For the Insect Pathogen Photorhabdus luminescens, Which End of a Nematode Is Out?

The nematode Heterorhabditis bacteriophora is the vector for transmitting the entomopathogenic bacterium Photorhabdus luminescens between insect larvae. The dauer juvenile (DJ) stage nematode selectively retains P. luminescens in its intestine until it releases the bacteria into the hemocoel of an insect host. We report the results of studying the transmission of the bacteria by its nematode vector. Cells of P. luminescens labeled with green fluorescent protein preferentially colonized a region of the DJ intestine immediately behind the basal bulb, extending for various distances toward the anus. Incubation of DJ nematodes in vitro in insect hemolymph induced regurgitation of the bacteria. Following a 30-min lag, the bacteria migrated in a gradual and staggered movement toward and ultimately exited the mouth. This regurgitation reaction was induced by a low-molecular-weight, heat- and protease-stable, anionic component present in arthropod hemolymph and in supernatants from insect cell cultures. Nematodes anesthetized with levamisole or treated with the antihelmenthic agent ivermectin did not release their bacteria into hemolymph. The ability to visualize P. luminescens in the DJ nematode intestine provides the first clues to the mechanism of release of the bacteria during infection of insect larvae. This and the partial characterization of a component of hemolymph triggering release of the bacteria render this fascinating example of both a mutualistic symbiosis and disease transmission amenable to future genetic and molecular study.     

Stages of Infection during the Tripartite Interaction between Xenorhabdus nematophila, Its Nematode Vector, and Insect Hosts

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.     

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.     

Feeding of the Nematode Acrobeloides nanus on Bacteria

Information on the effect of bacteria-feeding nematodes on bacterial populations in the soil is sparse. We have isolated, cultured, and microscopically examined bacteria and nematodes coexisting within an agricultural soil and have studied their feeding relationship. The bacterium Pseudomonas corrugata isolate 2140R is a biocontrol agent against the pathogenic fungus Gaeumannomyces graminis var. tritici. The nematode Acrobeloides nanus is a cosmopolitan, bacteria-feeding organism widespread in agricultural and arid soils throughout Australia. Using light and electron microscopy, we observed the ingestion and breakdown of P. corrugata in the pharynx of A. nanus and bacterial passage through the nematode intestine as well as the accumulation of fluorescent compounds from ingested and broken P. fluorescens in the lumen of the nematode''s intestine. We also observed A. nanus feeding, growing, and reproducing on the Gram-positive bacterium Clavibacter toxicus, the causative agent of the disease annual ryegrass toxicity, and detected crushed bacteria in the nematode''s intestine.     

A Lover and a Fighter: The Genome Sequence of an Entomopathogenic Nematode Heterorhabditis bacteriophora

Heterorhabditis bacteriophora are entomopathogenic nematodes that have evolved a mutualism with Photorhabdus luminescens bacteria to function as highly virulent insect pathogens. The nematode provides a safe harbor for intestinal symbionts in soil and delivers the symbiotic bacteria into the insect blood. The symbiont provides virulence and toxins, metabolites essential for nematode reproduction, and antibiotic preservation of the insect cadaver. Approximately half of the 21,250 putative protein coding genes identified in the 77 Mbp high quality draft H. bacteriophora genome sequence were novel proteins of unknown function lacking homologs in Caenorhabditis elegans or any other sequenced organisms. Similarly, 317 of the 603 predicted secreted proteins are novel with unknown function in addition to 19 putative peptidases, 9 peptidase inhibitors and 7 C-type lectins that may function in interactions with insect hosts or bacterial symbionts. The 134 proteins contained mariner transposase domains, of which there are none in C. elegans, suggesting an invasion and expansion of mariner transposons in H. bacteriophora. Fewer Kyoto Encyclopedia of Genes and Genomes Orthologies in almost all metabolic categories were detected in the genome compared with 9 other sequenced nematode genomes, which may reflect dependence on the symbiont or insect host for these functions. The H. bacteriophora genome sequence will greatly facilitate genetics, genomics and evolutionary studies to gain fundamental knowledge of nematode parasitism and mutualism. It also elevates the utility of H. bacteriophora as a bridge species between vertebrate parasitic nematodes and the C. elegans model.     

For the insect pathogen Photorhabdus luminescens,which end of a nematode is out?

The nematode Heterorhabditis bacteriophora is the vector for transmitting the entomopathogenic bacterium Photorhabdus luminescens between insect larvae. The dauer juvenile (DJ) stage nematode selectively retains P. luminescens in its intestine until it releases the bacteria into the hemocoel of an insect host. We report the results of studying the transmission of the bacteria by its nematode vector. Cells of P. luminescens labeled with green fluorescent protein preferentially colonized a region of the DJ intestine immediately behind the basal bulb, extending for various distances toward the anus. Incubation of DJ nematodes in vitro in insect hemolymph induced regurgitation of the bacteria. Following a 30-min lag, the bacteria migrated in a gradual and staggered movement toward and ultimately exited the mouth. This regurgitation reaction was induced by a low-molecular-weight, heat- and protease-stable, anionic component present in arthropod hemolymph and in supernatants from insect cell cultures. Nematodes anesthetized with levamisole or treated with the antihelmenthic agent ivermectin did not release their bacteria into hemolymph. The ability to visualize P. luminescens in the DJ nematode intestine provides the first clues to the mechanism of release of the bacteria during infection of insect larvae. This and the partial characterization of a component of hemolymph triggering release of the bacteria render this fascinating example of both a mutualistic symbiosis and disease transmission amenable to future genetic and molecular study.     

Photobactin: a Catechol Siderophore Produced by Photorhabdus luminescens, an Entomopathogen Mutually Associated with Heterorhabditis bacteriophora NC1 Nematodes

The nematode Heterorhabditis bacteriophora transmits a monoculture of Photorhabdus luminescens bacteria to insect hosts, where it requires the bacteria for efficient insect pathogenicity and as a substrate for growth and reproduction. Siderophore production was implicated as being involved in the symbiosis because an ngrA mutant inadequate for supporting nematode growth and reproduction was also deficient in producing siderophore activity and ngrA is homologous to a siderophore biosynthetic gene, entD. The role of the siderophore in the symbiosis with the nematode was determined by isolating and characterizing a mini-Tn5-induced mutant, NS414, producing no detectable siderophore activity. This mutant, being defective for growth in iron-depleted medium, was normal in supporting nematode growth and reproduction, in transmission by the dauer juvenile nematode, and in insect pathogenicity. The mini-Tn5 transposon was inserted into phbH; whose protein product is a putative peptidyl carrier protein homologous to the nonribosomal peptide synthetase VibF of Vibrio cholerae. Other putative siderophore biosynthetic and transport genes flanking phbH were characterized. The catecholate siderophore was purified, its structure was determined to be 2-(2,3-dihydroxyphenyl)-5-methyl-4,5-dihydro-oxazole-4-carboxylic acid [4-(2,3-dihydroxybenzoylamino)-butyl]-amide, and it was given the generic name photobactin. Antibiotic activity was detected with purified photobactin, indicating that the siderophore may contribute to antibiosis of the insect cadaver. These results eliminate the lack of siderophore activity as the cause for the inadequacy of the ngrA mutant in supporting nematode growth and reproduction.     

Symbiont-mediated competition: Xenorhabdus bovienii confer an advantage to their nematode host Steinernema affine by killing competitor Steinernema feltiae

Bacterial symbionts can affect several biotic interactions of their hosts, including their competition with other species. Nematodes in the genus Steinernema utilize Xenorhabdus bacterial symbionts for insect host killing and nutritional bioconversion. Here, we establish that the Xenorhabdus bovienii bacterial symbiont (Xb-Sa-78) of Steinernema affine nematodes can impact competition between S. affine and S. feltiae by a novel mechanism, directly attacking its nematode competitor. Through co-injection and natural infection assays we demonstrate the causal role of Xb-Sa-78 in the superiority of S. affine over S. feltiae nematodes during competition. Survival assays revealed that Xb-Sa-78 bacteria kill reproductive life stages of S. feltiae. Microscopy and timed infection assays indicate that Xb-Sa-78 bacteria colonize S. feltiae nematode intestines, which alters morphology of the intestine. These data suggest that Xb-Sa-78 may be an intestinal pathogen of the non-native S. feltiae nematode, although it is a nonharmful colonizer of the native nematode host, S. affine. Screening additional X. bovienii isolates revealed that intestinal infection and killing of S. feltiae is conserved among isolates from nematodes closely related to S. affine, although the underlying killing mechanisms may vary. Together, these data demonstrate that bacterial symbionts can modulate competition between their hosts, and reinforce specificity in mutualistic interactions.     

New Insights into the Colonization and Release Processes of Xenorhabdus nematophila and the Morphology and Ultrastructure of the Bacterial Receptacle of Its Nematode Host, Steinernema carpocapsae

We present results from epifluorescence, differential interference contrast, and transmission electron microscopy showing that Xenorhabdus nematophila colonizes a receptacle in the anterior intestine of the infective juvenile (IJ) stage of Steinernema carpocapsae. This region is connected to the esophagus at the esophagointestinal junction. The process by which X. nematophila leaves this bacterial receptacle had not been analyzed previously. In this study we monitored the movement of green fluorescent protein-labeled bacteria during the release process. Our observations revealed that Xenorhabdus colonizes the distal region of the receptacle and that exposure to insect hemolymph stimulated forward movement of the bacteria to the esophagointestinal junction. Continued exposure to hemolymph caused a narrow passage in the distal receptacle to widen, allowing movement of Xenorhabdus down the intestine and out the anus. Efficient release of both the wild type and a nonmotile strain was evident in most of the IJs incubated in hemolymph, whereas only a few IJs incubated in nutrient-rich broth released bacterial cells. Incubation of IJs in hemolymph treated with agents that induce nematode paralysis dramatically inhibited the release process. These results suggest that bacterial motility is not required for movement out of the distal region of the receptacle and that hemolymph-induced esophageal pumping provides a force for the release of X. nematophila out of the receptacle and into the intestinal lumen.     

The bacterial community of entomophilic nematodes and host beetles

Insects form the most species‐rich lineage of Eukaryotes and each is a potential host for organisms from multiple phyla, including fungi, protozoa, mites, bacteria and nematodes. In particular, beetles are known to be associated with distinct bacterial communities and entomophilic nematodes. While entomopathogenic nematodes require symbiotic bacteria to kill and reproduce inside their insect hosts, the microbial ecology that facilitates other types of nematode–insect associations is largely unknown. To illuminate detailed patterns of the tritrophic beetle–nematode–bacteria relationship, we surveyed the nematode infestation profiles of scarab beetles in the greater Los Angeles area over a five‐year period and found distinct nematode infestation patterns for certain beetle hosts. Over a single season, we characterized the bacterial communities of beetles and their associated nematodes using high‐throughput sequencing of the 16S rRNA gene. We found significant differences in bacterial community composition among the five prevalent beetle host species, independent of geographical origin. Anaerobes Synergistaceae and sulphate‐reducing Desulfovibrionaceae were most abundant in Amblonoxia beetles, while Enterobacteriaceae and Lachnospiraceae were common in Cyclocephala beetles. Unlike entomopathogenic nematodes that carry bacterial symbionts, insect‐associated nematodes do not alter the beetles' native bacterial communities, nor do their microbiomes differ according to nematode or beetle host species. The conservation of Diplogastrid nematodes associations with Melolonthinae beetles and sulphate‐reducing bacteria suggests a possible link between beetle–bacterial communities and their associated nematodes. Our results establish a starting point towards understanding the dynamic interactions between soil macroinvertebrates and their microbiota in a highly accessible urban environment.     

Regulation of Phenotypic Switching and Heterogeneity in Photorhabdus luminescens Cell Populations

Phenotypic heterogeneity in bacterial cell populations allows genetically identical organisms to different behavior under similar environmental conditions. The Gram‐negative bacterium Photorhabdus luminescens is an excellent organism to study phenotypic heterogeneity since their life cycle involves a symbiotic interaction with soil nematodes as well as a pathogenic association with insect larvae. Phenotypic heterogeneity is highly distinct in P. luminescens. The bacteria exist in two phenotypic forms that differ in various morphologic and phenotypic traits and are therefore distinguished as primary (1°) and secondary (2°) cells. The 1 cells are bioluminescent, pigmented, produce several secondary metabolites and exo-enzymes, and support nematode growth and development. The 2° cells lack all these 1°-specific phenotypes. The entomopathogenic nematodes carry 1° cells in their upper gut and release them into an insect's body after slipping inside. During insect infection, up to the half number of 1° cells undergo phenotypic switching and convert to 2° cells. Since the 2° cells are not able to live in nematode symbiosis any more, they cannot re-associate with their symbiosis partners after the infection and remain in the soil. Phenotypic switching in P. luminescens has to be tightly regulated since a high switching frequency would lead to a complete break-down of the nematode-bacteria life cycle. Here, we present the main regulatory mechanisms known to-date that are important for phenotypic switching in P. luminescens cell populations and discuss the biological reason as well as the fate of the 2° cells in the soil.     

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species^1-5, as well as the wealth of information available regarding the insect''s immune system^6-8, and the pending genome sequence⁹ make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter¹⁰. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking¹¹ and oral toxicity assays¹². Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides⁷; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR¹³. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes⁶. To analyze these responses, injected insects can be dissected and visualized by microscopy^{13, 14}.     

The reproduction potentials of four entomopathogenic nematode strains related to cost-effective production for biological control

Bioassays to evaluate the mortality, virulence and reproduction potentials of four indigenous EPN strains, S-PQ16, S-BM12, H-KT3987 and H-CB3452 on insect larvae of mealworm (Tenebrio molitor) and greater wax moth (Galleria mellonella) revealed the highest mortality rates of two insect larvae at the highest inoculation dose of 100 IJs to range from 89 to 100 percent and 94.3–100 percent at 48 h after inoculation, respectively. Virulence was high for all nematode strains, with LC₅₀ values between 29.6 and 47.3 IJs/insect host. The highest IJ yields were different between nematode strains and insect host, from 66.8 × 10³ IJs (S-PQ16) to 118.6 × 10³ IJs (H-KT3987) on T. molitor, and from 54.2 × 10³ IJs (S-BM12) to 163.3 × 10³ IJs (H-KT3987) on G. mellonella. The culturing cost in terms of food expenditure for rearing insect larvae varied between insect larvae and nematode strains, from 6.76 to 26.63 USD per billion IJs for nematode strains cultured on T. molitor larvae and from 3.54 to 7.81 USD per billion IJs for nematode strains cultured on G. mellonella larvae. The full cost for a nematode product of 2.5 × 10⁹ IJs per hectare, produced through in vivo mass culturing, of the most efficient nematode strain, H-KT3987, was 191.3 USD, slightly cheaper than 199.4 USD for the same nematode product produced through in vitro mass culturing.     

Bacteria associated with the nematode Neoaplectana carpocapsae and the pathogenicity of this complex for Galleria mellonella larvae

The microflora of the nematode Neoaplectana carpocapsae and its host Galleria mellonella was examined. In predominating quantity, Alcaligenes odorans, Pseudomonas fluorescens, P. maltophilia, P. alcaligenes, and Acinetobacter sp. were present in the nematodes. The quantitative ratio between the surface and the gut microflora was established. Virulence of the bacteria for Galleria larvae was determined, as was the degree of mortality of Galleria larvae caused by axenic nematodes infected with pure culture of these bacteria. In this system, nematodes do not merely work as a “living syringe” for bacteria when invading the host and thus introducing bacteria into the insect.     

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic ^1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis ⁴. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects ⁵. For transmission between insect hosts, the bacteria colonize the intestine of the nematode''s infective juvenile stage ^6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects ^9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems ⁹.We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila¹⁴. Similar methods have been used to investigate other nematode-bacterium associations ^9,15-18and the approach therefore is generally applicable.The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization ^14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues ^14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication ²²or grinding ²³, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes ^21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization ^17,18, and is less laborious than other methods, including sonication ^22,25-27and individual nematode dissection ^28,29.     

昆虫病原线虫共生细菌共生性的分子生物学研究进展

嗜线虫致病杆菌属Xenorhabdus和发光杆菌属Photorhabdus细菌隶属肠杆菌科Enterobacteriaceae,对多种害虫致病能力强,分别与斯氏属Steinernema和异小杆属Heterorhabditis昆虫病原线虫互惠共生。该两属共生细菌既存在对昆虫寄主的病原性,又存在与线虫寄主的共生性。共生细菌与其线虫寄主的共生性主要表现以下4方面：（1）细菌产生食物信号诱导滞育不取食的感染期线虫恢复;（2）细菌为线虫生长与繁殖提供营养;（3）细菌能于感染期线虫的肠道定殖与生长;（4）细菌产生杀线虫毒素杀死非共生线虫。本文综述了共生菌以上4方面的共生性及其相关的分子机制。     

Partitioning Yield Loss on Yellow Squash into Nematode and Insect Components

The effect of a contplex of several insect and nematode pests on yield of yellow squash (Cucurbita pepo L.) was examined in two field tests in southern Florida. Applications of permethrin for insect control and oxamyl primarily for nematode control plus some insect control were made alone and in combination to achieve differential reduction of various insect and nematode components contributing to yield loss. The effect of these components on yield was further analyzed by multiple regression. Yield losses in weight of small fruit to nematode and insect pests together were estimated at 23.4% and 30.4% in each of the two tests, respectively. In the first test, this loss was attributed to the melonworm, Diaphania hyalinata, while in the second test, it was attributed to D. hyalinata and the nematodes Quinisulcius acutus and particularly Rotylenchulus reniforrnis. D. hyalinata accounted for further losses of 9.0% and 10.3%, respectively, from direct damage to the fruit. Despite the presence of low levels of Diabrotica balteata, Liriomyza sativae, and Myzus persicae, yields were little affected by these pests. Prediction of yield loss by multiple regression analysis was more accurate when both insect and nematode populations were present in the plots than when nematodes alone were present.     

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.