Effectiveness of different species of entomopathogenic nematodes for biocontrol of the Mediterranean flatheaded rootborer, Capnodis tenebrionis (Linné) (Coleoptera: Buprestidae) in potted peach tree

The susceptibility of larvae of the Mediterranean flatheaded rootborer (Capnodis tenebrionis) to 13 isolates of entomopathogenic nematodes was examined using GF-677 potted trees (peachxalmond hybrid) as the host plant. The nematode strains tested included nine Steinernema feltiae, one S. affine, one S. carpocapsae and two Heterorhabditis bacteriophora. Nematodes showed the ability to locate and kill larvae of C. tenebrionis just after they enter into the roots of the tree. S. feltiae strains provided an efficacy ranging from 79.68% to 88.24%. H. bacteriophora strains resulted in control of 71.66-76.47%. S. carpocapsae (B14) and S. affine (Gspe3) caused lower control of C. tenebrionis larvae (62.03% and 34.76%, respectively). The influence of foraging strategy and the use of autochthonous nematodes to control C. tenebrionis larvae inside the roots is discussed.     

Virulence of entomopathogenic nematodes to pecan weevil larvae, Curculio caryae (Coleoptera: Curculionidae), in the laboratory

The pecan weevil, Curculio caryae (Horn), is a key pest of pecans in the Southeast. Entomopathogenic nematodes have been shown to be pathogenic toward the larval stage of this pest. Before this research, only three species of nematodes had been tested against pecan weevil larvae. In this study, the virulence of the following nine species and 15 strains of nematodes toward fourth-instar pecan weevil was tested: Heterorhabditis bacteriophora Poinar (Baine, HP88, Oswego, NJ1, and Tf strains), H. indica Poinar, Karunakar & David (original and Homl strains), H. marelatus Liu & Berry (IN and Point Reyes strains), H. megidis Poinar, Jackson & Klein (UK211 strain), H. zealandica Poinar (NZH3 strain), Steinernema riobrave Cabanillas, Poinar & Raulston (355 strain), S. carpocapsae (Weiser) (All strain), S. feltiae (Filipjev) (SN strain), and S. glaseri (Steiner) (NJ43 strain). No significant difference in virulence was detected among nematode species or strains. Nematode-induced mortality was not significantly greater than control mortality (in any of the experiments conducted) for the following nematodes: H. bacteriophora (Baine), H. zealandica (NZH3), S. carpocapsae (All), S. feltiae (SN), S. glaseri (NJ43), and S. riobrave (355). All other nematodes caused greater mortality than the control in at least one experiment. Heterorhabditis megidis (UK211) but not H. indica (original) displayed a positive linear relationship between nematode concentration and larval mortality. Results suggested that, as pecan weevil larvae age, they may have become more resistant to infection with entomopathogenic nematodes.     

Compatibility of entomopathogenic nematodes with fipronil

The survival and infectivity of infective juveniles (IJs) of three species of entomopathogenic nematodes, Steinernema carpocapsae Weiser, S. arenarium (Artyukhovsky) (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), were determined after exposure to different concentrations (250, 500, 1000 and 2000 ppm) of fipronil, an insecticide acting on the GABA receptors to block the chloride channel. Heterorhabditis bacteriophora was very tolerant to all concentrations of fipronil, with the highest mortality of 17% being observed at 2000 ppm of fipronil after 72 h exposure. Steinernema carpocapsae showed a similar response, with the highest mortality of 11.25% of IJs being observed after 72 h exposure to 2000 ppm of fipronil. Steinernema arenarium was, however, more sensitive to fipronil, and at 2000 ppm mortality rates of 94.6% and 100% were observed after 24 and 72 h, respectively. Fipronil had negligible effects on the infectivity of the three nematode species tested. The IJs which survive exposure to all concentrations of fipronil tested can infect and reproduce in Galleria larvae. The moderate effects on entomopathogenic nematodes of a lower fipronil concentration (250 ppm) and the field rates (12-60 ppm) of fipronil used as insecticide, suggest that direct mixing of entomopathogenic nematodes and fipronil at field rates is a viable integrated pest management option.     

Host cadavers protect entomopathogenic nematodes during freezing

The entomopathogenic nematodes Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema glaseri, and Steinernema feltiae were exposed to freezing while inside their hosts. Survival was assessed by observing live and dead nematodes inside cadavers and by counting the infective juveniles (IJs) that emerged after freezing. We (1) measured the effects of 24h of freezing at different times throughout the course of an infection, (2) determined the duration of freezing entomopathogenic nematodes could survive, (3) determined species differences in freezing survival. Highest stage-specific survival was IJs for S. carpocapsae, and adults for H. bacteriophora. When cadavers were frozen two or three days after infection, few IJs emerged from them. Freezing between five and seven days after infection had no negative effect on IJ production. No decrease in IJ production was measured for H. bacteriophora after freezing. H. bacteriophora also showed improved survival inside versus outside their host when exposed to freezing.     

Characterization of New Entomopathogenic Nematodes from Thailand: Foraging Behavior and Virulence to the Greater Wax Moth, Galleria mellonella L. (Lepidoptera: Pyralidae)

Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis and their associated bacteria (Xenorhabdus spp. and Photorhabdus spp., respectively) are lethal parasites of soil dwelling insects. We collected 168 soil samples from five provinces, all located in southern Thailand. Eight strains of EPNs were isolated and identified to species using restriction profiles and sequence analysis. Five of the isolates were identified as Heterorhabditis indica, and one as Heterorhabditis baujardi. Two undescribed Steinernema spp. were also discovered which matched no published sequences and grouped separately from the other DNA restriction profiles. Behavioral tests showed that all Heterorhabditis spp. were cruise foragers, based on their attraction to volatile cues and lack of body-waving and standing behaviors, while the Steinernema isolates were more intermediate in foraging behavior. The infectivity of Thai EPN strains against Galleria mellonella larvae was investigated using sand column bioassays and the LC(50) was calculated based on exposures to nematodes in 24-well plates. The LC(50) results ranged from 1.99-6.95 IJs/insect. Nine centimeter columns of either sandy loam or sandy clay loam were used to determine the nematodes' ability to locate and infect subterranean insects in different soil types. The undescribed Steinernema sp. had the greatest infection rate in both soil types compared to the other Thai isolates and three commercial EPNs (Heterorhabditis bacteriophora, Steinernema glaseri and Steinernema riobrave).     

Susceptibility of diamond back moth,Plutella xylostella (L) to entomopathogenic nematodes

Eight entomopathogenic nematode species / strains, Steinernema glaseri (steiner), S. carpocapsae (Weiser), S. feltiae (Filipjev), Steinernema sp. Ecomax strain, Heterorhabditis bacteriophora (Pioner), Heterorhabditis sp. Ecomax strain, two locally isolated strains called as JFC and TFC were tested against the final instar larvae of diamond back moth, Plutella xylostella (L.). All nematodes were found pathogenic. However, H. bacteriophora was adjudged the most pathogenic amongst the test nematodes on the basis of LD50 (9.16 IJS/larva), LT50 (43.26 hr), Lex T50 (3.24 hr) and the propagation potential (average of 271.42 IJS/mg) on the host body weight.     

Virulence of entomopathogenic nematodes to Diaprepes abbreviatus (Coleoptera: Curculionidae) in the laboratory

The Diaprepes root weevil, Diaprepes abbreviatus (L.) is the most severe weevil pest in Florida citrus. Entomopathogenic nematodes have effectively suppressed larval populations of D. abbreviatus. Our objective was to conduct a broad laboratory comparison of entomopathogenic nematodes for virulence toward larvae of D. abbreviatus. The study was conducted at three temperatures (20, 24, and 29 degrees C) and included nine entomopathogenic species and 17 strains: Heterorhabditis bacteriophora Poinar (Baine, NJl, Hb, Hbl, HP88, and Lewiston strains), H. indica Poinar, Karunakar & David (original and Homl strains), H. marelatus Liu & Berry (IN and Point Reyes strains), H. megidis Poinar, Jackson & Klein (UK21l strain), H. zealandica Poinar (NZH3 strain), Steinernema riobrave Cabanillas, Poinar & Raulston (355 strain), S. carpocapsae (Weiser) (All strain), S. feltiae (Filipjev) (SN and UK76 strains), and S. glaseri (Steiner) (NJ43 strain). At 20 degrees C, the greatest mortality was caused by S. riobrave although it was not significantly greater than H. bacteriophora (Baine), H. bacteriophora (Hb), H. bacteriophora (Hbl), and H. indica (original). At 24 and 29 degrees C, S. riobrave caused greater larval mortality than other nematodes tested. Two strains of H. indica, H. bacteriophora (Baine), and S. glaseri were next in terms of virulence at 29 degrees C. Our results suggest that S. riobrave has the greatest potential for control of D. abbreviatus.     

Parasitism of bark scorpion Centruroides exilicauda (Scorpiones: Buthidae) by entomopathogenic nematodes (Rhabditida: Steinernematidae; Heterorhabditidae)

In laboratory bioassays, Steinernema glaseri Steiner, Steinernema riobrave Cabanillas, Poinar & Raulston, Heterorhabditis bacteriophora Poinar, and Heterorhabditis marelatus Liu & Berry were capable of infecting and killing the bark scorpion, Centruroides exilicauda (Wood). Steinernema feltiae (Filipjev) and Steinernema carpocapsae (Weiser) failed to infect C. exilicauda at 22 degrees C. S. glaseri, H. marelatus, and H. bacteriophora caused significant mortality at 22 degrees C, indicating the potential role of these parasites as a biocontrol option. Efficacy of S. glaseri and H. bacteriophora was reduced in an assay conducted at 25 degrees C. Only S. glaseri was able to reproduce in the target host. Dissection of scorpions at the end of the experimental periods revealed inactive juvenile S. riobrave, H. marelatus, and H. bacteriophora nematodes. Both mermithid and oxyurid nematodes have been documented as nematode parasites of scorpions, but rhabditids have not been reported until now. Field studies are warranted to assess the usefulness of entomopathogenic nematodes as biocontrol agents of bark scorpions.     

Rapid age-related changes in infection behavior of entomopathogenic nematodes

Nonfeeding infective juvenile (IJ) entomopathogenic nematodes (EPNs) are used as biological agents to control soil-dwelling insects, but poor storage stability remains an obstacle to their widespread acceptance by distributors and growers as well as a frustration to researchers. Age is one factor contributing to variability in EPN efficacy. We hypothesized that age effects on the infectiousness of IJs would be evident within the length of time necessary for IJs to infect a host. The penetration behavior of "young" (<1-wk-old) and "old" (2- to 4-wk-old) Heterorhabditis bacteriophora (GPS 11 strain), Steinernema carpocapsae (All strain), and Steinernema feltiae (UK strain) IJs was evaluated during 5 "exposure periods" to the larvae of the wax moth, Galleria mellonella. Individual larvae were exposed to nematode-infested soil for exposure periods of 4, 8, 16, 32, and 64 hr. Cadavers were dissected after 72 hr, and the IJs that penetrated the larvae were counted. Larval mortality did not differ significantly between 72- and 144-hr "observation periods," or points at which larval mortality was noted, for any age class or species. However, age and species effects were noted in G. mellonella mortality and nematode penetration during shorter time periods. Initial mortality caused by S. carpocapsae and H. bacteriophora IJs declined with nematode age but increased with S. feltiae IJ age. Young S. carpocapsae IJs penetrated G. mellonella larvae at higher rates than old members of the species (27-45% vs. 1-4%). Conversely, old S. feltiae IJs had higher penetration rates than young IJs (approximately 8 to 57% vs. 4 to approximately 31%), whereas H. bacteriophora IJs had very low penetration rates regardless of age (3-5.6%). Our results show that the effect of age on IJ infectiousness can be detected in IJs aged only 2 wk by a 4-hr exposure period to G. mellonella. These results have important implications for storage and application of EPNs and suggest the possibility of shortening the time required to detect nematodes in the soil.     

Susceptibility of Sap Beetles (Coleoptera: Nitidulidae) to Entomopathogenic Nematodes

Nitidulid beetles (Coleoptera) are considered to be serious pests of date palms throughout the world. They attack ripe fruit, causing it to rot, and damage is reflected in both reduced yield and lower fruit quality. The present study was aimed at an evaluation of the susceptibility to different sap beetles to entomopathogenic nematodes. We further tested nematode efficacy in pots filled with soil infested by third instar larvae of the two beetle species. In Petri dish assay, mortality levels of Carpophilus humeralis and C. hemipterus exposed to Heterorhabditis sp. IS-5 strain indicated that the latter is less susceptible to nematode infection. Exposure of both sap beetle species to different nematode strains gave moderate levels of mortality (35-65%) with the heterorhabditid strains HP88, IS-5 and IS-25. The IS-12 strain of Heterorhabditis sp. showed poor virulence (<35% mortality) against larvae of C. humeralis as well as larvae and pupae of C. humipterus. The nematode species S. riobrave showed moderate virulence (35-65%) mortality to larvae and pupae of S. humeralis as well as to larvae of C. hemipterus . Exposure of C. hemeralis to different concentrations of Heterorhabditis sp. IS-5 in pots containing soil resulted in high mortality (>65%). In contrast, the lower concentrations (500 and 1000 nematodes/pot) caused low mortality (35%) of C. hemipterus . Other heterorhabditid strains caused 95-100% mortality of C. humeralis in pot assay. The HP88 strain of H. bacteriophora and the Tx strain of Steinernema riobrave showed poor effectiveness. Incubation of different nematode strains with the C. humeralis larvae at high temperature (32 C) resulted in an increase in insect mortality with the IS-12 and IS-21 strains. Reduced mortality was recorded with the HP88 strain treatment at the higher temperature. The IS-5 and IS-12 strains were equally effective in all three soil types tested, whereas the IS-19 strain was more effective in the Almog type soil than in the others.     

Susceptibility of the boll weevil to Steinernema riobrave and other entomopathogenic nematodes

The susceptibility of the boll weevil (BW), Anthonomus grandis Boheman, to Steinernema riobrave and other nematode species in petri dishes, soil (Hidalgo sandy clay loam), and cotton bolls and squares was investigated. Third instar weevils were susceptible to entomopathogenic nematode (EN) species and strains in petri dish bioassays at 30 degrees C. Lower LC(50)'s occurred with S. riobrave TX- 355 (2 nematodes per weevil), S. glaseri NC (3), Heterorhabditis indicus HOM-1 (5), and H. bacteriophora HbL (7) than H. bacteriophora IN (13), S. riobrave TX (14), and H. bacteriophora HP88 (21). When infective juveniles (IJs) of S. riobrave were applied to weevils on filter paper at 25 degrees C, the LC(50) of S. riobrave TX for first, second, and third instars, pupae, and 1-day-old and 10-days-old adult weevils were 4, 5, 4, 12, 13, and 11IJs per weevil, respectively. The mean time to death, from lowest to highest concentration, for the first instar (2.07 and 1.27days) and second instar (2.55 and 1.39days) weevils were faster than older weevil stages. But, at concentrations of 50 and 100IJs/weevil, the mean time to death for the third instar, pupa and adult weevils were similar (1.84 and 2.67days). One hundred percent weevil mortality (all weevil stages) occurred 3days after exposure to 100IJs per weevil. Invasion efficiency rankings for nematode concentration were inconsistent and changed with weevil stage from 15 to 100% when weevils were exposed to 100 and 1IJs/weevil, respectively. However, there was a consistent relationship between male:female nematode sex ratio (1:1.6) and nematode concentration in all infected weevil stages. Nematode production per weevil cadaver increased with increased nematode concentrations. The overall mean yield of nematodes per weevil was 7680IJs. In potted soil experiments (30 degrees C), nematode concentration and soil moisture greatly influenced the nematode efficacy. At the most effective concentrations of 200,000 and 400,000IJs/m(2) in buried bolls or squares, higher insect mortalities resulted in pots with 20% soil moisture either in bolls (94 and 97% parasitism) or squares (92 and 100% parasitism) than those of 10% soil moisture in bolls (44 and 58% parasitism) or squares (0 and 13% parasitism). Similar results were obtained when nematodes were sprayed on the bolls and squares on the soil surface. This paper presents the first data on the efficacy of S. riobrave against the boll weevil, establishes the potential of EN to control the BW inside abscised squares and bolls that lay on the ground or buried in the soil.     

Relationship between the successful infection by entomopathogenic nematodes and the host immune response

Reproduction of entomopathogenic nematodes requires that they escape recognition by a host's immune system or that they have mechanisms to escape encapsulation and melanization. We investigated the immune responses of larvae for the greater wax moth (Galleria mellonella), tobacco hornworm (Manduca sexta), Japanese beetle (Popillia japonica), northern masked chafer (Cyclocephala borealis), oriental beetle (Exomala orientalis) and adult house crickets (Acheta domesticus), challenged with infective juveniles from different species and strains of entomopathogenic nematodes. The in vivo immune responses of hosts were correlated with nematode specificity and survival found by infection assays. In P. japonica, 45% of injected infective juveniles from Steinernema glaseri NC strain survived; whereas the hemocytes from the beetle strongly encapsulated and melanized the Heterorhabditis bacteriophora HP88 strain, S. glaseri FL strain, Steinernema scarabaei and Steinernema feltiae. Overall, H. bacteriophora was intensively melanized in resistant insect species (E. orientalis, P. japonica and C. borealis) and had the least ability to escape the host immune response. Steinernema glaseri NC strain suppressed the immune responses in susceptible hosts (M. sexta, E. orientalis and P. japonica), whereas S. glaseri FL strain was less successful. Using an in vitro assay, we found that hemocytes from G. mellonella, P. japonica, M. sexta and A. domestica recognized both nematode species quickly. However, many S. glaseri in M. sexta and H. bacteriophora in G. mellonella escaped from hemocyte encapsulation by 24h. These data indicate that, while host recognition underlies some of the differences between resistant and susceptible host species, escape from encapsulation following recognition can also allow successful infection. Co-injected surface-coat proteins from S. glaseri did not protect H. bacteriophora in M. sexta but did protect H. bacteriophora in E. orientalis larva; therefore, surface coat proteins do not universally convey host susceptibility. Comparisons of surface coat proteins by native and SDS-PAGE demonstrated different protein compositions between H. bacteriophora and S. glaseri and between the two strains of S. glaseri.     

Biological Control of the Pecan Weevil,Curculio caryae (Coleoptera: Curculionidae), with Entomopathogenic Nematodes

Steinernema carpocapsae (Weiser) strain A11, S. feltiae (Filipjev) strain SN, and Heterorhabditis bacteriophora Poinar strains HP88 and Georgia were tested for their efficacy as biological control agents of the pecan weevil, Curculio caryae (Horn), in pecan orchard soil-profile containers under greenhouse conditions. Percentage C. caryae parasitism by S. carpocapsae and H. bacteriophora strain HP88 and Georgia was consistently poor when applied either prior to or following C. caryae entry into the soil, suggesting that these nematode species and (or) their enterobacteria are poor biological control agents of weevil larvae. Soil taken 21 days following application of S. carpocapsae or H. bacteriophora strain HP88 induced a low rate of infection of Galleria mellonella larvae, whereas soil that had been similarily treated with H. bacteriophora strain Georgia induced a moderate rate of infection. Percentage C. caryae parasitism by S. feltiae was consistently low when applied following C. caryae entry into the soil and was inconsistent when applied as a barrier prior to entry of weevil larvae into the soil. Soil taken 21 days following application of S. feltiae induced a high rate of infection of G. mellonella larvae.     

Effect of soil type on infectivity and persistence of the entomopathogenic nematodes Steinernema scarabaei, Steinernema glaseri, Heterorhabditis zealandica, and Heterorhabditis bacteriophora

We tested the effect of soil type on the performance of the entomopathogenic pathogenic nematodes Steinernema scarabaei, Steinernema glaseri, Heterorhabditis zealandica, and Heterorhabditis bacteriophora. Soil types used were loamy sand, sandy loam, loam, silt loam, clay loam, acidic sand, and a highly organic potting mix. Infectivity was tested by exposing third-instar Anomala orientalis or Popillia japonica to nematodes in laboratory and greenhouse experiments and determining nematode establishment in the larvae and larval mortality. Infectivity of H. bacteriophora and H. zealandica was the highest in potting mix, did not differ among loamy sand and the loams, and was the lowest in acidic sand. Infectivity of S. glaseri was significantly lower in acidic sand than in loamy sand in a laboratory experiment but not in a greenhouse experiment, and did not differ among the other soils. Infectivity of S. scarabaei was lower in silt loam and clay loam than in loamy sand in a greenhouse experiment but not in a laboratory experiment, but was the lowest in acidic sand and potting mix. Persistence was determined in laboratory experiments by baiting nematode-inoculated soil with Galleria mellonella larvae. Persistence of both Heterorhabditis spp. and S. glaseri was the shortest in potting mix and showed no clear differences among the other substrates. Persistence of S. scarabaei was high in all substrates and its recovery declined significantly over time only in clay loam. In conclusion, generalizations on nematode performance in different soil types have to be done carefully as the effect of soil parameters including soil texture, pH, and organic matter may vary with nematode species.     

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.     

Soil Applications of Steinernematid and Heterorhabditid Nematodes for Control of Colorado Potato Beetles,Leptinotarsa decemlineata (Say)

Three strains of Steinernema feltiae Filipjev (All, Mexican, and Breton strains) and one of Heterorhabditis heliothidis (Khan, Brooks, and Hirschmann) were evaluated for their potential to control Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), larvae and pupae in the soil. In laboratory studies, H. heliothidis and S. feltiae (Mexican strain) produced the highest mortality (6 days posttreatment) of CPB when applied to the surface of a soil column containing mature CPB larvae 5 cm below. Mortality ranged from 80 to 90% at rates of 79-158 nematodes/cm². Similar results were seen in a field microplot study with all four nematodes; S. feltiae (Mexican strain) and H. heliothidis were most effective. Adult CPB emergence was reduced 86.5-100% after application of 31-93 H. heliothidis/cm² and 88.4-100% with 93-155 S. feltiae (Mexican strain)/cm². The All strain of S. feltiae was moderately effective (ca. 80% reduction at 93-155 nematodes/cm²), while the Breton strain was ineffective (< 40% reduction at 155 nematodes/cm²). In small plots of potatoes enclosed in field cages, application of H. heliothidis and S. feltiae (Mexican strain) at rates of 93-155 nematodes/cm² before larval CPB burial in the soil resulted in 66-77% reduction in adult CPB emergence. Soil applications of these nematodes show potential for biological control of CPB.     

Comparison of Bioassays to Measure Virulence of Different Entomopathogenic Nematodes

Five bioassays were compared for their usefulness to determine the virulence of four nematode strains. The objective of this study was to develop standard assays for particular nematode species. In all assays, the nematodes Steinernema feltiae (strain UK), S. riobravis, S. scapterisci Argentina and Heterorhabditis bacteriophora HP88 were exposed to Galleria mellonella larvae. All bioassays except the sand column assay were conducted in multi-well plastic dishes. In the penetration rate assay, the number of individual nematodes invading the insect was determined after a 48-h exposure to 200 infective juveniles (IJs). In the one-on-one assay, each larva was exposed to an individual nematode for 72 h before insect mortality was recorded. In the exposure time assay, insect mortality was recorded after exposure to 200 IJs for variable time periods. The dose-response assay involved exposing larvae to different nematode concentrations over the range 1-200 IJs/insect and recording mortality every 24 h for a 96-h period. In the sand columns assay, insects were placed in the bottom of a plastic cylinder filled with sand. Nematodes were applied on top of the sand and insect mortality was determined after IJs had migrated through the cylinder. The highest mortality level in the sand column assay was obtained with IJs of S. feltiae followed by H. bacteriophora; treatments with S. riobravis and S. scapterisci produced low levels of insect mortality. In the other four assays, S riobravis was the most virulent, followed by S. feltiae, H. bacteriophora and S. scapterisci. In the exposure time assay, rapid mortality was achieved when the insects were exposed to S. feltiae and S. riobravis. For these nematode species, a gradual increase in the number of individuals which penetrated into cadavers was recorded. Conversely, the number of nematodes in the cadavers of insects infected by H. bacteriophora and S. scapterisci remained low during the entire exposure period. In this assay, exposing the insects to these nematodes resulted in a gradual increase in mortality. In the dose-response assay, complete separation among nematode species was obtained only after 48 h of incubation at a concentration of 15 IJs/insect. LD and LD values were calculated from 50 90 dose-response assay data. However, these values did not indicate differences among the different nematode species. The present study demonstrated the variation in entomopathogenic nematode performance in different bioassays and supports the notion that one common bioassay cannot be used as a universal measure of virulence for all species and strains because nematodes differ in their behavior. Furthermore, particular assays should be used for different purposes. To select a specific population for use against a particular insect, assays that are more laborious but which simulate natural environmental conditions (e.g. the sand column assay) or invasion by the nematode (e.g. the penetration rate assay) should be considered. In cases where commercial production batches of the same nematode strains are compared, simple and fast assays are needed (e.g. the one-on-one and exposure time assays). Further studies are needed to determine the relationships between data obtained in each assay and nematode efficacy in the field.     

Infectivity and reproductive potential of the Oswego strain of Heterorhabditis bacteriophora associated with life stages of the clover root curculio,Sitona hispidulus

The infectivity and reproductive potential of the entomopathogenic nematode Heterorhabditis bacteriophora (Oswego strain), at different concentrations, was studied. Seventy to 80.0% mortality to late instar larvae of the clover root curculio, Sitona hispidulus, and 40.0-76.0% mortality to pupae, was observed at concentrations of 15-100 infective juveniles. There were no significant differences in mortality among nematode concentrations. LC(50) levels of 4.0 and 21.4 nematodes were determined for clover root curculio larvae and pupae, respectively. Nematodes did not cause significant mortality to adult or first instar clover root curculio. H. bacteriophora was able to complete its development and reproduce in 74.0-95.0% of clover root curculio late instar larvae and pupae. Reproductive potential in curculio larvae and pupae ranged from 0 to 7040 infective juveniles per host. Larvae exposed to 100 nematodes had a reproductive potential significantly higher than in those larvae exposed to 15 and 50 nematodes. Reproductive potential in pupae decreased with an increased nematode dose, indicating potential crowding effects. Host larval and pupal mass were positively correlated with nematode progeny production.     

Differences in penetration routes and establishment rates of four entomopathogenic nematode species into four white grub species

We compared the penetration of the entomopathogenic nematodes Steinernema scarabaei (AMK001 strain), S. glaseri (NC1 strain), Heterorhabditis zealandica (X1 strain), and H. bacteriophora (GPS11 strain) into third-instars of the scarabs Popillia japonica, Anomala orientalis, Cyclocephala borealis, and Rhizotrogus majalis. When larvae were exposed to nematodes for 6-72 h larval mortality and nematode establishment rate and occasionally speed of kill often showed the same pattern within nematode-white grub combinations. But no two nematodes or white grub species had the same pattern for these observations for all white grub or nematode species, respectively. Mortality, establishment, and speed of kill followed a similar pattern for H. zealandica, S. glaseri, and S. scarabaei, but there was no clear relationship for H. bacteriophora. Significant nematode establishment was only observed after at least 48 h exposure in most nematode-white grub combinations. Faster establishment was observed only for H. zealandica in A. orientalis and R. majalis (after 24 h) and for S. scarabaei in P. japonica and R. majalis (after 12 h). Nematode establishment after 72 h in the different scarab species was generally low for S. glaseri (<1.5%) and H. bacteriophora (<3%), higher for H. zealandica (2-5%), and the highest for S. scarabaei (1-14%). However, in another experiment establishment was generally higher after 96h exposure. Nematode penetration sites were determined by comparing nematode establishment in larvae with mouth, anus, mouth+anus, or none sealed with glue. The trends for each nematode species were very similar in the different white grub species. H. zealandica and H. bacteriophora showed excellent cuticular penetration ability but may also penetrate through mouth and/or anus. S. glaseri also penetrated through the cuticle but lower establishment in larvae with mouth or mouth+anus sealed suggested that the mouth is an important penetration site. S. scarabaei showed a preference for the mouth as a penetration site, but it showed some cuticular penetration ability and may also use the anus as a penetration site. The methodology used cannot exclude that cuticular penetration also included penetration through the spiracles. To fully understand the effect of nematode and white grub species on nematode virulence, future studies will have to compare host immune response to the penetrating IJs and the role of the symbiotic bacteria in these interactions.