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.     

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.     

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.     

Impact of the host cadaver on survival and infectivity of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) under desiccating conditions

Entomopathogenic nematode species of Steinernema carpocapsae, Steinernema riobrave, or Heterorhabditis bacteriophora were used to compare survival and infectivity among infective juveniles (IJs) emerging in water from hosts in White traps (treatment a), emerging in sand from hosts placed in sand (treatment c), and emerging from hosts placed on a mesh suspended over sand (treatment m). Nematode survival and infectivity was recorded in sand at three-day intervals during 21 days of storage in desiccators at 75% relative humidity and 25 degrees C. Infectivity was measured by exposing 5 Galleria mellonella for 16 h to IJs. Treatment did not affect percent survival of H. bacteriophora IJs. Percent survival of S. riobrave and S. carpocapsae IJs was lowest in treatment a. Across all treatments, by 10 days after the beginning of the experiments, IJ survival declined to 93, 43, and 28% of levels on day 1 for H. bacteriophora, S. riobrave, and S. carpocapsae, respectively. For the three treatments, infection rate over time was described by a negative exponential function for S. riobrave and S. carpocapsae and by a sigmoid function for H. bacteriophora.     

Susceptibility of the strawberry crown moth (Lepidoptera: Sesiidae) to entomopathogenic nematodes

The objective of this study was to determine the susceptibility of the strawberry crown moth, Synanthedon bibionipennis (Boisduval) (Lepidoptera: Sesiidae) larvae to two species of entomopathogenic nematodes. The entomopathogenic nematodes Steinernema carpocapsae (Weiser) strain Agriotos and Heterorhabditis bacteriophora (Steiner) strain Oswego were evaluated in laboratory soil bioassays and the field. Both nematode species were highly infective in the laboratory bioassays. Last instars were extremely susceptible to nematode infection in the laboratory, even in the protected environment inside the strawberry (Fragaria x ananassa Duch.) crown. Infectivity in the laboratory was 96 and 94% for S. carpocapsae and H. bacteriophora, respectively. Field applications in late fall (October) were less effective with S. carpocapsae and H. bacteriophora, resulting in 51 and 33% infection, respectively. Larval mortality in the field from both nematode treatments was significantly greater than the control, but treatments were substantially less efficacious than in the laboratory. Soil temperature after nematode applications in the field (11 degrees C mean daily temperature) was below minimum establishment temperatures for both nematode species for a majority of the post-application period. It is clear from laboratory data that strawberry crown moth larvae are extremely susceptible to nematode infection. Improved control in the field is likely if nematode applications are made in late summer to early fall when larvae are present in the soil and soil temperatures are more favorable for nematode infection.     

A Phosphopantetheinyl transferase homolog is essential for Photorhabdus luminescens to support growth and reproduction of the entomopathogenic nematode Heterorhabditis bacteriophora

The bacterium Photorhabdus luminescens is a symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora. The nematode requires the bacterium for infection of insect larvae and as a substrate for growth and reproduction. The nematodes do not grow and reproduce in insect hosts or on artificial media in the absence of viable P. luminescens cells. In an effort to identify bacterial factors that are required for nematode growth and reproduction, transposon-induced mutants of P. luminescens were screened for the loss of the ability to support growth and reproduction of H. bacteriophora nematodes. One mutant, NGR209, consistently failed to support nematode growth and reproduction. This mutant was also defective in the production of siderophore and antibiotic activities. The transposon was inserted into an open reading frame homologous to Escherichia coli EntD, a 4'-phosphopantetheinyl (Ppant) transferase, which is required for the biosynthesis of the catechol siderophore enterobactin. Ppant transferases catalyze the transfer of the Ppant moiety from coenzyme A to a holo-acyl, -aryl, or -peptidyl carrier protein(s) required for the biosynthesis of fatty acids, polyketides, or nonribosomal peptides. Possible roles of a Ppant transferase in the ability of P. luminescens to support nematode growth and reproduction are discussed.     

河北异小杆线虫一品系的分类鉴定及其对蛴螬致病力的测定

昆虫病原线虫是一种有应用潜力的地下害虫生物防治因子。本研究利用形态学特征和ITS-rDNA分析方法对从河北省沧州分离的异小杆线虫一品系进行了鉴定, 并在室内测定比较了其对蛴螬的致病力。通过对该线虫侵染期幼虫和雄性成虫主要形态学特征的参数测量, 发现其与嗜菌异小杆线虫Heterorhabditis bacteriophora的形态特征最为相近;同时ITS1-rDNA序列比对和系统发育学分析结果显示其与嗜菌异小杆线虫亲缘关系最近。结合形态学和分子生物学特征, 确定该线虫为嗜菌异小杆线虫沧州品系。该线虫对蛴螬（华北大黑鳃金龟Holotrichia oblita、暗黑鳃金龟H. parallela和铜绿丽金龟Anomala corpulenta 3种金龟子的2龄幼虫）的致病力研究结果表明：处理72 h后, 暗黑鳃金龟幼虫死亡率显著高于另外两种金龟子幼虫 (P<0.05);处理120 h后, 暗黑鳃金龟和铜绿丽金龟幼虫的死亡率分别达到93.3%和80.0%, 二者无显著差异 (P>0.05), 可见该线虫对它们有较强的致病力。不同线虫对暗黑鳃金龟幼虫的致病力结果显示, 嗜菌异小杆线虫沧州品系侵染96 h后, 幼虫的死亡率显著高于Steinernema feltiae和S. longicaudum两种线虫的处理 (P<0.05) , 说明嗜菌异小杆线虫沧州品系的致病力显著高于另外两种线虫。     

A novel ascaroside controls the parasitic life cycle of the entomopathogenic nematode Heterorhabditis bacteriophora

Entomopathogenic nematodes survive in the soil as stress-resistant infective juveniles that seek out and infect insect hosts. Upon sensing internal host cues, the infective juveniles regurgitate bacterial pathogens from their gut that ultimately kill the host. Inside the host, the nematode develops into a reproductive adult and multiplies until unknown cues trigger the accumulation of infective juveniles. Here, we show that the entomopathogenic nematode Heterorhabditis bacteriophora uses a small-molecule pheromone to control infective juvenile development. The pheromone is structurally related to the dauer pheromone ascarosides that the free-living nematode Caenorhabditis elegans uses to control its development. However, none of the C. elegans ascarosides are effective in H. bacteriophora, suggesting that there is a high degree of species specificity. Our report is the first to show that ascarosides are important regulators of development in a parasitic nematode species. An understanding of chemical signaling in parasitic nematodes may enable the development of chemical tools to control these species.     

Multiple-species natural enemy approach for biological control of alfalfa snout beetle (Coleoptera: Curculionidae) using entomopathogenic nematodes

Multiple-species natural enemy approach for the biological control of the alfalfa snout beetle, Otiorhynchus ligustici (L.) (Coleoptera: Curculionidae), was compared with using single-species of natural enemies in the alfalfa ecosystem by using entomopathogenic nematodes with different dispersal and foraging behaviors. Steinernema carpocapsae NY001 (ambush nematode), Heterorhabditis bacteriophora Oswego (cruiser nematode), and Steinernema feltiae Valko (intermediate nematode) were applied in single-species, two-species combinations, and one three-species combination treatments at 2.5 x 10(9) infective juveniles per hectare. All nematode species persisted for a full year (357 d). S. carpocapsae NY001 protected the plants from root-feeding damage better than H. bacteriophora Oswego but allowed for higher larval survival than all other nematode treatments. S. feltiae Valko protected the plants better than H. bacteriophora Oswego and controlled alfalfa snout beetle larvae better than S. carpocapsae NY001. H. bacteriophora Oswego allowed for similar root damage compared with control plots but reduced larval populations better than S. carpocapsae NY001. The combination of S. carpocapsae NY001 and H. bacteriophora Oswego provided significantly better protection for the plants than the control (unlike H. bacteriophora Oswego alone) and reduced host larva survival more than S. carpocapsae NY001 alone. The combination S. feltiae Valko and H. bacteriophora Oswego could not be statistically separated from the performance of S. feltiae Valko applied alone.     

Host range, specificity, and virulence of Steinernema feltiae, Steinernema rarum, and Heterorhabditis bacteriophora (Steinernematidae and Heterorhabditidae) from Argentina

Infections were carried out in the laboratory to determine the host range, specificity, and virulence of Steinernema rarum, S. feltiae, and Heterorhabditis bacteriophora that were isolated from different regions of Argentina. All insect orders showed a remarkable susceptibility to the three nematode species, showing mortality values higher than 66%, except for Anoplura. The mortality of the insects of agronomic interest was more than 90%. As for insects of sanitary importance, S. feltiae and S. rarum caused 60% of mortality, whereas H. bacteriophora caused 80%. The results fluctuated when considering the buccal apparatus function of the insects. Nematodes completed their cycle in anoplurans, orthopterans, lepidopterans, and hymenopterans, while the development varied in coleopterans, homopterans, hemipterans, and dipterans. S. rarum developed completely both in immature and adult, while S. feltiae and H. bacteriophora developed preferably in immature. The three isolates are capable of parasitizing a wide host range under laboratory conditions; H. bacteriophora is more virulent than the evaluated Steinernema spp. against Galleria mellonella larvae; the pathogenicity and specificity depend on the bioecological characteristics of nematodes and hosts.     

Assessing the Potential of Entomopathogenic Nematodes to Control the Grape Root Borer Vitacea polistiformis (Lepidoptera: Sesiidae) Through Laboratory and Greenhouse Bioassays

Seventeen entomopathogenic nematode species and strains were evaluated for virulence to the grape root borer, Vitacea polistiformis (Harris) in laboratory and greenhouse bioassays. Heterohabditis bacteriophora strain GPS11 and H. zealandica strain X1 produced a larval mortality rate of over 85% of larvae embedded in the root cambium in laboratory bioassays. The nematode species H. marelata and H. bacteriophora strain Oswego produced mortality rates of over 75%. Of the Steinernema species tested, S. carpocapsae strain 'All' performed the best with a mortality rate of 69%. All other nematode species and strains tested, with the exception of S. bicornutum , produced some degree of larval mortality. In the greenhouse bioassays, 93% control was achieved with H. zealandica strain X1 applied at 4 ×109 infective juveniles (IJs) acre1 -1 (9.88 ×10 9 IJs ha -1 ). H. bacteriophora strain GPS11 successfully reproduced in grape root borer larvae. The numbers of IJs produced within infected larvae were related to larval size. The survival rate of neonate larvae on grape root sections was 61%, which thus provides a means to rear the neonate larvae for bioassays.     

Infection success in novel hosts: an experimental and phylogenetic study of Drosophila-parasitic nematodes

Surprisingly little is known about what determines a parasite's host range, which is essential in enabling us to predict the fate of novel infections. In this study, we evaluate the importance of both host and parasite phylogeny in determining the ability of parasites to infect novel host species. Using experimental lab assays, we infected 24 taxonomically diverse species of Drosophila flies (Diptera: Drosophilidae) with five different nematode species (Tylenchida: Allantonematidae: Howardula, Parasitylenchus), and measured parasite infection success, growth, and effects on female host fecundity (i.e., virulence). These nematodes are obligate parasites of mushroom-feeding Drosophila, particularly quinaria and testacca group species, often with severe fitness consequences on their hosts. We show that the potential host ranges of the nematodes are much larger than their actual ranges, even for parasites with only one known host species in nature. Novel hosts that are distantly related from the native host are much less likely to be infected, but among more closely related hosts, there is much variation in susceptibility. Potential host ranges differ greatly between the related parasite species. All nematode species that successfully infected novel hosts produced infective juveniles in these hosts. Most novel infections did not result in significant reductions in the fecundity of female hosts, with one exception: the host specialist Parasitylenchus nearcticus sterilized all quinaria group hosts, only one of which is a host in nature. The large potential host ranges of these parasites, in combination with the high potential for host colonization due to shared mushroom breeding sites, explain the widespread host switching observed in comparisons of nematode and Drosophila phylogenies.     

Susceptibility of lady beetles (Coleoptera: Coccinellidae) to entomopathogenic nematodes

We investigated differential susceptibility of lady beetles to entomopathogenic nematodes, for two reasons: (1) to estimate potential nontarget effects on natural lady beetle populations, (2) to compare the susceptibility of exotic versus native lady beetle species. We hypothesize that successful establishment of some exotically introduced arthropods may be due, in part, to a lower susceptibility relative to competing native species. In laboratory studies, we compared the pathogenicity, virulence, and reproductive capacity of Heterorhabditis bacteriophora and Steinernema carpocapsae among two native (Coleomegilla maculata and Olla v-nigrum) and two successfully established exotic (Harmonia axyridis and Coccinella septempunctata) lady beetles, and a known susceptible lepidopteran host, Agrotis ipsilon. After 1 and 2 days of exposure to either nematode species, mortality of A. ipsilon was higher than in all lady beetles. Thus, we predict that nematode field applications would have significantly less impact on lady beetle populations than on a susceptible target pest. Additionally, the impact of soil-applied nematodes may be lower on lady beetles than on soil-dwelling hosts because the former spends relatively less time on the soil. Exotic lady beetles were less susceptible to nematode infection than native species. Reproductive capacity data also indicated lower host suitability in H. axyridis, but not in C. septempunctata. Overall, the hypothesis that low susceptibility to pathogens in certain exotic lady beetles may have contributed to competitive establishment was supported (especially for H. axyridis). Additional studies incorporating different hosts and pathogens from various geographic locations will be required to further address the hypothesis.     

嗜菌异小杆线虫侵染后暗黑鳃金龟和大黑鳃金龟幼虫脂肪体和中肠组织超微结构观察

【目的】探究昆虫病原线虫嗜菌异小杆线虫沧州品系 Heterorhabditis bacteriophora strain Cangzhou侵染对蛴螬脂肪体和中肠的影响,进一步明确其对蛴螬的致病机理。【方法】采用透射电镜技术,观察暗黑鳃金龟 Hololtrichia oblita (Faldermann)和大黑鳃金龟 H. parallela Motschulsky 2龄幼虫被嗜菌异小杆线虫沧州品系侵染后其脂肪体和中肠组织的病理变化。【结果】血腔注射感染期病原线虫嗜菌异小杆线虫沧州品系悬浮液24和48 h后,观察发现暗黑鳃金龟和大黑鳃金龟2龄幼虫脂肪体和中肠的组织结构均按时序逐渐发生变化,起初表现为脂肪球变形或变小,颜色变浅,脂肪体细胞和中肠细胞内质网、线粒体肿胀,中肠微绒毛变形脱落等现象,48 h后包裹脂肪球的膜结构破裂,脂肪体细胞和中肠细胞线粒体破裂,内质网数量减少,中肠微绒毛大量脱落,同时核内染色质大量解离,核膜破裂。【结论】经昆虫病原线虫嗜菌异小杆线虫沧州品系处理后,暗黑鳃金龟和大黑鳃金龟两种金龟甲2龄幼虫脂肪体和中肠细胞均出现明显的病理变化过程,这是嗜菌异小杆线虫高效致死蛴螬的原因之一。本研究可为昆虫病原线虫作为一种生物防治手段在蛴螬的综合防治中更好地发挥作用提供理论依据。     

The protein coats or ghosts or coliphage T2. III. Metabolic studies of Escherichia coli B infected with T2 bacteriophage ghosts

Assimilation of oxygen, inorganic phosphate, and ammonia nitrogen by normal T2 phage and T2 ghost-infected E. coli B was studied. The rate of oxygen and phosphorus uptake by ghost-infected bacteria is similar to that of normal and phage-infected cells. The R.Q. in glucose-salts medium remains approximately 1. Assimilation of ammonia nitrogen by ghost-infected bacteria is maintained at a rate approximately 80 per cent of normal. The inorganic phosphate which is assimilated was found to be incorporated into TCA-soluble compounds which were rapidly released into the medium. Within 5 minutes after absorption of the ghosts there was a loss from the cell of TCA-soluble constituents including organic phosphorus and compounds which absorb at 260 mmicro. No corresponding breakdown of nucleic acid present in the cell prior to infection could be detected. The incorporation of inorganic phosphate into organic linkages in the ghost-infected cell and its release into the medium were found to proceed at a rate approaching that of the incorporation of inorganic phosphorus into the nucleic acid of normal cells. The net increase in 260 mmicro absorbing compounds appeared to be inhibited.     

Plasticity demonstrated in the proteome of a parasitic nematode within the intestine of different host strains

The soluble global proteome of adult nematode Heligmosomoides polygyrus (H. p.) bakeri, a hookworm laboratory model was compared for the first time in the intestines of a slow-responder mouse host strain (C57/BL10) that is known to support a primary parasite infection for many months, and rapid-responder mouse host (SWR) that is known to eliminate the nematode infection by week 6 postinfection. At week 4 postinfection, major adult nematode proteins selectively produced following establishment of infection in C57/BL10 hosts include several globin forms, calreticulin and a phosphatidylethanolamine-binding protein. The increased synthesis of forms of myosin, actin and troponin in the nematode living in the rapid-responder SWR host may relate to the attempted reorganisation or repair of the cytoskeleton and/or muscle layer in the host immune initiated, increased mucus production and smooth muscle activity within intestinal environment. Initial evidence suggests weakly antigenic forms of globins dominant in the cytosol of H. p. bakeri adults in the intestinal environment compared to their low production in a related free-living nematode. The demonstration of considerable plasticity within a parasitic nematode proteome provides a molecular basis for the previously observed phenotypic plasticity within different host environments. Proteome plasticity has relevance to the efficiency of future vaccine and drug therapy, and the continued failure of defined antigen vaccines in mammalian populations.     

Dynamics of carbon dioxide release from insects infected with entomopathogenic nematodes

The quality of an insect as a host to an entomopathogenic nematode infective juvenile depends in part on whether or not the insect is already infected and on the stage of that infection. Previous research has shown that nematode response to hosts can change after infection and that, for uninfected hosts, CO(2) can be an important cue used by infective stage juveniles during attraction. We hypothesized that CO(2) production from an insect changes after it is infected, and that these changes could influence nematode infection decisions. Changes in CO(2) released by two insect species (Galleria mellonella and Tenebrio molitor) after infection by one of four nematode species (Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri, or Steinernema riobrave) were measured. Measurements were taken every 2h from time of initial exposure to nematodes up to 224 h after infection. Dead (freeze-killed) and live uninfected insects were used as controls. Infected G. mellonella showed two distinct peaks of CO(2) production: one between 20 and 30 h and the other between 70 and 115 h after exposure to the nematodes. Peaks were up to two times higher than levels produced by uninfected insects. Infected T. molitor showed only one peak between 25 and 50h. We found differences in peak height and timing among nematode and insect species combinations. The influence of these changes in CO(2) production on IJ attraction and infection behavior remains to be determined.     

Spatial and temporal distribution of endemic and released entomopathogenic nematode populations in turfgrass

Entomopathogenic nematodes (Rhabdita: Heterorhabditidae and Steinernematidae) have been effective as inundative biological control agents of scarab larvae (Coleoptera: Scarabaeidae) in turfgrass. Entomopathogenic nematodes also occur naturally in turfgrass and endemic or inoculated populations may be able to provide effective long-term control. Variation in Heterorhabditis bacteriophora and Steinernema carpocapsae spatial and temporal distribution along transects placed at different turfgrass sites in central New Jersey, USA, was investigated. H. bacteriophora tended to be recovered from fewer sections in a transect than S. carpocapsae, but the two species, overall, did not differ in patchiness of distribution. In one transect with a H. bacteriophora population S. feltiae was also recovered, but the two populations seldom overlapped spatially. In transects with adequate scarab larvae density for analysis, H. bacteriophora density and Popillia japonica larvae density were inversely correlated. This suggests that endemic H. bacteriophora populations may suppress P. japonica populations. In one transect, an epizootic of H. bacteriophora in an undetermined host may have occurred. Edaphic factors were relatively uniform along transects and were, at most, weakly correlated with nematode recovery. Uniform inoculative releases of H. bacteriophora tended to return to patterns of distribution typical of endemic populations.     

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.