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.     

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

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

Effect of Entomopathogenic Nematode Concentration on Survival during Cryopreservation in Liquid Nitrogen

Entomopathogenic nematodes are used for biological control of insect pests. A method for improved cryopreservation of infective juvenile stage nematodes has been developed using Steinernema carpocapsae and Heterorhabditis bacteriophora. Optimum survival for both species was achieved with 12,000 infective juveniles/ml in glycerol and 7,500/ml in Ringer''s solution. For S. carpocapsae, maximum survival also was observed with 60,000 infective juveniles/ml in glycerol and 25,000/ml in Ringer''s solution. These concentrations resulted in 100% post-cryopreservation survival of S. carpocapsae and 100% retention of original virulence to Galleria mellonella larvae. This is the first report of achieving 100% survival of an entomopathogenic nematode after preservation in liquid nitrogen. Maximum survival of H. bacteriophora following cryopreservation was 87%.     

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.     

Influence of Salinity on Survival and Infectivity of Entomopathogenic: Nematodes

Exposure to NaC1, KCI, and CaCl₂ affected the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema glaseri differently. Survival, virulence, and penetration efficiency of S. glaseri were not affected by these salts. At high concentrations, however, all three salts inhibited its ability to move through a soil column and locate and infect a susceptible host. Calcium chloride and KCl had no effect on H. bacteriophora survival, penetration efficiency, or movement through a soil column, but moderate concentrations of these salts enhanced H. bacteriophora virulence. NaCl, however, adversely affected each of these parameters at high salinities (>16 dS/m). Salt effects on S. glaseri are attributed solely to interference with nematode host-finding ability, whereas the NaCl effects on H. bacteriophora are attributed to its toxicity and possibly to interference with host-finding behavior.     

Variations in Immune Response of Popillia japonica and Acheta domesticus to Heterorhabditis bacteriophora and Steinernema Species

The infectivities of Steinernema carpocapsae, S. glaseri, S. scapterisci, and Heterorhabditis bacteriophora to Japanese beetle larvae, Popillia japonica, and house cricket adults, Acheta domesticus, were compared using external exposure and hemocoelic injection. Only H. bacteriophora and S. glaseri caused high P. japonica mortality after external exposure. When nematodes were injected, P. japonica had a strong encapsulation and melanization response to all species except S. glaseri. Heterorhabditis bacteriophora and S. carpocapsae were able to overcome the immune response, but S. scapterisci was not. All species except S. scapterisci were able to kill and reproduce within the host. Only S. scapterisci and S. carpocapsae caused A. domesticus mortality after external exposure. When nematodes were injected, A. domesticus had a strong immune response to all species except S. scapterisci. Steinernema carpocapsae effectively overcame the strong immune response and caused high host mortality, but S. glaseri and H. bacteriophora did not. Steinernema scapterisci caused high host mortality and reproduced, S. glaseri and H. bacteriophora caused low host mortality but only S. glaseri reproduced, and S. carpocapsae was able to kill the host but reproduced poorly. Most (ca. 90%) of the S. carpocapsae in the hemocoel of P. japonica became encapsulated and melanized within 8 hours postinjection. The symbiotic bacterium, Xenorhabduf nematophilus, was often released before this encapsulation and melanization.     

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.     

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

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

Gold-conjugated Reagents for the Labeling of Carbohydrate-Recognition Domains and Glycoconjugates on Nematode Surfaces

Various fluorescent conjugated lectins have been used for the detection of glycoconjugates on nematode surfaces under light microscopy. Several problems have been experienced with these reagents including penetration of the cuticle by fluorescent lectins, non-glycoconjugate specificity, strong nematode autofluorescence at the emission wavelength of the fluorescent dye, and prevention of persistent visualization due to rapid quenching of the fluorescent components. Gold-conjugated reagents combined with silver enhancement alleviated these difficulties when working with three phytonematode species (Heterodera avenae, H. latipons, and Meloidogyne javanica) and two entomopathogenic species (Steinernema carpocapsae and S. glaseri) under light-microscopy visualization of binding by fluorescent lectins and neoglycoproteins. Moreover, gold-conjugated reagents resulted in stable bindings that enabled long-term observations.     

Nonsusceptibility of the Honey Bee,Apis mellifera (Hymenoptera: Apidae), to Steinernematid and Heterorhabditid Nematodes

We exposed honey bee workers and brood to four entomopathogenic nematode species under conditions normally encountered in the hive by spraying nematodes onto combs. Mortality of adult bees exposed to any of the nematode species was less than 10%, and there was no evidence of nematode infection when dead adults were dissected. To assess the impact of nematodes on brood, we used smaller-size honey combs placed in the second story (super) of a hive and large brood combs placed in the main section of the hive. Our results were inconsistent between these two experimental designs. The smaller honey combs sprayed with Steinernema carpocapsae contained the largest number of uncapped ceils, those sprayed with Heterorhabditis baeteriophora or S. riobravis contained an intermediate number of uncapped cells, and control combs and those sprayed with S. glaseri contained the fewest nmnber of uncapped cells. Large combs sprayed with S. riobravis contained more uncapped ceils than controls or those sprayed with S. carpocapsae, although the differences were not significant. Our results do not support the hypothesis that high-temperature-tolerant species of nematodes are necessarily more infective to honey bees.     

Comparison of Assays for the Determination of Entomogenous Nematode Infectivity

Injection, contact, and soil assays were used to compare infectivity of Heterorhabditis bacteriophora strain HP88 and Steinernema carpocapsae strain All to final instar Galleria mellonella larvae. Under comparable assay conditions, H. bacteriophora produced less Galleria mortality and showed greater within-assay variability in infectivity than S. carpocapsae. Injection of individual S. carpocapsae or H. bacteriophora infective juveniles into Galleria indicated that a comparatively greater percentage of S. carpocapsae was capable of initiating infection. In addition to nematode species, other major components of variability in assay estimations of nematode infectivity were number of nematodes used in the assay, assay type, date of the assay, and possibly, Galleria age.     

Effects of Two Carbamates on Infective Juveniles of Stemernema carpocapsae All Strain and Steinernema feltiae Ume? Strain

Laboratory bioassays were conducted to determine the effects of two carbamates, carbofuran (an acetylcholinesterase inhibitor) and fenoxycarb (a juvenile hormone analog), on survival and infectivity of the infective juveniles (IJ) of Steinernema feltiae Umeå strain and Steinernema carpocapsae All strain. Both insecticides caused mortality of IJ in a dose-related fashion. The two nematode species were equally sensitive to fenoxycarb (LD₅₀ ca. 0.03mg/ml). Whereas IJ of S. feltiae were several orders of magnitude more sensitive to carbofuran (LD₅₀ ≤ 0.2 μg/ml) than to fenoxycarb, S. carpocapsae IJ displayed approximately the same degree of sensitivity to carbofuran (LD₅₀ 0.01-0.03 mg/ml) as they did toward fenoxycarb. Toxicity of the carbamates was the same at all exposure periods from 24 to 168 hours'' duration. Determinations of infective doses of nematodes required to cause 50% mortality of Galleria mellonella larvae showed that the infectivity of IJ that survived exposure to either of the two carbamates was not compromised by treatment.     

A Comparison of Entomopathogenic Nematode Longevity in Soil under Laboratory Conditions

We compared the longevity of 29 strains representing 11 entomopathogenic nematode species in soil over 42 to 56 d. A series of five laboratory experiments were conducted with six to eight nematode strains in each and one or more nematode strains in common, so that qualitative comparisons could be made across experiments. Nematodes included Heterorhabditis bacteriophora (four strains), H. indica (Homl), H. marelatus (Point Reyes), H megidis (UK211), H. mexicana (MX4), Steinernema carpocapsae (eight strains), S. diaprepesi, S. feltiae (SN), S. glaseri (NJ43), S. rarum (17C&E), and S. riobrave (nine strains). Substantial within-species variation in longevity was observed in S. carpocapsae, with the Sal strain exhibiting the greatest survival. The Sal strain was used as a standard in all inter-species comparisons. In contrast, little intra-species variation was observed in S. riobrave. Overall, we estimated S. carpocapsae (Sal) and S. diaprepesi to have the highest survival capability. A second level of longevity was observed in H. bacteriophora (Lewiston), H. megidis, S. feltiae, and S. riobrave (3–3 and 355). Lower levels of survivability were observed in other H. bacteriophora strains (Hb, HP88, and Oswego), as well as S. glaseri and S. rarum. Relative to S. glaseri and S. rarum, a lower tier of longevity was observed in H. indica and H. marelatus, and in H. mexicana, respectively. Although nematode persistence can vary under differing soil biotic and abiotic conditions, baseline data on longevity such as those reported herein may be helpful when choosing the best match for a particular target pest.     

Effects of Earthworms on the Dispersal of Steinernema spp.

Previous studies indicated that dispersal of S. carpocapsae may be enhanced in soil with earthworms. The objective of this research was to determine and compare the effects of earthworms on dispersal of other Steinernema spp. Vertical dispersal of Steinernema carpocapsae, S. feltiae, and S. glaseri was tested in soil columns in the presence and absence of earthworms (Lumbricus terrestris). Dispersal was evaluated by a bioassay and by direct extraction of nematodes from soil. Upward dispersal of S. carpocapsae and S. feltiae increased in the presence of earthworms, whereas upward dispersal of S. glaseri was not affected by earthworms. No significant differences were detected in downward dispersal of S. carpocapsae and S. feltiae in soil with earthworms compared to soil without earthworms. Downward dispersal of S. glaseri, however, was greater in soil without earthworms relative to soil with earthworms. In soil void of earthworms, dispersal of S. glaseri was greatest followed by dispersal of S. carpocapsae. The presence of earthworm burrows in soil did not influence nematode dispersal. Nematodes were recovered from the surface, interior, and casts of earthworms. Therefore, nematodes may have a phoretic association with 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.     

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.     

Factors influencing parasitism of adult Japanese beetles,Polillia japonica (Col.: Scarabaeidae) by entomopathogenic nematodes

Several factors that influence the activity of steinernematid and heterorhabditid nematodes against adult Japanese beetles were examined in the laboratory. The effect of nematode concentration on mortality of adult beetles was evaluated using a Petri plate bioassay. The adults were exposed to 1,000 to 10,000 infective stage juveniles (J3) ofSteinernema glaseri per 10 beetles with or without food for 24 hr after which they were held with food for an additional 6 days. The LC50s for males with and without food during exposure were 3,435 and 2,854 J3s/10 adults, respectively. The LC50s for mixtures of males and females with and without food were 5,228 and 1,762 J3s/10 adults respectively. Although mortality occurred during and shortly after exposure, significant additional mortality was observed 1–4 days following exposure. Exposure of males and females with food to 10,000 J3s/10 adults for 6, 12, 18 or 24 hr resulted in 47, 58, 72 and 77% mortality, respectively. Comparative activity ofS. glaseri, S. carpocapsae (All strain),S. feltiae (Biosys experimental cold adapted strain=bibionis),S. feltiae (Biosys experimental strain 27),Heterorhabditis bacteriophora, andHeterorhabditis sp. (Terceiran isolate) was evaluated against adult Japanese beetles using a 24 hr exposure to 8,000 J3s/10 adults. The most virulent species wereS. glaseri, S. feltiae (=bibionis), the Terceiran isolate ofHeterorhabditis andS. carpocapsae producing 55, 44, 36 and 34% mortality respectively. Our results indicate that adult Japanese beetles infected with entomopathogenic nematodes could serve as a mechanism for nematode dispersal.     

Use of Cellulose Acetate Electrophoresis in the Taxonomy of Steinernematids (Rhabditida,Nematoda)

A steinernematid nematode was isolated from soil samples collected near St. John''s, Newfoundland, Canada. On the basis of its morphometry and RFLPs in ribosomal DNA spacer, it was designated as a new strain, NF, of Steinernema feltiae. Cellulose acetate electrophoresis was used to separate isozymes of eight enzymes in infective juveniles of S. feltiae NF as well as four other isolates: S. feltiae Umeå strain, S. feltiae L1C strain, Steinernema carpocapsae All strain, and Steinernema riobravis TX strain. Based on comparisons of the relative electrophoretic mobilities (μ) of the isozymes, one of the eight enzymes (arginine kinase) yielded zymograms that were distinctive for each of the isolates, except for the Umeå and NF strains of S. feltiae, which had identical banding patterns. Four enzymes (fumarate hydratase, phosphoglucoisomerase, phosphoglucomutase, and 6-phosphogluconate dehydrogenase) yielded isozyme banding patterns that were characteristic for all isolates, except for the L1C and NF strains of S. feltiae, which were identical. Two enzymes (aspartate amino transferase and glycerol-3-phosphate dehydrogenase) yielded zymograms that permitted S. carpocapsae All strain to be discriminated from the other four isolates, while the remaining enzyme (mannose-6-phosphate isomerase) was discriminatory for S. riobravis TX strain. Except for one enzyme, the isozyme banding pattern of the NF isolate of S. feltiae was the same as in the L1C strain, isolated 13 years previously from Newfoundland. Cellulose acetate electrophoresis could prove invaluable for taxonomic identification of isolates of steinernematids, provided that a combination of enzymes is used.     

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.     

Susceptibility of the Plum Curculio,Conotrachelus nenuphar,to Entomopathogenic Nematodes

The plum curculio, Conotrachelus nenuphar, is a major pest of pome and stone fruit. Our objective was to determine virulence and reproductive potential of six commercially available nematode species in C. nenuphar larvae and adults. Nematodes tested were Heterorhabditis bacteriophora (Hb strain), H. marelatus (Point Reyes strains), H. megidis (UK211 strain), Steinernema riobrave (355 strain), S. carpocapsae (All strain), and S. feltiae (SN strain). Survival of C. nenuphar larvae treated with S. feltiae and S. riobrave, and survival of adults treated with S. carpocapsae and S. riobrave, was reduced relative to non-treated insects. Other nematode treatments were not different from the control. Conotrachelus nenuphar larvae were more susceptible to S. feltiae infection than were adults, but for other nematode species there was no significant insect-stage effect. Reproduction in C. nenuphar was greatest for H. marelatus, which produced approximately 10,000 nematodes in larvae and 5,500 in adults. Other nematodes produced approximately 1,000 to 3,700 infective juveniles per C. nenuphar with no significant differences among nematode species or insect stages. We conclude that S. carpocapsae or S. riobrave appears to have the most potential for controlling adults, whereas S. feltiae or S. riobrave appears to have the most potential for larval control.