Dauer Juvenile Research Articles

Single nucleotide polymorphism (SNP) marker-assisted breeding of Heterorhabditis bacteriophora for improvement of reproductive potential and stress tolerance

Summary The entomopathogenic nematode (EPN), Heterorhabditis bacteriophora, is an efficient biological control agent against several economically important insect pests. Recent research has assessed the possibility to correlate desired beneficial traits with genotype data to pave a way for marker-assisted breeding approaches. A collection of H. bacteriophora WT inbred lines has been phenotyped in this framework for stress- and virulence-related traits. However, these traits are rarely combined in a single line. Thus, unifying these traits in commercial strains is of high priority. This investigation unified beneficial traits in hybrid pools through marker-assisted breeding using single nucleotide polymorphisms (SNPs) associated with reproductive potential, longevity, virulence and cold tolerance. Recombinant inbred lines (RILs) generated from a cross between a stress tolerant WT inbred line (XX21) and a line high in in vitro reproduction potential (IL3) were genotyped via SeqSNP and screened for SNP markers associated with beneficial traits. Thereafter, a genotypic pool (X21L3) comprising 22 ILs was formed. The X21L3 pool was subsequently evaluated for the target traits in comparison with the cross parents and a commercial strain HB4. An improvement of oxidative stress tolerance at 2°C (cold tolerance) was recorded with X21L3 surviving 1 day longer than the best performing parent (XX21). The hybrid pool also survived 1 day longer than the least performing parent IL3 for the trait longevity at 25°C under oxidative stress conditions. A higher dauer juvenile (DJ) recovery (58%) and DJ yield (209 000 DJ ml−1) than the least performing parent XX21 was recorded for the pool. The storage stability in diatomaceous earth formulation at 2°C and 7.5°C was also improved by 2 and 5 days, respectively, in comparison to the least performing parent XX21. This study depicts the potential of precision marker-assisted breeding for beneficial trait improvement of H. bacteriophora.

Monitoring the Photorhabdus spp. bacterial load in Heterorhabditis bacteriophora dauer juveniles over different storage times and temperatures: A molecular approach

Biological control products based on the entomopathogenic nematode Heterorhabditis bacteriophora can vary in virulence (quality). The influence of their symbiotic bacteria Photorhabdus spp. inside the infective dauer juvenile (DJ) on DJ quality has not received much attention in the past. The presence of the bacteria in the DJ is crucial for its biocontrol potential. This investigation provides a method to quantify the bacterial load inside the DJ based on a qPCR technique. Information from the genome of Photorhabdus laumondii strain DE2 was used to identify single copy genes with no homology to any other bacterial accessions. One gene (hereby named CG2) was selected for primers design and for further qPCR experiments. Cross-amplification tests with P. thracensis and P. kayaii, also symbionts of H. bacteriophora, were positive, whereas no amplicons were produced for P. temperata or Xenorhabdus nematophila. We tested our qPCR system in DJ populations carrying defined proportions of bacteria-free (axenic) vs bacteria-carrying nematodes. With an increasing proportion of axenic DJ in a population, virulence declined, and the virulence was proportional to the amount of bacterial DNA detected in the population by qPCR. Along liquid storage over long time, virulence also decreased, and this factor correlated with the reduction of bacterial DNA on the respective DJ population. We observed that stored DJ kept virulent up to 90 days and thereafter the virulence as well as the amount of bacterial DNA drastically decreased. Storage temperature also influenced the bacterial survival. Inside formulated DJ, the loss of bacterial DNA on the DJ population was accelerated under storage temperatures below 7.5 °C, suggesting that reproduction of the bacterial cells takes place when growth temperature is favorable. The role of bacterial survival inside stored DJ can now be adequately addressed using this molecular quality-control technique.

Phenotyping dauer juvenile oxidative stress tolerance, longevity and persistence within wild type and inbred lines of the entomopathogenic nematode Heterorhabditis bacteriophora

The commercial use of the entomopathogenic nematodeHeterorhabditis bacteriophoraas a biocontrol agent against noxious insects is limited due to its relatively short shelf-life. Longevity of dauer juveniles (DJ) during storage and in transit to end users is considerably restricted by environmental stresses. As a derivative stress triggered by environmental factors, oxidative stress causes a strong internal metabolic imbalance leading to lifespan reduction. In this study, the relation between DJ oxidative stress tolerance and longevity inH. bacteriophorawas investigated at 25 and 7°C. A strong and significant correlation between DJ oxidative stress tolerance and longevity during storage in Ringer’s solution ( at 7°C; at 25°C) was recorded. Phenotyping of these traits was performed for 40H. bacteriophorawild type strain and inbred line collections. At 25°C, the mean time survived in Ringer’s by 50% of the DJ (MTS50) ranged from 21 to 57 days, whereas under oxidative stress, survival was from 3 to 22 days. At 7°C, a maximum MTS50of 94 days was assessed when DJ were stored in Ringer’s, while the maximum MTS50was only 25 days with oxidative stress induction. The heritability of DJ tolerance to oxidative stress, determined by using homozygous inbred lines, is high (), an indication of a high probability for successful selective breeding. In a subset of preselectedH. bacteriophorainbred lines, DJ oxidative stress tolerance correlated with the DJ survival (persistence) after application to sand (). The study provides fundamental data required for a genetic breeding programme to produce hybrids with improved stress tolerance and prolonged shelf-life and soil persistence.

Life history trait analysis of the entomopathogenic nematode Steinernema feltiae provides the basis for prediction of dauer juvenile yields in monoxenic liquid culture.

Entomopathogenic nematodes (Steinernema spp.) are used in integrated pest management to control insect pests in cryptic environments. The nematodes are mass produced in monoxenic liquid culture with their symbiotic bacteria Xenorhabdus spp. For a better understanding of nematode population dynamics, the life history traits (LHTs) of the entomopathogenic nematode Steinernema feltiae were assessed at 25°C by observing single pairs of male and female nematodes using a hanging drop technique. To investigate the influence of different food supplies on nematode reproduction, the LHTs were assessed with a daily supply of 5×, 10× and 20×10(9)cellsml(-1) of the nematode's bacterial symbiont Xenorhabdus bovienii in semi-solid nematode growth gelrite (NGG) medium. Increasing bacterial density had a significant positive influence on the average number of offspring produced, which ranged from 359 to 813 per female. The intrinsic rate of natural increase r m, which ranges from 1.10 to 1.19day(-1), was neither influenced by the bacterial density, nor was the mean generation time T (5.12-5.25days) and population doubling time (PDT) (0.64-0.59days). The average lifespan of reproductive females, which ranged from 6.7 to 7.3days, was positively correlated with bacterial density. A positive correlation between female body volume and bacterial density was recorded (R=0.67) as well as a significant positive correlation between female body size and offspring production (R=0.89) in hanging drops. Whether these data can be used to predict nematode yields in liquid culture was tested. The total female body volume calculated as the average female body volume × total number of parental females per millilitre 3days after nematode inoculation was positively correlated (R=0.72) with nematode yields. The total female body volume on process day 3 is thus a good indicator for the estimation of nematode yield at the end of the process (12-15days post dauer juvenile (DJ) inoculation) in both Erlenmeyer flasks and bioreactors. With a mean deviation of 9467DJsml(-1), the error resembles approximately 5% of the final DJ yields.

An Entomoparasitic Adult Form inBursaphelenchus doui(Nematoda: Tylenchomorpha) Associated withAcalolepta fraudatrix

The nematode family Aphelenchoididae (Rhabditida: Tylenchomorpha) includes species with various feeding habitats. Bursaphelenchus, a member of the family, has for a long time been considered as a home for plant parasitic or mycophagous species (or both). However, recent intensive biological studies on the family revealed that the genus contains several insect parasitic species. Dauer juveniles of Bursaphelenchus doui were isolated from Acalolepta fraudatrix during a field study of longhorn beetle-Bursaphelenchus nematode associations. Two different insect-associated forms, an "entomoparasitic adult form" and a regular dauer juvenile, were isolated from a single individual beetle in a subsequent laboratory investigation of the B. doui-A. fraudatrix relationship. Thus these 2 distinct, insect-associated forms were confirmed to occur simultaneously. The entomoparasitic form is morphologically similar to that of Bursaphelenchus luxuriosae, with a dome-shaped head and vacuole-like spots assumed to be an internal structure of sensory organ, a stylet, a metacorpus (median bulb), and a moderately-developed and seemingly fully functional reproductive system. It is distinguishable from B. luxuriosae based on male spicule morphology and female tail morphology. A degenerate ingestive-digestive system distinguishes the entomoparasitic form from the propagative form and, unlike dauer juveniles, it has a moderately-developed reproductive system. The presence of this characteristic parasitic adult form is known only in these 2 Bursaphelenchus species. However, these 2 species did not form a clear monophyletic clade within the Bursaphelenchus xylophilus group and, thus, this characteristic parasitic form may occur independently in each species.

The First Report of a Putative “Entomoparasitic Adult Form” in Bursaphelenchus

A putative "entomoparasitic adult form" of Bursaphelenchus luxuriosae was found in the tracheal system and body cavity of its cerambycid vector beetle, Acalolepta luxuriosa. Morphologically, this form is intermediate between the usual mycophagous adult and the phoretic dauer juvenile, i.e., it shares the primary and secondary reproductive features with the mycophagous phase but shares a degenerate digestive tract with JIV dauer juveniles. In addition, the "parasitic form" has specialized characters relative to the mycophagous phase analogous to dicyclic Hexatylina, e.g., vacuole-like dots (assumed to be sensory organs) at the anterior end, a very long vulval flap, conical female tail, and elongated male spicules. The presence of insect-parasitic juveniles has been reported in several Bursaphelenchus species, but this is the first confirmed report of a putative "entomoparasitic adult form" in the genus. Thus, the "parasitic adult form" of B. luxuriosae is hypothesized to be an autapomorphic character of this species in the genus. The physiological impact of the parasitism on the host beetle is assumed to be weak because no clear symptoms were observed in the infested beetles. Also, no nematode eggs or propagative juveniles were observed in the beetle host, suggesting that more research is needed to confirm the nature of the association (parasitic vs. endophoretic).

Seasonal and vertical distribution of the entomopathogenic nematodes Heterorhabditis bacteriophora (TUR-H2) and Steinernema feltiae (TUR-S3) in turf and fallow areas

AbstractTo determine the seasonal movement of the dauer juvenile (DJ) stage of entomopathogenic nematodes, DJ of Heterorhabditis bacteriophora (TUR-H2) and Steinernema feltiae (TUR-S3) were applied to field plots (turf and fallow) and their vertical distribution in soil was monitored for 8 months (October to June) in Turkey. Most S. feltiae remained near the soil surface (0-15 cm deep) in both kinds of plots and throughout the study. By contrast, H. bacteriophora moved to 40 cm depth in fallow plots during winter months but remained mostly near the soil surface (0-15 cm deep) in turf plots throughout the study.

Gene induction by desiccation stress in the entomopathogenic nematode Steinernema carpocapsae reveals parallels with drought tolerance mechanisms in plants

The dauer juvenile (DJ) stage of the insect parasitic nematode Steinernema carpocapsae is the only stage in the life cycle which is capable of surviving outside its host and it is adapted for tolerating environmental stresses and for host finding. We have isolated 45 unique expressed sequence tags (ESTs) that are up-regulated in response to desiccation in S. carpocapsae DJs. The majority of these ESTs were co-expressed in response to desiccation and osmotic stress and were generally not induced in response to heat and cold stress. Thirty-two ESTs showed similarity to known sequences. Among these were sequences which encode putative signalling molecules or transcription factors, sequences which detoxify reactive oxygen species, two C-type lectin sequences, ESTs which encode membrane-associated proteins and seven distinct late embryogenic abundant (LEA) sequences. We also isolated 13 novel ESTs. These data show that the molecular response to desiccation stress in entomopathogenic nematode DJs is complex and parallels many of the adaptive changes which occur in drought tolerant plants during exposure to desiccation and osmotic stress. A notable feature of the desiccation response of plants is the number and diversity of hydrophilic LEA proteins synthesised in response to desiccation. All of the LEA sequences detected in animals to date, including those reported in this study, belong to LEA3 group. We show that S. carpocapsae expresses several novel sequences which encode putative hydrophilic and natively unfolded proteins. It is likely that these novel and putative proteins play an important role in desiccation tolerance, possibly by carrying out analogous roles in nematodes to those carried out by the other LEA protein classes in plants.

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.

Detection of changes occurring during recovery from the dauer stage in Heterorhabditis bacteriophora.

Nematodes of the genus Heterorhabditis are insect parasites that are widely used as biological control agents. When conditions are unfavourable for reproduction in H. bacteriophora, a long-lived, non-feeding, survival and dispersal stage, the dauer juvenile (DJ), is formed. This DJ stage is also adapted for host finding and infection. When it infects a suitable host, the DJ recovers and resumes growth and development. We describe a series of methods for improved detection of recovery in H. bacteriophora. We also describe some of the physiological changes that occur immediately after the onset of recovery in these nematodes as revealed using fluorescent nucleic acid binding SYTO dyes. Although recovery could be monitored using morphological changes, we found that observation of the uptake of fluorescent latex microspheres by recovering nematodes was a far more sensitive and efficient means of detecting recovery. SYTO dyes were also found to be useful indicators of recovery, binding to the pharyngeal glands and genital primordia as little as 3 h after the onset of recovery. The use of SYTO dyes also indicated that the pharyngeal glands produce large quantities of RNA following the onset of recovery, implying that these structures may produce proteins important in the infection and/or feeding process of H. bacteriophora.

Dauer juvenile longevity and stress tolerance in natural populations of entomopathogenic nematodes: is there a relationship?

Qualitative and quantitative genetic analysis of life span in experimental adult animals predicts that resistance to stress and longevity are positively correlated, but such studies on field populations of animals are rare. We tested this hypothesis using dauer juveniles of 15 natural populations of the entomopathogenic nematode, Heterorhabditis bacteriophora, collected from diverse localities. Dauer juvenile longevity at 25 °C in autoclaved tap water and tolerance to major environmental stresses including heat (survival at 40 °C for 2 h), ultraviolet (UV) radiation (original virulence remaining after exposure to 302 nm UV for 5 min), hypoxia (survival at approximately 0% dissolved O 2 at 25 °C for 96 h), and desiccation (survival in 25% glycerol at 25 °C for 72 h) differed significantly among populations. Intrinsic dauer juvenile longevity, defined as the number of weeks to 90% mortality (LT 90) estimated using probit analysis of nematode survival data at 25 °C varied between 6 and 16 weeks among populations. Longevity was most strongly correlated with heat followed by UV and hypoxia tolerance, respectively, but showed no correlation with desiccation tolerance. The strong positive correlation of longevity with heat tolerance was further confirmed through principal components analysis which showed almost identical variance for heat and longevity. Among the stress factors, only UV tolerance was positively correlated with heat and hypoxia tolerance. Differences in longevity and stress tolerance in nematode populations isolated from a single 200 m 2 grassland locality further support another hypothesis that population structure of heterorhabditid nematodes is highly fragmented, thus suggesting the existence of metapopulation dynamics.

Heritability of the Liquid Culture Mass Production Potential of the Entomopathogenic Nematode Heterorhabditis bacteriophora

The infective stage of entomopathogenic nematodes ( Heterorhabditis spp.) is the mobile, but developmentally arrested dauer juvenile (DJ). For commercial application, nematodes are produced in liquid culture. Prior to the inoculation of the DJ, their symbiotic bacterium Photorhabdus luminescens is cultured. The DJ exit from the arrested stage (recovery) and develop to reproductive adults. Recovery is a response to bacterial food signals. In liquid culture the percentage of DJs recovering from the DJ stage is highly variable, which significantly influences the number of reproducing hermaphrodites and the final DJ yields. The liquid culture yield is defined by the number of DJ mL -1 harvested from the medium. The heritability of the disposition to recover from the DJ stage and of the final DJ yield in liquid culture has been evaluated. From a hybrid strain of H. bacteriophora 30 homozygous inbred lines were established by inbreeding over seven generations. These inbred lines were propagated in liquid culture and DJ recovery and yields were recorded. The calculated heritability for the DJ recovery was low ( h 2 = 0.38). No significant genetic variability could be detected for this trait. In contrast, a high heritability ( h 2 = 0.90) was found for the total number of DJs produced in the liquid medium.

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.

Physico-chemical properties and mode of action of a signal from the symbiotic bacterium Photorhabdus luminescens inducing dauer juvenile recovery in the entomopathogenic nematode Heterorhabditis bacteriophora

AbstractRecovery in entomopathogenic nematodes is the exit from the dauer juvenile stage. It is a response to environmental queues signalling the presence of food sources (e.g., insect haemolymph). The bacterium Photorhabdus luminescens excretes a signal which also induces recovery of its symbiotic Heterorhabditis bacteriophora dauer juveniles. This bacterial signal is composed of at least two compounds with different polarity. The symbiotic bacteria also secrete an antagonistic signal which inhibits nematode recovery. The recovery-inducing signal compounds have a molecular mass of less than 20 kDa and are negatively charged. The data indicate that at least one compound is smaller than 5 kDa. The bacterial signal triggers by receptor binding, the first step in a recovery-inducing muscarinic signalling pathway.

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%.

Liquid culture of the entomopathogenic nematode-bacterium-complex Heterorhabditis megidis/Photorhabdus luminescens

A process technology for production of the entomopathogenic biocontrol nematode-bacterium complex Heterorhabditis megidis/Photorhabdus luminescens in monoxenic liquid culture in laboratory scale bioreactors is described. Dauer juvenile yields varied between 21 and 68 million dauer juveniles/l medium. The maximum density was reached at 13 to 25 days after inoculation of P. luminescens. The reason for the high variability in yield was identified. After the 24-h bacterial preculture the bioreactor is inoculated with nematode dauer juveniles which develop to self-fertilizing hermaphrodites. The exit from enduring dauer stages (recovery) was between 18 and 90% of the inoculum density. Low dauer juvenile recovery resulted in the development of two-generations within 20 to 25 days. In contrast, high dauer juvenile recovery led to a one-generation process terminated within 15 days. Factors influencing dauer recovery are still unknown.

Pathogenicity of AxenicSteinernema feltiae, Xenorhabdus bovienii,and the Bacto–Helminthic Complex to Larvae ofTipula oleracea(Diptera) andGalleria mellonella(Lepidoptera)

The pathogenicity of the nematode–bacterium complexSteinernema feltiae–Xenorhabdus bovieniito larvae ofTipula oleraceaandGalleria mellonellawas investigated by injection of dauer juvenile nematodes carrying their bacterial symbiont cells (monoxenic nematodes). Axenic nematodes (free of bacteria) and the symbiotic bacteria themselves were tested. The LC50ofX. bovieniiinT. oleraceawas 15,700 colony forming units (CFU)/larva compared to ≤8 CFU inG. mellonella. Xenorhabdus bovieniiis apparently removed from the tipulids hemolymph, possibly by cellular defense mechanisms. Axenic nematodes were less pathogenic than monoxenic nematodes for both insects. The difference was less pronounced inG. mellonellalarvae: one axenic nematode was sufficient to kill 80% in 1 day. The remaining insects found dead after 50 days were developmentally arrested. InT. oleracea20 axenic nematodes caused 39% whereas 20 monoxenic dauer juveniles caused 90% mortality within 8 days. The data indicate that the virulence of theS. feltiae/X. bovieniicomplex is greater than the additive effect of the nematodes and their bacteria, further evidence for the synergistic activity of the symbiotic bacto–helminthic complex during pathogenesis.

Partially Characterized Components of the Epicuticle of Dauer Juvenile Steinernema feltiae and Their Influence on Hemocyte Activity in Galleria mellonella

The epicuticle of dauer juveniles of the DD 136 strain of Steinernemafeltiae prevents encapsulation of the nematodes in vivo by hemocytes of Galleria mellonella larvae. The chemical composition of the epicuticle, which provides this protection, was determined using fluorescein isothiocyanate-conjugated lectins (concanavalin A, soybean agglutinin, wheat germ agglutinin) in conjunction with carbohydrases, lipase, and proteases. a-Mannose, 3-N-acetyl-D-galactosamine, and f8-N-acetyl-D-glucosamine were detected on the nematode's epicuticle. The absence of these sugars from enzyme-modified epicuticle did not affect hemocyte attachment. There was no evidence of epicuticular glycoproteins. In general, proteolytic enzymes did not alter the dauer juvenile epicuticle such as to elicit hemocyte-mediated encapsulation. Digestion of the epicuticle of dauer juveniles with lipase rendered the nematodes susceptible to hemocyte-mediated encapsulation. Many entomophilic nematodes live part of their lives in the hemolymph of their insect hosts, where the nematode cuticle is the interface between the nematode and the humoral and cellular components of the hemolymph. The function of the cuticle of these nematodes is incompletely known, but studies have shown that aspects of the transcuticular uptake of nutrients by entomophilic parasitic nematodes (Rutherford and Webster, 1974; Chen and Howells, 1979; Gordon et al., 1982) are related to cuticular structure (Nicholas, 1972; Batson, 1979; Platzer and Platzer, 1985). During the parasitic phase, the cuticle thickness of many entomophilic nematodes decreases (Bailey and Gordon, 1973), and in others, an outer microvillar layer develops (Riding, 1970) and functions as a nutrient absorptive layer. These nematodes encounter humoral (phenol oxidases) and/or cellular (hemocyte-mediated encapsulation) nonself resistance systems in the insect hemolymph (Ratcliffe and Rowley, 1979), but the contribution of the nematode cuticle to either resisting or evading these defenses is unknown, despite the reported occurrence of humoral (Welch and Bronskill, 1962; Andreadis and Hall, 1976) and cellular encapsulation (Nappi, 1975; Ratcliffe and Rowley, 1979). Vinson (1977) proposed that in the absence of active suppression of insect defenses, successful insect Received 5 September 1986; revised 3 December 1986; accepted 5 December 1986. * Present address: Department of Entomology, Macdonald College, McGill University, Ste. Anne de Bellevue, Quebec, Canada H9X 1CO. parasitoids would not be encapsulated due to (1) t e acquisition of a coat of host hemolymph components presenting the nonself agent as self, (2) the possession of heterophilic antigens, (3) the possession ofnonreactive surfaces, or (4) molecular mimicry. Evidence of a protective coat, albeit from insect midgut, may explain the reduction of humoral encapsulation of Brugia pahangi mic ofilariae in mosquitoes (Sutherland et al., 1984; Lafond et al., 1985). Entomophilic nematodes are not known to use molecular mimicry, although it may be used by cestodes and ac nthocephala (Lackie and Lackie, 1979) in some insects. The DD136 strain of Steinernema feltiae evades the hemolymph resistance systems of the gr ater wax moth, Galleria mellonella (Lepidoptera) larvae by avoiding nonself recognition (Dunphy and Webster, 1985), but the means by which this is achieved are unknown. The present study examines some of the epicuticular components of sheathed dauer juvenile S. feltiae DD136, and assesses the contribution of these components in evasion of encapsulation by the nematodes in G. mellonella. MATERIALS AND METHODS

A Factor Inducing Dauer Juvenile Formation in Caenorhabditis Elegans

Caenorhabditis elegans were induced to form true dauer juveniles by the material lyophilized from a water solution in which living C. elegans (1 g fresh weight/50 ml distilled water) were incubated for 24 hr at 20°. The material best induced dauer formation at 23°, pH 6.0, 2% agar with 1/8 strength of Sayre et al.'s axenic medium. Overcrowding itself exerted no major effect. First and 2nd stage juveniles most actively secreted the dauer inducing substance into the incubating medium. Neither dauer juveniles nor other nematodes such as Bursaphelenchus xylophilus or Neoaplectana carpocapsae secreted the active substance.