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 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.     

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.     

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.     

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.     

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.     

Dispersal, Infectivity and Sex Ratio of Early- or Late-Emerging Infective Juveniles of the Entomopathogenic Nematode Steinernema carpocapsae

Differences in activity between infective juveniles (IJ) of the entomopathogenic nematode Steinernema carpocapsae that emerged directly from cadavers onto either a sand or agar substrate compared with those emerging from a cadaver into water and then being placed on the same substrate are known to occur. Differences between S. carpocapsae IJ that emerged directly from a cadaver vs. those that emerged from a cadaver and held in water were further elucidated. Dispersed and non-dispersed IJ from a cadaver were compared with those held in water between two time periods designated as early- (first two days) or late-emerging IJ (seventh day). A significantly greater proportion of early-emerging IJ from the cadaver treatment dispersed, compared with late-emerging IJ from a cadaver or either group of emerging IJ held in aqueous suspension. Moreover, IJ from cadavers were more infectious than those from the aqueous suspensions, and IJ that dispersed were less infectious than those that did not disperse. IJ that emerged early were mostly males, whereas those that emerged late were mostly females. For the non-dispersed IJ, most that emerged early were males, and those that emerged later were females, but among dispersing IJ, there was no difference in sex ratio between early- and late-emerging nematodes.     

Location of the Phasmids on Infective Juveniles of Steinernema glaseri

Scanning electron microscopy revealed the location of the phasmids on infective juveniles of Steinernema glaseri. The phasmids are located about 40% of the tail length posterior to the anus and are at or near the same level. Instead of being in the center of the lateral fields, they are located just ventral to the lateral fields, or interrupting the ventral-most lateral ridge. The phasmids were covered often by an exudate.     

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.     

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.     

"Pesta": New Granular Formulations for Steinernema carpocapsae

"Pesta," a new granular product for use with entrapped biocontrol agents, is based on a cohesive dough made of wheat flour, fillers, and other additives. Infective juveniles of the entomopathogen Steinernema carpocapsae strain All incorporated in Pesta granules emerged when the granules were softened by immersion in water. These granules may be useful for the biocontrol of insect pests in the soil. Storage temperature had the greatest effect on recovery of nematodes, followed by the moisture content of the granules. Recovery of nematodes was the same among the formulations tested and was unaffected by storage in nitrogen. Nematode recovery after storage at 21 C decreased to zero after 3-6 weeks. Storage of samples at 4 C and with a high moisture content (19.9-23.1%) greatly improved nematode viability.     

Bionomics of a Phoretic Association Between Paenibacillus sp. and the Entomopathogenic Nematode Steinernema diaprepesi

Spores of an unidentified bacterium were discovered adhering to cuticles of third-stage infective juvenile (IJ) Steinernema diaprepesi endemic in a central Florida citrus orchard. The spores were cup-shaped, 5 to 6 mm in length, and contained a central endospore. Based on 16S rDNA gene sequencing, the bacterium is closely related to the insect pathogens Paenibacillus popilliae and P. lentimorbus. However, unlike the latter bacteria, the Paenibacillus sp. is non-fastidious and grew readily on several standard media. The bacterium did not attach to cuticles of several entomopathogenic or plant-parasitic nematodes tested, suggesting host specificity to S. diaprepesi. Attachment of Paenibacillus sp. to the third-stage cuticle of S. diaprepesi differed from Paenibacillus spp. associated with heterorhabditid entomopathogenic nematodes, which attach to the IJ sheath (second-stage cuticle). The inability to detect endospores within the body of S. diaprepesi indicates that the bacterial association with the nematode is phoretic. The Paenibacillus sp. showed limited virulence to Diaprepes abbreviatus, requiring inoculation of larvae with 108 spores to achieve death of the insect and reproduction of the bacterium. The effect of the bacterium on the nematode population biology was studied in 25-cm-long vertical sand columns. A single D. abbreviatus larva was confined below 15-cm depth, and the soil surface was inoculated with either spore-free or spore-encumbered IJ nematodes. After 7 days, the proportion of IJ below 5-cm depth was seven-fold greater for spore-free IJ than for spore-encumbered nematodes. Mortality of D. abbreviatus larvae was 72% greater (P <= 0.01) for spore-free compared to spore-encumbered S. diaprepesi. More than 5 times as many progeny IJs (P <= 0.01) were produced by spore-free compared to spore-encumbered nematodes. These data suggest that the bacterium is a component of the D. abbreviatus food web with some potential to regulate a natural enemy of the insect.     

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.     

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%.     

Life Cycle of Steinernema scapterisci Nguyen &Smart, 1990

The life cycle of Steinernema scapterisci Nguyen and Smart, 1990 consists of an egg stage, four juvenile stages, and an adult stage (male and female). The cycle from IJ (third stage infective juveniles) to IJ may proceed by one of two routes. If the nutrient supply is sufficient and the population is not overcrowded, the IJ develop to adult males and females of the first generation. Most eggs from these adult females hatch and the juveniles develop through each life stage to become adult males and females of the second generation. Eggs produced by these females develop to IJ. This cycle takes 8-10 days (long cycle) at 24 C. If the nutrient supply is insufficient or if overcrowded, the IJ develop to adult males and females of the first generation, and eggs produced by the females develop directly to IJ. This cycle takes 6-7 days (short cycle). The nematode is less tolerant of lower temperatures and more tolerant of higher temperatures than are other species of the genus. The sex ratio is influenced by temperature. At 15 and 24 C, females constituted 54% and 60% of the population, respectively, but at 30 C females constituted 47% of the population.     

Steinernema feltiae Intraspecific Variability: Infection Dynamics and Sex-Ratio

Entomopathogenic nematodes (EPNs) from the Heterorhabditidae and Steinernematidae families are well-known biocontrol agents against numerous insect pests. The infective juveniles (IJs) are naturally occurring in the soil and their success in locating and penetrating the host will be affected by extrinsic/intrinsic factors that modulate their foraging behavior. Characterizing key traits in the infection dynamics of EPNs is critical for establishing differentiating species abilities to complete their life cycles and hence, their long-term persistence, in different habitats. We hypothesized that phenotypic variation in traits related to infection dynamics might occur in populations belonging to the same species. To assess these intraspecific differences, we evaluated the infection dynamics of 14 populations of Steinernema feltiae in two experiments measuring penetration and migration in sand column. Intraspecific variability was observed in the percentage larval mortality, time to kill the insect, penetration rate, and sex-ratio in both experiments (P < 0.01). Larval mortality and nematode penetration percentage were lower in migration experiments than in penetration ones in most of the cases. The sex-ratio was significantly biased toward female-development dominance (P < 0.05). When the populations were grouped by habitat of recovery (natural areas, crop edge, and agricultural groves), nematodes isolated in natural areas exhibited less larval mortality and penetration rates than those from some types of agricultural associated soils, suggesting a possible effect of the habitat on the phenotypic plasticity. This study reinforces the importance of considering intraspecific variability when general biological and ecological questions are addressed using EPNs.     

Energy Metabolism and Survival of the Infective Juveniles of Steinernema carpocapsae under Oxygen-Deficient Conditions

Energy metabolism and its relation to survival of the infective juveniles (IJ) of S. carpocapsae under anaerobic and oxygen-deficient conditions were studied by monitoring changes in survival rate, levels of key energy reserve materials, oxygen consumption, and respiratory quotient (RQ). The effects of various factors on the survival of IJ under anaerobic conditions were also investigated. Under anaerobic conditions, the IJ were inactivated but could survive for several days in an immobile state, using the carbohydrate reserves glycogen and trehalose for energy supply. The survival time of IJ was mainly dependent on the availability of energy supply, which, in turn, was influenced by factors such as temperature and metabolic by-products. Surviving, anaerobically incubated IJ fully recovered upon return to aerobic conditions. Recovering IJ were characterized by regaining mobility and restoration of carbohydrate reserves consumed during the anaerobic period. Carbohydrate reserves were restored by conversion from lipid reserves and possibly from anaerobic metabolic by-products. The infectivity of IJ recovered from the anaerobic state was not affected. At 1% oxygen level, IJ were also immobile and mainly depended on carbohydrate reserves for energy supply and the RQ was greater than 1. However, some oxygen was consumed; the survival time of these IJ was shorter than those kept in natural air but longer than those under anaerobic conditions. When IJ were incubated at oxygen levels of 3% to 21%, the RQs were maintained at 0.7 to 0.8. Oxygen consumption rates and the reduction in both mean dry weight and lipid levels were proportional to oxygen levels while the survival time of IJ was inversely proportional to oxygen levels.     

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.     

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.     

Curative Control of the Peachtree Borer Using Entomopathogenic Nematodes

The peachtree borer, Synanthedon exitiosa (Say 1823), is a major pest of stone fruit trees in North America. Current management relies upon preventative control using broad-spectrum chemical insecticides, primarily chlorpyrifos, applied in the late summer or early fall. However, due to missed applications, poor application timing, or other factors, high levels of S. exitiosa infestation may still occur and persist through the following spring. Curative treatments applied in the spring to established infestations would limit damage to the tree and prevent the next generation of S. exitiosa from emerging within the orchard. However, such curative measures for control of S. exitiosa do not exist. Our objective was to measure the efficacy of the entomopathogenic nematode, Steinernema carpocapsae, as a curative control for existing infestations of S. exitiosa. In peach orchards, spring applications of S. carpocapsae (obtained from a commercial source) were made to infested trees and compared with chlorpyrifos and a water-only control in 2014 and 2015. Additionally, types of spray equipment were compared: nematodes were applied via boom sprayer, handgun, or trunk sprayer. To control for effects of application method or nematode source, in vivo laboratory-grown S. carpocapsae, applied using a watering can, was also included. Treatment effects were assessed 39 d (2014) or 19 d (2015) later by measuring percentage of trees still infested, and also number of surviving S. exitiosa larvae per tree. Results indicated that S. carpocapsae provided significant curative control (e.g., >80% corrected control for the handgun application). In contrast, chlorpyrifos failed to reduce S. exitiosa infestations or number of surviving larvae. In most comparisons, no effect of nematode application method was detected; in one assessment, only the handgun and watering can methods reduced infestation. In conclusion, our study indicates that S. carpocapsae may be used as an effective curative measure for S. exitiosa infestations.