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

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 host-finding or other aspects of navigation in the soil.     

Magnetic and electric fields induce directional responses in Steinernema carpocapsae

Entomopathogenic nematode species respond directionally to various cues including electrical stimuli. For example, in prior research Steinernema carpocapsae was shown to be attracted to an electrical current that was applied to an agar dish. Thus, we hypothesised that these nematodes may use electromagnetic reception to assist in navigating through the soil and finding a host. In this study we discovered that S. carpocapsae also responds to electrical fields (without current) and to magnetic fields; to our knowledge this is the first report of nematode directional movement in response to a magnetic field. Our research expands on the range of known stimuli that entomopathogenic nematodes respond to. The findings may have implications for foraging behavior.     

Aggregative group behavior in insect parasitic nematode dispersal

Movement behavior of foraging animals is critical to the determination of their spatial ecology and success in exploiting resources. Individuals sometimes gain advantages by foraging in groups to increase their efficiency in garnering these resources. Group movement behavior has been studied in various vertebrates. In this study we explored the propensity for innate group movement behavior among insect parasitic nematodes. Given that entomopathogenic nematodes benefit from group attack and infection, we hypothesised that the populations would tend to move in aggregate in the absence of extrinsic cues. Movement patterns of entomopathogenic nematodes in sand were investigated when nematodes were applied to a specific locus or when the nematodes emerged naturally from infected insect hosts; six nematode species in two genera were tested (Heterorhabditis bacteriophora, Heterorhabditis indica, Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri and Steinernema riobrave). Nematodes were applied in aqueous suspension via filter paper discs or in infected insect host cadavers (to mimic emergence in nature). We discovered that nematode dispersal resulted in an aggregated pattern rather than a random or uniform distribution; the only exception was S. glaseri when emerging directly from infected hosts. The group movement may have been continuous from the point of origin, or it may have been triggered by a propensity to aggregate after a short period of random movement. To our knowledge, this is the first report of group movement behavior in parasitic nematodes in the absence of external stimuli (e.g., without an insect or other apparent biotic or abiotic cue). These findings have implications for nematode spatial distribution and suggest that group behavior is involved in nematode foraging.     

Effect of Exsheathment on Motility and Pathogenicity of Two Entomopathogenic Nematode Species

The effect of sheath loss on motility and pathogenicity of the entomopathogenic nematodes, Heterorhabditis bacteriophora and Steinernema carpocapsae, was examined using both naturally and chemically exsheathed (desheathed) infective juveniles. Exsheathed S. carpocapsae showed increased motility on agar compared to sheathed nematodes. The presence of a host increased motility threefold in all S. carpocapsae treatments. These results suggest that activation of S. carpocapsae host finding may result from sheath loss in addition to host stimuli. Desheathed H. bacteriophora were significantly less motile than the sheathed or exsheathed groups. The decreased motility may be due to adverse effects of the chemical treatment for desheathment. Sheath loss did not affect the pathogenicity of either species.     

Steinernema feltiae (DD-136) and S. glaseri: Persistence in Soil and Bark Compost and Their Influence on Native Nematodes

Infective juveniles (J3) of the entomogenous nematodes Steinernema feltiae DD-136 (ca. 10,000 J3/100 ml) and S. glaseri (ca. 2,500 J3/100 ml) were incubated in steam-sterilized and nonsterilized sandy soil and bark compost for 8 weeks at 25 C. The nematodes were recovered by a two-step extraction procedure at 1-week intervals, and their infectivity to lepidopterous larvae (Spodoptera litura and Galleria mellonella) and their effect on the population and community of native nematodes in soil were determined. Survival of inoculated nematodes and mortality of insects were enhanced in sterilized media. Nonsterilized bark compost proved to be equally as suitable a medium as sterilized compost. In nonsterilized soil, the survival curve of S.feltiae declined more rapidly than that or S. glaseri which was less infective to insects despite its greater persistence even in nonsterilized soil. Soon after the addition of steinernematids to soil, the population of native nematodes showed a fluctuation with an increase in rhabditids and a decrease in other kinds of nematodes.     

Natural occurrence of entomopathogenic nematodes in Liaoning (Northeast China)

Entomopathogenic nematodes (EPNs) can provide effective biological control of pest. In order to contribute to knowledge on these organisms for regional biological control programs, we studied EPN distribution and ecological requirements in Liaoning Province, Northeast China. One hundred and forty-nine soil samples were taken from 36 locations. EPNs were recovered from 22 of the 36 locations (61.11%). Forty-four samples contained steinernematids (89.80%) and 5 samples contained heterorhabditids (10.20%). EPN recovery varied among the different soil and habitat type. Most EPNs were isolated from sandy loam, and most of the samples containing EPNs were collected from woodland and fruit crop habitats. The morphological characters of infective juveniles were used for preliminary species diagnosis. We preliminarily identified 15 species of Steinernematidae (Steinernema litorale, Steinernema silvaticum, Steinernema feltiae, Steinernema bicornutum, Steinernema robustispiculum, Steinernema affine, Steinernema riobrave, Steinernema yirgalemense, Steinernema kushidai, Steinernema scapterisci, Steinernema carpocapsae, Steinernema ritteri, Steinernema tami, Steinernema rarum and Steinernema sasonense) and 4 species of Heterorhabditidae (Heterorhabditis megidis, Heterorhabditis zealandica, Heterorhabditis brevicaudis and Heterorhabditis bajardi).     

Attraction of entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora to the red palm weevil (Rhynchophorus ferrugineus)

The red palm weevil (RPW), Rhynchophorus ferrugineus, is a serious pest of date palms. Its larvae bore deep into the trunk disrupt the vascular tissues and kill the infested trees. Behavioral features of entomopathogenic nematodes (EPNs), reflected by attraction and distribution patterns, are fundamental aspect in determining their parasitic ability and potential management of RPW. We studied the attraction behavior of the EPNs Steinernema carpocapsae and Heterorhabditis bacteriophora to the RPW under simulated natural conditions in tubes to evaluate their infective potential. In all experiments, a certain proportion of infective juveniles (IJs) (16–20%) stayed near the inoculated site and a major proportion (38–48%) was attracted to the host end. Both H. bacteriophora and S. carpocapsae were efficient crawlers, climbing up and descending when locating their insect host. They were efficiently attracted to the various larval sizes and stages of the RPW life cycle. Host localization by ascending movement was more prominent in S. carpocapsae than in H. bacteriophora. In general, H. bacteriophora is classified as a cruiser forager and S. carpocapsae as an ambusher. However, in this study, we discovered a higher percentage of cruiser foragers among S. carpocapsae IJs. They dispersed much faster and their cruising behavior was prominent characteristic in controlling the cryptic RPW concealed in organic habitats.     

Recycling Potential and Fitness of Steinernematid Nematodes Cultured in Curculio caryae and Galleria mellonella

The recycling potential of entomopathogenic nematodes in the pecan weevil, Curculio caryae, following inundative applications is an important factor in considering whether nematodes could be incorporated into a C. caryae management strategy. Our objective was to determine the recycling potential and fitness of Steinernema carpocapsae and S. riobrave cultured in C. caryae. To estimate fitness and quality, we reared nematodes in larvae of C. caryae and in the commonly used standard host, the greater wax moth, Galleria mellonella. Nematode lipid content, infectivity (power to invade), virulence (power to kill), and reproductive capacity (yield per insect) in C. caryae larvae were compared with G. mellonella data. Lipid content was higher in S. carpocapsae cultured in C. caryae than in G. mellonella, but S. riobrave lipid content was not affected by host source. Host source did not affect subsequent infectivity or virulence to C. caryae (P > 0.05) but did affect reproductive capacity (P < 0.0001). Both nematode species produced more progeny in C. caryae when they were first cultured in G. mellonella than when they were first passed through C. caryae. In terms of potential to recycle under field conditions, we predict that nematodes resulting from one round of recycling in C. caryae larvae would be equally capable of infecting and killing more weevils, but the potential to continue recycling in C. caryae would diminish over time due to reduced reproduction in that host.     

Attraction of four entomopathogenic nematodes to four white grub species

To better understand the differences in the efficacy of entomopathogenic nematode species against white grub species, we are studying the various steps of the infection process of entomopathogenic nematodes into different white grub species using nematode species/strains with particular promise as white grub control agents. In this study we compared the attraction of the entomopathogenic nematodes Steinernema scarabaei (AMK001 strain), Steinernema glaseri (NC1 strain), Heterorhabditis zealandica (X1 strain), and Heterorhabditis bacteriophora (GPS11 strain) to third-instars of the scarabs Popillia japonica, Anomala orientalis, Cyclocephala borealis, and Rhizotrogus majalis, and late-instar greater wax moth, Galleria mellonella, larvae. Individual larvae were confined at the bottom of 5.5 cm vertical sand columns, nematodes added to the sand surface after 24 h, and nematodes extracted after another 24 h. Nematode attraction to hosts was strongly affected by nematode species but the effect of insect species varied with nematode species. S. glaseri had a high innate dispersal rate (i.e., in absence of insects) and was strongly attracted to insects without significant differences among insect species. S. scarabaei had a very low innate dispersal rate so that even a strong relative response to insects resulted in low absolute dispersal rates toward insects. S. scarabaei tended to be most attracted to G. mellonella and least attracted to C. borealis. H. zealandica had a high innate dispersal rate but only responded weakly to insects without significant differences among species. H. bacteriophora had limited innate dispersal and only weakly responded to insects with G. mellonella tending to be the most attractive and C. borealis the least attractive insect. It has to be noted that we cannot exclude that the use of different rearing hosts (A. orientalis and P. japonica larvae for S. scarabaei, G. mellonella larvae for the other nematodes) might have had an impact on the nematodes dispersal and relative attraction behavior. This study indicates that host attractiveness and nematode dispersal rates may contribute but do not play a major role in the variability in white grub susceptibility and/or nematode virulence.     

Viability and Virulence of Entomopathogenic Nematodes Exposed to Ultraviolet Radiation

Entomopathogenic nematodes (EPNs) can be highly effective biocontrol agents, but their efficacy can be reduced due to exposure to environmental stress such as from ultraviolet (UV) radiation. Our objectives were to 1) compare UV tolerance among a broad array of EPN species, and 2) investigate the relationship between reduced nematode viability (after exposure to UV) and virulence. Nematodes exposed to a UV radiation (254 nm) for 10 or 20 min were assessed separately for viability (survival) and virulence to Galleria mellonella. We compared 9 different EPN species and 15 strains: Heterorhabditis bacteriophora (Baine, fl11, Oswego, and Vs strains), H. floridensis (332), H. georgiana (Kesha), H. indica (HOM1), H. megidis (UK211), Steinernema carpocapsae (All, Cxrd, DD136, and Sal strains), S. feltiae (SN), S. rarum (17C&E), and S. riobrave (355). In viability assessments, steinernematids, particularly strains of S. carpocapsae, generally exhibited superior UV tolerance compared with the heterorhabditids. However, some heterorhabditids tended to be more tolerant than others, e.g., H. megidis and H. bacteriophora (Baine) were most susceptible and H. bacteriophora (Vs) was the only heterorhabditid that did not exhibit a significant effect after 10 min of exposure. All heterorhabditids experienced reduced viability after 20 min exposure though several S. carpocapsae strains did not. In total, after 10 or 20 min exposure, the viability of seven nematode strains did not differ from their non-UV exposed controls. In virulence assays, steinernematids (particularly S. carpocapsae strains) also tended to exhibit higher UV tolerance. However, in contrast to the viability measurements, all nematodes experienced a reduction in virulence relative to their controls. Correlation analysis revealed that viability among nematode strains is not necessarily related to virulence. In conclusion, our results indicate that the impact of UV varies substantially among EPNs, and viability alone is not a sufficient measure for potential impact on biocontrol efficacy as other characters such as virulence may be severely affected even when viability remains high.     

Effect of Steinernema glaseri-infected host exudates on movement of conspecific infective juveniles

The entomopathogenic nematode's decision to infect a host is paramount because once the decision is made it is irrevocable; nematodes that invade a host either develop and achieve reproductive success, or they die. Entomopathogenic nematodes that have a cruiser foraging behavior, such as Steinernema glaseri, follow host-associated cues to locate insects to infect. Most of the host finding and infection dynamics research has focused on the infective juvenile nematodes' responses to cues from live insects such as host-associated volatiles and host contact cues. Few studies focus on how previously infected hosts influence infective juvenile infection behaviors. We investigated how exudates from nematode-infected hosts affect the behavior of S. glaseri infective juveniles. We hypothesized that the infective juvenile's behavioral response to cadavers would change as the state of a nematode-infected host changes during pathogenesis. We examined the effect of exudates collected from infected hosts on infective juvenile locomotory behavior. We detected no effects on nematode repulsion or attraction from exudates produced within the first 48h post-infection. We observed repulsion from accumulated exudates during the 3-48, 3-72, 3-120, and 3-144h intervals. Repulsion from exudates was observed during the 48-66, 72-90, and 120-138h intervals in experiments evaluating daily exudate emissions. The repellent effect of infected host exudates may result in an infective juvenile discriminating between suitable and unsuitable hosts.     

Effectiveness of Steinernema spp. and Heterorhabditis bacteriophora against Popillia japonica in the Azores

Steinernema carpocapsae (Breton strain), S. glaseri, and Heterorhabditis bacteriophora were evaluated for their potential to control immature stages of the Japanese beetle, Popillia japonica, on Terceira Island (the Azores). In bioassays carried out at temperatures higher than 15 C, S. glaseri and H. bacteriophora caused 100% mortality of larvae, whereas S. carpocapsae caused 56% larval mortality. At temperatures slightly below 15 C, only S. glaseri remained effective. In field plots, in September, S. glaseri and S. carpocapsae reduced larval populations by 91% and 44%, respectively, when applied at the rate of 10⁶ nematodes/m². In April, S. glaseri caused 31% reduction in numbers of larvae, but S. carpocapsae was ineffective. In colder months (November-February) neither steinernematids nor H. bacteriophora reduced larval populations. Increasing the application rate from 10⁶ to 5 x 10⁶ infective stage S. glaseri per m² increased efficacy from 63% to 79% mortality.     

Mating and Sexual Communication by Steinernema carpocapsae (Nemata: Steinernematidae)

Entomopathogenic nematodes are lethal insect parasites that reproduce exclusively inside their hosts in nature. Infection decisions made by the free-living infective-stage juveniles have an impact on reproductive success, but it is likely that mating decisions are made by adults while inside their host. We investigated sexual communication between male and female adult stages of Steinernema carpocapsae (Rhabditida: Steinernematidae) to assess whether mating is chemically mediated during the adult stage or results from incidental encounters between adults inside the insect host. To assess chemical communication, we measured the behavioral response of adult male S. carpocapsae to several different potential sources of chemical information. Male S. carpocapsae responded to virgin females only and were not influenced by mated conspecific females, conspecific males, or heterospecific females. These results show that species-specific communication takes place between adult entomopathogenic nematodes within the host cadaver just prior to mating.     

Does scavenging extend the host range of entomopathogenic nematodes (Nematoda: Steinernematidae)?

Living and freeze-killed natural and laboratory hosts, with different susceptibility to entomopathogenic nematodes, were exposed to the larvae of Steinernema affine and Steinernema kraussei in two different experimental arenas (Eppendorf tubes, Petri dishes), and the success of the colonisation and eventual progeny production were observed. Both nematodes were able to colonise both living and dead larvae of Galleria mellonella (Lepidoptera) and adult Blatella germanica (Blattodea) even though the progeny production in dead hosts was lower on average. Living carabid beetles, Poecilus cupreus, and elaterid larvae (Coleoptera) were resistant to the infection, however, both nematodes were able to colonise and multiply in several dead P. cupreus and in a majority of dead elaterid larvae. By scavenging, EPNs can utilise cadavers of insects that are naturally resistant to EPN infection, and so broaden their host range.     

Sex ratios and sex-biased infection behaviour in the entomopathogenic nematode genus Steinernema

In experimentally infected insects, the sex ratio of first generation nematodes of five species of Steinernema was female-biased (male proportion 0.35-0.47). There was a similar female bias when the worms developed in vitro (0.37-0.44), indicating that the bias in these species is not due to a lower rate of infection by male infective juveniles (IJs). Experimental conditions influenced the proportion of males establishing in insects, indicating that male and female IJs differ in their behaviour. However, there was no evidence that males are the colonising sex in any species, contrary to what has previously been proposed. Time of emergence from the host in which the nematodes had developed influenced sex ratios in experimental infections. In three species (Steinernema longicaudum, Steinernema glaseri and Steinernema kraussei), early emerged nematodes had a higher proportion of males than those that emerged later, with the reverse trend for Steinernema carpocapsae and Steinernema feltiae. In a more detailed in vitro study of S. longicaudum, the proportion of males was similar whether or not the nematodes passed through the developmentally arrested IJ stage, indicating that the female bias is not due to failure of males to exit this stage. The sex ratio in vitro was independent of survival rate from juvenile to adult, and was female-biased even when all juveniles developed, indicating that the bias is not explained by failure of males to develop to adults. The female-biased sex ratio characteristic of Steinernema populations appears to be present from at least the early juvenile stage. We hypothesise that the observed female bias is the population optimal sex ratio, a response to cycles of local mate competition experienced by nematodes reproducing within insect hosts interspersed with periods of outbreeding with less closely related worms following dispersal.     

Effect of insect cadaver desiccation and soil water potential during rehydration on entomopathogenic nematode (Rhabditida: Steinernematidae and Heterorhabditidae) production and virulence

We examined the influence of insect cadaver desiccation on the virulence and production of entomopathogenic nematodes (EPNs), common natural enemies of many soil-dwelling insects. EPNs are often used in biological control, and we investigated the feasibility of applying EPNs within desiccated insect cadavers. Desiccation studies were conducted using the factitious host, Galleria mellonella (Lepidoptera: Pyralidae, wax moth larvae) and three EPN species (Heterorhabditis bacteriophora ‘HB1’, Steinernema carpocapsae ‘All’, and Steinernema riobrave). Weights of individual insect cadavers were tracked daily during the desiccation process, and cohorts were placed into emergence traps when average mass losses reached 50%, 60%, and 70% levels. We tracked the proportion of insect cadavers producing infective juveniles (IJs), the number and virulence of IJs produced from desiccated insect cadavers, and the influence of soil water potentials on IJ production of desiccated insect cadavers. We observed apparent differences in the desiccation rate of the insect cadavers among the three species, as well as apparent differences among the three species in both the proportion of insect cadavers producing IJs and IJ production per insect cadaver. Exposure of desiccated insect cadavers to water potentials greater than −2.75 kPa stimulated IJ emergence. Among the nematode species examined, H. bacteriophora exhibited lower proportions of desiccated insect cadavers producing IJs than the other two species. Desiccation significantly reduced the number of IJs produced from insect cadavers. At the 60% mass loss level, however, desiccated insect cadavers from each of the three species successfully produced IJs when exposed to moist sand, suggesting that insect cadaver desiccation may be a useful approach for biological control of soil insect pests.     

The lethal and sub-lethal consequences of entomopathogenic nematode infestation and exposure for adult pine weevils, Hylobius abietis (Coleoptera: Curculionidae)

Entomopathogenic nematodes (EPN) frequently kill their host within 1-2 days, and interest in EPN focuses mainly on their lethality. However, insects may take longer to die, or may fail to die despite being infected, but little is known about the effects of EPN infection on insects, other than death. Here we investigate both lethal and sub-lethal effects of infection by two EPN species, Steinernema carpocapsae and Heterorhabditis downesi, on adults of the large pine weevil, Hylobius abietis. Following 12 h nematode-weevil contact in peat, S. carpocapsae killed a significantly higher proportion of weevils (87-93%) than H. downesi (43-57%) at all concentrations tested. Less than 10% of weevils were dead within 2 days, and weevils continued to die for up to 10 days after exposure (LT₅₀ of 3 days or more). In a separate experiment, live weevils dissected 6 days after a 24 h exposure to nematodes on filter paper harbored encapsulated and dead nematodes, showing that weevils could defend themselves against infection. Some live weevils also harbored live nematodes 6 days after they had been removed from the nematode infested medium. Feeding by weevils was not affected by infection with, or exposure to, either species of EPN. We discuss these results in relation to the use of EPN in biological control against H. abietis.     

Effects of Culture Method and Formulation on the Virulence of Steinernema riobrave (Rhabditida: Steinernematidae) to Diaprepes abbreviatus (Coleoptera: Curculionidae)

The Diaprepes root weevil, Diaprepes abbreviatus, is a pest of vegetables, ornamental plants, sugarcane, and citrus in Florida and the Caribbean. The entomopathogenic nematode, Steinernema riobrave, can reduce larval populations of D. abbreviatus substantially. Efficacy of entomopathogenic nematodes, however, may be affected by culture method and formulation. Using D. abbreviatus as the host, we compared the efficacy of two commercial S. riobrave formulations, a liquid and a waterdispersible granule (WDG), with each other and with in vivo produced S. riobrave. Nematodes in the commercial formulations were produced in vitro through liquid fermentation; the in vivo nematodes were cultured in Galleria mellonella and applied in aqueous suspension. Laboratory experiments measured nematode virulence in plastic cups containing soil and seventh-eighth instar D. abbreviatus. One laboratory experiment was conducted using only fresh nematodes (less than 5 days old); another experiment included WDG nematodes that were stored for 25 days at 10 °C. Two field experiments were conducted in which nematodes were applied either to potted citrus (containing D. abbreviatus larvae) placed beneath mature citrus trees or to soil directly beneath the tree. In the latter experiment, efficacy was determined by measuring mortality of caged D. abbreviatus larvae that were buried beneath the soil surface prior to application. Mortality of D. abbreviatus treated with nematodes ranged from 80-98% and 50-75% in laboratory and field experiments, respectively. In all experiments, we did not detect any significant effects of culture method or formulation.     

Effect of Soil Depth and Moisture on the Vertical Distribution of Steinernema riobrave (Nematoda: Steinernematidae)

The effect of soil moisture on the distribution of Steinernema riobrave in a sand column was determined. Larvae of Pectinophora gossypiella were used to detect S. riobrave infective juveniles (IJ) in each 2.5-cm section of 30-cm-long soil columns. Soil moisture was determined for each section and related to the numbers of nematodes recovered from infected insect baits. Infective juveniles of S. riobrave applied on the sand column surface showed some degree of positive geotaxis. IJ in soil columns with a consistent moisture gradient grouped in the upper 12.7 cm within a water potential range of ¯40 to ¯0.0055 MPa (2% to 14% moisture). Nematodes in sand columns that were gradually dehydrating moved down the soil column, aggregating on the 28th day between 15-23 cm in depth. Nematode redistribution over time allowed IJ to remain within a water potential range of ¯0.1 to ¯0.012 MPa (5.2% to 9.5% moisture).