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.

Toxic Factor Produced by a Granulosis Virus in Armyworm Larva: Effect on Apanteles militaris.

The internal parasitoid Apanteles militaris is affected by a proteinaceous toxic factor in the hemolymph of granulosis virus-infected armyworm larva. The hemolymph, after centrifugation to remove the virus particles and inoculation into the larval hemocoel, is still toxic to the parasitoids.

Entomopathogenic nematodes: natural enemies of root-feeding caterpillars on bush lupine.

A new species of soil-dwelling entomopathogenic nematode Heterorhabditis hepialus killed up to 100% (mean=72%) of root-boring caterpillars of a ghost moth Hepialus californicus in coastal shrub lands. When unchecked, ghost moth caterpillars killed bush lupine, Lupinus arboreus. Here we describe this strange food chain. Although unappreciated by ecologists, entomopathogenic nematodes are widespread and probably one of the most important groups of natural enemies for underground insects. The free-living infective juvenile (IJ) of entomopathogenic nematodes searches for host insects in the soil. A single IJ can kill a host, although several often invade together. After entering the host through a spiracle or other orifice, the IJ regurgitates its symbiotic bacterium, Photorhabdus luminescens, which kills the host within 48 h. The bacteria digest the cadaver and provide food for the exponentially growing nematode population inside. The bacteria produce antibiotics and other noxious substances that protect the host cadaver from other microbes in the soil. When the cadaver is exhausted of resources, IJs break the host integument and can disperse. As many as 420,000 IJs can be produced within a large ghost moth caterpillar. Surface soil of the lupine rhizosphere is the primary habitat of IJs of H. hepialus. Attracted to waste gases emitted by insects, the 0.5-mm-long IJs can move 6 cm/day through moist soil. Prevalences of H. hepialus ranged from as high as 78% of rhizospheres in some lupine stands to almost zero in others, but it was absent from no stand at our study site. Field intensities ranged from 0.003 IJs/cm3 of soil to 7.5 IJs/cm3, and correlated roughly with prevalences among sites. Few ghost moth caterpillars (mean=6.7) succeeded in entering lupine roots where prevalence of H. hepialus was highest, and this stand had lowest mortality (0.02) of mature bush lupine. In the three stands with lowest prevalence (mean = 2%) of this nematode, many caterpillars (mean = 38.5) entered roots, and lupine mortality was high (range = 0.41-1.0). Old aerial photographs indicate that the stands with highest recent nematode prevalence have had little or no mass die-off of lupine over the past 40 years. The photos depict repeated die-offs of lupine during the past four decades in stands with lowest recent prevalence of the nematode. This pattern leads us to entertain the hypothesis that the nematode affects vegetation dynamics indirectly through a trophic cascade. Dispersal of entomopathogenic nematodes is little understood. We found that air drying of soil extirpates H. hepialus and speculate that this nematode is dispersed during the wet season in moist soil bits on the exterior of fossorial insects and mammals. H. hepialus colonized some previously unoccupied lupine rhizospheres during the wet winter-spring season and, obversely, became extinct from some rhizosperes as soil dried in summer. Root-feeding insects have only recently been recognized as a force in communities, and the regulation of these important herbivores is still largely an ecological terra incognita. All evidence indicates that entomopathogenic nematodes are found throughout terrestril ecosystems, and we propose that trophic chains similar to those described in this report should not be uncommon.

Density-dependent effects on Steinernema glaseri (Nematoda: Steinernematidae) within an insect host.

Increasing densities of Steinernema glaseri infective juveniles (IJs) in soil affected penetration efficiency and reproduction of the nematodes in larvae of the greater wax moth Galleria mellonella. The penetration efficiency and the proportion of penetrated IJs developing to adults decreased significantly with increasing numbers of IJs present in the soil and entering the hosts, respectively. The number of progeny produced/host cadaver initially increased, with the highest production being between 20.7 +/- 3.3 and 58.0 +/- 6.6 IJs established/host +/- SE and decreased at higher densities. Above 184.4 +/- 37.7 IJs established/host, no progeny emerged from the cadavers.

A multivariate analysis of morphometric characters of Heterorhabditis species (Nemata: Heterorhabditidae) and the role of morphometrics in the taxonomy of species of the genus.

A multivariate analysis on the morphometrics of 7 Heterorhabditis species was conducted to review the status of the traditional morphometric characterization methods for differentiating species of these nematodes. Results from this study showed consistency in the selection of morphometric characters for discriminating among males and infective juveniles of Heterorhabditis species. For the males, testis reflexion (TREF) and total length (LENGTH) were the variables that contributed most in the discrimination among the different species, and for the infective juveniles, tail length (TAILL) and total length (LENGTH) were the variables that contributed most. Therefore, we consider that these morphometric characters are useful and reliable, and that they should be used for the identification of Heterorhabditis species/isolates.

A new species of Heterorhabditis from the Hawaiian Islands.

A new species of nematode of the genus Heterorhabditis (Nemata: Heterorhabditidae) was found during a survey of the soil entomopathogenic nematode fauna of the Hawaiian Islands. Heterorhabditis hawaiiensis sp. n. can be separated from all other species of Heterorhabditis by the length of the infective juvenile and the morphological characters of the spicules, gubernaculum, and bursa. Random amplified polymorphic DNA (RAPD) fragment analysis showed that this species also has a distinct genetic pattern in RAPD bands relative to the other 6 species or isolates of Heterorhabditis that were compared.

Competition between two steinernematid nematode species for an insect host at different soil depths.

We studied interactions between 2 entomopathogenic nematode species, Steinernema carpocapsae, an ambusher forager, and Steinernema glaseri, a cruiser forager, when they were provided wax moth larvae as hosts at 0, 2, or 10 cm soil depth. Populations of infective juvenile nematodes in soil were monitored at 30-day intervals over 120 days using wax moth larvae as baits. After application of S. carpocapsae, S. glaseri, or the combination of both species, hosts were added at 30-day intervals. With hosts at 0 cm depth, each nematode species was negatively affected by the presence of the other species at the 30- and 60-day samples. At 90 and 120 days, S. carpocapsae numbers in the combined treatment were as high as in the single species treatment, whereas only few S. glaseri were recovered. With hosts at 2 or 10 cm depth, the presence of S. glaseri had a strong negative effect on S. carpocapsae, but S. glaseri was not affected by the presence of S. carpocapsae. In another experiment, S. carpocapsae dominated over S. glaseri in hosts located at 0 cm depth as measured by penetration efficiency into hosts and progeny production. In contrast, S. glaseri dominated at 2 cm depth. At 2 cm depth, S. carpocapsae penetrated into hosts too slowly to compete successfully with S. glaseri. Steinernema carpocapsae is superior to S. glaseri when competing for a host on the soil surface; however, below the surface S. glaseri is superior to S. carpocapsae.

Efficacy of a dehydrated steinernematid nematode against black cutworm (Lepidoptera: Noctuidae) and diamondback moth (Lepidoptera: Plutellidae).

We compared the ability of in vitro-produced, commercially formulated with in vivo-produced, nonformulated Steinernema carpocapsae (Weiser) Poinar. All strain to infect and kill larvae of black cutworm, Agrotis ipsilon (Hufnagel), and diamondback moth, Plutella xylostella (L.). In vitro-produced nematodes formulated in wettable dispersible granules, which were stored dry, were rehydrated in water for 0-72 h before application. Against black cutworms, the efficacy of nematodes was (from most to least effective): in vivo > in vitro rehydrated for 72 h > in vitro rehydrated for 48 h > in vitro rehydrated for 24 h > dehydrated (0 h). Nematodes rehydrated for 72 h in water or moist soil were equally effective against black cutworm larvae, and both were significantly more effective than nematodes without rehydration. These results indicated that nematodes in the wettable dispersible granule formulation required time to rehydrate in the soil before infecting black cutworm larvae. Nematode treatments described above were applied to radish plants held at 100 or 75% RH and tested against diamondback moth larvae. At 100% RH, nematode efficacy was (from most to least effective): in vitro rehydrated for 72 h > in vivo > in vitro rehydrated 48 h > in vitro rehydrated 24 h > dehydrated (0 h). The efficacy of all treatments was lower at 75% than at 100% RH, and the ranking of in vivo and in vitro nematodes rehydrated for 72 h was reversed. The nematodes in the wettable dispersible granule formulation were effective for foliar treatments when humidity was high and nematodes were rehydrated for at least 48 h before application. The data show that nematode infectivity was reduced unless nematodes were rehydrated.

Biological control agents for white grubs (Coleoptera: Scarabaeidae) in anticipation of the establishment of the Japanese beetle in California.

We tested biological control agents for the control of 3rd-instar scarab turfgrass pests, both for the masked chafer Cyclocephala hirta LeConte and the Japanese beetle, Popillia japonica Newman. The former species is endemic in California whereas the latter, although not yet established, constitutes a permanent serious threat to agriculture and horticulture in California. We conducted experiments using C. hirta in California and P. japonica in New Jersey. A field trial conducted in 2 different California turfgrass sites compared the field persistence in the absence of hosts of Bacillus thuringiensis Berliner subspecies japonensis Buibui strain, the milky disease bacterium, Paenibacillus (=Bacillus) popilliae (Dutky), and the entomopathogenic nematodes Steinernema kushidai Mamiya and Heterorhabditis bacteriophora Poinar to that of the organophosphate diazinon. Soil samples taken 0-70 d after applications were bio-assayed with P. japonica. Only diazinon and the entomopathogenic nematode S. kushidai caused substantial mortality and S. kushidai activity persisted significantly longer than diazinon activity. In greenhouse experiments, combinations of entomopathogenic nematode species usually resulted in additive mortality of scarab larvae. Combinations of S. kushidai and diazinon also resulted in additive mortality. In field trials, the efficacy of H. bacteriophora and especially S. kushidai and S. glaseri, was comparable to that of diazinon over 14-18 d. However, it is likely that at least S. kushidai would have outperformed diazinon over an extended period because of its longer persistence and potential for recycling in the hosts. S. kushidai, should it become commercially available, deserves further examination as an alternative to chemical white grub control especially as a highly compatible component of sustainable turfgrass management.

Suppression of diamondback moth (Lepidoptera: Plutellidae) with an entomopathogenic nematode (Rhabditida: Steinernematidae) and Bacillus thuringiensis Berliner.

We tested the efficacy of the All strain of Steinernema carpocapsae (Weiser) against larvae of the diamondback moth, Plutella xylostella (L.). In laboratory bioassays we found that (1) commercially formulated nematodes produced in vitro were as effective as nematodes produced in vivo, (2) resistance of P. xylostella to Bacillus thuringiensis Berliner subsp. kurstaki did not confer cross-resistance to nematodes, (3) mortality caused by nematodes was higher for early than late 3rd-instar P. xylostella larvae, and (4) no interaction occurred when B. thuringiensis and nematodes were combined against a susceptible strain of P. xylostella, but an antagonistic interaction occurred between the 2 pathogens against a strain of P. xylostella resistant to B. thuringiensis. In field trials conducted on 2 watercress [Rorippa Nasturtium-aquaticum (L.) Hayek] farms in Hawaii, nematodes provided 41% control, B. thuringiensis subsp. aizawai gave 44% control, and the combined treatment (B. thuringiensis plus nematodes both at half rate) resulted in 58% control. Using nemodes to control diamondback moth can theoretically reduce resistance development in diamondback moth populations to B. thuringiensis products, but repeated applications of nematodes will probably be ineffective in attaining control (suggested in simulation model). The results of this study demonstrate that nematodes may be a useful component of integrated pest management programs if efficacy can be increased, especially for populations of P. xylostella that are resistant to B. thuringiensis.

Effect of the Entomogenous Nematode Nemplectana carpocapsae on the Tachinid Parasite Compsilura concinnata (Diptera: Tachinidae).

The entomogenous nematode Neoaplectana carpocapsae and its associated bacterium, Xenorhabdus nematophilus, could not infect the pupal stage of the tachinid Compsilura concinnata through the puparium. N. carpocapsae had an adverse effect on 1-, 2- and 3-day-old C. concinnata larvae within the armyworm host in petri dish tests. All 1-day-old larvae treated with nematodes died in their hosts, whereas 61% and 69% of 2- and 3-day-old larvae treated with nematodes, respectively, died. However, the survivors developed to adults. Nine to thirty-seven percent of adult tachinids which emerged from nematode-treated soil (50 nematodes/cm(2)) were infected with N. carpocapsae. The nematode adversely affects C. concinnata directly by the frank infection of the tachinid and indirectly by causing the premature death of the host which results in tachinid death.

Development of the DD-136 strain of Neoaplectana carpocapsae at constant temperatures.

The development of the DD-136 strain of Neoaplectana carpocapsae was studied on three food sources at 10, 15, 20, 25, 30, 33, 35, and 37 C. No growth occurred at 10 or above 33 C. At 15, 20. and 25 C, growth and reproduction occurred. The most favorable growth occurred at 25 C. At 30 C, N. carpocapsae developed to adults but did not reproduce. Key Words: temperature-growth effects, DD-136 strain.

Infectivity of Neoaplectana carpocapsae and Heterorhabditis heliothidis to pupae of the parasite Apanteles militaris.

The infectivity of Neoaplectana carpocapsae and Heterorhabditis heliothidis to Apanteles militaris, a gregarious parasite of the armyworm, was deterntined at 100. 1,000, 5,000, and 10,000 nematodes per petri dish. For both nematode species, the percentage of infected A. militaris within a cocoon cluster decreased as inoculum levels decreased. At the highest inoculum level, N. carpocapsae infected an average of 32% of the parasite pupae within a cocoon cluster, whereas H. heliothidis infected an average of 22%. Covariance analysis indicated, however, that N. carpocapsae had significantly greater infectivity than did H. heliothidis. Some of the dauer juveniles of N. carpocapsae on the body of the armyworm contacted the emerging parasites and eventually became enveloped within the silken cocoons. Dauer juveniles produced by N. carpocapsae in parasite pupae could not penetrate and escape from silken cocoons even when the cocoons were placed in a moist environment.

Impact of a Nematode-parasitic Fungus on the Effectiveness of Entomopathogenic Nematodes.

The impact of the nematode-parasitic fungus Hirsutella rhossiliensis on the effectiveness of Steinernema carpocapsae, S. glaseri, and Heterorhabditis bacteriophora against Galleria mellonella larvae was assessed in the laboratory. The presence of Hirsutella conidia on the third-stage (J3) cuticle of S. carpocapsae and H. bacteriophora interfered with infection of insect larvae. Conidia on the J3 cuticle of S. glaseri and on the ensheathing second-stage cuticle of H. bacteriophora did not reduce the nematodes' ability to infect larvae. The LD values for S. carpocapsae, S. glaseri, and H. bacteriophora in sand containing H. rhossiliensis were not different from those in sterilized sand when Galleria larvae were added at the same time as the nematodes. However, when Galleria larvae were added 3 days after the nematodes, the LD of S. glaseri was higher in Hirsutella-infested sand than in sterilized sand, whereas the LD of H. bacteriophora was the same in infested and sterilized sand. Although the LD of S. carpocapsae was much higher in Hirsutella-infested sand than in sterilized sand, the data were too variable to detect a significant difference. These data suggest that H. bacteriophora may be more effective than Steinernema species at reducing insect pests in habitats with abundant nematode-parasitic fungi.

The nematode Heterotylenchus autumnalis and face fly Musca autumnalis: a field study in northern California.

Helerolylenchus aulumnalis was found in six northern California counties surveyed, and the incitlence of nematode infection of face flies ranged from 4.7 to 43.8%. Intensive studies at a cattle ranch in Yuba County showed that population densities of the host and nematode infections were highest in flies from cow pats receiving full sun. Average host population density was 105.7 puparia per pat, and nematode infection averaged 38.6%. Pats in partial sun averaged 13.5 puparia and 13,1% nematode infection. No face fly was recovered from shaded pats. When data from pats first exposed during day or night were compared, no significant differences in host population density or nematode infection rates were apparent. Uninfected and superinfected flies were more frequent than predicted by a Poisson distribution.Infected and uninfected female flies of all ages captured on white sticky traps appeared to feed with similar frequency upon a creatny substance which was probably acquired from cattle, However, older infected females fed less on blood and more upon dung than older uninfected females. Percent nematode infection and host population densities were highest in spring and early summer, declined to a midsummer low, and then increased slightly. Both dung-reared flies and captured females showed similar trends in abundance anti infection rates. Regression analysis indicated that H. autumnalis may not be regulating face fly population density.

Interaction between Neoaplectana carpocapsae and a granulosis virus of the armyworm Pseudaletia unipuncta.

Neoaplectaua carpocapsae developed and reproduced in armyworm hosts infected with a granulosis virus (GV). Macerated tissues of dauer juveniles from GV-infected hosts had sufficient GV to infect 1st and 2nd instar armyworms. Electron-microscope examination of dauer juveniles and adult female nematodes confirmed the presence of GV in the lumen of the intestine. No GV was observed in other tissues of the nematode.

Susceptibility of Various Species of Lepidopterous Pupae to the Entomogenous Nematode Neoaplectana carpocapsae.

The susceptibility of certain species of lepidopterous pupae occurring in different ecological situations to the entomogenous nematode, Neoaplectana carpocapsae, was tested. Soil- or litter-pupating lepidopterous insects were not highly susceptible to N. carpocapsae. The most susceptible insect pupating in the soil was Spodoptera exigua with 63% pupal mortality, while Harrisinia brillians, which pupates in litter, had 55% mortality. Other soil- and litter-pupating insects had mortalities of less than 25%. Some insect species that pupate above ground were highly susceptible (> 84% mortality) to N. carpocapsae infection.

Dispersal and Infectivity of the Entomogenous Nematode, Neoaplectana carpocapsae Weiser (Rhabditida: Steinernematidae), in Sand.

Laboratory tests determined the lateral and vertical dispersal patterns of Neoaplectana carpocapsae in sand. In the vertical tests, placement of infective juveniles 15 cm below the sand's surface resulted in the majority (77%) being recovered above the point of placement after 48 h. Placement of the nematodes on the sand's surface resulted in the majority (90.4%) remaining within 1 cm of the sand's surface. Placement of nematodes at depths of 2.5 cm and 5.0 cm below the sand's surface also resulted in little nematode dispersal. However, vertical hioassay tests showed that juvenile nematodes placed on the sand's surface dispersed 12 cm down to infect 67% of the Galleria mellonella pupae placed at the depth. Conversely, when nematodes were placed 11 cm below the insect pupae no infection was observed, but 53% infection occurred when nematodes were 7 cm below the site of the insect pupae. In lateral dispersal, 87% of the nematodes rentained within 2 cm of the placement site, although 0.5% were recovered at 12-14 cm away from the point of placement. Lateral bioassay tests indicated that the nematodes were capable of infecting 90, 35, and 5% of the G. mellonella pupae at 7 cm, 10 cm, and 14 cm from the point of placement, respectively.

Effects of Selected Insecticides and Nematicides on the In Vitro Development of the Entomogenous Nematode Neoaplectana carpocapsae.

The effects of organophosphates (mevinphos, phenamiphos, trichlorfon), carbamates (carbofuran, methomyl, oxamyl), a formamidine (chlordimeform), a synthetic pyrethroid (fenvalerate), a chlorinated hydrocarbon (methoxychlor). and an insect growth regulator (diflubenzuron) on in vitro development and reproduction of Neoaplectana carflocapsae were tested by incorporating each chemical into a nematode rearing medium. Organophosphates and carbamates adversely affected development and reproduction at concentrations >/= 0.1 mg/ml. Phenamiphos was the most toxic, with no nematode reproduction at 0.01 mg/ml. Inoculated infective juveniles developed to adults with some of the organophosphates and carbamates, but limited or no reproduction occurred. Chlordimeform inhibited development at 1.0 mg/ml, while diflubenzuron, fenvalerate, and methoxychlor did not significantly (P > 0.05) reduced reproduction at 1.0 mg/ml. The organophosphate and carbamate nematicides in use for control of plant-parasitic nematodes may be toxic to N. carpocapsae in the soil.

Parasitism of Brown Planthopper and Whitebacked Planthopper by Agamermis unka in Korea.

Adults of the brown planthopper (BPH) and the whitebacked planthopper (WBPH) that migrated into Korea from China were not parasitized by the mermithid, Agamermis unka. BPH and WBPH collected from Korean rice fields were parasitized. Parasitism of BPH in the lst-3rd, 4th, and 5th instars, and adults was 31.5%, 61.5%, 66.4%, and 45.5%, respectively, whereas parasitism of the same stages of WBPH was 50%, 50%, 100% and 90.7%, respectively. Parasitism of BPH by A. unka significantly reduced the number of eggs. Only 4.2% of the parasitized females contained eggs, whereas 85.6% of unparasitized females had eggs. Tilling of rice fields significantly increased mermithid parasitism of BPH. Mermithids parasitized 39.3% of caged adults in the untilled field and 77.8% in the tilled field.