtained last instar Galleria mellonella (L.) according to proce-dures described in Kaya and Stock (1997). Unless indicatedotherwise (see below), the nematodes were stored at 13 �C for lessthan two weeks prior to experimentation. All experiments wereconducted at room temperature (approximately 23–25 �C).

2.2. Diversity of response to electrical fields among EPN species

The measurement of directional response was based on proce-dures described by Shapiro-Ilan et al. (2009a). Experiments wereconducted in plastic Petri dishes (90 mm diam.) with 2% agar (AgarN.F., ICN Biomedicals, Inc., Aurora, OH) at approximately 1 cmdepth. Sodium Chloride (0.01%) was incorporated into the agar tofacilitate conductance of electricity (Sukul et al., 1975); the low saltconcentration we used was not adverse to steinernematids(Thurston et al. 1994). An electric field was generated across the agar

plate using an electrophoresis power supply (model EC 105, E–CApparatus Corporation, Holbrook, NY). An electrode from the cath-ode (negative on the power supply) with copper wire (0.5 mmdiam.) was inserted vertically on one side of dish (approximately1 mm from edge), and the other electrode from the anode was placedon the opposite side. Approximately 4000 IJs were concentrated byvacuum filter and placed in the middle of the agar dish, except2000 IJs were used for S. glaseri and S. feltiae because these specieswere more dispersive (Shapiro-Ilan et al., 2009a; personal observa-tion). The power supply was set at 36 V (3.0 milliamps) and appliedfor 30 min, at which time the number of IJs within a 2.5 cm semicir-cle around the cathode or anode was counted. Controls consisted ofplates treated identically but with no applied voltage. Movement to-ward the cathode indicates a response toward a lower potentialwhereas movement to the anode indicates a response in the direc-tion of a higher potential. The experiments were conducted sepa-rately for each of the nine EPN species tested (see list of speciesand strains above). There were eight replicates for each speciesand each experiment was conducted three times (resulting in 24plates with voltage and 24 control plates for each species).

2.3. Estimation of the lowest voltage eliciting a response

Experiments to determine the lowest magnitude of voltageneeded to elicit a directional response were conducted with S.carpocapsae and S. feltiae. The methods to determine directional re-sponse were as described above. Two experiments were conductedfor each species, one experiment tested IJ response when the low-est voltage applied to the agar was 1.8 V, and the other was con-ducted using 0.9 V as the lowest voltage applied. These voltagelevels were chosen based on preliminary trials indicating that thelowest voltage response would be below 3 V (unpublished data).In each experiment, in addition to the 1.8 or 0.9 V plate, one platemeasured IJ directional response when no voltage was applied (a

36 D.I. Shapiro-Ilan et al. / Journal of Invertebrate Pathology 109 (2012) 34–40

negative control) and one plate measured response when 36 V wasapplied (positive control). Each species was tested separately.There were two plates for each voltage level (run simultaneously)and the experiment was repeated six times (hence 12 plates pertreatment within each test).

Due to the resistance and dielectric constant of the material, theactual potential through the agar plates is expected to be less thanthe applied voltage. A voltmeter (Fluke 87 True RMS Multimeter,Fluke Corp., Everett, WA) was used to determine the actual poten-tial through the agar when the lowest voltage was applied (0.9 V).Measurements were taken 1.25 cm and 3.25 cm from the edge ofthe agar dish (three replicates each).

2.4. The effects of IJ age on response to electrical fields

Experiments were conducted to determine if sensitivity to elec-trical field is affected by IJ age. The experiments used S. carpocapsae

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Fig. 1. Mean percentage (± SEM) of infective juvenile nematodes (IJs) that moved fromwhen voltage (36 V) was applied (‘‘with voltage’’, i.e., the treatment), or no voltage was aHeterorhabditis georgiana, Hi = Heterorhabditis indica, Hm = Heterorhabditis megidis, ScSteinernema riobrave, Ss = Steinernema siamkayai. ⁄ indicates a statistically significant difplate (paired t-test, a = 0.05); different letters above bars indicate statistically significan

as a model because this nematode was one of the species that con-sistently showed a strong response to electrical fields. Four exper-iments were conducted containing nematodes that were stored at10 �C for 7, 14, 21, or 28 d. All tests compare aged IJs directly tofresh IJs (62-d-old). Assays testing directional response on agarplates followed procedures described above and were conductedwith 0, 1.8 V and 36 V. Assays were repeated 12–18 times for eachage interval and voltage level.

2.5. Statistical analyses

For all treatments and controls, preferential movement of nem-atodes to one side of the dish (i.e., a directional response) wasdetermined through paired t-tests comparing the number of IJsat the anode side versus cathode side (SAS, 2002; Shapiro-Ilanet al., 2009a; a = 0.05). Additionally, to compare the level ofresponse among treatments and controls, the percentage of

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the center of an agar plate to the cathode side of the plate during a 30 min intervalpplied (‘‘without voltage’’, i.e., the control). Hb = Heterorhabditis bacteriophora, Hg == Steinernema carpocapsae, Sf = Steinernema feltiae, Sg = Steinernema glaseri, Sr =

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Fig. 2. Mean percentage (± SEM) of S. carpocapsae infective juvenile nematodes (IJs)that moved from the center of an agar plate to the cathode side of the plate during a30 min interval when varying voltage levels were applied. ⁄ indicates a statisticallysignificant difference between the number of nematodes at the cathode versusanode side of the plate (paired t-test, a = 0.05); different letters above bars indicatestatistically significant differences among the treatments (ANOVA and SNK test,a = 0.05).

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Fig. 3. Mean percentage (± SEM) of S. feltiae infective juvenile nematodes (IJs) thatmoved from the center of an agar plate to the cathode side of the plate during a30 min interval when varying voltage levels were applied. ⁄ indicates a statisticallysignificant difference between the number of nematodes at the cathode versusanode side of the plate (paired t-test, a = 0.05); different letters above bars indicatestatistically significant differences among the treatments (ANOVA and SNK test, a =0.05).

Table 2Statistical results from an experiment investigating the lowest current that wouldelicit a directional response in Steinernema carpocapsae (Sc) and S. feltiae (Sf).

Nematode Experiment Voltage Statistic Value df P

Sc 1.8 V test 0 t 1.11 11 0.2915Sc 1.8 V test 1.8 t 9.70 11 0.0001Sc 1.8 V test 36 t 4.76 11 0.0006Sc 0.9 V test 0 t 1.89 11 0.0850Sc 0.9 V test 0.9 t 1.43 11 0.1804Sc 0.9 V test 36 t 2.62 11 0.0238Sc 1.8 V test F 41.99 2, 28 0.0001Sc 0.9 V test F 9.67 2, 28 0.0006Sf 1.8 V test 0 t 1.11 11 0.2915Sf 1.8 V test 1.8 t 3.58 11 0.0043Sf 1.8 V test 36 t 3.18 11 0.0088Sf 0.9 V test 0 t 0.44 11 0.6660Sf 0.9 V test 0.9 t 1.82 11 0.0968Sf 0.9 V test 36 t 4.01 11 0.0021Sf 1.8 V test F 25.47 2, 28 0.0001Sf 0.9 V test F 11.60 2, 28 0.0002

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nematodes that moved to the cathode side (relative to the totalthat moved to either side) was calculated for each dish and averagepercentages were compared among voltage levels through analysisof variance (ANOVA, a = 0.05) (SAS, 2002), or in the case of the age-sensitivity experiment percentage moving to the cathode wascompared between fresh and aged IJs. Variation among repeatedexperiments (e.g., trials conducted on different dates) was ac-counted for as a block effect. In the experiment estimating the low-est voltage that elicited a response (with three voltage levels), theStudent–Newman–Keuls’ test was used to further elucidate treat-ment effects when a significant F value was detected in the ANOVA(SAS, 2002). Percentage data were arcsine transformed prior toanalysis (Steel and Torrie, 1980).

3. Results

3.1. Diversity of response to electrical fields among EPN species

Directional movement in response to electrical fieldsdiffered among species (Table 1; Fig. 1). Based on t-test resultsthat compared the number of IJs on each side of the dish, thepositive controls responded as expected based on previousobservations (Shapiro-Ilan et al., 2009a), i.e., a greater propor-tion of S. carpocapsae moved toward the cathode relative tothe anode, and S. glaseri moved away from the cathode. Amongother species that responded directionally, H. georgiana, andS. riobrave moved away from cathode, whereas S. feltiae andS. siamkayai, moved toward the cathode. Directional responseto electrical field was not detected in H. bacteriophora, H. indica,and H. megidis. No significant differences in movement to thecathode versus anode were detected on the control plates (nocurrent) for any species (Table 1; Fig 1). Additionally, basedon ANOVA, for each nematode species that responded direction-ally, the percentage of nematodes that moved to the cathodewas different between treatment and control plates, and nodifferences between control and treatment plates were observedfor species that did not exhibit a directional response (Table 1;Fig 1).

3.2. Estimation of the lowest voltage eliciting a response

Both S. carpocapsae and S. feltiae exhibited a directionalresponse at 1.8 V, but the level of response was less than at 36 V(Table 2; Figs. 2 and 3). The level of response at 1.8 V was interme-diate between 36 V and the control (no current). At 0.9 V, a direc-tional response was not detected in both nematode species and the

38 D.I. Shapiro-Ilan et al. / Journal of Invertebrate Pathology 109 (2012) 34–40

percentages of IJs at the cathode were not different from the con-trols (Table 2; Figs. 2 and 3). In the instances where directionalmovement was observed it was consistent with previous observa-tions, i.e., the majority of nematodes moved toward the cathode(Figs. 2 and 3). When 0.9 V was applied to the plates the measuredvoltage across the agar was 0.123 ± 0.015 V and 0.103 ± 0.011 V at1.25 cm and 3.25 cm from the edge of the dish, respectively.

3.3. The effects of IJ age on response to electrical fields

Age effects on the response of S. carpocapsae to electrical fieldswere observed and were more pronounced at the lower voltagelevel (1.8 V) than the higher level (36 V) (Table 3; Fig. 4). FreshIJs exhibited a directional response in all tests at both voltage lev-els; more S. carpocapsae moved to the cathode side of the platethan the anode (Table 3; Fig. 4). Nematodes that were stored fordifferent durations also exhibited a directional response to electri-cal fields generated at 1.8 and 36 V, except 28-d-old IJs did not re-spond to either voltage level (Table 3; Fig. 4). At 1.8 V, when thelevel of movement was compared between the fresh and aged Ijs,nematodes stored for 7, 14, or 21 d exhibited a lower directionalresponse than fresh IJs, yet no difference was detected at 28 d.No differences in directional response were detected between freshand aged IJs at 36 V for any tests. Additionally no differences be-tween aged and fresh IJs were detected in all controls (0 V) andno directional responses within the plates were detected in anycontrol plates except aged IJs in the 14 d test (out of 21 analyses

Table 3Statistical results from an experiment investigating the impact of infective juvenileage on Steinernema carpocapsae’s directional response to electrical fields.

Test Treatmenta Voltage Statistic Value df P

7d 7-d-old 0 t 1.08 11 0.3097d Fresh 0 t 0.29 11 0.77867d 7-d-old 1.8 t 5.15 11 0.00037d Fresh 1.8 t 7.40 11 0.00017d 7-d-old 36 t 6.52 11 0.00017d Fresh 36 t 5.80 11 0.00017d 0 F 0.13 1, 22 0.72337d 1.8 F 5.81 1, 22 0.02487d 36 F 0.54 1, 22 0.4699

14d 14-d-old 0 t 2.59 17 0.019114d Fresh 0 t 0.02 17 0.985314d 14-d-old 1.8 t 2.27 11 0.044714d Fresh 1.8 t 4.85 11 0.000514d 14-d-old 36 t 4.72 17 0.000214d Fresh 36 t 4.89 17 0.000114d 0 F 3.63 1, 34 0.065214d 1.8 F 6.70 1, 22 0.016814d 36 F 2.01 1, 34 0.165021d 21-d-old 0 t 1.77 11 0.10721d Fresh 0 t 0.55 11 0.592321d 21-d-old 1.8 t 2.64 11 0.023221d Fresh 1.8 t 5.5 11 0.000221d 21-d-old 36 t 2.3 11 0.041921d Fresh 36 t 4.13 11 0.001721d 0 F 0.33 1, 22 0.573021d 1.8 F 13.27 1, 22 0.001421d 36 F 3.59 1, 22 0.0714

28d 28-d-old 0 t 1.96 11 0.075828d Fresh 0 t 1.29 11 0.222728d 28-d-old 1.8 t 0.87 11 0.400528d Fresh 1.8 t 2.58 11 0.025528d 28-d-old 36 t 1.55 11 0.148928d Fresh 36 t 2.74 11 0.019328d 0 F 0.08 1, 22 0.781328d 1.8 F 3.47 1, 22 0.075828d 36 F 1, 22 0.2263

a Infective juveniles nematodes were either fresh (62-d-old) or stored at 10 �C forthe time indicated (7, 14, 21, or 28 d).

of control plates, this was the only instance where a controlshowed a response).

4. Discussion

Our hypothesis that EPN response to electrical fields variesamong species was supported. Furthermore, response appears tobe related to foraging strategy, at least for the steinermatids, i.e.both ambusher-type foragers (S. carpocapsae and S. siamkayai)moved to a lower electrical potential, S. glaseri (a cruiser) movedto a higher electrical potential, and the intermediate foragerstested (S. feltiae and S. riobrave) were split in their directional re-sponse. We did not detect a directional response to electrical cur-rent in the heterorhabditd species tested, except for H. georgiana (acruiser), which moved to a higher electrical potential.

Similar to our findings on EPNs, the response of plant parasiticnematodes to electrical field varies among species, e.g., some spe-cies move to a higher electrical potential and others move to a low-er potential (Viglierchio and Yu, 1983; Riga, 2004). The reasons fordifferential response to electrical fields among EPN species are un-known. Insects are associated with electrical potentials that canvary among different species and circumstances (e.g., movementthrough different substrates) (Scheie and Smyth, 1967; McGonigleand Jackson, 2002); perhaps EPNs orient to electrical fields differ-entially in order to find suitable hosts or optimum areas to forage.For example, ambusher-type nematodes may be adapted tomoving to a lower electrical potential because ambushers tend toforage on soil surfaces more so than cruisers (Lewis, 2002) andmovement of insects on surfaces (e.g., soil) leads to an accumula-tion of electrical charge (McGonigle and Jackson, 2002; Jacksonand McGonigle, 2005).

The lower levels of electrical potential that elicited a directionalresponse in S. carpocapsae and S. feltiae correspond to electricalpotentials associated with the surface of some insects (whichmay reach P1 V) (Warnke 1976; Colin et al. 1992). This findingsupports the hypothesis that EPNs may use electrical fields inhost-finding. The lower level that elicited a response is also compa-rable with the membrane potential of some plant roots (e.g., up to0.24 V) (Weisenseel and Meyer 1997; Toko et al., 1990; Ivashikinaet al., 2001). EPNs have been observed to migrate toward plantroots, a behavior that can enhance host finding (Kanagy and Kaya,1996; Wang and Gaugler, 1998). Thus, EPNs may orient to plantroots based on attraction to electrical fields. The ability of plantparasitic nematodes to sense electrical fields has also been sug-gested to aid in the location of roots (their primary host) (Bird,1959; Riga, 2004). The attraction of certain EPNs to roots for thepurpose of finding insect hosts is facilitated, at least in part, bythe release of volatile chemicals from the plant that is being at-tacked, a kind of distress call (Wang and Gaugler 1998; Rasmannet al., 2005). Given that roots also react to stress such as woundswith changes in electric potential (Filek and Koscielniak, 1997), itis conceivable that EPNs’ orientation to electrical signals contrib-utes to their migration toward damaged roots as well.

Our results also indicated that S. carpocapsae’s response to elec-trical fields diminishes with IJ age. Conceivably, the importance ofdirectional response in foraging strategies may be most importantearly in the nematode’s life-cycle. Alternatively, sensitivity to elec-trical fields simply degenerates with age. In future research, theimpact of current intensity and IJ age on the response to electricalfields should be tested in other EPN species. Additionally, in abroader sense, given that differing substrates may affect EPN re-sponse (e.g., agar versus soil; El-Borai et al. 2011), future researchshould investigate further the role of electrical fields in EPN forag-ing or other aspects of fitness maintenance in the context of vari-ous field soils.

Fig. 4. Mean percentage (± SEM) of S. carpocapsae infective juvenile nematodes that moved from the center of an agar plate to the cathode side of the plate during a 30 mininterval when 0, 1.8 or 36 V were applied through the agar. Nematodes were either 6 2-d-old (‘‘Fresh’’) or stored (‘‘Aged’’) for 7, 14, 21, or 28 d prior to the experiment.⁄ indicates a statistically significant difference between the number of nematodes at the cathode versus anode side of the plate (paired t-test, a = 0.05); different letters abovebars within each voltage indicate statistically significant differences between aged and fresh nematodes (ANOVA, a = 0.05).

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Acknowledgments

We thank Terri Brearley, Kathy Halat and Kent Owusu for tech-nical assistance, and Matt Halat and Todd Brearley for helpful ad-vice on electrical conductance.

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