Tank-Mix compatibility of the entomopathogenic nematodes, Heterorhabditis bacteriophora and Steinernema carpocapsae, with selected chemical pesticides used in turfgrass

We evaluated the compatibility of two entomopathogenic nematodes, Heterorhabditis bacteriophora HP88 and Steinernema carpocapsae All strain with selected pesticide formulations used in turfgrass in tank-mixes under laboratory conditions. The nematodes were exposed to the recommended rates of pesticides applied in either 100, 300, or 500 L/ha tank-mix volumes in 24-well plates at room temperature for 3 h and infective juveniles (IJ) viability determined, and then tested against Galleria mellonella larvae at 22-26°C for 96 h to assess IJ pathogenicity. We found that S. carpocapsae viability was not affected by any of the pesticides, while aluminum tris and trichlorfon significantly reduced S. carpocapsae pathogenicity at all concentrations. Thiamethoxam and trichlorfon significantly reduced H. bacteriophora viability, while halofenozide, aluminum tris, trichlorfon, and carbaryl significantly reduced H. bacteriophora pathogenicity. Imidacloprid, at the recommended rate 330-440 g AI/ha, significantly increased H. bacteriophora pathogenicity at 500 and 300 L/ha application volume. The integration of these nematode pesticide combinations in turf pest management programs is discussed.     

Biocontrol potential of the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae on cucurbit fly,Dacus ciliatus (Diptera: Tephritidae)

Entomopathogenic nematodes (EPNs) from the families Steinernematidae and Hererorhabditidae are considered excellent biological control agents against many insects that damage the roots of crops. In a regional survey, native EPNs were isolated, and laboratory and greenhouse experiments were conducted to determine the infectivity of EPNs against the cucurbit fly, Dacus ciliatus Loew (Diptera: Tephritidae). Preliminary experiments showed high virulence by a native strain of Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) and a commercial strain of Steinernema carpocapsae Weiser (Rhabditida: Steinernematidae). These two strains were employed for further analysis while another native species, Steinernema feltiae, was excluded due to low virulence. In laboratory experiments, larvae and adult flies were susceptible to nematode infection, but both nematode species induced low mortality on pupae. S. carpocapsae had a significantly lower LC₅₀ value against larvae than H. bacteriophora in filter paper assays. Both species of EPNs were effective against adult flies but S. carpocapsae caused higher adult mortality. When EPN species were applied to naturally infested fruit (150 and 300 IJs/cm²), the mortality rates of D. ciliatus larvae were 28% for S. carpocapsae and 12% for H. bacteriophora. Both EPN strains successfully reproduced and emerged from larvae of D. ciliates. In a greenhouse experiment, H. bacteriophora and S. carpocapsae had similar effects on fly larvae. Higher rates of larval mortality were observed in sandy loam and sand soils than in clay loam. The efficacy of S. carpocapsae and H. bacteriophora was higher at 25 and 30°C than at 19°C. The results indicated that S. carpocapsae had the best potential as a biocontrol agent of D. ciliatus, based on its higher virulence and better ability to locate the fly larvae within infected fruits.     

Cryopreservation of Steinernema carpocapsae and Heterorhabditis bacteriophora

A method for the cryopreservation of third-stage infective juveniles (IJ) of Steinernema carpocapsae and Heterorhabiditis bacteriophora was developed. Cryoprotection was achieved by incubating the nematodes in 22% glycerol (S. carpocapsae) or 14% glycerol (H. bacteriophora) for 24 hours, followed by 70% methanol at 0 C for 10 minutes. The viability of S. carpocapsae frozen in liquid nitrogen as 20 μl volumes spread over cover slip glass was > 80%. Survival of H. bacteriophora frozen on glass varied from 10 to 60% but was improved to > 80% by replacing the glass with filter paper. Cryopreservation and storage of 1-ml aliqots of S. carpocapsae IJ resulted in > 50% survival after 8 months; pathogenicity was retained and normal in vitro development took place. Trehalose and glycerol levels increased and glycogen levels decreased during incubation of S. carpocapsae IJ in glycerol. Normal levels of trehalose, glycerol and glycogen were restored during post freezing rehydration.     

Field efficacy of entomopathogenic nematodes against the beetle Maladera matrida (Coleoptera: Scarabaeidae)

Single, double and triple releases of the entomopathogenic nematode Heterorhabditis bacteriophora Poinar, reduced the population of the beetle Maladera matrida Argaman, infesting peanuts (’Shulamit’ cv.) by 70, 75 and 93% respectively in microplot tests. Simultaneous and late (2 weeks after infestation) applications reduced beetle numbers by 63 and 79% respectively, in the microplots, while early application (2 weeks prior to infestation) did not reduce the beetle population. In a field trial, reductions in insect population and damage to the crop were achieved by early treatment with the nematode as well as by Heptachlor, leading to reductions in the insect population of 60 and 90% respectively, when recorded 4 weeks after nematode application. However, the nematode treatment did not maintain its effectiveness for a longer period and pest damage increased to the same level as the untreated control after 7 weeks. When the nematodes were applied at different concentrations (0.25–1.0 x 10⁶ infective juveniles (IJs) m^‐2) their effectiveness was not related to the concentration level. The only significant (P < 0.05) reduction in insect levels was recorded in the treatment with 0.5 X 10⁶ IJs m^‐2. In a second field trial, both H. bacteriophora and Steinernema glaseri reduced insect populations significantly (P < 0.05) by approximately 50% in comparison to the control. In the third trial, treatment with H. bacteriophora resulted in a decrease in insect population of 90% while treatment with S. carpocapsae reduced the grub numbers by 40% in comparison to the control. A differential susceptibility of various grub developmental stages was recorded in the field. The small grubs (I‐4 mm long, lst‐2nd larval stage) were not affected by the nematode treatments while the numbers of medium and large size grubs were reduced by 2‐ and 3‐fold respectively in the various tests. Nematodes were recovered by ‘nematode traps’ containing Galleria mellonella larvae from treated field plots 78 days after application. The implications of the results from the present studies on the use of entomopathogenic nematodes are discussed in relation to the development of an integrated pest management programme.     

Pathogenicity of three entomopathogenic nematodes against the onion thrips,Thrips tabaci Lind. (Thys.; Thripidae)

Pathogenicity of a native isolate of Steinernema feltiae (H1) and two exotic strains, Heterorhabditis bacteriophora and Steinernema carpocapsae was assessed under laboratory conditions using different concentrations i.e. 4000, 6000, 8000 and 10,000 infective juveniles/ml against second instar larvae, prepupa and pupa of Thrips tabaci Lindeman. The mortality data were recorded 24 and 48?h post-inoculation. The highest mortality rate was recorded for prepupa (62%) than second instar (12.5%) by H. bacteriophora and S. carpocapsae, respectively, 24?h after treatment. No significant differences were found in mortality between prepupa and pupa with increasing the nematodes concentrations (from 4000 to 10,000 nematode/ml) but increasing nematode concentrations increased the mortality of second instar. At the end of the experiment (48?h.), S. feltiae H1 caused the highest mortality on second instar larvae (74%), whereas all other species caused 80–83% mortalities on pupa. This study suggests that native isolate of S. feltiae (H1) had high potential to infect soil-dwelling stages of T. tabaci.     

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.     

Effectiveness of entomopathogenic nematodes Steinernema feltiae and Heterorhabditis bacteriophora on the Colorado potato beetle Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) under laboratory and greenhouse conditions

In laboratory and greenhouse studies, the invading ability, virulence, and mortality caused by Stinernema feltiae and Heterorhabditis bacteriophora were compared. After one and two days of exposure to either nematode species, the mortality of Colordo potato beetle (CPB) Leptinotarsa decemlineata larvae at different instars, third and fourth, was recorded and the number of nematodes invading cadavers was more than the number of nematodes inside the larvae at the late last instar (one day before pre-pupa). Two concentrations, 250 and 500 IJs/dish, infective juvenile nematodes/0.5 ml were tested on different CPB larval instar. S. feltiae was more effective, with fourth instar rather than third and late last instar. On the other hand, H. bacteriophora showed a very weak effect with L. decemlineata. Also it was clear that S. feltiae was more effective and faster than H. bacteriophora: more than 70% of larvae were killed within 24 hours compared with H. bacteriophora which killed 40% of larvae within 48–72 hours. A significant difference in invading efficiency was observed with concentration 2500 IJs/pot in the greenhouse test. The number of adult females found in the cadavers of L. decemlineata larvae was always higher than the number of males. Foliage application of S. feltiae and H. bacteriophora resulted in a significant reduction of the number of damaged leaves and a lower index of damage compared with that in the control. We conclude that S. feltiae has significant potential and can help in the management of the Colorado potato beetle.     

Storage of osmotically treated entomopathogenic nematode Steinernema carpocapsae

The infective juveniles (IJs) of Steinernema carpocapsae‘All’ were osmotically stressed by a mixture of ionic (fortified artificial seawater) and non‐ionic (3.2 mol/L glycerol) solutions to establish a method for osmotic storage of entomopathogenic nematodes. Seven combinations (termed solution A to G) with different proportions of these two solutions were tested, with sterile extra pure water (sepH₂O, termed solution H) as a control. The mortality of the IJs at a concentration of 5 × 10⁵ IJ/mL in the solutions A to G, and H were 13.2%, 16.2%, 16.7%, 13.5%, 25.2%, 31.6%, 44.6%, and 1.0%, respectively, after 21 days storage at 25°C. Most of the IJs shrunk and stopped motility after 6–9 hours incubation at 25°C in solutions A to D. Based on the results, solutions A to D and H were chosen to further test the osmotic survival of the IJs at different IJ concentrations (5 × 10⁵, 2.5 × 10⁵, 2 000 IJ/mL) and incubation temperature (30°C, 25°C, 10°C). The resulting IJs were exposed to a high temperature assay (45°C for 4 h, HTA). Osmotically stressed IJs showed improved heat tolerance. The mortality of the IJs increased with the increasing concentrations of the test IJs and the storage temperatures after exposing to the HTA. More than 88.4%, 62.3% or 2.4% of the treated IJs died at the above three IJ concentrations, respectively. At the three IJ concentrations (2 000 IJs/mL, 2.5 × 10⁵ IJs/mL or 5 × 10⁵ IJs/mL), the highest mortality was recorded in solution D (11.6%, 85.9% or 98.0%, respectively), and the lowest mortality in solution B (2. 4%, 62.3% or 86.6%, respectively). No untreated IJs survived after the heat treatment. During 42 days storage at 10°C, the IJs mortality in the solutions A to D and H were 7.19%, 5.97%, 4.41%, 4.34%, and 4.34% respectively, and showed no significant differences. In conclusion, osmotic treatment of the IJs of S. carpocapsae‘All’ in a mixture of ionic and non‐ionic solutions enhances the heat tolerance. The mortality of the IJs after HTA increased with the increasing concentrations of the test IJs and the storage temperatures after exposure to the HTA. The result is promising for the osmotic storage of the entomopathogenic nematodes.     

病原线虫对桔小实蝇种群的控制作用

通过室内和田间实验研究了昆虫病原线虫对桔小实蝇Bactrocera (Bactrocera) dorsalis (Hendel)的控制作用。室内实验结果表明,供试的3种线虫的4个品系（小卷蛾斯氏线虫Steinernema carpocapsae All品系与A24品系,夜蛾斯氏线虫Steinernema feltiae SN品系和嗜菌异小杆线虫Heterorhabditis bacteriophora H06品系）,以小卷蛾斯氏线虫All品系对桔小实蝇的侵染力最强,其3天的LD₅₀和LD₉₅分别为35.0和257.1条/cm²土壤。按300条/cm²土壤的量施用,小卷蛾斯氏线虫All品系对当代桔小实蝇的控制效果为86.3%。用以虫期作用因子组建的生命表方法评价了小卷蛾斯氏线虫All品系对田间桔小实蝇下代种群的控制作用,结果表明,按300条/cm²土壤的量施用线虫,对照杨桃园的桔小实蝇种群趋势指数为105.9,而处理杨桃园的桔小实蝇种群趋势指数下降为15.5;小卷蛾斯氏线虫All品系对桔小实蝇的干扰控制指数为0.146,即线虫处理果园的下代种群密度仅为对照果园的14.6%。     

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.     

Isolation and characterization of microsatellite loci in the entomopathogenic nematode Heterorhabditis bacteriophora

Twenty microsatellite loci were identified from genomic DNA enrichment and expressed sequence tags of entomopathogenic nematode Heterorhabditis bacteriophora. Eight loci were found to be polymorphic in a Northeast Ohio H. bacteriophora population. Levels of polymorphism were evaluated in 31-56 individuals and the number of alleles ranged from two to three. The values of observed and expected heterozygosities ranged from 0 to 0.536 and from 0.223 to 0.616, respectively. All eight loci showed heterozygote deficiencies, but three conformed to Hardy-Weinberg equilibrium at the subpopulation level. This is the first set of microsatellite markers in entomopathogenic nematodes.     

Pathogenicity,reproduction and foraging behaviours of some entomopathogenic nematodes on a new turf pest,Dorcadion pseudopreissi (Coleoptera: Cerambycidae)

Entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematidae have considerable potential as biological control agents of soil-inhabiting insect pests. In the present study, the control potential of the EPNs Steinernema carpocapsae (TUR-S4), S. feltiae (Nemaplus), S. carpocapsae (Nemastar), S. feltiae (TUR-S3) and Heterorhabditis bacteriophora (Nematop) against a new longicorn pest, Dorcadion pseudopreissi Breuning, 1962 (Coleoptera: Cerambycidae), on turf was examined in laboratory studies. Pathogenicity tests were performed at the following doses: 50, 100 and 150 Dauer Juveniles (DJs)/larva at 25°C. Highest mortalities (75–92%) of the larvae were detected at the dose of 150 DJs/larva for all nematodes used. Reproduction capabilities of the used EPNs were examined at doses of 50, 75, 100 and 150 DJs/larva at 25°C. S. carpocapsae (TUR-S4) had the most invasions (32 DJs/larva) and reproduction (28042 DJs/larva) at the dose of 100 DJs, and the highest reproduction (per invaded DJ into a larva) was observed in H. bacteriophora (Nematop) (2402.85 DJs) at a dose of 50 DJs. The foraging behaviour of the nematodes in the presence of D. pseudopreissi and Galleria mellonella L. (Lepidoptera: Galleriidae) larvae was studied using a Petri dish filled with sand at 20°C. All of the used nematodes accumulated near the larvae section of both insect species (32–53% of recovered DJs) with a higher percentage of S. carpocapsae (TUR-S4) (53%) and H. bacteriophora (48%) (Nematop) moving towards larvae of D. pseudopreissi, than the S. feltiae strains.     

Assessment of establishment and persistence of entomopathogenic nematodes for biological control of western corn rootworm

Abstract:  The use of entomopathogenic nematodes (EPN) is potentially one ecological approach to control the invasive alien western corn rootworm ( Diabrotica virgifera virgifera LeConte, Col., Chrysomelidae) in Europe. This study investigated the establishment and the short- and long-term persistence of Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), Heterorhabditis megidis Poinar, Jackson and Klein (Rh., Heterorhabditidae) and Steinernema feltiae Filipjev (Rh., Steinernematidae) in three maize fields in southern Hungary, using the insect-baiting technique. All three EPN species equally established and persisted in maize fields. The timing of application (April or June) did not influence the establishment of EPN species. EPNs persisted for 2–5 months, i.e. they survived up to and throughout D. v. virgifera larval occurrence in the soil. Results demonstrate that D. v. virgifera larvae can potentially be controlled by EPNs during the same year of EPN application but no long-term control effect is expected under intensive maize cultivation practices.     

Assessing immunocompetence in red palm weevil adult and immature stages in response to bacterial challenge and entomopathogenic nematode infection

Parasites and pathogens can follow different patterns of infection depending on the host developmental stage or sex. In fact, immune function is energetically costly for hosts and trade‐offs exist between immune defenses and life history traits as growth, development and reproduction and organisms should thus optimize immune defense through their life cycle according to their developmental stage. Identifying the most susceptible target and the most virulent pathogen is particularly important in the case of insect pests, in order to develop effective control strategies targeting the most vulnerable individuals with the most effective control agent. Here, we carried out laboratory tests to identify the most susceptible target of infection by infecting different stages of the red palm weevil Rhynchophorus ferrugineus (larvae, pupae, male, and female adults) with both a generic pathogen, antibiotic‐resistant Gram‐negative bacteria Escherichia coli XL1‐Blue, and two specific strains of entomopathogenic nematodes (EPNs), Steinernema carpocapsae ItS‐CAO1 and Heterorhabditis bacteriophora ItH‐LU1. By evaluating bacterial clearance, host mortality and parasite progeny release, we demonstrate that larvae are more resistant than adults to bacterial challenge and they release less EPNs progeny after infection despite a higher mortality compared to adults. Considering the two EPN strains, S. carpocapsae was more virulent than H. bacteriophora both in terms of host mortality and more abundant progeny released by hosts after death. The outcomes attained with unspecific and specific pathogens provide useful information for a more efficient and sustainable management of this invasive pest.     

Post-application of anti-desiccant agents improves efficacy of entomopathogenic nematodes in formulated host cadavers or aqueous suspension against diapausing codling moth larvae (Lepidoptera: Tortricidae)

Codling moth (CM), Cydia pomonella (L.) is the most serious pest of apple and other pome fruit worldwide. In temperate climates, diapausing cocooned larvae make up 100% of the population. Control of this stage would reduce or eliminate damage by first generation CM in late spring and early summer. Entomopathogenic nematodes (EPNs) are good candidates for control of CM in the cryptic habitats where the larvae overwinter. The two predominant limiting factors for EPNs are adequate moisture and temperatures below 15°C. Formulation that maintains moisture and enables survival of EPN infective juveniles (IJs) until they can infect overwintering larvae would significantly improve their utility for protection of apple, pear and walnut. In laboratory studies conducted in moist mulch (consisting of apple and conifer wood), Galleria mellonella (L.) larvae infected with Steinernema carpocapsae (Weiser), S. feltiae (Filipjev), or Heterorhabditis bacteriophora Poinar and coated with starch and clay, produced mean mortalities of 42, 88, and 24%, respectively in CM larvae. Mulched field plots treated with formulated S. carpocapsae- or S. feltiae-infected G. mellonella larvae, then followed by an application of wood flour foam as an anti-desiccant, resulted in 56 and 86% mortality, respectively. Comparative tests of aqueous suspensions of S. carpocapsae IJs applied to cardboard bands on apple tree trunks followed by water, fire retardant gel or foam resulted in 11, 35, and 85% respective mortalities. Identical tests with S. feltiae resulted in 20, 19, and 97% respective mortalities. Our research with cadaver formulations of EPNs in mulch and aqueous suspensions on tree trunks combined with anti-desiccant agents, demonstrated significant improvement in larvicidal activity for diapausing cocooned CM larvae.     

Enhanced biological control potential of the entomopathogenic nematode,Steinernema carpocapsae,applied with a protective gel formulation

Entomopathogenic nematodes (EPNs) are adapted to subterranean environments, and are prone to damage by UV light and desiccation. EPNs, Steinernema carpocapsae, combined with a protective gel, and anti-UV ingredients, have potential for above-ground pest management. We (1) ascertained whether the gel could provide protection to EPNs at low concentrations when applied in direct sun, (2) determined if other ingredients added to the gel increased efficacy, and (3) quantified retention and survival of EPNs applied to foliage with the gel. EPNs in 1% protective gel caused higher host mortality (60%) than other treatments (2–37%). UV protection provided by titanium dioxide (TD) and octyl methoxycinnamate with 1% protective gel solution was tested outdoors; these formulations resulted in higher host mortality (43% and 25%) than other treatments (2–7%). After 8?h in the greenhouse, 0.25% protective gel solution had the highest percentage of live EPNs on leaves. The gel at low concentration protects EPNs, and addition of TD enhanced the protective properties of the formulation. It is important to continually improve options for different growing systems and insect pest behaviour. TD added to a low-concentration formulation of the protective gel makes this application technique more viable for growers to use.     

Host density effects on efficacy of entomopathogenic nematodes against white grub (Coleoptera: Scarabaeidae) species

We tested the effect of host density on entomopathogenic nematode efficacy in 1-L pots with grass and soil. In four experiments, combinations ranged from somewhat resistant hosts (oriental beetle, Anomala orientalis, or northern masked chafer, Cyclocephala borealis, with Heterorhabditis bacteriophora) over more susceptible hosts (Japanese beetle, Popillia japonica, with Steinernema glaseri) to a highly susceptible host (P. japonica and S. scarabaei). In each experiment, four larval densities were exposed to two nematode rates over a 14-day period. A significant effect of host density on nematode efficacy occurred only in the A. orientalis–H. bacteriophora combination, but there was no clear trend in the data. This suggests that an exhaustion of available nematode populations to less lethal levels by high host numbers was counteracted by other factors such as increased chances for nematode-host contact and increased host susceptibility due to stress via reduced food resources and increased aggression between larvae.     

Dispersal Behaviour and Transmission Strategies of the Entomopathogenic Nematodes Heterorhabditis and Steinernema

Entomopathogenic nematodes of the Heterorhabditidae and Steinernematidae appear to be capable of long-distance dispersal and local migration. Their transmission strategies include both highly active seek-and-destroy behaviours and ambusher strategies, and they may be sensitive to sex-related factors in their own populations. Their host-finding abilities are poorly understood, despite the fact that these abilities are fundamental to their success as biocontrol agents in soil. Like the vast numbers of exotic hymenopterans and other natural enemies that have been released for biological control over the past century, they may be used in their ecologically competent wild-type form. On the other hand, because they are applied inundatively, they may be tailored, by breeding or transformation, to their intended purpose and to ecological incompetence, improving both their efficacy and their ecological safety.     

Specific oxygen uptake of the entomopathogenic nematode,Steinernema carpocapsae CABA01, in submerged culture

The specific oxygen uptake rate (qO₂) of stages of the entomopathogenic nematode (EPN) Steinernema carpocapsae CABA01 in liquid culture was measured. Nematodes were grown into previously pasteurised culture broths of their symbiotic bacterium, Xenorhabdus nematophila, in orbitally agitated flask cultures (V_L = 125 mL) at N = 150 rpm and T = 25°C. The basal medium contained 3% (w/v) soy trypticase broth and 0.5% (w/v) yeast extract. The EPNs developed from the egg stage to the adult stage exhibiting qO₂ values of 1.92, 5.48, 0.48, 0.28 and 0.0014 [10^?1 mmolO₂/(g_{nematode-wet base} h)] for the egg-Juvenile 1 (J1), J2, J3, J4 and the adult stages, respectively.     

Overwintering behavior of the entomopathogenic nematodes Steinernema scarabaei and Heterorhabditis bacteriophora and their white grub hosts

Entomopathogenic nematodes (EPNs) are obligate pathogens known to naturally persist in many habitats. Because survival is a fundamental component of persistence, we investigated whether vertical movement and other avoidance behaviors (i.e., in‐host survival and latent infection), previously speculated as viable survival mechanisms, are exhibited during the cooler months in a temperate turfgrass habitat. The vertical distribution of populations of two EPN species, Steinernema scarabaei Stock & Koppenhöfer (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), and two important hosts of these EPN species, the white grub species Popillia japonica Newman and Anomala orientalis Waterhouse (both Coleoptera: Scarabaeidae), were regularly monitored in turf plots from October through April in two consecutive years. Entomopathogenic nematode vertical distribution showed limited changes for H. bacteriophora but none for S. scarabaei. Recovery of H. bacteriophora showed a strong and consistent decline at 0–4 cm depth in the 1st year and a weaker decline at 4–10 cm in the 1st year and at 0–4 cm in the 2nd year. Due to high variability in the data, it was not possible to determine whether the decline in the upper soil layers was due to downward migration or attrition of infective juvenile nematodes. The decline occurred mostly during the first half of the season before the soil froze to any significant extent. The vertical distribution of both white grub species changed with temperature during fall and spring, but not during winter. Overwintering infective juveniles were only recovered in the soil. There was no evidence for successful in‐host survival or latent infection by the nematodes in endemic white grub populations.