Efficacy of the Entomopathogenic Nematodes Heterorhabditis megidis and Heterorhabditis bacteriophora for the control of grubs (Phyllopertha horticola and Aphodius contaminatus) in Golf Course Turf

Liquid culture-produced entomopathogenic nematodes, Heterorhabditis megidis and Heterorhabditis bacteriophora, were applied at 0.5 and 1.5 million dauer juveniles m-2 against Aphodius contaminatus and Phyllopertha horticola on a golf course. The reduction of A. contaminatus was found to be between 40 and 62%. P. horticola reduction reached 70% with H. megidis and 83% with H. bacteriophora. Turf damage caused by birds preying on the grubs was successfully prevented.     

Effect of soil type on infectivity and persistence of the entomopathogenic nematodes Steinernema scarabaei, Steinernema glaseri, Heterorhabditis zealandica, and Heterorhabditis bacteriophora

We tested the effect of soil type on the performance of the entomopathogenic pathogenic nematodes Steinernema scarabaei, Steinernema glaseri, Heterorhabditis zealandica, and Heterorhabditis bacteriophora. Soil types used were loamy sand, sandy loam, loam, silt loam, clay loam, acidic sand, and a highly organic potting mix. Infectivity was tested by exposing third-instar Anomala orientalis or Popillia japonica to nematodes in laboratory and greenhouse experiments and determining nematode establishment in the larvae and larval mortality. Infectivity of H. bacteriophora and H. zealandica was the highest in potting mix, did not differ among loamy sand and the loams, and was the lowest in acidic sand. Infectivity of S. glaseri was significantly lower in acidic sand than in loamy sand in a laboratory experiment but not in a greenhouse experiment, and did not differ among the other soils. Infectivity of S. scarabaei was lower in silt loam and clay loam than in loamy sand in a greenhouse experiment but not in a laboratory experiment, but was the lowest in acidic sand and potting mix. Persistence was determined in laboratory experiments by baiting nematode-inoculated soil with Galleria mellonella larvae. Persistence of both Heterorhabditis spp. and S. glaseri was the shortest in potting mix and showed no clear differences among the other substrates. Persistence of S. scarabaei was high in all substrates and its recovery declined significantly over time only in clay loam. In conclusion, generalizations on nematode performance in different soil types have to be done carefully as the effect of soil parameters including soil texture, pH, and organic matter may vary with nematode species.     

Occurrence and distribution of the entomopathogenic nematodes Steinernema spp. and Heterorhabditis indica in Indonesia

Soil samples from 79 sites on five islands of Indonesia were baited with insects for the recovery of entomopathogenic nematodes. Heterorhabditis and Steinernema were equally prevalent, and were recovered from 11.7% of samples representing 20.3% of sites sampled. Both genera were recovered from coastal sites only. Entomopathogenic nematodes were more prevalent on the Moluccan islands of Ambon and Seram than on Java or Bali. They were not detected on Sulawesi, where non-coastal sites only were sampled. RFLP analysis was used in the identification of nematode isolates. Heterorhabditis indica was the only heterorhabditid identified. Two RFLP types of Steinernema were identified.     

Phylogenetic and cophylogenetic relationships of entomopathogenic nematodes (Heterorhabditis: Rhabditida) and their symbiotic bacteria (Photorhabdus: Enterobacteriaceae)

Mutualistic association between entomopathogenic Photorhabdus bacteria and Heterorhabditis nematodes represents one of the emerging model systems in symbiosis studies, yet little is known about this partnership from a coevolutionary perspective. Herein, we investigated phylogenetic and cophylogenetic relationships of Heterorhabditis and Photorhabdus strains using molecular markers Internal Transcribed Spacer and gyrase B gene sequences, respectively. The phylogenies presented consistent, well supported, monophyletic groups in the parsimonious and likelihood analyses for both the nematode and bacterial strains and supported the placement of currently recognized taxa, from which a potentially new Heterorhabditis species represented by a Thailand strain MP68 was identified. While the nematode strains with distant geographic distributions showed no detectable phylogenetic divergence within H. bacteriophora or H. georgiana monophyletic groups, their respective symbiotic bacteria speciated into two Photorhabdus species: P. luminescens and P. temperata, indicating the occurrence of duplication. Although such evolutionary process reduces the phylogenetic congruence between Heterorhabditis nematodes and Photorhabdus bacteria, global cophylogenetic tests using ParaFit detected a highly significant correlation between the two phylogenies (ParaFitGlobal = 0.001). Further, the associations between H. zealandica, H. indica and H. megidis strains and their symbiotic bacteria exhibited significant contribution to the overall cophylogenetic structure. Overall, this study reveals evidence of coevolution between Photorhabdus bacteria and Heterorhabditis nematodes and provides a framework for further examination of the evolution of these associations.     

Mass Production of the Beneficial Nematode Heterorhabditis bacteriophora and Its Bacterial Symbiont Photorhabdus luminescens

Entomoparasitic nematodes (EPNs) are being commercialized as a biocontrol measure for crop insect pests, as they provide advantages over common chemical insecticides. Mass production of these nematodes in liquid media has become a major challenge for commercialization. Producers are not willing to share the trade secrets of mass production and by doing so, have made culturing EPNs extremely difficult to advance existing technologies. Theoretically, mass production in liquid media is an ideal culturing method as it increases cost efficiency and nematode quantity. This paper will review current culturing methodologies and suggest basic culturing parameters for mass production. This review is focused on Heterorhabditis bacteriophora; however, this information can be useful for other nematode species.     

Isolation of insect pathogenic bacteria, Providencia rettgeri, from Heterorhabditis spp.

Nematodes of the genus Heterorhabditis carry bacteria of the genus Photorhabdus into insects including pests of horticultural crops. The bacteria kill the insect and provide conditions which allow for the growth and development of the nematodes. It is reported here that the majority of Heterorhabditis spp. strains tested contained a second bacterial species which was identified as Providencia rettgeri. Injection of the bacteria into waxmoth larvae showed that P. rettgeri was at least as pathogenic as Photorhabdus sp. K122. Both had LD₅₀ values of less than one bacterial cell/larva, but P. rettgeri killed the insects at a considerably faster rate than K122 at both 28°C and 9°C. Since Photorhabdus kills very slowly at low temperatures, it appeared that P. rettgeri might be a better pest control agent under these conditions. However, P. rettgeri was not pathogenic when carried into insect larvae by the nematode, indicating that the nematode suppressed either its release or pathogenicity. It will be necessary to find ways of bypassing or inhibiting this suppression for P. rettgeri to fulfil its potential in pest control.     

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.     

Isolation and characterization of microsatellite loci in the entomopathogenic nematode Heterorhabditis bacteriophora

Twenty microsatellite loci were identified from genomic DNA enrichment and expressed sequence tags of entomopathogenic nematode Heterorhabditis bacteriophora. Eight loci were found to be polymorphic in a Northeast Ohio H. bacteriophora population. Levels of polymorphism were evaluated in 31-56 individuals and the number of alleles ranged from two to three. The values of observed and expected heterozygosities ranged from 0 to 0.536 and from 0.223 to 0.616, respectively. All eight loci showed heterozygote deficiencies, but three conformed to Hardy-Weinberg equilibrium at the subpopulation level. This is the first set of microsatellite markers in entomopathogenic nematodes.     

Effect of Photorhabdus luminescens phase variants on the in vivo and in vitro development and reproduction of the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae

Photorhabdus luminescens (Enterobacteriaceae) is a symbiont of entomopathogenic nematodes Heterorhabditis spp. (Nematoda: Rhabditida) used for biological control of insect pests. For industrial mass production, the nematodes are produced in liquid media, pre-incubated with their bacterial symbiont, which provides nutrients essential for the nematode's development and reproduction. Particularly under in vitro conditions, P. luminescens produces phase variants, which do not allow normal nematode development. The phase variants were distinguished based on dye absorption, pigmentation, production of antibiotic substances, occurrence of crystalline inclusion proteins and bioluminescence. To understand the significance of the phase shift for the symbiotic interaction between the bacterium and the nematode, feeding experiments tested the effect of homologous and heterologous P. luminescens phase variants isolated from a Chinese Heterorhabditis bacteriophora (HO6), the Heterorhabditis megidis type strain from Ohio (HNA) and the type strain of Heterorhabditis indica (LN2) on the in vivo and in vitro development and reproduction of the nematode species H. bacteriophora (strain HO6) and another rhabditid and entomopathogenic nematode, Steinernema carpocapsae (A24). In axenically cultured insect larvae (Galleria mellonella) and in vitro in liquid media, H. bacteriophora produced offspring on phase I of its homologous symbiont and on the heterologous symbiont of H. megidis, but not on the two corresponding phase II variants. In solid media, nematode yields were much lower on phase II than on phase I variants. On the heterologous phase I symbiont isolated from H. indica the development of H. bacteriophora was not beyond the fourth juvenile stage of the nematode in any of the media tested, but further progressed on phase II with even a small amount of offspring recorded in solid media. Infective juveniles of S. carpocapsae did not develop beyond the J3 stage on all phase I P. luminescens. They died in phase I P. luminescens isolated from H. bacteriophora. Development to adults was recorded for S. carpocapsae on all phase II symbionts and offspring were produced in all media except in liquid. It is concluded that a lack of essential nutrients or the production of toxins is not responsible for the negative impact of homologous phase II symbiont cells on the development and reproduction of H. bacteriophora. The infective juveniles of H. bacteriophora retained cells of the homologous phase I symbiont, but not phase II cells and cells from heterologous symbionts, indicating that the transmission of the symbiont by the infective juvenile is selective for phase I cells and the homologous bacterial associate.     

The Natural Host Range of Steinernema and Heterorhabditis spp. and Their Impact on Insect Populations

The natural host range of entomopathogenic nematodes of the genera Steinernema and Heterorhabditis can be defined as the range of insects which indigenous nematode populations use for propagation. Information on the natural host range is rare. However, based on records of insects found to be naturally infected with nematodes, some conclusions regarding the natural host range of some Steinernema spp. and Heterorhabditis spp. are presented. Reports of indigenous nematode populations impacting on insect populations can be divided between relatively balanced, long-lasting nematode-host associations and unbalanced, short-lasting epizootics. Examples of the augmentation and inoculative introduction of nematodes in agriculture and forestry ecosystems are presented. Based on current knowledge, nematode reproduction strategies are discussed and indications of the risk involved in the release of non-indigenous nematodes are given.     

Effects of Density, Age and Host Cues on the Dispersal of Heterorhabditis megidis

Agar plate assays were used to assess the effect of density, incubation time and age of nematodes and the presence of insect hosts on the dispersal of infective juveniles (IJs) of Heterorhabditis megidis (strain NLH-E87.3). IJs dispersed faster and further at high densities than at low densities. Dispersal was also influenced by the age of the IJs. Individuals stored for a period of 1.5 and 4.5 weeks showed to be more active than those stored for 2.5 and 3.5 weeks. The presence of a host insect enhanced the dispersion of nematodes. The increasing in the incubation period showed that IJs responded positively to host cues from Galleria mellonella but poorly to cues from Otiorhynchus sulcatus larvae.     

Environmental factors affecting sexual differentiation in the entomopathogenic nematode Heterorhabditis bacteriophora

The present study was aimed at determining the influence of various environmental factors on sex differentiation (SD) in the entomopathogenic nematode Heterorhabditis bacteriophora HP88 strain, under in vivo and in vitro culture conditions. Injection of individual nematodes into last instars of Galleria mellonella resulted in development of a similar number of females and hermaphrodites (35-40%) and 20-25% males. Increasing the number of nematodes injected into the insect did not change these proportions. In smaller insects (0.7-1.5 cm long), an increase in the proportion of hermaphrodites was recorded as compared with larger size cadavers (2.4-2.7 cm long). When individual hermaphrodites were placed on NGM, the proportion of hermaphrodites, females and male progeny was 63%, 31%, and 6%, respectively. Rearing on richer medium ("Dog-food" agar) resulted in reduction in the proportion of hermaphrodites. Nematodes introduced to the symbiotic bacterium obtained from other nematode strains (IS-5 and IS-33) developed similarly to the culture reared on the HP88 bacteria. Rearing the nematodes at a temperature range between 21 degrees C to 30 degrees C also did not have a significant effect on the sexual differentiation among nematodes cultured on NGM. The proportion of hermaphrodites increased as the starvation period of hatching nematode juveniles lengthened (>6 hr). The data obtained in the present study strongly suggest that the main factor affecting sex differentiation in H. bacteriophora is the nutrition source. The practical and biological implications of the results are discussed.     

Fatty acid composition of Heterorhabditis sp. during storage

The fatty acid composition of three North West European isolates of Heterorhabditis sp. from different geographical origins, UK211 (England), HF85 (The Netherlands) and EU17 (Estonia) was assessed directly after harvest and, for UK211 and HF85, after 5 weeks storage in water at 20 degrees C. Lipid represented 34-43% of the dry weight of fresh nematodes. Of this, neutral lipid (NL) comprised from 70% (HF85) to over 90% (UK211, EU17). The fatty acid patterns were similar between the three isolates. Oleic (C18:In-9), palmitic (C16:0), and linoleic (C18:2n-6) acid predominated with 51, 13 and 12%, respectively in the total lipid (TL) of fresh nematodes (average for the three isolates). Levels of unsaturation (U.I.) of fresh nematodes were on average 110, 112, 113 and 152 for the TL, NL, phospholipid and free fatty acid fractions, respectively. EU17 had a slightly lower U.I than the other two strains, despite its more northern origin. Changes in fatty acid composition due to storage were most significant in the NL fraction. The U.I. for the NL fraction increased during storage, suggesting a preferential use of saturated fatty acids.     

河北异小杆线虫一品系的分类鉴定及其对蛴螬致病力的测定

昆虫病原线虫是一种有应用潜力的地下害虫生物防治因子。本研究利用形态学特征和ITS-rDNA分析方法对从河北省沧州分离的异小杆线虫一品系进行了鉴定, 并在室内测定比较了其对蛴螬的致病力。通过对该线虫侵染期幼虫和雄性成虫主要形态学特征的参数测量, 发现其与嗜菌异小杆线虫Heterorhabditis bacteriophora的形态特征最为相近;同时ITS1-rDNA序列比对和系统发育学分析结果显示其与嗜菌异小杆线虫亲缘关系最近。结合形态学和分子生物学特征, 确定该线虫为嗜菌异小杆线虫沧州品系。该线虫对蛴螬（华北大黑鳃金龟Holotrichia oblita、暗黑鳃金龟H. parallela和铜绿丽金龟Anomala corpulenta 3种金龟子的2龄幼虫）的致病力研究结果表明：处理72 h后, 暗黑鳃金龟幼虫死亡率显著高于另外两种金龟子幼虫 (P<0.05);处理120 h后, 暗黑鳃金龟和铜绿丽金龟幼虫的死亡率分别达到93.3%和80.0%, 二者无显著差异 (P>0.05), 可见该线虫对它们有较强的致病力。不同线虫对暗黑鳃金龟幼虫的致病力结果显示, 嗜菌异小杆线虫沧州品系侵染96 h后, 幼虫的死亡率显著高于Steinernema feltiae和S. longicaudum两种线虫的处理 (P<0.05) , 说明嗜菌异小杆线虫沧州品系的致病力显著高于另外两种线虫。     

Effects of Paenibacillus nematophilus on the entomopathogenic nematode Heterorhabditis megidis

The insect parasitic nematodes Heterorhabditis spp. are mutualistically associated with entomopathogenic bacteria, Photorhabdus spp. A novel association has been detected between H. megidis isolate EU17 and the endospore-forming bacterium Paenibacillus nematophilus. P. nematophilus sporangia adhere to infective juveniles (IJs) of H. megidis and develop in insect hosts along with the nematodes and their symbiont. We tested the effects of P. nematophilus on H. megidis. The yield and quality (size, energy reserves, and storage survival) of IJs were not affected by co-culture in insects with P. nematophilus. Dispersal of IJs in sand and on agar was inhibited by adhering P. nematophilus sporangia: fewer than 2% of IJs with P. nematophilus sporangia reached the bottom of a sand column, compared to 30% of the control treatment. Sporangia significantly reduced infectivity of H. megidis for wax moth larvae in sand, but not in a close contact (filter paper) assay. The results suggest that P. nematophilus may reduce the transmission potential of H. megidis through impeding the motility of IJs.     

Genetics of the Nematode Heterorhabditis bacteriophora Strain HP88: The Diversity of Beneficial Traits

Genotypic variation among infective juveniles of Heterorhabditis bacteriophora (strain HP88) in heat, desiccation, ultraviolet tolerance, and host-finding ability was assessed by comparing the performance of inbred lines of this entomopathogenic nematode in laboratory assays. Each line consisted of highly homozygous offspring originating from one individual obtained from a natural population. Considerable variation in all four traits was detected among the different inbred lines. The heritability values for heat or ultraviolet tolerance and for host-finding ability were high, indicating that selection should be an efficient way for improving these traits in the population. The results for desiccation tolerance varied considerably within each line. Heritability value was low, indicating that the results were influenced mainly by environmental variation and suggesting that selective breeding for higher desiccation tolerance would be inefficient. Improvement through induction of mutations may be a better alternative in this population.     

Host influences on the pathogenicity of Heterorhabditis megidis

The infectivity of infective juveniles(IJs) of Heterorhabditis megidis (strain NLH-E87.3) produced on small, medium and large larvae ofGalleria mellonella, and on medium and largelarvae of Otiorhynchus sulcatus was tested underlaboratory conditions against G. mellonella andO. sulcatus larvae. Infective juvenilesoriginating from small G. mellonella exposed toan initial dose of one IJ were more infectious thanthose from small cadavers exposed to a dose of 30 IJs.Independent of the initial inoculum size, IJs fromsmall cadavers of G. mellonella were moreinfectious than those from medium and large cadavers.At a dose of one IJ per larva, IJs originating frommedium size O. sulcatus cadavers were moreinfective against G. mellonella than againstO. sulcatus larvae. Large G. mellonellalarvae were less susceptible to all IJ batches thanmedium and small sized larvae.     

Diet Composition and Lipids of In Vitro-Produced Heterorhabditis bacteriophora

Several entomopathogenic nematode species are currently under evaluation for mass production and field efficacy for biological control of insect pests. However, quality and quantity of in vitro-produced entomopathogenic nematodes vary considerably, depending on media, temperature, and production method. In addition, nematode production should be cost effective. We investigated nematode yield, production time, total lipid content, and fatty acid composition of Heterorhabditis bacteriophora produced in artificial media supplemented with different lipid sources. Lipid source significantly affected lipid quantity and quality in H. bacteriophora. Media supplemented with extractable insect lipids produced yields 1.9 times higher than did beef fat- or lard-supplemented media. Moreover, the developmental rate in media supplemented with host lipids was 1.7 times faster than that in media supplemented with beef fat or lard. Nematodes grown in media supplemented with insect lipids accumulated significantly higher lipid proportion per dry biomass than those grown in media supplemented with other lipid sources. H. bacteriophora produced in media supplemented with insect lipids, olive oil, or canola oil had similar fatty acid patterns, with oleic (18:1) acid as the major lipid fatty acid. Media supplemented with other lipid sources produced nematodes with fatty acid patterns different from those of media supplemented with insect lipids. We recommend addition of fatty acid mixtures that resemble natural host lipids for mass-producing entomopathogenic nematodes. This could provide nematode quality similar to in vivo-produced nematodes and could improve yield.     

Purification and characterization of an acetylcholinesterase from the infective juveniles of Heterorhabditis bacteriophora

Acetylcholinesterases (AChEs) have been estimated in the infective juveniles (IJs) of eight different strains of heterorhabditid nematodes. The enzyme content ranged from 45.6 to 421.3 units/10(5) IJs with specific activity 34.0 to 82.6 units/mg protein. The isoenzyme patterns revealed the existence of two-slow-moving isoforms. Heterorhabditis bacteriophora AChE1A has been purified from the IJs of the heterorhabditid nematode strain of the highest enzymatic activity to homogeneity by ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and DEAE-Sepharose. The specific activity of the purified enzyme was 1378.1 units/mg protein with purification fold 17.5 over crude extract. The enzyme has a pH optimum at 7.5. The optimum temperature for enzyme activity and stability was 35 degrees C. The activation energy was calculated to be 9.0 kcal/mol. The enzyme hydrolyzes acetylthiocholine (AcSCh), propionylthiocholine (PrSCh), S-butyrylthiocholine (BuSCh) and benzoylthiocholine (BzSCh) iodides with relative rate 100, 74.6, 41.7 and 22.2%, respectively. It displayed an apparent Michaelis-Menten behavior in the concentration range from 0.1 to 2 mM for the three former substrates with Km values 0.27, 0.42 and 0.59 mM, respectively. H. bacteriophora ChE1A is an AChE since it hydrolyzed AcSChI at higher rate than the other substrates and displayed excess substrate inhibition with AcSChI at concentrations over 2 mM. It was inhibited by eserine and BW284C51, but not by iso-OMPA. Its biochemical properties were compared with those reported for different species of insects as target hosts for heterorhabditid nematodes and animal parasitic nematodes.