Host range, specificity, and virulence of Steinernema feltiae, Steinernema rarum, and Heterorhabditis bacteriophora (Steinernematidae and Heterorhabditidae) from Argentina

Infections were carried out in the laboratory to determine the host range, specificity, and virulence of Steinernema rarum, S. feltiae, and Heterorhabditis bacteriophora that were isolated from different regions of Argentina. All insect orders showed a remarkable susceptibility to the three nematode species, showing mortality values higher than 66%, except for Anoplura. The mortality of the insects of agronomic interest was more than 90%. As for insects of sanitary importance, S. feltiae and S. rarum caused 60% of mortality, whereas H. bacteriophora caused 80%. The results fluctuated when considering the buccal apparatus function of the insects. Nematodes completed their cycle in anoplurans, orthopterans, lepidopterans, and hymenopterans, while the development varied in coleopterans, homopterans, hemipterans, and dipterans. S. rarum developed completely both in immature and adult, while S. feltiae and H. bacteriophora developed preferably in immature. The three isolates are capable of parasitizing a wide host range under laboratory conditions; H. bacteriophora is more virulent than the evaluated Steinernema spp. against Galleria mellonella larvae; the pathogenicity and specificity depend on the bioecological characteristics of nematodes and hosts.     

Effects of Microbial and Other Antagonistic Organism and Competition on Entomopathogenic Nematodes

Antagonistic factors, broadly identified as antibiosis, competition and natural enemies, impact on entomopathogenic nematodes. Antibiosis can occur through the release of plant chemicals from the roots into the soil, which may adversely affect the host-finding behavior of the infective stage nematode, or the presence of these chemicals in the host insect may negatively affect nematode reproduction. In laboratory studies, intra-specific and inter-specific competition reduces nematode fitness, and inter-specific competition can cause local extinction of a nematode species. For example, after concomitant infection of a host, a steinernematid species usually excludes a heterorhabditid species. The mechanism for the steinernematid superiority has been postulated to be a bacteriocin(s) produced by Xenorhabdus, the symbiotic bacterium of the steinernematid, which prevents Photorhabdus, the symbiotic bacterium of the heterorhabditid, from multiplying. Inter-specific competition between two steinernematid species shows that both can co-exist in a host, but one species will eventually prevail in the environment. By having different foraging strategies, however, both steinermatid species may co-exist in the same habitat. An important issue is whether the introduction of an exotic entomopathogenic nematode species will competitively displace an indigenous nematode species. Although the environmental risks are small, the recommended policy is that the introduction of exotic nematodes be regulated. With other pathogens, entomopathogenic nematodes can out-compete entomopathogenic fungi, but not Bacillus thuringiensis, for the same host individual when both the nematode and entomopathogen are applied simultaneously. The best studied natural enemy is the nematophagous fungus, Hirsutella rhossiliensis, which causes higher mortality in Steinernema glaseri compared with Heterorhabditis bacteriorphora. Differential susceptibility to the fungus may be associated with the retention of the second-stage cuticle by H. bacteriophora. Invertebrate predators including mites and collembolans feed on entomopathogenic nematodes. Although a number of studies have been conducted with antagonists, there is a dearth of field data. We suggest that long-term research plots be established where natural populations of entomopathogenic nematodes occur and include antagonists as a component of such studies.     

Characterization of New Entomopathogenic Nematodes from Thailand: Foraging Behavior and Virulence to the Greater Wax Moth, Galleria mellonella L. (Lepidoptera: Pyralidae)

Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis and their associated bacteria (Xenorhabdus spp. and Photorhabdus spp., respectively) are lethal parasites of soil dwelling insects. We collected 168 soil samples from five provinces, all located in southern Thailand. Eight strains of EPNs were isolated and identified to species using restriction profiles and sequence analysis. Five of the isolates were identified as Heterorhabditis indica, and one as Heterorhabditis baujardi. Two undescribed Steinernema spp. were also discovered which matched no published sequences and grouped separately from the other DNA restriction profiles. Behavioral tests showed that all Heterorhabditis spp. were cruise foragers, based on their attraction to volatile cues and lack of body-waving and standing behaviors, while the Steinernema isolates were more intermediate in foraging behavior. The infectivity of Thai EPN strains against Galleria mellonella larvae was investigated using sand column bioassays and the LC(50) was calculated based on exposures to nematodes in 24-well plates. The LC(50) results ranged from 1.99-6.95 IJs/insect. Nine centimeter columns of either sandy loam or sandy clay loam were used to determine the nematodes' ability to locate and infect subterranean insects in different soil types. The undescribed Steinernema sp. had the greatest infection rate in both soil types compared to the other Thai isolates and three commercial EPNs (Heterorhabditis bacteriophora, Steinernema glaseri and Steinernema riobrave).     

Directional movement of entomopathogenic nematodes in response to electrical field: effects of species, magnitude of voltage, and infective juvenile age

Entomopathogenic nematodes respond to a variety of stimuli when foraging. Previously, we reported a directional response to electrical fields for two entomopathogenic nematode species; specifically, when electrical fields were generated on agar plates Steinernema glaseri (a nematode that utilizes a cruiser-type foraging strategy) moved to a higher electric potential, whereas Steinernema carpocapsae, an ambush-type forager, moved to a lower potential. Thus, we hypothesized that entomopathogenic nematode directional response to electrical fields varies among species, and may be related to foraging strategy. In this study, we tested the hypothesis by comparing directional response among seven additional nematode species: Heterorhabditis bacteriophora, Heterorhabditis georgiana, Heterorhabditis indica, Heterorhabditis megidis, Steinernema feltiae, Steinernema riobrave, and Steinernema siamkayai. S. carpocapsae and S. glaseri were also included as positive controls. Heterorhabditids tend toward cruiser foraging approaches whereas S. siamkayai is an ambusher and S. feltiae and S. riobrave are intermediate. Additionally, we determined the lowest voltage that would elicit a directional response (tested in S. feltiae and S. carpocapsae), and we investigated the impact of nematode age on response to electrical field in S. carpocapsae. In the experiment measuring diversity of response among species, we did not detect any response to electrical fields among the heterorhabditids except for H. georgiana, which moved to a higher electrical potential; S. glaseri and S. riobrave also moved to a higher potential, whereas S. carpocapsae, S. feltiae, and S. siamkayai moved to a lower potential. Overall our hypothesis that foraging strategy can predict directional response was supported (in the nematodes that exhibited a response). The lowest electric potential that elicited a response was 0.1 V, which is comparable to electrical potential associated with some insects and plant roots. The level of response to electrical potential diminished with nematode age. These results expand our knowledge of electrical fields as cues that may be used by entomopathogenic nematodes for host-finding or other aspects of navigation in the soil.     

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.     

昆虫病原线虫资源概况和分类技术进展

昆虫病原线虫是具有重要潜在应用价值的害虫生物防治资源,主要包括斯氏线虫科（Steinernematidae）的斯氏线虫属Steinernema与新斯氏线虫属Neosteinernema线虫和异小杆线虫科（Heterorhabditidae）的异小杆线虫属Heterorhabditis线虫。近10年来,分子生物学方法与传统的形态学方法相结合应用到线虫的鉴定与分类,昆虫病原线虫的分类进入稳定与发展时期,越来越多的新种或品系被发现及应用于生物防治。目前已描述的昆虫病原线虫种类达65种,其中斯氏线虫属52种,新斯氏线虫属1种,异小杆线虫属12种。本文整理列出了迄今报道的昆虫病原线虫种类及其来源,并综述了昆虫病原线虫分类现状以及鉴定与分类方法上的研究进展,重点阐述了分子生物学技术在昆虫病原线虫鉴定与分类的应用状况。     

Present and future prospects for entomopathogenic nematode products

Entomopathogenic nematodes in the genera Steinernema and Heterorhabditis have emerged as excellent candidates for biological control of insect pests. Attributes making the nematodes ideal biological insecticides include their broad host range, high virulence, safety for nontarget organisms and high efficacy in favourable habitats. Progress achieved in liquid fermentation, formulation stability and application strategy has allowed nematode‐based products to become competitive with chemical insecticides in medium‐ and high‐value crops on the basis of cost/benefit ratio and ease of application. Further technological advancements are needed to expand and improve the market potential of the nematode‐based products.     

Aggregative group behavior in insect parasitic nematode dispersal

Movement behavior of foraging animals is critical to the determination of their spatial ecology and success in exploiting resources. Individuals sometimes gain advantages by foraging in groups to increase their efficiency in garnering these resources. Group movement behavior has been studied in various vertebrates. In this study we explored the propensity for innate group movement behavior among insect parasitic nematodes. Given that entomopathogenic nematodes benefit from group attack and infection, we hypothesised that the populations would tend to move in aggregate in the absence of extrinsic cues. Movement patterns of entomopathogenic nematodes in sand were investigated when nematodes were applied to a specific locus or when the nematodes emerged naturally from infected insect hosts; six nematode species in two genera were tested (Heterorhabditis bacteriophora, Heterorhabditis indica, Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri and Steinernema riobrave). Nematodes were applied in aqueous suspension via filter paper discs or in infected insect host cadavers (to mimic emergence in nature). We discovered that nematode dispersal resulted in an aggregated pattern rather than a random or uniform distribution; the only exception was S. glaseri when emerging directly from infected hosts. The group movement may have been continuous from the point of origin, or it may have been triggered by a propensity to aggregate after a short period of random movement. To our knowledge, this is the first report of group movement behavior in parasitic nematodes in the absence of external stimuli (e.g., without an insect or other apparent biotic or abiotic cue). These findings have implications for nematode spatial distribution and suggest that group behavior is involved in nematode foraging.     

Susceptibility of the Carrot Weevil (Coleoptera: Curculionidae) to Steinernema feltiae,S. bibionis,and Heterorhabditis heliothidis

Larvae, pupae, and adults of the carrot weevil (Listronotus oregonensis) were infected and killed by the three entomophagous nematodes (Steinernema feltiae, S. bibionis, and Heterorhabditis heliothidis) under controlled conditions. Third-stage larvae were more susceptible than pupae or adults. S. feltiae and S. bibionis were the most aggressive nematode species, causing larval mortality after 24-48 hours in both continuous and 2-hour contact with nematode suspension. The nematodes multiplied sufficiently in all insects at all stages of development; however, production of infective-stage larvae per host cadaver was variable.     

Susceptibility of diamond back moth,Plutella xylostella (L) to entomopathogenic nematodes

Eight entomopathogenic nematode species / strains, Steinernema glaseri (steiner), S. carpocapsae (Weiser), S. feltiae (Filipjev), Steinernema sp. Ecomax strain, Heterorhabditis bacteriophora (Pioner), Heterorhabditis sp. Ecomax strain, two locally isolated strains called as JFC and TFC were tested against the final instar larvae of diamond back moth, Plutella xylostella (L.). All nematodes were found pathogenic. However, H. bacteriophora was adjudged the most pathogenic amongst the test nematodes on the basis of LD50 (9.16 IJS/larva), LT50 (43.26 hr), Lex T50 (3.24 hr) and the propagation potential (average of 271.42 IJS/mg) on the host body weight.     

Entomopathogenic Nematode Infectivity Assay: Comparison of Penetration Rate into Different Hosts

Penetration rate (the percentage of the initial infective juvenile inoculum that invades an insect host) was tested as an indicator of entomopathogenic nematode infectivity. Several host-parasite-substrate combinations were evaluated for penetration rate. Four steinernematids, Steinernema carpocapsae, S. glaseri, S. feltiae, S. riobravis and two strains of Heterorhabditis bacteriophora were tested in a contact bioassay against the wax moth, Galleria mellonella, the yellow meal worm, Tenebrio molitor, the beet armyworm, Spodoptera exigua, the black cutworm, Agrotis ipsilon, and the European corn borer, Ostrinia nubilalis. The insect larvae were confined individually in sand and filter paper arenas and exposed to 200 infective juveniles. After incubation, dead insects were dissected in order to count the nematodes penetrated. The data were analyzed for the effects of nematode strain and substrate on penetration rate. The bioassay substrate had a variable effect depending on the insect species. The nematode effect was highly significant for all insects tested. The penetration rate therefore allowed comparisons among nematode strains invading a host. Nematode ranking for infectivity differed according to the insect tested.     

Diversity and phylogenetic relationships of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) from the Sky Islands of Southern Arizona

A survey for entomopathogenic nematodes was conducted in oak-juniper woodlands of four mountain ranges (Santa Rita, Santa Catalina, Pinale?o, and Chiricahuas), in southeastern Arizona. From a total of 120 soil samples, 23.3% were EPN-positive. Of them 78.5% were positive for Steinernema spp. and 21.5% were positive for Heterorhabditis spp. An integrated approach, combining both traditional (morphological) and molecular methods, was used for examining the diversity of species of these entomopathogenic nematodes. Two named-species S. oregonense and S. riobrave are reported for the first time in Arizona, expanding their currently known geographic range. In addition to this, three undescribed Steinernema and three Heterorhabditis spp. were recovered. Insular evolution, in part, could account for the geographic distribution of entomopathogenic nematodes in Arizona.     

Legislation on the Introduction of Exotic Entomopathogenic Nematodes into Australia and New Zealand

There are stringent requirements for the importation of all exotic organisms into Australia and New Zealand but since both countries have already permitted the importation and release of some species of both Heterorhabditis and Steinernema , the difficulties of the importation of entomopathogenic nematodes are reduced. In both countries, a series of authorities must be consulted before importation is permitted but only in New Zealand must entomopathogenic nematodes be registered before commercial trials and sales are allowed. Registration not only entails a thorough evaluation of the nematode species and its formulation for a wide range of possible harmful effects to humans, crops and the environment, but efficacy must be demonstrated for each species of nematode in each type of formulation against each pest.     

A sensory code for host seeking in parasitic nematodes

Parasitic nematode species often display highly specialized host-seeking behaviors that reflect their specific host preferences. Many such behaviors are triggered by host odors, but little is known about either the specific olfactory cues that trigger these behaviors or the underlying neural circuits. Heterorhabditis bacteriophora and Steinernema carpocapsae are phylogenetically distant insect-parasitic nematodes whose host-seeking and host-invasion behavior resembles that of some devastating human- and plant-parasitic nematodes. We compare the olfactory responses of Heterorhabditis and Steinernema infective juveniles (IJs) to those of Caenorhabditis elegans dauers, which are analogous life stages. The broad host range of these parasites results from their ability to respond to the universally produced signal carbon dioxide (CO(2)), as well as a wide array of odors, including host-specific odors that we identified using thermal desorption-gas chromatography-mass spectroscopy. We find that CO(2) is attractive for the parasitic IJs and C. elegans dauers despite being repulsive for C. elegans adults, and we identify a sensory neuron that mediates CO(2) response in both parasitic and free-living species, regardless of whether CO(2) is attractive or repulsive. The parasites' odor response profiles are more similar to each other than to that of C. elegans despite their greater phylogenetic distance, likely reflecting evolutionary convergence to insect parasitism.     

Parasitism of bark scorpion Centruroides exilicauda (Scorpiones: Buthidae) by entomopathogenic nematodes (Rhabditida: Steinernematidae; Heterorhabditidae)

In laboratory bioassays, Steinernema glaseri Steiner, Steinernema riobrave Cabanillas, Poinar & Raulston, Heterorhabditis bacteriophora Poinar, and Heterorhabditis marelatus Liu & Berry were capable of infecting and killing the bark scorpion, Centruroides exilicauda (Wood). Steinernema feltiae (Filipjev) and Steinernema carpocapsae (Weiser) failed to infect C. exilicauda at 22 degrees C. S. glaseri, H. marelatus, and H. bacteriophora caused significant mortality at 22 degrees C, indicating the potential role of these parasites as a biocontrol option. Efficacy of S. glaseri and H. bacteriophora was reduced in an assay conducted at 25 degrees C. Only S. glaseri was able to reproduce in the target host. Dissection of scorpions at the end of the experimental periods revealed inactive juvenile S. riobrave, H. marelatus, and H. bacteriophora nematodes. Both mermithid and oxyurid nematodes have been documented as nematode parasites of scorpions, but rhabditids have not been reported until now. Field studies are warranted to assess the usefulness of entomopathogenic nematodes as biocontrol agents of bark scorpions.     

Ecology in the service of biological control: the case of entomopathogenic nematodes

 Biological control manipulations of natural enemies to reduce pest populations represent large-scale ecological experiments that have both benefited from and contributed to various areas of modern ecology. Unfortunately, economic expediency and the need for rapid implementation often require that biological control programs be based more on trial and error than on sound ecological theory and testing. This approach has led to some remarkable successes but it has also produced dismal failures. This point is particularly well illustrated in the historical development and use of entomopathogenic nematodes for the biological control of insect pests. Intense effort has focused on developing these natural enemies as alternatives to chemical insecticides, in part because laboratory assays indicated that these nematodes possess a broad host range. This illusory attribute launched hundreds of field releases, many of which failed due to ecological barriers to infection that are not apparent from laboratory exposures, where conditions are optimal and host-parasite contact assured. For example, the entomopathogenic nematode Steinernema carpocapsae is a poor choice to control scarab larvae because this nematode uses an ambusher foraging strategy near the soil surface whereas the equally sedentary scarab remains within the soil profile, shows a weak host recognition response to scarabs, has difficulty overcoming the scarab immune response, and has low reproduction in this host. Conversely, two other nematodes, Heterorhabditis bacteriophora and S. glaseri, are highly adapted to parasitize scarabs: they use a cruising foraging strategy, respond strongly to scarabs, easily overcome the immune response, and reproduce well in these hosts. Increased understanding of the ecology of entomopathogenic nematodes has enabled better matches between parasites and hosts, and more accurate predictions of field performance. These results underline the importance of a strong partnership between basic and applied ecology in the area of biological control. Received: 15 July 1996 / Accepted: 5 November 1996     

Susceptibility of lady beetles (Coleoptera: Coccinellidae) to entomopathogenic nematodes

We investigated differential susceptibility of lady beetles to entomopathogenic nematodes, for two reasons: (1) to estimate potential nontarget effects on natural lady beetle populations, (2) to compare the susceptibility of exotic versus native lady beetle species. We hypothesize that successful establishment of some exotically introduced arthropods may be due, in part, to a lower susceptibility relative to competing native species. In laboratory studies, we compared the pathogenicity, virulence, and reproductive capacity of Heterorhabditis bacteriophora and Steinernema carpocapsae among two native (Coleomegilla maculata and Olla v-nigrum) and two successfully established exotic (Harmonia axyridis and Coccinella septempunctata) lady beetles, and a known susceptible lepidopteran host, Agrotis ipsilon. After 1 and 2 days of exposure to either nematode species, mortality of A. ipsilon was higher than in all lady beetles. Thus, we predict that nematode field applications would have significantly less impact on lady beetle populations than on a susceptible target pest. Additionally, the impact of soil-applied nematodes may be lower on lady beetles than on soil-dwelling hosts because the former spends relatively less time on the soil. Exotic lady beetles were less susceptible to nematode infection than native species. Reproductive capacity data also indicated lower host suitability in H. axyridis, but not in C. septempunctata. Overall, the hypothesis that low susceptibility to pathogens in certain exotic lady beetles may have contributed to competitive establishment was supported (especially for H. axyridis). Additional studies incorporating different hosts and pathogens from various geographic locations will be required to further address the hypothesis.     

Seasonal dynamics of entomopathogenic nematodes of the genera Steinernema and Heterorhabditis as a response to abiotic factors and abundance of insect hosts

The seasonal dynamics of entomopathogenic nematodes (EPNs) of the genus Steinernema and Heterorhabditis were studied during one season in meadow and oak wood habitats, in the vicinity of Ceské Budejovice, Czech Republic. The influences of soil temperature, moisture, and abundance of suitable hosts on EPN dynamics were investigated. The host range of these nematodes, in both habitats was also observed. A total of four EPN species were found in both habitats. Steinernema affine was the dominant species both in oak wood and in meadow. Additionally, the oak wood habitat was inhabited by S. kraussei and S. weiseri; the meadow habitat by Heterorhabditis bacteriophora. The mean abundance of total EPN community was 28,000ind./m(2) in oak wood and 11,000ind./m(2) in meadow. The seasonal dynamics of entomopathogenic nematodes in both habitats were characterized by high nematode densities in the beginning of the season, followed by a rapid decrease, and then stabilization. EPN abundances did not show any apparent correlation with soil temperature and moisture, but they were negatively correlated with the abundance of suitable insect hosts. Inter- and intraspecific competition for limited nutrients (hosts) probably played a major role in EPN seasonal dynamics. Broad host range of entomopathogenic nematodes in both habitats was predominantly represented by dipteran and coleopteran larvae. Most common hosts belonged to the families Asilidae, Bibionidae, and Empididae (Diptera), as well as Carabidae and Curculionidae (Coleoptera).     

Attraction of four entomopathogenic nematodes to four white grub species

To better understand the differences in the efficacy of entomopathogenic nematode species against white grub species, we are studying the various steps of the infection process of entomopathogenic nematodes into different white grub species using nematode species/strains with particular promise as white grub control agents. In this study we compared the attraction of the entomopathogenic nematodes Steinernema scarabaei (AMK001 strain), Steinernema glaseri (NC1 strain), Heterorhabditis zealandica (X1 strain), and Heterorhabditis bacteriophora (GPS11 strain) to third-instars of the scarabs Popillia japonica, Anomala orientalis, Cyclocephala borealis, and Rhizotrogus majalis, and late-instar greater wax moth, Galleria mellonella, larvae. Individual larvae were confined at the bottom of 5.5 cm vertical sand columns, nematodes added to the sand surface after 24 h, and nematodes extracted after another 24 h. Nematode attraction to hosts was strongly affected by nematode species but the effect of insect species varied with nematode species. S. glaseri had a high innate dispersal rate (i.e., in absence of insects) and was strongly attracted to insects without significant differences among insect species. S. scarabaei had a very low innate dispersal rate so that even a strong relative response to insects resulted in low absolute dispersal rates toward insects. S. scarabaei tended to be most attracted to G. mellonella and least attracted to C. borealis. H. zealandica had a high innate dispersal rate but only responded weakly to insects without significant differences among species. H. bacteriophora had limited innate dispersal and only weakly responded to insects with G. mellonella tending to be the most attractive and C. borealis the least attractive insect. It has to be noted that we cannot exclude that the use of different rearing hosts (A. orientalis and P. japonica larvae for S. scarabaei, G. mellonella larvae for the other nematodes) might have had an impact on the nematodes dispersal and relative attraction behavior. This study indicates that host attractiveness and nematode dispersal rates may contribute but do not play a major role in the variability in white grub susceptibility and/or nematode virulence.