Low structural diversity of the O-polysaccharides of Photorhabdus asymbiotica subspp. asymbiotica and australis and their similarity to the O-polysaccharides of taxonomically remote bacteria including Francisella tularensis

The O-polysaccharides were isolated from the lipopolysaccharides of emerging human pathogens Photorhabdus asymbiotica subsp. asymbiotica US-86 and US-87 and subsp. australis AU36, AU46, and AU92. Studies by sugar analysis and ¹H and ¹³C NMR spectroscopy before and after O-deacetylation showed that the O-polysaccharide structures are essentially identical within, and only slightly different between, the subspecies. The following structures of the repeating units of the O-polysaccharides were established:→3)-β-d-Quip4NGlyFo-(1→4)-α-d-GalpNAcAN3Ac-(1→4)-α-d-GalpNAcA3R-(1→3)-α-d-QuipNAc-(1→where GalNAcA stands for 2-acetamido-2-deoxygalacturonic acid, GalNAcAN for amide of GalNAcA, QuiNAc for 2-acetamido-2,6-dideoxyglucose, and Qui4NGlyFo for 4,6-dideoxy-4-(N-formylglycyl)aminoglucose; R = Ac in subsp. asymbiotica or H in subsp. australis. The structures established resemble those of a number of taxonomically remote bacteria including Francisella tularensis (Vinogradov, E. V.; Shashkov, A. S.; Knirel, Y. A.; Kochetkov, N. K.; Tochtamysheva, N. V.; Averin, S. P.; Goncharova, O. V.; Khlebnikov, V. S. Carbohydr. Res.1991, 214, 289–297), which differs in (i) the presence of a formyl group on Qui4N rather than the N-formylglycyl group, (ii) the mode of the linkage between the repeating units (β1→2 vs α1→3), (iii) amidation of both GalNAcA residues rather than one residue, and iv) the lack of O-acetylation.     

Antagonism between entomopathogenic fungi and bacterial symbionts of entomopathogenic nematodes

Mutual effects between the symbiotic bacteria of entomopathogenic nematodes, Photorhabdus luminescens and Xenorhabdus poinarii, and entomopathogenic fungi were investigated in vitro. A dual culture assay on nutrient agar supplemented with bromothymol blue and triphenyltetrazolium chloride (NBTA) medium revealed that P. luminescens is antagonistic to Metarhizium anisopliae, Beauveria bassiana, B. brongniartii and Paecilomyces fumosoroseus by inhibiting their growth and conidial production; the fungal growth was not inhibited by X. poinarii. In a second laboratory experiment, crude extract produced by M. anisopliae was tested for its activity against P. luminescens and X. poinarii. Crude extract from M. anisopliae was antibacterial to P. luminescens and X. poinarii at 1000 g/ml and inhibited their growth on NBTA, but had no effect at 100 or 10 g/ml. The influence of the crude extract of M. anisopliae on the dispersal of infective juveniles (IJs) of Heterorhabditis megidis and Steinernema glaseri was assayed on Sabouraud Dextrose Agar (SDA) plates. Results showed that the crude extract of M. anisopliae had no toxic effects even at highest concentration (1000 g/ml).     

Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria

Galleria mellonella L. larvae were infected with three species (seven strains) of Steinernema spp. or three species (three strains) of Heterorhabditis spp. Infected larvae were incubated at 22, 27, and 32 degrees C. Larvae were dorsally dissected every 6h over a 48-h period. Hemolymph was collected and streaked on tryptic soy agar plates. Several non-symbiotic bacterial species were identified from infected insect cadavers: Enterobacter gergoviae, Vibrio spp., Pseudomonas fluorescens type C, Serratia marcescens, Citrobacter freundii, and Serratia proteomaculans. At 18-24 h incubation, the nematode-associated symbiont occurred almost exclusively. Bacterial associates generally appeared outside the 18-24 h window. Infective juveniles of Steinernema feltiae (Filipjev) (27), Steinernema riobrave Cabanillas, Poinar, and Raulston (Oscar), or Steinernema carpocapsae (Weiser) (Kapow) were left untreated, or surface sterilized using thimerosal, then pipetted under sterile conditions onto tryptic soy agar plates. Several additional species of associated bacteria were identified using this method compared with the less extensive range of species isolated from infected G. mellonella. There was no difference in bacterial species identified from non-sterile or surface sterilized nematodes, suggesting that the bacteria identified originated from either inside the nematode or between second and third stage juvenile cuticles. Infective juveniles of S. feltiae (Cowles), S. carpocapsae (Cowles), and H. bacteriophora Poinar (Cowles) were isolated from field samples. Nematodes were surface-sterilized using sodium hypochlorite, mixed with G. mellonella hemolymph, and pipetted onto Biolog BUG (with blood) agar. Only the relevant symbionts were isolated from the limited number of samples available. The nematodes were then cultured in the laboratory for 14 months (sub-cultured in G. mellonella 7-times). Other Enterobacteriaceae could then be isolated from the steinernematid nematodes including S. marcescens, Salmonella sp., and E. gergoviae, indicating the ability of the nematodes to associate with other bacteria in laboratory culture.     

Evaluation of entomopathogenic nematodes against the olive fruit fly, Bactrocera oleae (Diptera: Tephritidae)

Infectivity of six entomopathogenic nematode (EPNs) species against Bactrocera oleae was compared. Similar infection levels were observed when third-instar larvae were exposed to infective juveniles (IJs) on a sand-potting soil substrate. When IJs were sprayed over naturally infested fallen olives, many larvae died within treated olives as well as in the soil; Steinernema feltiae caused the highest overall mortality of 67.9%. In addition, three laboratory experiments were conducted to optimize a time period for S. feltiae field application. (1) Abundance of fly larvae inside fallen olives was estimated over the 2006–2007 season with the highest number of susceptible larvae (3 mm and larger) per 100 olives being observed during December, 2006. (2) S. feltiae efficacy against fly larvae dropped to the soil post-IJ-application was determined. B. oleae added to the substrate before and after nematode application were infected at similar levels. (3) Effect of three temperature regimes (min–max: 10–27, 6–18, and 3–12 °C) corresponding to October through December in Davis, California on S. feltiae survival and infectivity was determined. After 8 weeks, the IJs at the 3–12 °C treatment showed the highest survival rate. However, the cold temperature significantly limited S. feltiae infectivity. Our results demonstrate that B. oleae mature larvae are susceptible to EPN infection both in the soil and within infested olives. Being the most effective species, S. feltiae may have the potential to suppress overwintering populations of B. oleae. We suggest that November is the optimal time for S. feltiae field application in Northern California.     

Effects of entomopathogenic bacterium Photorhabdus temperata infection on the intestinal microbiota of the sugarcane stalk borer Diatraea saccharalis (Lepidoptera: Crambidae)

Photorhabdus temperata is an entomopathogenic bacterium that is associated with nematodes of the Heterorhabditidae family in a symbiotic relationship. This study investigated the effects of P. temperata infection on the intestinal microbiota of the sugarcane stalk borer Diatraea saccharalis. Histopathology of the infection was also investigated using scanning electron microscopy. Groups of 20 larvae were infected by injection of approximately 50 bacterial cells directly into the hemocoel. After different periods of infection, larvae were dissected and different tissues were used for bacterial cell quantification. P. temperata was highly virulent with an LD₅₀ of 16.2 bacterial cells at 48 h post-infection. Infected larvae started dying as soon as 30 h post-infection with a LT₅₀ value of 33.8 h (confidence limits 32.2–35.6) and an LT₉₀ value of 44.8 h (CL 40.8–51.4). Following death of the larvae, bacteria from the midgut did not invade the hemocoel. In the midgut epithelium, P. temperata occupied the space underneath the basal lamina. The cultivable intestinal bacterial populations decreased as soon as 1 h post-infection and at 48 h post-infection, 90% of the gut microbiota had died. The role of P. temperata in control of the midgut microbiota was discussed.     

Biological control of tobacco cutworm,Spodoptera litura Fabricius with entomopathogenic nematodes

The efficacies of several entomopathogenic nematodes ofSteinernema andHeterorhabditis spp. were examined against tobacco cutworm,Spodoptera litura Fabricius.H. bacteriophora HY showed 100% mortality after 20 h against 2nd instar of tobacco cutworm. In the case of 3–4th instar,S. carpocapsae PC.,H. bacteriophora HY andS. monticola CR showed 100% mortality after 47 h. In the case of 5–6th instar,S. carpocapsae PC proved more effective than the others. Generally, the number of nematodes harvested increased as their size decreased. Also, the highest number of nematodes was obtained in the 5–6th instar ofS. litura byH. bacteriophora HY, showing about 1.3×10⁶ nematodes per larva.In vitro culturedS. carpocapsae PG showed 100% mortality after 73 h against 5–6th instar tobacco cutworm, indicating that nematodes producedin vitro can be potentially used for the biological control ofS. litura instead of nematodesin vivo.     

Isolation and identification of entomopathogenic nematodes from citrus orchards in South Africa and their biocontrol potential against false codling moth

A survey was conducted to determine the diversity and frequency of endemic entomopathogenic nematodes (EPN) in citrus orchards in the Western Cape, Eastern Cape and Mpumalanga provinces of South Africa. The main aim of the survey was to obtain nematodes as biological control agents against false codling moth (FCM), Thaumatotibia leucotreta, a key pest of citrus in South Africa. From a total of 202 samples, 35 (17%) tested positive for the presence of EPN. Of these, four isolates (11%) were found to be steinernematids, while 31 (89%) were heterorhabditids. Sequencing and characterisation of the internal transcribed spacer (ITS) region was used to identify all nematode isolates to species level. Morphometrics, morphology and biology of the infective juvenile (IJ) and the first-generation male were used to support molecular identification and characterisation. The Steinernema spp. identified were Steinernema khoisanae, Steinernema yirgalemense and Steinernema citrae. This is the first report of S. yirgalemense in South Africa, while for S. citrae it is the second new steinernematid to be identified from South Africa. Heterorhabditis species identified include Heterorhabditis bacteriophora, Heterorhabditis zealandica and an unknown species of Heterorhabditis. Laboratory bioassays, using 24-well bioassay disks, have shown isolates of all six species found during the survey, to be highly virulent against the last instar of FCM larvae. S. yirgalemense, at a concentration of 50 IJs/FCM larva caused 100% mortality and 74% at a concentration of 200 IJs/pupa. Using a sand bioassay, S. yirgalemense gave 93% control of cocooned pupae and emerging moths at a concentration of 20 IJs/cm². This is the first report on the potential use of EPN to control the soil-borne life stages of FCM, which includes larvae, pupae and emerging moths. It was shown that emerging moths were infected with nematodes, which may aid in control and dispersal.     

Facultative scavenging as a survival strategy of entomopathogenic nematodes

Entomopathogenic nematodes cannot be considered only as parasitic organisms. With dead Galleria mellonella larvae, we demonstrated that these nematodes use scavenging as an alternative survival strategy. We consider scavenging as the ability of entomopathogenic nematodes to penetrate, develop and produce offspring in insects which have been killed by causes other than the nematode-bacteria complex. Six Steinernema and two Heterorhabditis species scavenged but there were differences among them in terms of frequency of colonisation and in the time after death of G. mellonella larvae that cadavers were penetrated. The extremes of this behaviour were represented by Steinernema glaseri which was able to colonise cadavers which had been freeze-killed 240 h earlier and Heterorhabditis indica which only colonised cadavers which had been killed up to 72 h earlier. Also, using an olfactometer, we demonstrated that entomopathogenic nematodes were attracted to G. mellonella cadavers.     

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.     

A novel approach to biological control with entomopathogenic nematodes: Prophylactic control of the peachtree borer, Synanthedon exitiosa

Generally, microbial control agents such as entomopathogenic nematodes are applied in a curative manner for achieving pest suppression; prophylactic applications are rare. In this study, we determined the ability of two Steinernema carpocapsae strains (All and Hybrid) to prophylactically protect peach trees from damage caused by the peachtree borer, Synanthedon exitiosa, which is a major pest of stone fruit trees in North America. In prior studies, the entomopathogenic nematodes S. carpocapsae and Heterorhabditis bacteriophora caused field suppression when applied in a curative manner to established S. exitiosa populations. In our current study, nematodes were applied three times (at 150,000–300,000 infective juveniles/tree) during September and October of 2005, 2006, and 2007. A control (water only) and a single application of chlorpyrifos (at the labeled rate) were also made each year. The presence of S. exitiosa damage was assessed each year in the spring following the treatment applications. Following applications in 2006, we did not detect any differences among treatments or the control (possibly due to a low and variable S. exitiosa infestation of that orchard). Following applications in 2005 and 2007, however, the nematode and chemical treatments caused significant damage suppression. The percentage of trees with S. exitiosa damage in treated plots ranged from 0% damage in 2005 to 16% in plots treated with S. carpocapsae (Hybrid) in 2007. In control plots damage ranged from 25% (2005) to 41% (2007). Our results indicate that nematodes applied in a preventative manner during S. exitios’s oviposition period can reduce insect damage to levels similar to what is achieved with recommended chemical insecticide treatments.     

Differences in susceptibility of introduced and native white grub species to entomopathogenic nematodes from various geographic localities

Invasive, non-native, white grubs (Coleoptera: Scarabaeidae) cause significant damage in urban landscapes. Although the lack of natural enemies in their new home is often suggested as an important factor in the establishment and spread of invasive species, the potential of incumbent generalist parasites and pathogens to delay their establishment and spread has not been explored. We compared the susceptibility of the introduced Popillia japonica and the native Cyclocephala borealis to 16 species and strains of entomopathogenic nematodes isolated from within or outside the geographic ranges of the two scarabs. We found large variation in the virulence of the species/strains of nematodes with over 50% mortality of P. japonica produced by Heterorhabditis zealandica strain X1 and H. bacteriophora strain GPS11 and of C. borealis by H. zealandica and H. bacteriophora strains KMD10 and NC1. Heterorhabditis indica and H. marelatus caused less than 20% mortality of both scarab species. When considered as a group the nematode species and strains from within and outside the geographic ranges of either P. japonica or C. borealis did not differ in virulence towards either scarab species. Dose response studies with selected nematode species and strains against P. japonica and two additional non-native species Anomala (Exomala) orientalis and Rhizotrogus majalis and the native C. borealis indicated that R. majalis was the least susceptible and P. japonica and A. orientalis were as susceptible as the native C. borealis. Heterorhabditis zealandica was significantly more virulent than any other species or strain against P. japonica with a LC₅₀ of 272 IJs/grub. The LC₃₀ and LC₅₀ values for H. zealandica were also the lowest among the four nematode species/strains tested against A. orientalis and C. borealis. The LC₅₀ values for H. zealandica and H. megidis (UK strain) were significantly lower for the native C. borealis than the introduced A. orientalis. H. zealandica also showed the highest penetration efficiency and the lowest encapsulation in P. japonica and C. borealis grubs. Results suggest that the introduction of the exotic H. zealandica into the front-line states with respect to the movement of P. japonica and A. orientalis should be explored as a tactic to delay their establishment and spread. The results also suggest that the manipulation of the indigenous H. bacteriophora populations may help in delaying spread and mitigating losses caused by the invasive grub species.     

Susceptibility of Hoplia philanthus (Coleoptera: Scarabaeidae) larvae and pupae to entomopathogenic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae)

Biological control potential of nine entomopathogenic nematodes, Heterorhabditis bacteriophora CLO51 strain (HbCLO51), H. megidis VBM30 strain (HmVBM30), H. indica, Steinernema scarabaei, S. feltiae, S. arenarium, S. carpocapsae Belgian strain (ScBE), S. glaseri Belgian strain (SgBE) and S. glaseri NC strain (SgNC), was tested against second-, and third-instar larvae and pupae of Hoplia philanthus in laboratory and greenhouse experiments. The susceptibility of the developmental stages of H. philanthus differed greatly among tested nematode species/strains. In the laboratory experiments, SgBE, SgNC, HbCLO51 and HmVBM30 were highly virulent to third-instar larvae and pupae while SgBE was only virulent to second-instar larvae. Pupae were highly susceptible to HbCLO51, HmVBM30, SgBE and SgNC (57–100%) followed by H. indica and S. scarabaei (57–76%). In pot experiments, HbCLO51, SgBE and S. scarabaei were highly virulent to the third-instar larvae compared to the second-instar larvae. Our observations, combined with those of previous studies on other nematode and white grub species, show that nematode virulence against white grub developmental stages varies with white grub and nematode species.     

Virulence of entomopathogenic nematodes to larvae of the guava weevil, Conotrachelus psidii (Coleoptera: Curculionidae), in laboratory and greenhouse experiments

The guava weevil, Conotrachelus psidii, is a major pest of guava in Brazil and causes severe reduction in fruit quality. This weevil is difficult to control with insecticides because adults emerge over a long period, and larvae develop to the fourth-instar inside the fruit and move to the soil for pupation. We assessed the virulence of entomopathogenic nematodes to fourth-instar larvae in soil by comparing their susceptibility to nine species or strains: Heterorhabditis bacteriophora HP88, H. baujardi LPP7, and LPP1, H. indica Hom1, Steinernema carpocapsae All and Mexican, S. feltiae SN, S. glaseri NC, and S. riobrave 355. In petri dish assays with sterile sand at a concentration of 100 infective juveniles (IJs) of a given nematode species/strain, larval mortality ranged from 33.5 to 84.5%, with the heterorhabditids being the most virulent. In sand column assays with H. baujardi LPP7, H. indica Hom1, or S. riobrave 355 at concentrations of 100, 200, and 500 IJs, mortality was greater than the control only for H. baujardi (62.7%) and H. indica (68.3%) at the highest concentration. For H. baujardi LPP7 in a petri dish assay, the time required to kill 50 and 90% of the larvae (LT₅₀ and LT₉₀) for 100 IJs was 6.3 and 9.9 days, whereas the lethal concentration required to kill 50 and 90% of the larvae (LC₅₀ and LC₉₀) over 7 days was 52 and 122.2 IJs. In a greenhouse study with guava trees in 20-L pots, 10 weevil larvae per pot, and concentrations of 500, 1000 or 2000 IJs, H. baujardi LPP7 caused 30 and 58% mortality at the two highest concentrations. These results show that H. baujardi is virulent to fourth-instar larvae and has potential as a biological control agent in IPM programs.     

Pathogenic effect of entomopathogenic nematode-bacterium complexes on terrestrial isopods

In this study, we evaluated the effect of entomopathogenic nematodes (EPNs) Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora, symbiotically associated with bacteria of the genera Xenorhabdus or Photorhabdus, on the survival of eight terrestrial isopod species. The EPN species S. carpocapsae and H. bacteriophora reduced the survival of six isopod species while S. feltiae reduced survival for two species. Two terrestrial isopod species tested (Armadillidium vulgare and Armadillo officinalis) were found not to be affected by treatment with EPNs while the six other isopod species showed survival reduction with at least one EPN species. By using aposymbiotic S. carpocapsae (i.e. without Xenorhabdus symbionts), we showed that nematodes can be isopod pathogens on their own. Nevertheless, symbiotic nematodes were more pathogenic for isopods than aposymbiotic ones showing that bacteria acted synergistically with their nematodes to kill isopods. By direct injection of entomopathogenic bacteria into isopod hemolymph, we showed that bacteria had a pathogenic effect on terrestrial isopods even if they appeared unable to multiply within isopod hemolymphs. A developmental study of EPNs in isopods showed that two of them (S. carpocapsae and H. bacteriophora) were able to develop while S. feltiae could not. No EPN species were able to produce offspring emerging from isopods. We conclude that EPN and their bacteria can be pathogens for terrestrial isopods but that such hosts represent a reproductive dead-end for them. Thus, terrestrial isopods appear not to be alternative hosts for EPN populations maintained in the absence of insects.     

Entomopathogenic symbiotic bacteria,Xenorhabdus and Photorhabdus of nematodes

Xenorhabdus and Photorhabdus species are entomopathogenic bacteria with a wide insect host range, that belong to the family Enterobacteriaceae. Xenorhabdus and Photorhabdus species symbiotically associate with nematodes of the families Steinernematidae and Heterorhabditidae respectively. The factor(s) determining the symbiotic interaction between nematodes and bacteria are yet to be identified. Xenorhabdus and Photorhabdus species exist in two main phenotypic forms, a phenomenon known as phase variation. The phase I (or primary form) varies from phase II (or secondary form) in certain physiological and morphological characteristics. There is no variation in the DNA integrity of phase I and phase II and this supports epigenetic regulatory mechanism in phase variation. Certain pathogenic determinants such as pili, lipopolysaccharides and toxins contribute to the pathogenicity of Xenorhabdus and Photorhabdus species, and both appear to be equally pathogenic to insects. The observed similarity in their virulence to insect hosts may reflect possible in vivo conversion of phase II to phase I, however the host cellular invasion and virulence is yet to be properly understood. The virulence of Xenorhabdus variants varies among insects apparently due to factors which include the feeding habits of the insects. The molecular mechanism and biological significance of phase variation are presently unknown.     

First record of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) in Costa Rica

A survey of entomopathogenic nematodes was conducted in the north Pacific (Guanacaste Conservation Area) and southeast Caribbean (Gandoca-Manzanillo Natural Refuge) regions of Costa Rica. Out of a total of 41 soil samples, 5 were positive for entomopathogenic nematodes (20.5%), with 3 (12.3%) containing Steinernema and 2 (8.2%) Heterorhabditis isolates. Morphological and molecular studies were undertaken to characterize these isolates. The Heterorhabditis isolates were identified as Heterorhabditis indica and the three Steinernema isolates were identified as two new undescribed species. H. indica was recovered from a coastal dry forest. Steinernema n. sp. 1 was isolated from a rainforest valley, between volcanoes. Steinernema sp. n. 2 was isolated from sand dunes in the Caribbean Coast (Punta Uva) near the rainforest strip along the coast. Although limited to two geographic regions, this study suggests entomopathogenic nematodes may be diverse and perhaps widely distributed in Costa Rica. A more intensive survey, covering all geographic regions is currently undergoing.     

Pathogenic potential of six isolates of entomopathogenic nematodes (Rhabditida: Steinernematidae) from Vietnam

The potential of six Steinernema isolates, isolated from different provinces in Vietnam, was evaluated in the laboratory against Galleria mellonella and Spodoptera littoralis. Steinernema sangi and S. robustispiculum TN24 had the highest penetration rate in both hosts according to a penetration rate assay. The virulence assay showed that S. sangi had a high virulence to both hosts and along with isolate TN38 it was the most mobile among the isolates tested. The migration of S. sangi in sand columns with an insect host at the bottom was significantly higher than in sand columns without insect host. This Steinernema species was the only one that penetrated a host in 24h after migrating 10cm in sand columns at 25°C. Moreover, a multiplication assay showed that S. sangi produced a high number of infective juveniles in G. mellonella. However, all Steinernema isolates tested had low multiplication rates in S. littoralis.     

Effect of entomopathogenic nematodes on Plectrodera scalator (Fabricius) (Coleoptera: Cerambycidae)

Entomopathogenic nematodes were screened for efficacy against the cottonwood borer, Plectrodera scalator (Fabricius). Steinernema feltiae SN and S. carpocapsae All killed 58 and 50% of larvae, respectively, in filter paper bioassays but less than 10% in diet cup bioassays. S. glaseri NJ, S. riobrave TX, and H. indica MG-13 killed less than 10% of larvae in both assays. H. marelata IN was ineffective in the diet cup bioassay and killed 12.9% of larvae in a filter paper bioassay. The nematode isolates we tested are not suitable for use as biological control agents against P. scalator.     

Susceptibility of Meligethes spp. and Dasyneura brassicae to entomopathogenic nematodes during pupation in soil

The susceptibility of pupating larvae of pollen beetles, Meligethes spp. Stephens (Coleoptera: Nitidulidae) and brassica pod midges, Dasyneura brassicae Winnertz (Diptera: Cecidomyidae) to entomopathogenic nematodes (Nematoda: Rhabditida) was studied in the laboratory. The results showed that brassica pod midge larvae were almost unaffected by the tested nematodes (Steinernema bicornutum, S. feltiae and Heterorhabditis bacteriophora) whereas successful pupation of pollen beetle larvae was reduced with increasing number of nematodes (S. bicornutum, S. carpocapsae, S. feltiae and H. bacteriophora). The exposed larvae had been collected in the field and some of the pollen beetle larvae were parasitised by parasitoid wasps. It appeared that parasitised larvae were less affected by nematodes than non-parasitised larvae.