The entomopathogenic nematodes (EPN) Heterorhabditis and Steinernema are widely used for the biological control of insect pests and are gaining importance as model organisms for studying parasitism and symbiosis. In this paper recent advances in the understanding of EPN behavior are reviewed. The “foraging strategy” paradigm (distinction between species with ambush and cruise strategies) as applied to EPN is being challenged and alternative paradigms proposed. Infection decisions are based on condition of the potential host, and it is becoming clear that already-infected and even long-dead hosts may be invaded, as well as healthy live hosts. The state of the infective juvenile (IJ) also influences infection, and evidence for a phased increase in infectivity of EPN species is mounting. The possibility of social behavior - adaptive interactions between IJs outside the host - is discussed. EPNs’ symbiotic bacteria (Photorhabdus and Xenorhabdus) are important for killing the host and rendering it suitable for nematode reproduction, but may reduce survival of IJs, resulting in a trade-off between survival and reproduction. The symbiont also contributes to defence of the cadaver by affecting food-choice decisions of insect and avian scavengers. I review EPN reproductive behavior (including sperm competition, copulation and evidence for attractive and organizational effects of pheromones), and consider the role of endotokia matricida as parental behavior exploited by the symbiont for transmission.
Behavior; biological control; ecology; endotokia matricida; entomopathogenic nematode; fitness trade-off; foraging strategy; Heterorhabditis; phased infectivity; Photorhabdus; reproduction; scavenging; sexual maturation; symbiosis; Steinernema; Xenorhabdus
Xenorhabdus and Photorhabdus spp. are bacterial symbionts of entomopathogenic nematodes (EPNs). In this study, we isolated and characterized Xenorhabdus and Photorhabdus spp. from across Thailand together with their associated nematode symbionts, and characterized their phylogenetic diversity. EPNs were isolated from soil samples using a Galleria-baiting technique. Bacteria from EPNs were cultured and genotyped based on recA sequence. The nematodes were identified based on sequences of 28S rDNA and internal transcribed spacer regions. A total of 795 soil samples were collected from 159 sites in 13 provinces across Thailand. A total of 126 EPNs isolated from samples taken from 10 provinces were positive for Xenorhabdus (n = 69) or Photorhabdus spp. (n = 57). Phylogenetic analysis separated the 69 Xenorhabdus isolates into 4 groups. Groups 1, 2 and 3 consisting of 52, 13 and 1 isolates related to X. stockiae, and group 4 consisting of 3 isolates related to X. miraniensis. The EPN host for isolates related to X. stockiae was S. websteri, and for X. miraniensis was S. khoisanae. The Photorhabdus species were identified as P. luminescens (n = 56) and P. asymbiotica (n = 1). Phylogenenic analysis divided P. luminescens into five groups. Groups 1 and 2 consisted of 45 and 8 isolates defined as subspecies hainanensis and akhurstii, respectively. One isolate was related to hainanensis and akhurstii, two isolates were related to laumondii, and one isolate was the pathogenic species P. asymbiotica subsp. australis. H. indica was the major EPN host for Photorhabdus. This study reveals the genetic diversity of Xenorhabdus and Photorhabdus spp. and describes new associations between EPNs and their bacterial symbionts in Thailand.
Entomopathogenic nematodes (EPNs) are obligate parasites of insects that are widely distributed in soils throughout the world. They have great potential for use as biological control agents for insect pests. It is known that strains of Steinernema and Heterorhabditis isolated from different geographical regions exhibit differences in their ecological traits, such as infectivity, establishment, survival, reproduction, etc. A precise knowledge of these factors is therefore an essential pre-requisite for devising successful strategies to use these nematodes in biological control programmes. The present study investigated the effect of soil moisture on the activity (as measured by number of nematodes established in hosts) of three entomopathogenic nematode species (Heterorhabditis indica Poinar, Karunakar & David; Steinernema thermophilum Ganguly & Singh; Steinernema glaseri Steiner), isolated from forest soils in Meghalaya, India, under laboratory conditions. The experiments for EPNs were conducted at 25 ± 2°C (30 ± 2°C for S. thermophilum) in a sandy loam soil (85% sand, 12% silt and 3% clay, pH 6.54). Last instar larvae of wax moth, Galleria mellonella served as the experimental insect host. The soil moistures tested were 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 25% (w/w). The study revealed that soil moisture has marked influences on establishment of infective juveniles of different nematode species in insect host. While, S. thermophilum showed establishment at 4% and above soil moistures, H. indica and S. glaseri showed establishment at 5% and above soil moistures. The optimum soil moisture for different nematode species were noted as: H. indica 8–18%, S. thermophilum 6–20%, and S. glaseri 8–25%. Further, a minimum of 6% soil moisture was noted to be essential for achieving 100% host mortality for all the three nematode species.
Entomopathogenic nematodes; Soil moisture; Heterorhabditis indica; Steinernema thermophilum; Steinernema glaseri; Galleria mellonella; Biological control; Meghalaya
Entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematidae have a mutualistic–symbiotic association with enteric γ-Proteobacteria (Steinernema–Xenorhabdus and Heterorhabditis–Photorhabdus), which confer high virulence against insects. EPNs have been studied intensively because of their role as a natural mortality factor for soil-dwelling arthropods and their potential as biological control agents for belowground insect pests. For many decades, research on EPNs focused on the taxonomy, phylogeny, biogeography, genetics, physiology, biochemistry and ecology, as well as commercial production and application technologies. More recently, EPNs and their bacterial symbionts are being viewed as a model system for advancing research in other disciplines such as soil ecology, symbiosis and evolutionary biology. Integration of existing information, particularly the accumulating information on their biology, into increasingly detailed population models is critical to improving our ability to exploit and manage EPNs as a biological control agent and to understand ecological processes in a changing world. Here, we summarize some recent advances in phylogeny, systematics, biogeography, community ecology and population dynamics models of EPNs, and describe how this research is advancing frontiers in ecology.
biodiversity; entomopathogenic nematodes; Heterorhabditis; multivariate analysis; Photorhabdus; soil ecology; soil food web; Steinernema; Xenorhabdus
Hemocyte encapsulation reactions of infective juveniles of two Iranian isolates of the entomopathogenic nematodes, Heterorhabditis bacteriophora Poinar (Rhabditina: Heterorhabditidae) and Steinernema feltiae Filipjev (Tylenchina: Steinernematidae), were compared in the economic pest Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), and the greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae). The former was a more responsive host than the latter and the hemocyte responses occurred sooner and more extensively. Complete encapsulation of some of the nematodes occurred by 4 h post injection for H. bacteriophora in both L. decemlineata and G. mellonella, and by 2 h pi for S. feltiae in L. decemlineata. The percentage of encapsulation from 24 h to 72 h pi in L. decemlineata was 86.2% for S. feltiae and 39% for H. bacteriophora. In G. mellonella there were no encapsulation or melanization responses against S. feltiae, whereas when H. bacteriophora was encapsulated and melanized (16.7%) the encapsulation level was lower than in L. decemlineata. This study may contribute to effectively selecting entomopathogenic nematode species active against significant economic pests based on the latter's cellular immune response.
cellular encapsulation; insect; melanization; resistant host
A survey was done in the summer months along the Alaska Highway, in other parts of British Columbia, in northern Alberta, and in the Yukon Territory for steinernematid and heterorhabditid nematodes occurring in the top 10 cm of soil. Steinernema feltiae and Steinernema spp. were found at 18 and Heterorhabditis megidis at 7 sites of 125 sampled. Most nematodes were found where visible insect infestation occurred and where human influence on the habitat was substantial (e.g., agricultural, forested and bush-hedgerow habitats); none was found in grassland or virgin forests. Heterorhabditis megidis occurred in only the southern, warmer, drier region of British Columbia. In the laboratory some steinernematid isolates and H. megidis killed Galleria mellonella larvae at 13 and 22 C, whereas some isolates of Steinernema killed the larvae at only 13 C. Steinernema spp. from three high altitude sites with low, average July temperatures (13-14 C) are cold-active in that they produced infective juveniles at 13 C and killed G. mellonella at 6 C.
biological control; entomopathogenic nematode; Heterorhabditidae; low-temperature activity; nematode; Steinernenaatidae; survey; temperature
A soil survey for entomopathogenic nematodes was conducted throughout the nine islands of the Azorean archipelago. Forty-six out of 1,180 samples (3.9%) were positive, with Heterorhabditis spp. isolated from 30 sites on six islands and Steinernema spp. isolated from 16 sites on three islands. São Miguel and Terceira Islands were positive for both genera, and Pico Island was positive only for Steinernema. Entomopathogenic nematodes were found from sea level up to 750 m. Seventy percent of the samples positive for Heterorhabditis were collected below 150 m, whereas 62.5% of the samples positive for Steinernema were collected above 300 m. Heterorhabditis was not isolated above 450 m. Steinernema was collected mostly in loamy-sand and sandy-loam soils with a pH below 6, whereas Heterorhabditis was mostly collected in sandy and loamy-sand soils with pH higher than 6. Steinernema and Heterorhabditis were found in cropland, orchards, and pastures, while Heterorhabditis was found also in woodland and native vegetation.
azores; biological control; entomopathogenic nematodes; Heterorhabditis spp.; insecta; island; natural occurrence; Steinernema spp.; survey
To isolate potential insect biocontrol agents, entomogenous nematodes were surveyed in Tennessee plant nurseries in 1991. Soil samples from 113 nursery sites were baited with greater wax moth (Galleria mellonella) larvae, house cricket (Acheta domesticus) adults, lesser mealworm (Alphitobius diaperings) adults, and house fly (Musca domestica) larvae. Heterorhabditis bacteriophora and Steinernema carpocapsae were each recovered from 17 soil samples. Heterorhabditis bacteriophora was more common in habitats with crape myrtle (Lagerstroemia indica) and Chinese juniper (Juniperus chinensis) than other nursery plants, and S. carpocapsae was more frequently recovered from habitats with juniper and Southern magnolia (Magnolia grandiflora). Bulk density, electrical conductivity, organic matter, pH, temperature, and moisture content of the entomogenous-nematode positive soil samples were compared. Other nematode genera recovered with insect baits included Rhabditis sp., Pelodera sp., Cryptaphelenchoides sp., and Mesodiplogaster sp., which was recovered from a greater percentage of soil samples than the other five genera.
Acheta; Alphitobius; biological control; Cryptaphelenchoides; distribution; entomogenous nematode; Heterorhabditis; Mesodiplogaster; Musca; nursery plant; Pelodera; Rhabditis; Steinernema; survey
Control of Diaprepes abbreviatus by endemic and exotic entomopathogenic nematodes (EPN) was monitored during 2000-2001 in two citrus orchards in central Florida (Bartow and Poinciana). Caged sentinel insect larvae were buried beneath citrus trees for 7 days at 1 to 2-month intervals from April to October each year. At Bartow, the survey occurred in experimental plots that were (i) not treated with commercial EPN, (ii) treated twice annually since 1998 with commercially formulated Steinernema riobrave, or (iii) treated twice annually with S. riobrave and liquid fertilization (15 times/year) occurred in place of dry fertilizer (3 times/year) used in the other treatments. Four endemic EPN species, in addition to S. riobrave, were recovered from the sandy soil at Bartow: S. diaprepesi, Heterorhabditis zealandica, H. indica, and H. bacteriophora. Mean insect mortality in control plots was 39.4% (range = 13% to 74%), with seasonal maxima in May to July each year. Endemic EPN were recovered from 55% (range = 22% to 81%) of the cadavers each month. Total numbers of endemic EPN recovered in all plots during 2 years were directly related to the numbers of adult weevils (D. abbreviatus and Pachnaeus litus) captured in modified Tedder's traps and inversely related to recovery of S. riobrave. Insect mortality was higher and cadavers containing endemic EPN were more numerous in untreated control plots than in S. riobrave-treated plots, except during months in which S. riobrave was applied. In treated plots, endemic EPN were recovered from cadavers at twice the rate of S. riobrave. Suppression of endemic EPN in plots treated with S. riobrave, combined with inferior persistence by the introduced species, may have attenuated the net efficacy of S. riobrave against D. abbreviatus. In contrast, H. indica was the only endemic nematode recovered from the sandy clay loam soil at Poinciana, where the average mortality of D. abbreviatus was 12% (range 3% to 20%) and incidence of H. indica did not exceed 8%. Results of these surveys suggest that the regional patterns in the abundance and damage to citrus caused by D. abbreviatus in Florida are regulated by endemic EPN and other soilborne enemies of the weevil.
Abbott's formula; biological control; competition; entomopathogenic nematodes; Heterorhabditis; natural control; seasonality; Steinernema
Laboratory experiments were conducted to study non-target effects of augmenting entomopathogenic nematode (EPN)communities in soil. When raw soil from a citrus orchard was augmented with either 2,000 Steinernema riobrave or S. diaprepesi, fewer EPN (P ≤ 0.05) survived if the soil had also been treated with 2,000 S. riobrave 7 d earlier (i.e., two augmentation events rather than one). EPN survival was unaffected by treatment (P ≤ 0.05) in soil that was air-dried to disrupt antagonist activity prior to the experiment. When S. diaprepesi, S. riobrave, Heterorhabditis zealandica or no EPN were added to raw soil and S. diaprepesi was added 5 d later, the survival of both S. diaprepesi and of total EPN was greater (P ≤ 0.05) in soil that received no pretreatment than in soilpre treated with S. riobrave. Pretreatment of soil with H. zealandica or S. diaprepesi had less or no affect on survival of S. diaprepesi or total EPN. When nematodes were recovered from soil and placed on water agar, the number of S. diaprepesi that were killed by endoparasitic and trapping nematophagous fungi was greater (P ≤ 0.05) if soil was pretreated with steinernematid species than if the soil was not pretreated or was pretreated with H. zealandica. The adverse effects of pretreating soil on EPN survival were density dependent within a range of pretreatment dosages (20–100 IJ/cm2 soil surface), and the treatment effects required more time to become evident at lower than at higher dosages. These experiments suggest that non-target effects of augmenting the EPN community in soil vary among EPN species and have the potential to temporarily reduce EPN numbers below the natural equilibrium density.
Antagonism; nematophagous fungi; numerical response; post-application biology; predation; survival
The objective of this study was to evaluate the efficacy of three indigenous strains of entomopathogenic nematodes (EPN) from Meghalaya, India, namely Heterorhabditis indica Poinar, Karunakar and David, Steinernema thermophilum Ganguly and Singh, and Steinernema glaseri (Steiner) against the last instar larva of mustard sawfly, Athalia lugens proxima Klug, a serious pest of mustard and radish in India. The larvae of A. lugens proxima were exposed to 10, 25, 50, 75 and 100 infective juveniles (IJs) concentration of each nematode species in Petri dishes. Percentage larval mortality and nematode reproduction in insect larvae was studied. The sawfly larvae were found to be susceptible to all the three EPNs tested, but the degree of susceptibility to infection varied from among nematode species. Based on LC50 value, H. indica was the most pathogenic species. Nevertheless, S. thermophilum and S. glaseri also showed a high insect mortality. This study also revealed that all the three test nematodes are also able to propagate in the host cadaver and produce first generation infective juveniles. However, H. indica produced significantly more number of IJs per insect larva than the other two nematode species. The progeny production was recorded to be the least in case of S. glaseri. In conclusion, our findings suggest that of the three indigenous EPNs studied, H. indica and S. thermophilum have good potential as biological control agents against mustard sawfly, A. lugens proxima.
Entomopathogenic nematodes; Biological control; Mustard sawfly; Athalia lugens proxima Klug; Heterorhabditis indica; Steinernema thermophilum; Steinernema glaseri; Meghalaya; India
Some studies suggest that entomopathogenic nematodes (EPN) affect plant-parasitic nematode populations. Here, the effects of live and dead IJ of Heterorhabditis bacteriophora JPM4, H. baujardi LPP7, Steinernema feltiae SN and S. carpocapsae All were evaluated against eggs and J2 of the plant-parasitic nematode Meloidogyne mayaguensis. According to treatment, 100 IJ were applied with 350 eggs, 350 J2 or 175 eggs + 175 J2 to tomato plants. Bioassays were conducted in March to May and repeated in September to November 2005. Both experiments lasted 9 weeks, and the variable evaluated was number of galls per plant. When eggs were used for infections in the first trial, plants exhibited lower gall number compared to control when live and dead H. baujardi IJ and live S. feltiae IJ were added (9.7, 4.5, 7.3 and 85.7 galls, respectively). In the second trial, live S. feltiae and S. carpocapasae IJ influenced gall formation compared to control (14.33, 14.57 and 168.02 galls, respectively). When J2 were used for infections, plants with live H. baujardi IJ presented less galls when compared to control in both trials (38.3 and 355.7 galls in the first trial and 145.2 and 326.2 in the second one, respectively). Infection with a mixture of J2 and eggs resulted in fewer galls than when live S. feltiae IJ were present in both trials, compared to control (38.3 and 44.2 galls vs. 275.3 and 192.2 galls, respectively). We conclude that H. baujardi and S. feltiae apparently may be inhibiting egg hatching and J2 infection.
Entomopathogenic nematodes; nematode-nematode interaction; biological control; plant-parasitic nematode; Meloidogyne mayaguensis
The oriental fruit moth (OFM), Grapholita molesta (Busck), which is among the most important insect pests of peaches and nectarines, has developed resistance to a wide range of insecticides. We investigated the ability of the entomopathogenic nematodes (EPN) Steinernema carpocapsae (Weiser), S. feltiae (Filipjev), S. riobrave (Cabanillas et al.), and Heterorhabditis marelatus (Liu and Berry) to control OFM under laboratory and fruit bin conditions. At a dosage of 10 infective juveniles (IJ)/cm2 in the laboratory, S. carpocapsae caused 63%, S. feltiae 87.8%, S. riobrave 75.6%, and H. marelatus 67.1% OFM mortality. All four nematode species caused significant OFM larval mortality in comparison to the nontreated controls. Steinernema feltiae was used for the bin assays due to the higher OFM mortality it caused than the other tested EPN species and to its ability to find OFM under cryptic environments. Diapausing cocooned OFM larvae in miniature fruit bins were susceptible to IJ of S. feltiae in infested corner supports and cardboard strips. Treatment of bins with suspensions of 10 or 25 S. feltiae IJ/ml water with wetting agent (Silwet L77) resulted in 33.3 to 59% and 77.7 to 81.6% OFM mortality in corner supports and cardboard strips, respectively. This paper presents new information on the use of EPN, specifically S. feltiae, as nonchemical means of OFM control.
Biological control; cardboard strips; fruit bins; Grapholita molesta; entomopathogenic nematodes; Heterorhabditis marelatus; oriental fruit moth; Steinernema carpocapsae; S. feltiae; S. riobrave; wetting agent
Understanding the desiccation survival attributes of infective juveniles of entomopathogenic nematodes (EPN) of the genera Steinernema and Heterorhabditis, is central to evaluating the reality of enhancing the shelf-life and field persistence of commercial formulations. Early work on the structural and physiological aspects of desiccation survival focused on the role of the molted cuticle in controlling the rate of water loss and the importance of energy reserves, particularly neutral lipids. The accumulation of trehalose was also found to enhance desiccation survival. Isolation of natural populations that can survive harsh environments, such as deserts, indicated that some populations have enhanced abilities to survive desiccation. However, survival abilities of EPN are limited compared with those of some species of plant-parasitic nematodes inhabiting aerial parts of plants. Research on EPN stress tolerance has expanded on two main lines: i) to select strains of species, currently in use commercially, which have increased tolerance to environmental extremes; and ii) to utilize molecular information, including expressed sequence tags and genome sequence data, to determine the underlying genetic factors that control longevity and stress tolerance of EPN. However, given the inherent limitations of EPN survival ability, it is likely that improved formulation will be the major factor to enhance EPN longevity and, perhaps, increase the range of applications.
biocontrol; bioinsecticides; dauer; desiccation; Heterorhabditis; longevity; Steinernema
Field and laboratory experiments were conducted to determine the degree to which free-living, bactivorous nematodes (FLBN) are able to competitively displace entomopathogenic nematodes (EPN) from insect cadavers. Two hundred larvae of the insect Diaprepes abbreviatus were buried at regular intervals during 2 years in experimental plots that were untreated or treated twice annually with Steinernema riobrave. Larvae were recovered after 7 days, and nematodes emerging from cadavers during the next 30 days were identified. The monthly prevalence of FLBN was directly related to that of S. riobrave (r = 0.38; P = 0.001) but was not related to the prevalence of the endemic EPN, S. diaprepesi, Heterorhabditis zealandica, H. indica, or H. bacteriophora (r = 0.02; P = 0.80). In a second experiment, treatment of small field plots with S. riobrave increased the prevalence of insect cadavers in which only FLBN were detected compared to untreated controls (30% vs. 14%; P = 0.052), and increased numbers of FLBN per buried insect by more than 10-fold. In the laboratory, sand microcosms containing one D. abbreviatus larva were treated with (i) the FLBN, Pellioditis sp.; (ii) S. riobrave; (iii) S. riobrave + Pellioditis; or (iv) neither nematode. Insect mortality was higher in the presence of both nematodes (57%) than when S. riobrave was alone (42%) (P = 0.01). An average of 59.2 Pellioditis sp. g-1 insect body weight emerged in the presence of S. riobrave, whereas 6.2 nematodes g-1 insect were recovered in the absence of the EPN (P = 0.01). Pellioditis sp. reduced the number of S. riobrave per cadaver by 84%; (P = 0.03), and per available insect by 82% (P = 0.001), compared to S. riobrave alone. Population size of S. diaprepesi was not affected by Pellioditis sp. in experiments of the same design. Faster development (P = 0.05) and nutrient appropriation within the insect cadaver by S. diaprepesi compared to S. riobrave may increase the fitness of the former species to compete with Pellioditis sp. The results of these studies demonstrate the potential of FLBN to regulate population densities of EPN and to dampen estimates of EPN-induced mortality of insect pests in the field.
free-living nematodes; microbivorous nematodes; Pellioditis; Steinernema diaprepesi; Steinernema riobrave; Steinernematidae
The efficacy of three entomopathogenic nematode (EPN) species, Heterorhabditis indica, Steinernema thermophilum, and S. glaseri, from Meghalaya, India was studied against the larvae of taro leaf beetle, Aplosonyx chalybaeus (Hope) (Coleoptera: Chrysomelidae), under the laboratory conditions. The beetle larvae (grubs) were exposed to 25, 50, 75, 100 and 200 infective juveniles (IJs) of each nematode species for different time periods and they were found to be susceptible to all the EPNs tested. However, the susceptibility of grubs to nematode infection varied according to the dosages of IJs and their exposure periods. Appreciably good performance was achieved by S. glaseri, which showed 100 % mortality of insect larvae in 48 h exposure time. At 48 h of incubation, its LC50 value was 90.3 IJs/larva, which was lower than that of S. thermophilum (115.0 IJs/larva) and H. indica (186.0 IJs/larva), at the same exposure time. All the tested nematode species were also found to reproduce within the host and produced infective juveniles. H. indica, however, showed comparatively more production of IJs per cadaver of infected host (168.9 × 103 IJs/larva), as compared to the other two tested nematode species. The production of IJs per cadaver of infected host by S. thermophilum was recorded to be 82.0 × 103 IJs/larva. In case of S. glaseri, while production of IJs increased initially to 18.9 × 103 IJs/larva at concentration of 100 IJs/larva, it declined thereafter to 14.7 × 103 IJs/larva at the dose of 200 IJs/larva. In conclusion, the evidence obtained in this study suggests that all the three indigenous EPN species are virulent enough to produce 100 % mortality in the last instar larvae of A. chalybaeus. These EPN species thus have potential scope for the management of A. chalybaeus in taro crops.
Aplosonyx chalybaeus; Biological control; Entomopathogenic nematodes; Heterorhabditis indica; Steinernema glaseri; Steinernema thermophilum; Taro
The plum curculio, Conotrachelus nenuphar, is a major pest of stone and pome fruit (e.g., apples, pears, peaches, cherries, etc.). Entomopathogenic nematodes (Steinernema spp. and Heterorhabditis spp.) may be used to control the larval stage of C. nenuphar following fruit drop. Indeed, certain entomopathogenic nematodes species have previously been shown to be highly effective in killing C. nenuphar larvae in laboratory and field trials. In field trials conducted in the Southeastern, USA, Steinernema riobrave has thus far been shown to be the most effective species. However, due to lower soil temperatures, other entomopathogenic nematode strains or species may be more appropriate for use against C. nenuphar in the insect’s northern range. Thus, the objective of this study was to conduct a broad screening of entomopathogenic nematodes. Under laboratory conditions, we determined the virulence of 13 nematode strains (comprising nine species) in two different soils (a loam and clay-loam) and three different temperatures (12°C, 18°C, and 25°C). Superior virulence was observed in S. feltiae (SN strain), S. rarum (17 C&E strain), and S. riobrave (355 strain). Promising levels of virulence were also observed in others including H. indica (HOM1 strain), H. bacteriophora (Oswego strain), S. kraussei, and S. carpocapsae (Sal strain). All nematode treatments were affected by temperature with the highest virulence observed at the highest temperature (25°C). In future research, field tests will be used to further narrow down the most suitable nematode species for C. nenuphar control.
biological control; Conotrachelus nenuphar; entomopathogenic nematode; Heterorhabditis; plum curculio; Steinernema
The history of entomopathogenic nematology is briefly reviewed. Topic selections include early descriptions of members of Steinernema and Heterorhabditis, how only morphology was originally used to distinguish between the species; descriptions of the symbiotic bacteria and elucidating their role in the nematode- insect complex, including antibiotic properties, phase variants, and impeding host defense responses. Other topics include early solutions regarding production, storage, field applications and the first commercial sales of entomopathogenic nematodes in North America. Later studies centered on how the nematodes locate insect hosts, their effects on non-target organisms and susceptibility of the infective juveniles to soil microbes. While the goals of early workers was to increase the efficacy of entomopathogenic nematodes for pest control, the increasing use of Heterorhabditis and Photorhabdus as genetic models in molecular biology is noted.
History; entomopathogenic nematodes; Heterorhabditis spp.; Steinernema spp
Entomopathogenic nematodes (EPNs) from the Heterorhabditidae and Steinernematidae families are well-known biocontrol agents against numerous insect pests. The infective juveniles (IJs) are naturally occurring in the soil and their success in locating and penetrating the host will be affected by extrinsic/intrinsic factors that modulate their foraging behavior. Characterizing key traits in the infection dynamics of EPNs is critical for establishing differentiating species abilities to complete their life cycles and hence, their long-term persistence, in different habitats. We hypothesized that phenotypic variation in traits related to infection dynamics might occur in populations belonging to the same species. To assess these intraspecific differences, we evaluated the infection dynamics of 14 populations of Steinernema feltiae in two experiments measuring penetration and migration in sand column. Intraspecific variability was observed in the percentage larval mortality, time to kill the insect, penetration rate, and sex-ratio in both experiments (P < 0.01). Larval mortality and nematode penetration percentage were lower in migration experiments than in penetration ones in most of the cases. The sex-ratio was significantly biased toward female-development dominance (P < 0.05). When the populations were grouped by habitat of recovery (natural areas, crop edge, and agricultural groves), nematodes isolated in natural areas exhibited less larval mortality and penetration rates than those from some types of agricultural associated soils, suggesting a possible effect of the habitat on the phenotypic plasticity. This study reinforces the importance of considering intraspecific variability when general biological and ecological questions are addressed using EPNs.
entomopathogenic nematode; female-biased; migration assay; penetration assay; sex ratio; Steinernema feltiae
We describe the isolation and characterization of an insect pathogenic bacterium from the entomopathogenic nematode Heterorhabditis indica (Karnataka strain), an isolate from the southern regions of India. The strain has been identified and characterized by phenotypic, biochemical tests and PCR-RFLP analysis of the 16S rRNA gene as Photorhabdus luminescens subsp. akhurstii. The insecticidal toxin complex produced by this bacterium has been purified through a series of steps including ultrafiltration, anion exchange chromatography, and gel filtration chromatography. The toxin consists of two protein complexes of approximately 1,000 kD and was active against the larvae of Spodoptera litura and Galleria mellonella.
bacteria; entomopathogenic; Galleria mellonella; Heterorhabditis; insect; insecticidal; nematode; Photorhabdus; Spodoptera litura; toxin
Rearing conditions have been shown to affect several aspects of entomopathogenic nematode biology, including dispersal behavior and infectivity. The present study explores the differences in development rate of Heterorhabditis bacteriophora and Steinernema carpocapsae when infective juveniles (IJ) were collected in water using the standard White trap method vs. natural emergence from cadavers into sand. We exposed Galleria mellonella to IJ entompopathogenic nematodes treated in one of three ways: collected in a White trap, allowed to emerge directly into sand, or collected in a White trap and treated with a cadaver homogenate. When S. carpocapsae IJ were allowed to emerge from cadavers directly into sand and then allowed to infect new hosts, they developed into adults at a faster rate than IJ that were collected with White traps. The difference in development was not due to differential infection rates. No difference in development stages was detected amount the same H. bacteriophora treatments.
entomopathogenic nematodes; Heterorhabditis bacteriophora; host; rearing conditions; Steinernema carpocapsae
Infective-stage juveniles of Steinernema and Heterorhabditis spp. were cryopreserved using two-stage incubation in glycerol and 70% methanol before storage in cryotubes in liquid nitrogen. Optimal glycerol concentrations and incubation times for survival were determined for different species, but acceptable survival of all species and isolates of entomopathogenic nematodes can be obtained using 15% (w/w) glycerol and incubation for 48 hours. Mean survival was 69% for isolates of Steinernema and 68% for isolates of Heterorhabditis (n = 84). The maximum survival recorded was 97% for S. feltiae K254 stored in liquid nitrogen for 12 months.
cryopreservation; Heterorhabditis; nematode; Steinerneraa
Rifampin resistant (RifR) mutants of the insect pathogenic bacterium Photorhabdus luminescens LN2 from entomopathogenic nematode Heterorhabditis indica LN2 were genetically and proteomically characterized. The RifR mutants showed typical phase one characters of Photorhabdus bacteria, and insecticidal activity against Galleria mellonella larvae, but surprisingly influenced their nematicidal activity against axenic infective juveniles (IJs) of H. bacteriophora H06, an incompatible nematode host. 13 out of 34 RifR mutants lost their nematicidal activity against H06 IJs but supported the reproduction of H06 nematodes. 7 nematicidal-producing and 7 non-nematicidal-producing RifR mutants were respectively selected for rpoB sequence analysis. rpoB mutations were found in all 14 RifR mutants. The rpoB (P564L) mutation was found in all 7 mutants which produced nematicidal activity against H06 nematodes, but not in the mutants which supported H06 nematode production. Allelic exchange assays confirmed that the Rif-resistance and the impact on nematicidal activity of LN2 bacteria were conferred by rpoB mutation(s). The non-nematicidal-producing RifR mutant was unable to colonize in the intestines of H06 IJs, but able to colonize in the intestines of its indigenous LN2 IJs. Proteomic analysis revealed different protein expression between wild-type strain and RifR mutants, or between nematicidal-producing and non nematicidal-producing mutants. At least 7 putative proteins including DsbA, HlpA, RhlE, RplC, NamB (a protein from T3SS), and 2 hypothetical proteins (similar to unknown protein YgdH and YggE of Escherichia coli respectively) were probably involved in the nematicidal activity of LN2 bacteria against H06 nematodes. This hypothesis was further confirmed by creating insertion-deletion mutants of three selected corresponding genes (the downregulated rhlE and namB, and upregualted dsbA). These results indicate that the rpoB mutations greatly influence the symbiotic association between the symbionts and their entomopathogenic nematode hosts.
Photorhabdus and Xenorhabdus are Gram-negative, phylogenetically related, enterobacteria, forming mutualism with the entomopathogenic nematodes Heterorhabditis and Steinernema, respectively. The mutualistic bacteria living in the intestines of the nematode infective juveniles are pathogenic to the insect upon release by the nematodes into the insect hemolymph. Such a switch needs activation of genes that promote bacterial virulence. We studied in vivo gene expression in Photorhabdus temperata and Xenorhabdus koppenhoeferi upon infection of the white grub Rhizotrogus majalis using selective capture of transcribed sequences technique.
A total of 40 genes in P. temperata and 39 in X. koppenhoeferi were found to be upregulated in R. majalis hemolymph at 24 h post infection. Genomic presence or upregulation of these genes specific in either one of the bacterium was confirmed by the assay of comparative hybridization, and the changes of randomly selected genes were further validated by quantitative real-time PCR. The identified genes could be broadly divided into seven functional groups including cell surface structure, regulation, virulence and secretion, stress response, intracellular metabolism, nutrient scavenging, and unknown. The two bacteria shared more genes in stress response category than any other functional group. More than 60% of the identified genes were uniquely induced in either bacterium suggesting vastly different molecular mechanisms of pathogenicity to the same insect host. In P. temperata lysR gene encoding transcriptional activator was induced, while genes yijC and rseA encoding transcriptional repressors were induced in X. koppenhoeferi. Lipopolysaccharide synthesis gene lpsE was induced in X. koppenhoeferi but not in P. temperata. Except tcaC and hemolysin related genes, other virulence genes were different between the two bacteria. Genes involved in TCA cycle were induced in P. temperata whereas those involved in glyoxylate pathway were induced in X. koppenhoeferi, suggesting differences in metabolism between the two bacteria in the same insect host. Upregulation of genes encoding different types of nutrient uptake systems further emphasized the differences in nutritional requirements of the two bacteria in the same insect host. Photorhabdus temperata displayed upregulation of genes encoding siderophore-dependent iron uptake system, but X. koppenhoeferi upregulated genes encoding siderophore-independent ion uptake system. Photorhabdus temperata induced genes for amino acid acquisition but X. koppenhoeferi upregulated malF gene, encoding a maltose uptake system. Further analyses identified possible mechanistic associations between the identified gene products in metabolic pathways, providing an interactive model of pathogenesis for each bacterium species.
This study identifies set of genes induced in P. temperata and X. koppenhoeferi upon infection of R. majalis, and highlights differences in molecular features used by these two closely related bacteria to promote their pathogenicity in the same insect host.
The genetic diversity of symbiotic Xenorhabdus and Photorhabdus bacteria associated with entomopathogenic nematodes was examined by a restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes (rDNAs). A total of 117 strains were studied, most of which were isolated from the Caribbean basin after an exhaustive soil sampling. The collection consisted of 77 isolates recovered from entomopathogenic nematodes in 14 Caribbean islands and of 40 reference strains belonging to Xenorhabdus and Photorhabdus spp. collected at various localities worldwide. Thirty distinctive 16S rDNA genotypes were identified, and cluster analysis was used to distinguish the genus Xenorhabdus from the genus Photorhabdus. The genus Xenorhabdus appears more diverse than the genus Photorhabdus, and for both genera the bacterial genotype diversity is in congruence with the host-nematode taxonomy. The occurrence of symbiotic bacterial genotypes was related to the ecological distribution of host nematodes.