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-infecled 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.     

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.     

Interaction between Neoaplectana carpocapsae (Nematoda: Steinernematidae) and Apanteles militaris (Hymenoptera: Bracondiae), a parasitoid of the armyworm, Pseudaletia unipuncta

The DD-136 strain of Neoaplectana carpocapsae adversely affected the development of immature stages of Apanteles militaris, a gregarious internal parasitoid of the armyworm, Pseudaletia unipuncta. The adverse effect of the nematode-bacterium complex was indirect, i.e., the infection by the nematode killed the host before A. militaris could complete its development. However, if armyworms containing 10- or 11-day-old A. militaris were exposed to dauer juveniles in Petri dishes, 48.1 and 94.4%, respectively, produced normal cocoons. If hosts containing 9- or 10-day-old A. militaris were fed dauer juveniles, 42.6 and 73.4% of the armyworms, respectively, produced A. militaris which formed normal cocoons. Cocoon-spinning A. militaris larvae were infected by the nematode. After cocoon formation was completed, the dauer juveniles could not penetrate the cocoon and infect the pupa. However, pupae in cocoons which had been deliberately cut open at one end became infected. A. militaris adults were infected when exposed to dauer juveniles in Petri dishes. After 3 days of exposure to dauer juveniles, 25.0, 44.2, and 7.0% of the adults in three trials were alive, whereas 100, 100, and 96.7% of the control adults were alive. Examination of dead adults in the nematode treatment showed that 67.6% contained nematodes. N. carpocapsae developed and reproduced in unparasitized armyworms, in armyworms containing 9-day-old A. militaris, and in those from which A. militaris had emerged. Production of dauer juveniles was significantly higher in unparasitized armyworms and in armyworms containing 9-day-old A. militaris than in those from which A. militaris had emerged.     

Interaction of the Microbivorous Nematode Teratorhabditis dentifera and the Nematode-Pathogenic Fungus Hirsutella rhossiliensis

The fungus Hirsutella rhossiliensis is an obligate pathogen with a broad host range among nematodes. Microbivorous nematodes are abundant around plant roots and may serve as hosts for the fungus. Our objective was to determine the influence of the bacterial-feeding nematode Teratorhabditis dentifera on the abundance of H. rhossiliensis. Experiments were conducted in a growth chamber with pots containing pasteurized soil, the fungus, and potato plants. The abundance of infectious conidia was compared in pots with and without T. dentifera after 50 or 70 days. The nematode reached high densities (10-40/cm³ soil) but had no effect on the abundance of conidia. Many individuals were dauer juveniles, a stage that acquired conidia but did not become infected. To test whether this life stage could deplete the pool of conidia in soil, different proportions of dauer juveniles with (resistant) and without (susceptible) a sheath were added to H. rhossiliensis-infested soil. The number of conidia in the soil decreased with an increasing proportion of resistant nematodes. Different stages of T. dentifera appear to have opposing effects on H. rhossiliensis; while adults and regular juveniles acquire conidia, become infected, and produce new infectious conidia, dauer juveniles can deplete the supply of conidia.     

Biological control potentials of insect-parasitic nematode Rhabditis blumi (Nematoda: Rhabditida) for major cruciferous vegetable insect pests

Pathogenicity of Rhabditis blumi Sudhaus against major cruciferous insect pests was evaluated in the lab and greenhouse. In Petri-dish tests against the insects, including Artogeia rapae L., Mamestra brassicae L., and Plutella xylostella L., insect mortality by R. blumi and its associated bacteria was dose and time dependent, which increased with dose (0?C80 dauer juveniles/larva) and time increments. Pathogenicity against fourth-instar larvae was higher than the rate of corresponding third-instar larvae. The highest insect mortality rate was observed in fourth-instar larvae of P. xylostella, followed by A. rapae, and M. brassicae, with mortality rates of 93.5, 88.2, and 77.8?%, respectively. Lethal dose values at 50?% (LD₅₀) of R. blumi were 25.7 dauer juveniles/larva on P. xylostella; 28.0 dauer juveniles/larva on A. rapae; and 40.6 dauer juveniles/larva on M. brassicae, respectively. In greenhouse tests, P. xylostella larvae were most susceptible to nematodes, with insect reduction rate of 88.0?%. The rate varied with vegetable species and persistence time of live nematodes on vegetable leaves after spraying. Nematodes established in cadavers showed positive correlation with nematode dose, whereas nematode persistence on the leaf was inversely related to hours after treatment.     

Biochemical properties of viral envelopes of insect baculoviruses and their role in infectivity

The enveloped virions of a nuclear polyhedrosis virus (NPV) and those of a granulosis virus (GV) of the armyworm, Pseudaletia unipuncta, were isolated and purified from their inclusion bodies. The enveloped virion of NPV contained a large amount of phosphatidyl choline which was not detected in that of GV. The total electric charges distributed on the surface of the envelopes of NPV and GV were negative in neutral and alkaline solutions. Although there was little difference in charges between NPV and GV, the charge was less negative in NPV than in GV. When the negative charges were neutralized by cationic detergents, the NPV infectivity was enhanced.     

Persistence of Four Heterorhabditis spp. Isolates in Soil: Role of Lipid Reserves

Infective juveniles of four Heterorhabditis isolates (H. bacteriophora HI, H. megidis UK211 and HF85, and H. downesi M245) were stored in moist (pF 1.7) and dry (pF 3.3) loam soil at 20°C for up to 141 days. Survival, assessed by the number of nematodes extracted by centrifugal flotation, declined over time, reaching fewer than 18% alive by day 141 for all but one treatment (H. bacteriophora HI in dry soil). The infectivity of nematodes in soil for Tenebrio molitor also declined over time, roughly in accordance with the decline in numbers of nematodes. Energy reserves of extracted nematodes were assessed by image analysis densitometry. There were differences among isolates both in survival and in the depletion of reserves, and there was a significant correlation between these two parameters, suggesting that the extent to which energy reserves are depleted affects survival or that a common factor influences both. However, significant nematode mortality occurred while levels of reserves remained high, and the maximum reduction in utilizable body content for any treatment was 51%, well above starvation level. Therefore, the decline in numbers of living nematodes and the reduced nematode infectivity in soil cannot directly result from starvation of the nematodes. Survival and infectivity declined more rapidly in moist than in dry soil; one isolate, H. downesi M245, was less affected by soil moisture content than the other three isolates.     

Sensitivity of Meloidogyne incognita and Rotylenchulus reniformis to Abamectin

Avermectins are macrocyclic lactones produced by Streptomyces avermitilis. Abamectin is a blend of B_1a and B_1b avermectins that is being used as a seed treatment to control plant-parasitic nematodes on cotton and some vegetable crops. No LD₅₀ values, data on nematode recovery following brief exposure, or effects of sublethal concentrations on infectivity of the plant-parasitic nematodes Meloidogyne incognita or Rotylenchulus reniformis are available. Using an assay of nematode mobility, LD₅₀ values of 1.56 μg/ml and 32.9 μg/ml were calculated based on 2 hr exposure for M. incognita and R. reniformis, respectively. There was no recovery of either nematode after exposure for 1 hr. Mortality of M. incognita continued to increase following a 1 hr exposure, whereas R. reniformis mortality remained unchanged at 24 hr after the nematodes were removed from the abamectin solution. Sublethal concentrations of 1.56 to 0.39 μg/ml for M. incognita and 32.9 to 8.2 μg/ml for R. reniformis reduced infectivity of each nematode on tomato roots. The toxicity of abamectin to these nematodes was comparable to that of aldicarb.     

Structural proteins of Amsacta moorei,Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses

The structural proteins of Amsacta moorei, Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses (EPVs) were separated by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels. More than 35 structural proteins were detected in each virus. Based on the distribution and the variation in the molecular weights of the virus structural proteins little homology was detected between the EPVs and vaccinia virus. The molecular weight of Amsacta EPV occlusion body matrix protein (110,000) was determined by SDS-acrylamide gel electrophoresis. The occlusion body matrix protein of Amsacta EPV occluded virus isolated from infected E. acrea larvae was rapidly degraded at pH 10.6 to peptides of approximately 94,000 and 60,000 daltons. After 2 hr incubation at alkaline pH, Amsacta EPV occlusion body protein was degraded to approximately 56,000 daltons. Proteolysis of occlusion body protein was inhibited by SDS. No proteolytic degradation was detected in occlusion body matrix protein isolated from Amsacta EPV infected BTI-EAA cells. Amino acid analysis indicates that entomopoxvirus occlusion body matrix protein consists of approximately 20% acidic amino acids and 9% of the sulfur-containing amino acids cysteine and methionine.     

Immunogold Localization of Tobacco Rattle Virus Particles within Paratrichodorus anemones

Unequivocal evidence of the viral nature of virus-like particles observed at the specific site of retention of tobacco rattle virus (TRV) in Paratrichodorus and Trichodorus nematodes has not previously been available. A new staining technique using safranin-O, which does not affect viral antigenicity, was used with an antiserum raised against the coat protein of TRV and prepared for use with immunogold labelling. Application of this method enabled the occurrence and localization of particles of TRV to be confirmed in the pharynx of the natural vector of the virus, Paratrichodorus anemones, and provided unequivocal evidence that the particles observed were TRV particles. The TRV particles were observed attached only to the cuticle lining the posterior tract of the pharyngeal lumen of the vector. Therefore, the specific site of retention of TRV particles in P. anemones is apparently more localized than reported to occur in other vector trichodorid species.     

Ultrastructural changes during recovery from anabiosis in the plant parasitic nematode, Ditylenchus

Ultrastructural changes after desiccation and rehydration of the anabiotic fourth-stage juveniles of the plant parasitic nematode Ditylenchus dipsaci (Kuhn) Filipjev are described and quantified. Anabiotic juveniles retain their structural integrity, although the cuticle decreases in thickness and the muscle cell sarcoplasm condenses. In contrast the structure of the non-anabiotic nematode Panagrellus silusae is completely disorganized by desiccation. Following rehydration of D. dipsaci there is a lag phase of 2-3 hr before the nematodes become active. During this period the juveniles undergo an ordered series of morphological changes. The lipid droplets within the intestinal cells coalesce and the cuticle increases in thickness. The muscle cell sarcoplasm expands, the spacing of the thick myofilaments increases and the mitochondria swell before recovering a more normal appearance. These morphological changes, together with earlier metabolic studies, indicate that repair occurs during the lag phase prior to recovery. This may involve membrane repair and the re-establishment of the ionic gradients essential for normal muscle and nerve function.     

Application of the Laurell immunoelectrophoresis technique to the study of serological relationships between granulosis viruses

The Laurell technique of two-dimensional immunoelectrophoresis was used to distinguish between isolates of granulosis virus (GV) from Plodia interpunctella (GV strains A and B), Ephestia cautella, Spodoptera littoralis (GV strain 65), Pieris brassicae, and Cydia pomonella. Granules, alkali-soluble proteins, and virus particles of P. interpunctella GV strain A and granules of P. interpunctella GV strain B were used as sources of antigens. They were reacted with the immunoglobulins of antisera prepared against whole granules of each strain of virus. Peaks of precipitation were most clearly defined when antigens were pretreated with 0.1 m Na₂CO₃, 2% Triton X, and succinic anhydride, Granules and alkali-soluble proteins of P. interpunctella GV strain A treated in this way exhibited at least one peak of precipitation when reacted with each of the antisera studied. Four peaks were observed in both the homologous reaction and in the heterologous reaction with antiserum prepared against granules of P. interpunctella GV strain B. Four different peaks were present in the homologous reaction between immunoglobulins and virus particles of P. interpunctella GV strain A. Two peaks were present in the heterologous reaction with antiserum prepared against granules of P. interpunctella GV strain B and one in that with the antiserum prepared against granules of S. littoralis GV strain 65.     

Ingestion,Retention, and Transmission of two Strains of Raspberry Ring-spot Virus by Longidorus macrosoma

The transmission of two strains of raspberry ringspot virus (RRV) by small numbers of nematodes was compared. A strain of RRV from Scotland (RRV-S), originally found in the field associated with Longidorus elongatus, was transmitted frequently by L. elongatus but only once by L. macrosoma. A strain from England (RRV-E) associated with L. macrosoma in the field was transmitted infrequently by each species of nematode. The reasons why L. macrosoma infected only a small proportion of bait plants with virus were investigated, and it was found that most of the nematodes tested had fed on the source plants and many had ingested virus. Most nematodes exposed to RRV-E or RRV-S had fed on the roots of the bait plants and, when thin sections were examined by electron microscope, had retained particles (thought to be those of the virus) in the region of the anterior odontostyle, Thus, most nematodes seem to have had ample opportunity to transmit virus, and the low frequency of transmission may have been due to a failure of the virus particles to be released from the site of retention or to a lack of infectivity of the virus when L. macrosoma was the vector and Petunia hybrida was the host.     

Adhesion of conidia of the fungus Dilophospora alopecuri to the cuticle of the nematode Anguina agrostis,the vector in annual ryegrass toxicity

The adhesion of conidia of the fungus Dilophospora alopecuri to the surface of the second stage dauer larva (DL₂) of the nematode Anguina agrostis (syn. A. funesta) was examined using both light and electron optics. The process of attachment does not lead to any apparent damage to the epicuticle of the nematode. Photographs of sections cut tangentially through the setulose appendages of the conidia show that a mucilagenous fibrillar material appears to be exuded from the highly convoluted surface of these appendages. This material adheres to the surface of the nematode cuticle and is deposited in the transverse annulations. The adhesion of these spores to DL₂ of A. agrostis was examined in 4822 nematodes from four galls. The mean percentage of DL₂ with spores adhering was 64% and ranged from 43 to 85%. This adhesion was compared with that of Corynebacterium rathayi from bacterial galls and was found to coincide. Thus, bacteria adhere to nematodes with D. alopecuri conidia attached and these conidia adhere to nematodes with C rathayi attached. Furthermore, DL₂ that are free from conidial adhesion appear to be free from bacterial adhesion and, in most instances, DL₂ that remain from from bacterial adhesion remain free from conidial adhesion. These observations draw attention to the potential of D. alopecuri as an agent for the biological control of annual ryegrass toxicity. Conidial adhesion to A. agrostis differs from bacterial adhesion to this nematode in that no visible damage to the cuticle takes place. The concept that adhesion of microorganisms to nematodes occurs in two phases, one involving site recognition and the other, if it occurs, involving physiological interaction and morphological change is discussed.     

Pathogenicity, development, and reproduction of Heterorhabditis bacteriophora and Steinernema carpocapsae under axenic in vivo conditions

Galleria mellonella larvae cultured axenically were treated with axenic dauer juveniles of Heterorhabditis bacteriophora and Steinernema carpocapsae. After 3 days S. carpocapsae had killed all insects, with 9.4 +/- 4.3 nematodes per larva. H. bacteriophora were unable to kill G. mellonella, although 13.3 +/- 6.4 nematodes per Galleria were found in the hemocoel. Invading nematodes of both strains recovered from the dauer stage. H. bacteriophora developed into hermaphrodites with eggs and J1 in the uterus and in the hemolymph of the living insects. Development beyond the J1 stage was not recorded. An injection of supernatants from different Photorhabdus luminescens cultures killed the insects but could not provide nutrients to support a further development. Only the injection of bacterial cells supported production of dauers in the axenic insects. Axenic S. carpocapsae developed to adults and produced offspring. After 3 weeks an average of 5275 nematodes per larva were counted, of which 6.7% were dauer juveniles, 39.2% other juvenile stages, 11.9% males, and 42.2% females. Compared to in vivo reproduction in the presence of the symbiotic bacterium Xenorhabdus nematophilus the dauer juvenile yields were low. Even after 5 weeks the percentage of dauer juveniles did not surpass 10%.     

Infection behavior of the rhabditid nematode Phasmarhabditis hermaphrodita to the grey garden slug Deroceras reticulatum.

Infection behavior of the rhabditid nematode Phasmarhabditis hermaphrodita to the grey garden slug Deroceras reticulatum was studied. The dauer (enduring or nonaging) juveniles of P. hermaphrodita invade D. reticulatum within 8-16 hr following external exposure, with the posterior mantle region containing the shell cavity serving as the main portal of entry. The dauer juveniles can recover, multiply, and produce new dauer juveniles in the slug and slug feces homogenates, but not in the soil extract. These results demonstrate that P. hermaphrodita is a facultative parasite of the slug and can complete its life cycle under nonparasitic conditions associated with the host. Although the juvenile and adult nematodes can kill the slug if injected into the shell cavity of the host, only the dauer juvenile can serve as an infective stage in the natural environment.     

Storage and formulation of the entomopathogenicnematodes Heterorhabditis indica and H. bacteriophora

Successful control of insect pests through theapplication of entomopathogenic nematode dauerjuveniles of H. bacteriophora and H.indica can only be achieved when the nematodematerial reaches the end user in good condition.Storage and formulation techniques must provideoptimum conditions to guarantee a maximum survival andinfectivity of the nematodes. Nematode survival wastested at temperatures ranging between 5–25 °C.A maximum survival of H. indica was achieved at15 °C and the highest mortality at 5 °C.H. bacteriophora survived best at 7.5 °Cand least at 25 °C. An increase of the saltconcentration had positive effects on dauer juvenilesurvival in aqueous suspensions. Low pH between 6 and4 reduced the bacterial growth and prolonged survivalof stored dauer juveniles. Of the organic acidsascorbic, benzoic, citric and sorbic acid, onlyascorbic acid had a positive effect on H. indicasurvival. Extracts of the dried spice plants cinnamon,cloves, rosemary and oregano were tested. Enhancementof H. indica survival was recorded for cinnamonand cloves. Survival and infectivity of nematodesstored in attapulgite and bentonite clays and spongewere recorded over several weeks at different storagetemperatures. Infectivity was not influenced by thedifferent formulation materials. When stored insponge at 25 °C nematodes survived less than 1week and the formulation in clay could only prolongthis period for another week. At 5 °C thesurvival of H. bacteriophora in sponge wassuperior to that in clay, whereas H. indicasurvived less well in sponge than in clay at15 °C. Storage in aerated water at 5 °Cfor H. bacteriophora and at 15 °C for H. indica resulted in the lowest mortality. Forstorage at controlled conditions (temperature, pH andosmolarity), aerated water is superior to all othermethods tested and the addition of preservatives willincrease survival.     

Comparison of the methods applicable for the pathogenicity assessment of entomopathogenic nematodes

Single infective juveniles of Heterorhabditis bacteriophora, H. megidis (Nematoda: Heterorhabditidae), Steinernema arenarium, S. carpocapsae and S. feltiae (Nematoda: Steinernematidae) were used to infect single Galleria mellonella (Lepidoptera: Pyralidae) larvae. Four parameters of entomopathogenic nematodes pathogenicity were assessed: the mortality of insects, infectivity of nematodes, number of nematodes established per single G. mellonella, and degree of infective juveniles colonization (percent of infective juveniles which intestine was colonized by symbiotic bacteria). The accuracy, repeatability, and versatility for different species of EPNs in bioassay arenas were compared. Our modifications of the original methods yielded ~ 50% higher efficiency of infective juveniles in cell culture plates and > 20% higher efficiency in centrifuge test tubes. The efficiency of nematodes in cell culture plates (39–77%) was relatively low, especially in the case of Heterorhabditis spp. In the bioassay arena, infective juveniles migrated between cells. The results of our studies indicate that the pathogenicity of EPNs should be assessed in centrifuge test tubes. In these arenas, the infectivity of single IJs was ~ 90% for Heterorhabditis spp. and ~ 95% for Steinernema spp. The degree of colonization of the EPN isolates by symbiotic bacteria was in the range of 96–98%.     

Comparison of Three Methods for Estimating the Number of Entomopathogenic Nematodes Present in Soil Samples

Numbers of Steinernema sp. (CB2B) and S. carpocapsae (Agriotos) exponentially declined after application into a clay loam soil. Over a 35-day sampling period, Steinernema sp. (CB2B) was more persistent than S. carpocapsae (Agriotos). The presence or absence of the second-stage cuticle on the third-stage juveniles (J3) at the time of application did not alter the rate of population decline of Steinernema sp. (CB2B). Nearly all J3 of Steinernema sp. (CB2B) and S. carpocapsae (Agriotos) lost their cuticle within 24 hours of being in soil. Centrifugal flotation recovered the greatest number of nematodes, with a lower variance than either the live bait or Baermann funnel techniques. A strong positive linear relationship was evident between numbers of nematodes present in the soil and the numbers that established in a bait insect. Approximately 40% of Steinernema sp. (CB2B) and 30% of the S. carpocapsae (Agriotos) present in the soil established in Galleria mellonella larvae. The extraction techniques had different efficiencies and gave different relative estimates of persistence for the two species. Persistence and infectivity was best measured using a combination of live bait and flotation techniques.     

Isolation of a factor, from the capsule of a granulosis virus, synergistic for a nuclear-polyhedrosis virus of the armyworm

When the capsules of a granulosis virus are fed together with the polyhedra of a nuclear-polyhedrosis virus to larvae of the armyworm, Pseudaletia unipuncta, the former enhances the infectivity of the latter virus, a synergistic interaction. The enhancement of infectivity depends upon the concentration of the polyhedra and the capsules. The factor responsible for the synergistic activity in the capsule can be dissolved in alkaline solution, separated from the virus particles by centrifugation, and further purified by Sephadex G-200 gel filtration with 4 m urea. The fraction obtained from Sephadex filtration and containing the synergistic factor can be separated into two components by disc-electrophoresis with 8 m urea. Both components possess synergistic activity. The ID₅₀ of the synergistic factor corresponds to 0.0015 OD₂₈₀. Its optimum pH is 8.5. Synergism is most evident when the factor is fed to larvae together with the polyhedra or is fed 24 hr prior to the ingestion of the polyhedra. The factor appears to be a simple or a conjugated protein of the capsule.