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

Journal of Invertebrate Pathology 109 (2012) 34–40

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Directional movement of entomopathogenic nematodes in response to electrical field: 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 States UC Davis, Department of Nematology, Department of Entomology, University of California, Davis, CA 95616, United States c USDA-ARS, Center for Grain and Animal Health, Manhattan, KS 66502, United States d Wake Forest University, Department of Physics, Winston-Salem, NC 27109, United States b

a r t i c l e

i n f o

Article history: Received 1 July 2011 Accepted 6 September 2011 Available online 12 September 2011 Keywords: Electricity Entomopathogenic nematode Heterorhabditis Host finding Steinernema

a b s t r a c t Entomopathogenic nematodes respond to a variety of stimuli when foraging. Previously, we reported a directional response to electrical fields for two entomopathogenic nematode species; specifically, when electrical fields were generated on agar plates Steinernema glaseri (a nematode that utilizes a cruiser-type foraging strategy) moved to a higher electric potential, whereas Steinernema carpocapsae, an ambush-type forager, moved to a lower potential. Thus, we hypothesized that entomopathogenic nematode directional response 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, Steinernema feltiae, Steinernema riobrave, and Steinernema siamkayai. S. carpocapsae and S. glaseri were also included as positive controls. Heterorhabditids tend toward cruiser foraging approaches whereas S. siamkayai is an ambusher and S. feltiae and S. riobrave are intermediate. Additionally, we determined the lowest voltage that would elicit a directional response (tested in S. feltiae and S. carpocapsae), and we investigated the impact of nematode age on response to electrical field in S. carpocapsae. In the experiment measuring diversity of response among species, we did not detect any response to electrical fields among the heterorhabditids except for H. georgiana, which moved to a higher electrical potential; S. glaseri and S. riobrave also moved to a higher potential, whereas S. carpocapsae, S. feltiae, and S. siamkayai moved to a lower potential. Overall our hypothesis that foraging strategy can predict directional response was supported (in the nematodes that exhibited a response). The lowest electric potential that elicited a response was 0.1 V, which is comparable to electrical potential associated with some insects and plant roots. The level of response to electrical potential diminished with nematode age. These results expand our knowledge of electrical fields as cues that may be used by entomopathogenic nematodes for hostfinding or other aspects of navigation in the soil. Published by Elsevier Inc.

1. Introduction Entomopathogenic nematodes (EPNs) in the genera Heterorhabditis and Steinernema are important regulators of natural insect populations and are used as biocontrol agents in a variety of urban and agricultural systems (Shapiro-Ilan et al., 2002; Grewal et al., 2005; Stuart et al., 2006). These nematodes have a mutualistic symbiosis with a bacterium (Xenorhabdus spp. and Photorhabdus spp. for steinernematids and heterorhabditids, respectively) (Poinar, 1990). Infective juveniles (IJs), the only free-living stage, enter hosts through natural openings (mouth, anus, and spiracles), or in some cases, through the cuticle. After entering the host’s

⇑ Corresponding author. Fax: +1 478 956 2929. E-mail address: [email protected] (D.I. Shapiro-Ilan). 0022-2011/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.jip.2011.09.004

hemocoel, nematodes release their bacterial symbionts, which are primarily responsible for killing the host within 24–48 h, defending against secondary invaders, and providing the nematodes with nutrition (Dowds and Peters, 2002). The nematodes molt and, depending on the suitability of host resources, complete up to three generations within the host after which IJs exit the cadaver to find new hosts (Kaya and Gaugler, 1993). Elucidation of mechanisms in host-finding and foraging strategies is needed to enhance our ability to use EPNs in biocontrol programs and understand their role in nature. Foraging strategies among EPN species vary along a continuum between ambushers, which generally sit and wait for a passing host, and cruisers that actively search for hosts. EPNs can exhibit a combination of these behaviors to locate hosts and although some species exhibit primarily ambusher-type behaviors and others are mainly cruiser types, others are considered intermediate in their foraging

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behavior (Campbell and Kaya, 1999; Lewis, 2002; Kruitbos et al., 2010). Additionally, IJs respond to a variety of stimuli such as CO2 (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. Response to stimuli may also be useful in other aspects of fitness such as 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 and Bleckmann, 1998). Additionally, certain plant and animal parasitic, and free living nematodes respond to electrical fields, and in the case of parasitic nematodes, the response has been linked to foraging behavior (Sukul et al., 1975; Croll and Matthews, 1977; Viglierchio and Yu, 1983; Riga, 2004). Recently, EPNs were also found to respond directionally to electrical fields (Shapiro-Ilan et al., 2009a). When an electric potential was generated across an agar plate Steinernema carpocapsae (Weiser) (an ambusher) moved toward a lower electric potential and Steinernema glaseri (a cruiser) moved in the opposite direction. Given that the two nematodes tested showed differing responses we hypothesized that directional response to electrical fields varies across EPN species and the response may be related to foraging strategy. Thus, the primary objective of this study was to test that hypothesis by investigating the response to electrical field in a broader array of EPN species. Specifically, we measured directional response to electrical fields in seven nematode species that had not been tested previously: Heterorhabditis bacteriophora Poinar, Heterorhabditis georgiana Nguyen, Shapiro-Ilan and Mbata, Heterorhabditis indica Poinar, Karunkar and David, Heterorhabditis megidis Poinar, Jackson and Klein, Steinernema feltiae (Filipjev), Steinernema riobrave Cabanillas, Poinar and Raulston, and Steinernema siamkayai Stock, Somsook and Reid. S. carpocapsae and S. glaseri were also included as positive controls. Heterorhabditids tend toward cruiser foraging approaches whereas S. siamkayai is an ambusher and S. feltiae and S. riobrave are intermediate (Lewis, 2002; Shapiro-Ilan et al., 2009b). In addition to the characterization of species-specific response, our objectives were to estimate the lowest potential that would elicit a directional response, and to determine if response is affected by IJ age. 2. Materials and Methods 2.1. Nematodes and experimental conditions All nematodes (H. bacteriophora [Hb strain], H. georgiana [Kesha strain], H. indica [Hom-1 strain], H. megidis [UK211 strain], S. carpocapsae [All strain], S. feltiae [SN strain], S. glaseri [NJ43 strain]), S. riobrave [355 strain], and S. siamkayai) were cultured in commercially obtained last instar Galleria mellonella (L.) according to procedures described in Kaya and Stock (1997). Unless indicated otherwise (see below), the nematodes were stored at 13 °C for less than two weeks prior to experimentation. All experiments were conducted 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 procedures described by Shapiro-Ilan et al. (2009a). Experiments were conducted in plastic Petri dishes (90 mm diam.) with 2% agar (Agar N.F., ICN Biomedicals, Inc., Aurora, OH) at approximately 1 cm depth. Sodium Chloride (0.01%) was incorporated into the agar to facilitate conductance of electricity (Sukul et al., 1975); the low salt concentration we used was not adverse to steinernematids (Thurston et al. 1994). An electric field was generated across the agar

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

Statistic

Value

df

P

Hb with eHb without eHb Hg with eHg without eHg Hi with eHi without eHi Hm with eHm without eHm Sc with eSc without eSc Sf with eSf without eSf Sg with eSg without eSg Sr with eSr without eSr Ss with eSs without eSs

t t F t t F t t F t t F t t F t t F t t F t t F t t F

0.74 0.70 1.50 3.26 0.32 5.57 0.32 2.02 0.63 0.80 1.26 2.24 5.58 0.00 83.41 5.40 0.63 82.04 6.30 1.63 104.84 3.04 0.97 6.58 6.69 0.43 47.82

23 23 1, 44 23 23 1, 44 23 23 1, 44 23 23 1, 44 23 23 1, 44 23 23 1, 44 23 23 1, 44 23 23 1, 44 23 23 1, 44

0.4679 0.4895 0.2269 0.0034 0.7494 0.0228 0.7492 0.0556 0.4307 0.4293 0.2212 0.1416 0.0001 1.0000 0.0001 0.0001 0.5336 0.0001 0.0001 0.1165 0.0001 0.0058 0.3410 0.0138 0.0001 0.4293 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.

plate using an electrophoresis power supply (model EC 105, E–C Apparatus Corporation, Holbrook, NY). An electrode from the cathode (negative on the power supply) with copper wire (0.5 mm diam.) was inserted vertically on one side of dish (approximately 1 mm from edge), and the other electrode from the anode was placed on the opposite side. Approximately 4000 IJs were concentrated by vacuum filter and placed in the middle of the agar dish, except 2000 IJs were used for S. glaseri and S. feltiae because these species were more dispersive (Shapiro-Ilan et al., 2009a; personal observation). The power supply was set at 36 V (3.0 milliamps) and applied for 30 min, at which time the number of IJs within a 2.5 cm semicircle around the cathode or anode was counted. Controls consisted of plates treated identically but with no applied voltage. Movement toward the cathode indicates a response toward a lower potential whereas movement to the anode indicates a response in the direction of a higher potential. The experiments were conducted separately for each of the nine EPN species tested (see list of species and strains above). There were eight replicates for each species and each experiment was conducted three times (resulting in 24 plates 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 voltage needed to elicit a directional response were conducted with S. carpocapsae and S. feltiae. The methods to determine directional response were as described above. Two experiments were conducted for each species, one experiment tested IJ response when the lowest voltage applied to the agar was 1.8 V, and the other was conducted using 0.9 V as the lowest voltage applied. These voltage levels were chosen based on preliminary trials indicating that the lowest voltage response would be below 3 V (unpublished data). In each experiment, in addition to the 1.8 or 0.9 V plate, one plate measured IJ directional response when no voltage was applied (a

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

as a model because this nematode was one of the species that consistently showed a strong response to electrical fields. Four experiments were conducted containing nematodes that were stored at 10 °C for 7, 14, 21, or 28 d. All tests compare aged IJs directly to fresh IJs (62-d-old). Assays testing directional response on agar plates followed procedures described above and were conducted with 0, 1.8 V and 36 V. Assays were repeated 12–18 times for each age interval and voltage level.

negative control) and one plate measured response when 36 V was applied (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 per treatment within each test). Due to the resistance and dielectric constant of the material, the actual potential through the agar plates is expected to be less than the applied voltage. A voltmeter (Fluke 87 True RMS Multimeter, Fluke Corp., Everett, WA) was used to determine the actual potential 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 of the agar dish (three replicates each).

2.5. Statistical analyses For all treatments and controls, preferential movement of nematodes to one side of the dish (i.e., a directional response) was determined through paired t-tests comparing the number of IJs at the anode side versus cathode side (SAS, 2002; Shapiro-Ilan et al., 2009a; a = 0.05). Additionally, to compare the level of response among treatments and controls, the percentage of

2.4. The effects of IJ age on response to electrical fields Experiments were conducted to determine if sensitivity to electrical field is affected by IJ age. The experiments used S. carpocapsae 100

100

A

40 20 0

80 60

20

With voltage

20

100

Hm

80

A A

40 20

Without voltage

With voltage

Treatment

% IJs at Cathode

% IJs at Cathode

100

100

Sc

Without voltage

B

40 20

Without voltage

With voltage

100

100

80

A

40

B 20 0

% IJs at Cathode

Sg

A

With voltage

Treatment

B

40 20

Without voltage

Ss

100

B

40 20

With voltage

Treatment

Sr

A

80 60

B

40 20 0

0 Without voltage

60

0

With voltage

80 60

A

80

Treatment

Treatment

Sf

A

80 60

With voltage

Treatment

0

0

% IJs at Cathode

40

0 Without voltage

Treatment

60

B

40

A A

60

0 Without voltage

60

A

Hi

80

% IJs at Cathode

60

A

100

Hg

% IJs at Cathode

% IJs at Cathode

80

% IJs at Cathode

Hb

% IJs at Cathode

36

Without voltage

With voltage

Treatment

Without voltage

With voltage

Treatment

Fig. 1. Mean percentage (± SEM) of infective juvenile nematodes (IJs) that moved from the center of an agar plate to the cathode side of the plate during a 30 min interval when voltage (36 V) was applied (‘‘with voltage’’, i.e., the treatment), or no voltage was applied (‘‘without voltage’’, i.e., the control). Hb = Heterorhabditis bacteriophora, Hg = Heterorhabditis georgiana, Hi = Heterorhabditis indica, Hm = Heterorhabditis megidis, Sc = Steinernema carpocapsae, Sf = Steinernema feltiae, Sg = Steinernema glaseri, Sr = Steinernema riobrave, Ss = Steinernema siamkayai. ⁄ 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 above bars indicate statistically significant differences between the treatment and control (ANOVA, a = 0.05).

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Experiment

Voltage

Statistic

Value

df

P

Sc Sc Sc Sc Sc Sc Sc Sc Sf Sf Sf Sf Sf Sf Sf Sf

1.8 V 1.8 V 1.8 V 0.9 V 0.9 V 0.9 V 1.8 V 0.9 V 1.8 V 1.8 V 1.8 V 0.9 V 0.9 V 0.9 V 1.8 V 0.9 V

0 1.8 36 0 0.9 36

t t t t t t F F t t t t t t F F

1.11 9.70 4.76 1.89 1.43 2.62 41.99 9.67 1.11 3.58 3.18 0.44 1.82 4.01 25.47 11.60

11 11 11 11 11 11 2, 28 2, 28 11 11 11 11 11 11 2, 28 2, 28

0.2915 0.0001 0.0006 0.0850 0.1804 0.0238 0.0001 0.0006 0.2915 0.0043 0.0088 0.6660 0.0968 0.0021 0.0001 0.0002

test test test test test test test test test test test test test test test test

0 1.8 36 0 0.9 36

nematodes that moved to the cathode side (relative to the total that moved to either side) was calculated for each dish and average percentages were compared among voltage levels through analysis of variance (ANOVA, a = 0.05) (SAS, 2002), or in the case of the agesensitivity experiment percentage moving to the cathode was compared between fresh and aged IJs. Variation among repeated experiments (e.g., trials conducted on different dates) was accounted for as a block effect. In the experiment estimating the lowest voltage that elicited a response (with three voltage levels), the Student–Newman–Keuls’ test was used to further elucidate treatment effects when a significant F value was detected in the ANOVA (SAS, 2002). Percentage data were arcsine transformed prior to analysis (Steel and Torrie, 1980).

3.2. Estimation of the lowest voltage eliciting a response Both S. carpocapsae and S. feltiae exhibited a directional response 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 intermediate between 36 V and the control (no current). At 0.9 V, a directional response was not detected in both nematode species and the

100 90 80 70 60 50 40 30 20 10 0

C

0

1.8

36

A

B

0

B

0.9

36

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 a 30 min interval when varying voltage levels were applied. ⁄ 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 above bars indicate statistically significant differences among the treatments (ANOVA and SNK test, a = 0.05).

% IJs at Cathode

100 90 80 70 60 50 40 30 20 10 0

A B* C

0

1.8

36

Voltage

% IJs at Cathode

Directional movement in response to electrical fields differed among species (Table 1; Fig. 1). Based on t-test results that compared the number of IJs on each side of the dish, the positive controls responded as expected based on previous observations (Shapiro-Ilan et al., 2009a), i.e., a greater proportion of S. carpocapsae moved toward the cathode relative to the anode, and S. glaseri moved away from the cathode. Among other species that responded directionally, H. georgiana, and S. riobrave moved away from cathode, whereas S. feltiae and S. siamkayai, moved toward the cathode. Directional response to electrical field was not detected in H. bacteriophora, H. indica, and H. megidis. No significant differences in movement to the cathode versus anode were detected on the control plates (no current) for any species (Table 1; Fig 1). Additionally, based on ANOVA, for each nematode species that responded directionally, the percentage of nematodes that moved to the cathode was different between treatment and control plates, and no differences between control and treatment plates were observed for species that did not exhibit a directional response (Table 1; Fig 1).

A B*

Voltage

3. Results 3.1. Diversity of response to electrical fields among EPN species

100 90 80 70 60 50 40 30 20 10 0

Voltage

% IJs at Cathode

Nematode

% IJs at Cathode

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

100 90 80 70 60 50 40 30 20 10 0

A B

0

B

0.9

36

Voltage Fig. 3. Mean percentage (± SEM) of S. feltiae infective juvenile nematodes (IJs) that moved from the center of an agar plate to the cathode side of the plate during a 30 min interval when varying voltage levels were applied. ⁄ 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 above bars indicate statistically significant differences among the treatments (ANOVA and SNK test, a = 0.05).

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percentages of IJs at the cathode were not different from the controls (Table 2; Figs. 2 and 3). In the instances where directional movement was observed it was consistent with previous observations, i.e., the majority of nematodes moved toward the cathode (Figs. 2 and 3). When 0.9 V was applied to the plates the measured voltage across the agar was 0.123 ± 0.015 V and 0.103 ± 0.011 V at 1.25 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 fields were observed and were more pronounced at the lower voltage level (1.8 V) than the higher level (36 V) (Table 3; Fig. 4). Fresh IJs exhibited a directional response in all tests at both voltage levels; more S. carpocapsae moved to the cathode side of the plate than the anode (Table 3; Fig. 4). Nematodes that were stored for different durations also exhibited a directional response to electrical fields generated at 1.8 and 36 V, except 28-d-old IJs did not respond to either voltage level (Table 3; Fig. 4). At 1.8 V, when the level of movement was compared between the fresh and aged Ijs, nematodes stored for 7, 14, or 21 d exhibited a lower directional response than fresh IJs, yet no difference was detected at 28 d. No differences in directional response were detected between fresh and aged IJs at 36 V for any tests. Additionally no differences between aged and fresh IJs were detected in all controls (0 V) and no directional responses within the plates were detected in any control plates except aged IJs in the 14 d test (out of 21 analyses

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

Treatmenta

Voltage

Statistic

df

P

7d 7d 7d 7d 7d 7d 7d 7d 7d

7-d-old Fresh 7-d-old Fresh 7-d-old Fresh

0 0 1.8 1.8 36 36 0 1.8 36

t t t t t t F F F

1.08 0.29 5.15 7.40 6.52 5.80 0.13 5.81 0.54

11 11 11 11 11 11 1, 22 1, 22 1, 22

0.309 0.7786 0.0003 0.0001 0.0001 0.0001 0.7233 0.0248 0.4699

14d 14d 14d 14d 14d 14d 14d 14d 14d 21d 21d 21d 21d 21d 21d 21d 21d 21d

14-d-old Fresh 14-d-old Fresh 14-d-old Fresh

0 0 1.8 1.8 36 36 0 1.8 36 0 0 1.8 1.8 36 36 0 1.8 36

t t t t t t F F F t t t t t t F F F

2.59 0.02 2.27 4.85 4.72 4.89 3.63 6.70 2.01 1.77 0.55 2.64 5.5 2.3 4.13 0.33 13.27 3.59

17 17 11 11 17 17 1, 34 1, 22 1, 34 11 11 11 11 11 11 1, 22 1, 22 1, 22

0.0191 0.9853 0.0447 0.0005 0.0002 0.0001 0.0652 0.0168 0.1650 0.107 0.5923 0.0232 0.0002 0.0419 0.0017 0.5730 0.0014 0.0714

0 0 1.8 1.8 36 36 0 1.8 36

t t t t t t F F F

1.96 1.29 0.87 2.58 1.55 2.74 0.08 3.47

11 11 11 11 11 11 1, 22 1, 22 1, 22

0.0758 0.2227 0.4005 0.0255 0.1489 0.0193 0.7813 0.0758 0.2263

28d 28d 28d 28d 28d 28d 28d 28d 28d

21-d-old Fresh 21-d-old Fresh 21-d-old Fresh

28-d-old Fresh 28-d-old Fresh 28-d-old Fresh

Value

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

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

4. Discussion Our hypothesis that EPN response to electrical fields varies among species was supported. Furthermore, response appears to be 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) moved to a higher electrical potential, and the intermediate foragers tested (S. feltiae and S. riobrave) were split in their directional response. We did not detect a directional response to electrical current in the heterorhabditd species tested, except for H. georgiana (a cruiser), which moved to a higher electrical potential. Similar to our findings on EPNs, the response of plant parasitic nematodes to electrical field varies among species, e.g., some species move to a higher electrical potential and others move to a lower potential (Viglierchio and Yu, 1983; Riga, 2004). The reasons for differential response to electrical fields among EPN species are unknown. Insects are associated with electrical potentials that can vary among different species and circumstances (e.g., movement through different substrates) (Scheie and Smyth, 1967; McGonigle and Jackson, 2002); perhaps EPNs orient to electrical fields differentially in order to find suitable hosts or optimum areas to forage. For example, ambusher-type nematodes may be adapted to moving to a lower electrical potential because ambushers tend to forage on soil surfaces more so than cruisers (Lewis, 2002) and movement of insects on surfaces (e.g., soil) leads to an accumulation of electrical charge (McGonigle and Jackson, 2002; Jackson and McGonigle, 2005). The lower levels of electrical potential that elicited a directional response in S. carpocapsae and S. feltiae correspond to electrical potentials associated with the surface of some insects (which may reach P1 V) (Warnke 1976; Colin et al. 1992). This finding supports the hypothesis that EPNs may use electrical fields in host-finding. The lower level that elicited a response is also comparable with the membrane potential of some plant roots (e.g., up to 0.24 V) (Weisenseel and Meyer 1997; Toko et al., 1990; Ivashikina et al., 2001). EPNs have been observed to migrate toward plant roots, a behavior that can enhance host finding (Kanagy and Kaya, 1996; Wang and Gaugler, 1998). Thus, EPNs may orient to plant roots based on attraction to electrical fields. The ability of plant parasitic nematodes to sense electrical fields has also been suggested to aid in the location of roots (their primary host) (Bird, 1959; Riga, 2004). The attraction of certain EPNs to roots for the purpose of finding insect hosts is facilitated, at least in part, by the release of volatile chemicals from the plant that is being attacked, a kind of distress call (Wang and Gaugler 1998; Rasmann et al., 2005). Given that roots also react to stress such as wounds with changes in electric potential (Filek and Kos´cielniak, 1997), it is conceivable that EPNs’ orientation to electrical signals contributes to their migration toward damaged roots as well. Our results also indicated that S. carpocapsae’s response to electrical fields diminishes with IJ age. Conceivably, the importance of directional response in foraging strategies may be most important early in the nematode’s life-cycle. Alternatively, sensitivity to electrical fields simply degenerates with age. In future research, the impact of current intensity and IJ age on the response to electrical fields should be tested in other EPN species. Additionally, in a broader sense, given that differing substrates may affect EPN response (e.g., agar versus soil; El-Borai et al. 2011), future research should investigate further the role of electrical fields in EPN foraging or other aspects of fitness maintenance in the context of various field soils.

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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 min interval 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 above bars within each voltage indicate statistically significant differences between aged and fresh nematodes (ANOVA, a = 0.05).

Acknowledgments We thank Terri Brearley, Kathy Halat and Kent Owusu for technical assistance, and Matt Halat and Todd Brearley for helpful advice on electrical conductance.

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