Development Rates in Entomopathogenic Nematodes: Infected Hosts vs. Aqueous Suspension

Rearing conditions have been shown to affect several aspects of entomopathogenic nematode biology, including dispersal behavior and infectivity. The present study explores the differences in development rate of Heterorhabditis bacteriophora and Steinernema carpocapsae when infective juveniles (IJ) were collected in water using the standard White trap method vs. natural emergence from cadavers into sand. We exposed Galleria mellonella to IJ entompopathogenic nematodes treated in one of three ways: collected in a White trap, allowed to emerge directly into sand, or collected in a White trap and treated with a cadaver homogenate. When S. carpocapsae IJ were allowed to emerge from cadavers directly into sand and then allowed to infect new hosts, they developed into adults at a faster rate than IJ that were collected with White traps. The difference in development was not due to differential infection rates. No difference in development stages was detected amount the same H. bacteriophora treatments.     

Effect of insect cadaver desiccation and soil water potential during rehydration on entomopathogenic nematode (Rhabditida: Steinernematidae and Heterorhabditidae) production and virulence

We examined the influence of insect cadaver desiccation on the virulence and production of entomopathogenic nematodes (EPNs), common natural enemies of many soil-dwelling insects. EPNs are often used in biological control, and we investigated the feasibility of applying EPNs within desiccated insect cadavers. Desiccation studies were conducted using the factitious host, Galleria mellonella (Lepidoptera: Pyralidae, wax moth larvae) and three EPN species (Heterorhabditis bacteriophora ‘HB1’, Steinernema carpocapsae ‘All’, and Steinernema riobrave). Weights of individual insect cadavers were tracked daily during the desiccation process, and cohorts were placed into emergence traps when average mass losses reached 50%, 60%, and 70% levels. We tracked the proportion of insect cadavers producing infective juveniles (IJs), the number and virulence of IJs produced from desiccated insect cadavers, and the influence of soil water potentials on IJ production of desiccated insect cadavers. We observed apparent differences in the desiccation rate of the insect cadavers among the three species, as well as apparent differences among the three species in both the proportion of insect cadavers producing IJs and IJ production per insect cadaver. Exposure of desiccated insect cadavers to water potentials greater than −2.75 kPa stimulated IJ emergence. Among the nematode species examined, H. bacteriophora exhibited lower proportions of desiccated insect cadavers producing IJs than the other two species. Desiccation significantly reduced the number of IJs produced from insect cadavers. At the 60% mass loss level, however, desiccated insect cadavers from each of the three species successfully produced IJs when exposed to moist sand, suggesting that insect cadaver desiccation may be a useful approach for biological control of soil insect pests.     

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.     

Effect of Application Method on Fitness of Entomopathogenic Nematodes Emerging at Different Times

The entomopathogenic nematode species Steinernema feltiae and Heterorhabditis bacteriophora were compared for survival and infectivity of infective juveniles (IJ) collected with a standard White trap (i.e., emerging from hosts and accumulating in water) and later applied to sand (treatment A) to IJ allowed to emerge from hosts into sand (treatment C). Percentage IJ survival and infectivity was compared between treatments for S. feltiae IJ that emerged between days 1 to 3 and days 4 to 6. For H. bacteriophora, percentage IJ survival and infectivity was compared between treatments only for infective juveniles that emerged between days 4 to 6. For S. feltiae IJ percentage survival and infectivity decreased with time (P ≤ 0.05) and was greater (P ≤ 0.05) for IJ from treatment C than for IJ from treatment A. For H. bacteriophora IJ percentage survival decreased (P ≤ 0.05) and percentage infectivity increased (P ≤ 0.05) with time. While percent survival was higher (P ≤ 0.05) for treatment C than for A, percent infectivity was not different between treatments.     

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.     

Morphometrics of Infective Juveniles of Steinernema spp. and Heterorhabditis bacteriophora (Nemata: Rhabditida)

Selected morphometrics of Heterorhabditis bacteriophora and seven species of Steinernema from in vivo culture were compared in relation to time of harvest. In addition, five Steinernema species were reared in vitro and their morphometrics were compared with those from in vivo culture. With in vivo culture, there was generally a negative linear relationship between body length of infective juveniles (IJ) and time of harvest. The distance from the anterior end to the excretory pore (EP) and the tail length (T) of IJ also varied with time of harvest. The E percentage (= EP/T x 100) was the least variable. Body lengths of IJ reared in vitro were much less than those of IJ reared in vivo. The study suggests that IJ harvested from in vivo culture within 1 week of emergence from cadavers are best for species identification. Infective juveniles from in vitro culture should not be used for species identification.     

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.     

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.     

Effect of insect host age and diet on the fitness of the entomopathogenic nematode-bacteria mutualism

Insect host age and diet were evaluated as potential factors that could affect the fitness of the entomopathogenic nematode-bacterium mutualistic partnership. Two nematode species were considered: Steinernema carpocapsae and Heterorhabditis sonorensis, together with their symbionts Xenorhabdus nematophila and Photorhabdus luminescens, respectively. The tobacco hornworm, Manduca sexta, was used as the insect host. Insect developmental stage was a factor that impacted nematode virulence. Non-wandering 5th instar M. sexta were found to be more susceptible to nematode infection compared to wandering 5th instars. This was more noticeable for S. carpocapsae than for H. sonorensis. The nutritional status of the host also had an effect on the fitness of the two nematode species tested. In general, insects fed with the reduced diet content were less susceptible to nematode parasitism. The least observed mortality (0.5 %) was in those M. sexta larvae exposed to the low H. sonorensis dose. Host diet also had an effect on the production of IJ progeny in the insect cadavers. For both nematode species tested, the highest yield of emerging IJs was observed from those insect hosts fed with the low nutrient diet and exposed to the highest nematode inoculum. However, for both nematode species tested, the nutritional status of the host did not significantly affect time of emergence of IJ progeny or the reassociation with their bacterial symbionts (expressed as cfu/IJ). This is the first study on the effect of insect host physiology on both EPN and their symbiotic bacteria fitness.     

Control of the Oriental Fruit Moth, Grapholita molesta, Using Entomopathogenic Nematodes in Laboratory and Fruit Bin Assays

The oriental fruit moth (OFM), Grapholita molesta (Busck), which is among the most important insect pests of peaches and nectarines, has developed resistance to a wide range of insecticides. We investigated the ability of the entomopathogenic nematodes (EPN) Steinernema carpocapsae (Weiser), S. feltiae (Filipjev), S. riobrave (Cabanillas et al.), and Heterorhabditis marelatus (Liu and Berry) to control OFM under laboratory and fruit bin conditions. At a dosage of 10 infective juveniles (IJ)/cm² in the laboratory, S. carpocapsae caused 63%, S. feltiae 87.8%, S. riobrave 75.6%, and H. marelatus 67.1% OFM mortality. All four nematode species caused significant OFM larval mortality in comparison to the nontreated controls. Steinernema feltiae was used for the bin assays due to the higher OFM mortality it caused than the other tested EPN species and to its ability to find OFM under cryptic environments. Diapausing cocooned OFM larvae in miniature fruit bins were susceptible to IJ of S. feltiae in infested corner supports and cardboard strips. Treatment of bins with suspensions of 10 or 25 S. feltiae IJ/ml water with wetting agent (Silwet L77) resulted in 33.3 to 59% and 77.7 to 81.6% OFM mortality in corner supports and cardboard strips, respectively. This paper presents new information on the use of EPN, specifically S. feltiae, as nonchemical means of OFM control.     

Mating and Sexual Communication by Steinernema carpocapsae (Nemata: Steinernematidae)

Entomopathogenic nematodes are lethal insect parasites that reproduce exclusively inside their hosts in nature. Infection decisions made by the free-living infective-stage juveniles have an impact on reproductive success, but it is likely that mating decisions are made by adults while inside their host. We investigated sexual communication between male and female adult stages of Steinernema carpocapsae (Rhabditida: Steinernematidae) to assess whether mating is chemically mediated during the adult stage or results from incidental encounters between adults inside the insect host. To assess chemical communication, we measured the behavioral response of adult male S. carpocapsae to several different potential sources of chemical information. Male S. carpocapsae responded to virgin females only and were not influenced by mated conspecific females, conspecific males, or heterospecific females. These results show that species-specific communication takes place between adult entomopathogenic nematodes within the host cadaver just prior to mating.     

Effect of Entomopathogenic Nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) on Meloidogyne mayaguensis Rammah and Hirschmann (Tylenchida: Meloidoginidae) Infection in Tomato Plants

Some studies suggest that entomopathogenic nematodes (EPN) affect plant-parasitic nematode populations. Here, the effects of live and dead IJ of Heterorhabditis bacteriophora JPM4, H. baujardi LPP7, Steinernema feltiae SN and S. carpocapsae All were evaluated against eggs and J2 of the plant-parasitic nematode Meloidogyne mayaguensis. According to treatment, 100 IJ were applied with 350 eggs, 350 J2 or 175 eggs + 175 J2 to tomato plants. Bioassays were conducted in March to May and repeated in September to November 2005. Both experiments lasted 9 weeks, and the variable evaluated was number of galls per plant. When eggs were used for infections in the first trial, plants exhibited lower gall number compared to control when live and dead H. baujardi IJ and live S. feltiae IJ were added (9.7, 4.5, 7.3 and 85.7 galls, respectively). In the second trial, live S. feltiae and S. carpocapasae IJ influenced gall formation compared to control (14.33, 14.57 and 168.02 galls, respectively). When J2 were used for infections, plants with live H. baujardi IJ presented less galls when compared to control in both trials (38.3 and 355.7 galls in the first trial and 145.2 and 326.2 in the second one, respectively). Infection with a mixture of J2 and eggs resulted in fewer galls than when live S. feltiae IJ were present in both trials, compared to control (38.3 and 44.2 galls vs. 275.3 and 192.2 galls, respectively). We conclude that H. baujardi and S. feltiae apparently may be inhibiting egg hatching and J2 infection.     

Comparative study of the entomopathogenic nematode, Steinernema carpocapsae, reared on mutant and wild-type Xenorhabdus nematophila

The symbiotic interaction between Steinernema carpocapsae and Xenorhabdus nematophila was investigated by comparing the reproduction, morphology, longevity, behavior, and efficacy of the infective juvenile (IJ) from nematodes reared on mutant or wild-type bacterium. Nematodes reared on the mutant X. nematophila HGB151, in which an insertion of the bacterial gene, rpoS, eliminates the retention of the bacterium in the intestinal vesicle of the nematode, produced IJs without their symbiotic bacterium. Nematodes reared on the wild-type bacterium (HGB007) produced IJs with their symbiotic bacterium. One or the other bacterial strain injected into Galleria mellonella larvae followed by exposing the larvae to IJs that were initially symbiotic bacterium free produced progeny IJs with or without their Xenorhabdus-symbiotic bacterium. The two bacterial strains were not significantly different in their effect on IJ production, sex ratio, or IJ morphology. IJ longevity in storage was not influenced by the presence or absence of the bacterial symbiont at 5 and 15 °C, but IJs without their bacterium had greater longevity than IJs with their bacterium at 25 and 30 °C, suggesting that there was a negative cost to the nematode for maintaining the bacterial symbiont at these temperatures. IJs with or without their symbiotic bacterium were equally infectious to Spodoptera exigua larvae in laboratory and greenhouse and across a range of soil moistures, but the absence of the bacterial symbiont inhibited nematodes from producing IJ progeny within the host cadavers. In some situations, such as where no establishment of an alien entomopathogenic nematode is desired in the environment, the use of S. carpocapsae IJs without their symbiotic bacterium may be used to control some soil insect pests.     

Host cadavers protect entomopathogenic nematodes during freezing

The entomopathogenic nematodes Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema glaseri, and Steinernema feltiae were exposed to freezing while inside their hosts. Survival was assessed by observing live and dead nematodes inside cadavers and by counting the infective juveniles (IJs) that emerged after freezing. We (1) measured the effects of 24h of freezing at different times throughout the course of an infection, (2) determined the duration of freezing entomopathogenic nematodes could survive, (3) determined species differences in freezing survival. Highest stage-specific survival was IJs for S. carpocapsae, and adults for H. bacteriophora. When cadavers were frozen two or three days after infection, few IJs emerged from them. Freezing between five and seven days after infection had no negative effect on IJ production. No decrease in IJ production was measured for H. bacteriophora after freezing. H. bacteriophora also showed improved survival inside versus outside their host when exposed to freezing.     

Emergence and Dispersal Patterns of Two Isolates of the Entomopathogenic Nematode Steinernema feltiae

Few studies have addressed the role of the sexes in the emergence and dispersal of entomopathogenic nematodes from host insects. Individuals of two isolates of Steinernema feltiae, UK76 and SBIl, emerging from Galleria mellonella cadavers were classed as Non-Dispersed (remaining on the cadaver for up to nine days) and Dispersed (actively moving away from the cadaver). Sex ratios within both classes were examined in infective (individuals that successfully invaded bait G. mellonella) and entire (infective and noninfective individuals that matured in hanging drops of G. mellonella haemolymph) populations. Sex ratios differed significantly from 1:1 only in the SBIl Non-Dispersed entire population (female bias) and SBIl Non-Dispersed infective population (male bias). For each isolate, Dispersed individuals were significantly more infective than Non-Dispersed. However, only 11% of SBIl and 22% of UK76 Non-Dispersed individuals were found to be mature infective juveniles (IJ) compared with 78% of SBIl and 82% of UK76 Dispersed individuals (based on survival in SDS). Infective juveniles dispersing towards distant radial bait G. mellonella tended to migrate from the head region of the natal cadaver. For each isolate, a higher proportion of males than females arrived early at distant baits. SBIl males survived alone in G. mellonella cadavers for longer periods than did females, which supports the “male colonization” hypothesis.     

Effect of Soil Depth and Moisture on the Vertical Distribution of Steinernema riobrave (Nematoda: Steinernematidae)

The effect of soil moisture on the distribution of Steinernema riobrave in a sand column was determined. Larvae of Pectinophora gossypiella were used to detect S. riobrave infective juveniles (IJ) in each 2.5-cm section of 30-cm-long soil columns. Soil moisture was determined for each section and related to the numbers of nematodes recovered from infected insect baits. Infective juveniles of S. riobrave applied on the sand column surface showed some degree of positive geotaxis. IJ in soil columns with a consistent moisture gradient grouped in the upper 12.7 cm within a water potential range of ¯40 to ¯0.0055 MPa (2% to 14% moisture). Nematodes in sand columns that were gradually dehydrating moved down the soil column, aggregating on the 28th day between 15-23 cm in depth. Nematode redistribution over time allowed IJ to remain within a water potential range of ¯0.1 to ¯0.012 MPa (5.2% to 9.5% moisture).     

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.     

Competition Between Entomopathogenic and Free-Living Bactivorous Nematodes in Larvae of the Weevil Diaprepes abbreviatus

Field and laboratory experiments were conducted to determine the degree to which free-living, bactivorous nematodes (FLBN) are able to competitively displace entomopathogenic nematodes (EPN) from insect cadavers. Two hundred larvae of the insect Diaprepes abbreviatus were buried at regular intervals during 2 years in experimental plots that were untreated or treated twice annually with Steinernema riobrave. Larvae were recovered after 7 days, and nematodes emerging from cadavers during the next 30 days were identified. The monthly prevalence of FLBN was directly related to that of S. riobrave (r = 0.38; P = 0.001) but was not related to the prevalence of the endemic EPN, S. diaprepesi, Heterorhabditis zealandica, H. indica, or H. bacteriophora (r = 0.02; P = 0.80). In a second experiment, treatment of small field plots with S. riobrave increased the prevalence of insect cadavers in which only FLBN were detected compared to untreated controls (30% vs. 14%; P = 0.052), and increased numbers of FLBN per buried insect by more than 10-fold. In the laboratory, sand microcosms containing one D. abbreviatus larva were treated with (i) the FLBN, Pellioditis sp.; (ii) S. riobrave; (iii) S. riobrave + Pellioditis; or (iv) neither nematode. Insect mortality was higher in the presence of both nematodes (57%) than when S. riobrave was alone (42%) (P = 0.01). An average of 59.2 Pellioditis sp. g^-1 insect body weight emerged in the presence of S. riobrave, whereas 6.2 nematodes g^-1 insect were recovered in the absence of the EPN (P = 0.01). Pellioditis sp. reduced the number of S. riobrave per cadaver by 84%; (P = 0.03), and per available insect by 82% (P = 0.001), compared to S. riobrave alone. Population size of S. diaprepesi was not affected by Pellioditis sp. in experiments of the same design. Faster development (P = 0.05) and nutrient appropriation within the insect cadaver by S. diaprepesi compared to S. riobrave may increase the fitness of the former species to compete with Pellioditis sp. The results of these studies demonstrate the potential of FLBN to regulate population densities of EPN and to dampen estimates of EPN-induced mortality of insect pests in the field.     

Emergence of flies from overwintering populations of cabbage root fly pupae

ABSTRACT.

• 1 Once pupal diapause had been terminated, over-wintering cabbage root fly (Delia radicum (L.)) pupae from Wellesbourne required a further 14 days at 20°C for most of the flies to emerge.
• 2 There were considerable variations in the rates of fly emergence from thirteen populations of cabbage root fly pupae collected between latitudes 50° 42′ and 54° 59′ in England and Wales. These thirteen populations could be grouped into early-, intermediate- and late-emerging types. In the early-emerging type, flies emerged within 14 days at 20°C whereas in the late-emerging type emergence was protracted and was completed only after 100 days at 20°C in one population from Halsall, Lancashire. In the intermediateemerging type, approximately two-thirds of the flies emerged within 14 days at 20° C, the remainder taking considerably longer.
• 3 The intermediate-emerging types could be just mixtures of the early- and late-emerging types.
• 4 Subjecting pupae to diapause-breaking temperatures (4°C) for up to 1 year failed to shorten the time to subsequent fly emergence in any of the populations.
• 5 Populations of early, intermediate- and late-emerging fies could be selected from a parental population, heterogeneous with respect to emergence, within one generation.
• 6 The type of emergence that occurred in a locality was not correlated with latitude.
• 7 Any models developed for forecasting the most appropriate time to apply insecticide in a locality will have to include information about the emergence pattern of the fly population in that locality.

     

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%.