Temperature Effects on Heterorhabditis megidis and Steinernema carpocapsae Infectivity to Galleria mellonella

The effect of temperature on the infection of larvae of the greater wax moth, Galleria mellonella, by Heterorhabditis megidis H90 and Steinernema carpocapsae strain All, was determined. For both species, infection, reproduction, and development were fastest at 20 to 24 °C. Infection by both H. megidis and S. carpocapsae occurred between 8 and 16 °C; however, neither species reproduced at 8 °C. Among the nematodes used in experiments at 8 °C, no H. megidis and very few S. carpocapsae developed beyond the infective juvenile stage. Compared with H. megidis, S. carpocapsae invaded and killed G. mellonella larvae faster at 8 to 16 °C. By comparing invasion rates, differences in infectivity between the two nematode species were detected that could not be detected in conventional petri dish bioassays where mortality was measured after a specified period. Invasion of G. mellonella larvae by H. megidis was faster at 24 than at 16 °C.     

Parasitism of Western Corn Rootworm Larvae and Pupae by Steinernema carpocapsae

Virulence and development of the insect-parasitic nematode, Steinernema carpocapsae (Weiser) (Mexican strain), were evaluated for the immature stages of the western corn rootworm, Diabrotica virgifera virgifera LeConte. Third instar rootworm larvae were five times more susceptible to nematode infection than second instar larvae and 75 times more susceptible than first instar larvae and pupae, based on laboratory bioassays. Rootworm eggs were not susceptible. Nematode development was observed in all susceptible rootworm stages, but a complete life cycle was observed only in second and third instar larvae and pupae. Nematode size was affected by rootworm stage; the smallest infective-stage nematodes were recovered from second instar rootworm larvae. Results of this study suggest that S. carpocapsae should be applied when second and third instar rootworm larvae are predominant in the field.     

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.     

Effects of Pesta-Pelletized Steinernema carpocapsae (All) on Western Corn Rootworms and Colorado Potato Beetles

Pesta-pelletized Steinernema carpocapsae (All) nematodes were used in soil treatments in the greenhouse against larvae of Western corn rootworm and prepupae of Colorado potato beetle. The pesta-pellets delivered 100,000 living nematodes/g. Infective-stage nematodes and their associated bacteria survived the pesta-pellet process, emerged from the pellets in large numbers in the soil, and reduced adult emergence of both pest insects by more than 90%.     

Interactions between Nematodes and Earthworms: Enhanced Dispersal of Steinernema carpocapsae

Dispersal of the nematode Steinernema carpocapsae (All strain), applied on the top or the bottom of soil columns, was tested in the presence or absence of two earthworm species, Lumbricus terrestris or Aporrectodea trapezoides. Nematode dispersal was estimated after a 2-week period with a bioassay against the greater wax moth, Galleria mellonella. Vertical dispersal of nematodes was increased in the presence of earthworms. When nematodes were placed on the surface of soil columns, significantly more nematodes dispersed to the lower half of the columns when either earthworm species was present than when earthworms were not present. When nematodes were placed on the bottom of soil columns, significantly more nematodes dispersed to the upper half of the columns when L. terrestris was present than when A. trapezoides was present or in the absence of earthworms. Because nematodes were found on the exterior and in the interior of earthworms, nematode dispersal may be enhanced by direct contact with the earthworms.     

Simple In Vivo Production and Storage Methods for Steinernema carpocapsae Infective Juveniles

Methods are described for standardized in vivo production, rapid harvest, and storage, in a concentrated form, of infective juveniles of the entomopathogenic nematode, Steinernema carpocapsae Mexican strain Kapow selection. Nematodes were stored in nematode wool configurations, consisting of mats of intertwined infective juveniles. Freshly harvested nematodes are readily available in adequate quantities for laboratory and small-scale field evaluations as well as cottage industry production.     

Influence of Soil pH and Oxygen on Persistence of Steinernema spp.

Survival of infective juveniles of Steinernema carpocapsae and Steinernema glaseri gradually declined during 16 weeks of observation as the tested soil pH decreased from pH 8 to pH 4. Survival of both species of Steinernema dropped sharply after 1 week at pH 10. Survival or S. carpocapsae and S. glaseri was similar at pH 4, 6, and 8 during the first 4 weeks, but S. carpocapsae survival was significantly greater than S. glaseri at pH 10 through 16 weeks. Steinernema carpocapsae and S. glaseri that had been stored at pH 4, 6, and 8 for 16 weeks, and at pH 10 for 1 or more weeks were not infective to Galleria mellonella larvae. Steinernema carpocapsae survival was significantly greater than that of S. glaseri at oxygen:nitrogen ratios of 1:99, 5:95, and 10:90 during the first 2 weeks, and survival of both nematode species declined sharply to less than 20% after 4 weeks. Survival of both nematode species significantly decreased after 8 weeks as the tested oxygen concentrations decreased from 20 to 1%, and no nematode survival was recorded after 16 weeks. Steinernema carpocapsae pathogenicity was significantly greater than that of S. glaseri during the first 2 weeks. No nematode pathogenicity was recorded at oxygen concentrations of 1, 5, and 10% after 2 weeks and at 20% after 16 weeks.     

Growth and Virulence of Steinernema glaseri Influenced by Different Subspecies of Xenorhabdus nematophilus

Three Xenorhabdus nematophilus subspecies influenced Steinernema glaseri growth profiles and growth rates, but this was not necessarily because of different bacterial growth rates. Virulence of dauer nematodes in larval Galleria mellonella varied with the number of dauers retaining bacteria and the bacterial subspecies. Virulence was least for dauers grown on X. nematophilus subsp. bovienii because of the lack of retained bacteria. Virulence was subsequently restored by culturing these nematodes on X. nematophilus subsp. poinari.     

Effects of Crop Residue on the Persistence of Steinernema carpocapsae

We determined the effects of crop residue on the persistence of an entomopathogenic nematode, Steinernema carpocapsae. During 2 consecutive years, nematodes were applied at rates of 2.5 × 10₄ and 1.0 × 10⁵ infective juveniles/m² to small field plots planted with corn. Nematode persistence was monitored by exposing Galleria mellonella larvae to soil samples from plots with and without crop residue (approximately 75% coverage of soybean stubble). Persistence of S. carpocapsae was significantly greater in crop residue plots than in plots without residue. In crop residue plots that received the higher rate of nematode application, larval mortality did not significantly decrease during the study period (3 to 5 days) and remained above 85%. In nematode-treated plots without crop residue, however, larval mortality fell from over 96% to below 11% and 35% in the first and second trials, respectively. The increased crop residue may have benefited nematode persistence through protection from desiccation or ultraviolet light. We conclude that increased ground cover in cropping systems (e.g., due to reduced tillage) may lead to increased insect pest suppression with entomopathogenic nematodes.     

Effects of Insecticides on Movement,Nictation, and Infectivity of Steinernema carpocapsae

Movement, nictation, and infectivity of Steinernema carpocapsae strain All were compared for ensheathed (EnJ) and desheathed (DeJ) infective juveniles exposed to the insecticides acephate, dichlorvos, methomyl, oxamyl, or permethrin. Nematode response to various solutions included normal sinusoidal movement, uncoordinated motion, twitching, convulsion or formation of a pretzel shape, an inactive "S" posture with fine twitching, or a quiescent straight posture. The DeJ displayed these movements at lower concentrations of each insecticide than did EnJ. In petri dish bioassays, insecticide-treated EnJ caused generally lower mortality in the common cutworm, Spodoptera litura, than did EnJ alone but caused greater insect mortality than did insecticides alone. Nematode response to chemicals was more clearly demonstrated by nictating behavior than by the movement bioassay. Nictation of DeJ was suppressed by the test chemicals at low concentrations, except for acephate and permethrin. Nictating EnJ or DeJ, regardless of chemical treatment, killed host insects faster than did non-nictating juveniles. Insecticides that enhance nictating behavior at certain concentrations may be used for mixed applications with nematodes.     

Impacts of fluctuating temperature on the development and infectivity of entomopathogenic nematode Steinernema carpocapsae A10

Three different laboratory conditions were used to examine the impacts of fluctuating temperature on the development and infectivity of entomopathogenic nematode (EPN) Steinernema carpocaposae A10. Set I experiments focused on the impact of cold stress early in the development cycle. In these studies Galleria mellonella hosts were infected and incubated for 2 days at the control temperature of 23 degrees C and then subjected to lower temperatures of -10, 4, 10 or 14 degrees C, respectively, from days 3 to 36 post-infection (PI). Dissections of infected cadavers indicated arrested development at the adult stage at all lower temperatures tested. Set II experiments examined the impacts of cold stress early in the development followed by a return to 23 degrees C. Hosts were infected and incubated as in Set I and subjected to the same temperatures as above for 7 days, followed by incubation at 23 degrees C until 23 days PI. A limited number of EPN populations were able to complete development at 10 and 14 degrees C though emergent population numbers were significantly lower than those of control infections incubated continuously at 23 degrees C. In Set III experiments, infected hosts were subjected to cold stress later during development starting at day 4 post-infection followed by incubation at the control temperature. Population survival past first and second stage juveniles was reduced by at least 95% or more at the lower temperatures compared with controls. Emergent populations from the Set III cold-stressed hosts were not infectious. These studies may provide insights as to how EPN survive seasonal temperature fluctuations under natural environmental conditions.     

Use of a Stilbene Brightener,Tinopal LPW,as a Radiation Protectant for Steinernema carpocapsae

A stilbene fluorescent brightener, Tinopal LPW, was used as an ultraviolet (UV) protectant for the entomopathogenic nematode Steinernema carpocapsae (All strain). Irradiation of an aqueous suspension of nematodes produced a LC₅₀ in 15.7 minutes under a sunlamp and in 31.7 minutes in direct sunlight. Irradiation by both sunlamp and sunlight of a suspension of nematodes in Tinopal LPW did not reduce their biological activity as measured by their ability to parasitize wax moth larvae after exposure of 8 hours and 4 hours, respectively. Tinopal LPW appeared promising as a radiation protectant.     

Response of Steinernema carpocapsae Infective Juveniles to the Plasma of Three Insect Species

Exsheathed infective juveniles of Steinernema carpocapsae All strain were attracted to the plasma of three species of insects in agar plate bioassays. Plasma of Pieris rapae crucivora, Spodoptera litura, and Agrotis segetum attracted 88.6%, 80.4%, and 64.4%, respectively, of Steinernema carpocapsae juveniles added to plates. Autoclaved plasma of S. litura larvae attracted more juveniles than saline controls, but less than nonautoclaved plasma. The active agent passed through a 14,000 MW dialysis membrane.     

Isolation and identification of a symbiotic bacterium fromSteinernema carpocapsae

Xenorhabdus nematophilus sp., an insect-pathogenic bacterium, was newly isolated from Korean entomopathogenic nematode ofSteinernema carpocapsae, which can be used as a useful bioinsecticide. Primary and secondary form variants ofXenorhabdus nematophilus were observed when culturedin vitro. Primary form variants adsorbed bromothymol blue, while secondary form did not. However, many other characters of two variants were very similar. The variants were all rod-shaped and cell size was highly variable ranging from 0.5 by 2.0 μm to 1.0 by 5.0 μm. Both produced highly toxic substances and killed the insect larva within 20–38 hr, indicating that insect pathogenicity ofXenorhabdus is not directly associated with its phase variation. In addition, cell-free culture supernatant ofXenorhabdus was sufficient to kill the insect larva by injecting it into insect hemolymph; however, cell-harboring culture broth was more effective for killing the insect. The use ofXenorhabdus nematophilus may provide a potential alternative toBacillus thuringiensis (Bt) toxins.     

Characterization of Key Glycolytic and Oxidative Enzymes in Steinernema carpocapsae

The enzyme activities of isocitrate dehydrogenase (ICDH, NADP-specific), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), phosphoenolpyruvate carboxykinase (PEPCK), phosphofructokinase (PFK), pyruvate kinase (PK), and fructose-l,6-bisphosphatase (FBPase) were studied in the third-stage juveniles of Steinernema carpocapsae. Reaction requirements, pH optima, substrate and cofactor kinetic constants were similar to those reported previously from other parasitic helminths with the exception of LDH, which was unstable and could not be characterized for specific activity and kinetic constants. The respective pH optima were 7.5 for ICDH, 8.8 for MDH, 6.5 for PEPCK, 7.3 for PFK, 7.2 for PK, and 7.5 for FBPase. The specific activities for ICDH, MDH, PEPCK, PFK, PK, and FBPase at pH 7.5 were 4.8, 1,300, 22, 25, 35, and 6.8 (nmoles substrate ∙ min⁻¹ ∙ mg protein⁻¹), respectively. In summary, the infective juveniles of S. carpocapsae display the metabolism typical of a facultative aerobe.     

Effects of Enzymes,Chemicals, and Tempertaure on Steinernema carpocapsae Attraction to Host Plasma

Migration of exsheathed infective juveniles of Steinernema carpocapsae to plasma of the host insect Spodoptera litura was not affected by treatments with the lectins concanavalin A, soybean agglutinin, or wheat germ agglutinin; with the enzymes neuraminidase, α-mannosidase, lipase, pronase, or phospholipase C; or with cetyl trimethylammonium bromide or spermidine. Treatment with sodium metaperiodate or sodium hypochlorite inhibited nematode attraction towards insect plasma; numbers of randomly wandering nematodes increased. Nematode migration towards the source of attraction was unaffected by temperatures below 33 C but was impaired at 35 and 37 C. The adverse effect of 5 mM and 10 mM NaIO₄ on migratory behavior was reversed 24 hours after rinsing with buffered saline. The effect of NaOCl on nematode behavior was slightly reversible at concentrations of 0.2 and 0.4% (v/v) but apparently irreversible at 0.6 and 1.0%. The effect of heat treatment at 35 and 37 C was reversible.     

Quantification of Invasion of Two Strains of Steinernema carpocapsae (Weiser) into Three Lepidopteran Larvae

Studies with last instar larvae of the fall armyworm, Spodoptera frugiperda (J. E. Smith), the black cutworm, Agrotis ipsilon (Hufnagel), and the greater wax moth, Galleria mellonella (L.) were used to quantify the invasive ability of two strains (All and Mexican) of Steinernema carpocapsae and to determine how factors in the bioassay procedure affect both nematode invasion and host mortality. Nematode invasive ability was variable, with 10-50% of nematodes successfully infecting the host. The percentage of infectives invading the host (invasion efficiency) was positively related to increases in length of host exposure time and number of hosts per arena, negatively related to increases in substrate surface area per host, and not affected by nematode concentration. There was a direct relationship between concentration applied and the number of nematodes invading the host. Mortality was less affected than invasion efficiency by bioassay conditions and appears to be a much less sensitive index of nematode activity than invasive ability.     

Infection of the Entomopathogenic Nematode,Steinernema carpocapsae,as Affected by the Presence of Steinernema glaseri

The infection behavior of Steinernema carpocapsae infective juveniles (IJ) was investigated in the presence and absence of S. glaseri. Mixed inoculation of S. carpocapsae with S. glaseri IJ significantly raised the nictation rates of S. carpocapsae IJ. Significantly more S. carpocapsae IJ migrated to the host insect in the mixed inoculation with S. glaseri IJ on agar plates. More S. carpocapsae IJ penetrated into the host insect placed 2 cm below the surface in the mixed inoculation with S. glaseri IJ. More S. glaseri than S. carpocapsae IJ penetrated into hosts placed 7 cm deep. Irrespective of host location, the male ratio of S. carpocapsae IJ established in the host body was always higher in the mixed inoculation with S. glaseri IJ.     

Activity and Persistence of Steinernema carpocapsae and Spodoptera exigua Nuclear Polyhedrosis Virus against S. exigua Larvae on Soybean

Experiments were conducted to assess the effects of the entomopathogenic nematode Steinernema carpocapsae and Spodoptera exigua multinucleocapsid nuclear polyhedrosis virus (SeMNPV), alone and in combinations, on mortality of the beet armyworm, S. exigua, larvae on soybean. In 1991 tests, field-grown soybean plants were treated with S. carpocapsae at 0.3 and 0.6 nematodes/cm² of leaflet, SeMNPV at 20 and 40 polyhedral inclusion bodies (PIB)/cm², and all possible combinations. Treated leaflets were collected from plants and bioassayed with 5-day-old larvae. The combination of S. carpocapsae at 0.6 nematodes/cm² + SeMNPV at 40 PIB/cm² produced significantly higher larval mortality (61.7%) compared with either S. carpocapsae (24.8-35.1%) or SeMNPV (26.5-33.7%) alone. In 1992, similar tests were repeated using S. carpocapsae at 0.2 and 0.5 nematodes/cm², and SeMNPV at 14 and 35 PIB/cm². The combination of 0.5 nematodes/cm² + 35 PIB/cm² resulted in significantly higher larval mortality (64.0%) than either pathogen alone (41.5-49.0%). Steinernema carpocapsae and SeMNPV produced additive effects on beet arlnyworm mortality. Persistence of S. carpocapsae was 12-24 hours and SeMNPV was 96-120 hours on soybean.     

Granular Formulations of Steinernema carpocapsae (strain All) (Nematoda: Rhabditida) with Improved Shelf Life

Shelf life (nematode survival) of Steinernema carpocapsae (strain All) nematodes at 21 C in "Pesta" granules, made by a pasta-like process, was increased from 8 to 26 weeks by incorporating low concentrations of formaldehyde. Pesta samples containing an average of 427,000 nematodes/g were prepared with wheat flour (semolina or bread flour), kaolin, bentonite, peat moss, nematode slurry, and formaldehyde (0-1.4% w/w) and were dried to a water content of 23.6-26.9%. Nematodes emerged from Pesta (S. carpocapsae) granules when placed in water or on moist filter paper. Incorporation of 0.2% w/w formaldehyde (nominal; 0.05% by analysis) was optimum for increasing nematode survival in semolina-based Pesta, and also inhibited fungal growth on the granules. Bread flour Pesta samples prepared by formaldehyde addition to the nematode slurry prior to dough preparation, rather than by addition to a mixture of dry ingredients, had longer shelf life. Nematodes recovered from granules made with 0.2% formaldehyde and stored 20 weeks at 21 C caused 100% mortality of wax moth (Galleria mellonella) larvae.