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).     

Facultative scavenging as a survival strategy of entomopathogenic nematodes

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

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.     

Mutualistic association of Photorhabdus asymbiotica with Japanese heterorhabditid entomopathogenic nematodes

Gram-negative bacteria, Photorhabdus luminescens and P. temperata, form a mutualistic association with entomopathogenic heterorhabditid nematodes while P. asymbiotica is known as an opportunistic human pathogen that causes disseminated bacteremic spread on two continents, the United States and Australia. In the course of our phylogenetic study of Photorhabdus bacteria associated with Japanese Heterorhabditis nematodes, we found two Photorhabdus isolates (Photorhabdus sp. Cbkj163 and OnIr40) whose partial 16S rRNA gene sequence showed high similarities to clinical isolates of this pathogen from Heterorhabditis indica. The phylogenetic study, based upon the gyrase subunit B gene sequences of the two isolates, revealed clustering with these clinical isolates of P. asymbiotica from both the United States and Australia but not with other Photorhabdus bacteria associated with nematodes. The two bacterial isolates were also found to share microbiological and biochemical characteristics with clinical and entomopathogenic Photorhabdus strains. Moreover, not only the two novel Photorhabdus isolates but also an Australian clinical isolate of P. asymbiotica formed mutualistic association with H. indica isolates. These data suggest that the bacteria isolated from H. indica CbKj163 and OnIr40 are a novel subspecies of P. asymbiotica, and that some clinical isolates of P. asymbiotica could have originated from bacteria associated with entomopathogenic nematodes.     

Laboratory Evaluation of Seven Pakistani Strains of Entomopathogenic Nematodes Against a Stored Grain Insect Pest, Pulse beetle Callosobruchus chinensis (L.)

Seven Pakistani strains of entomopathogenic nematodes belonging to the genera Steinernema and Heterorhabditis were tested against last instar and adult stages of the pulse beetle, Callosobruchus chinensis (L.). These nematodes included Steinernema pakistanense Shahina, Anis, Reid and Maqbool (Ham 10 strain); S. asiaticum Anis, Shahina, Reid and Rowe (211 strain); S. abbasi Elawad, Ahmad and Reid (507 strain); S. siamkayai Stock, Somsook and Reid (157 strain); S. feltiae Filipjev (A05 strains); Heterorhabditis bacteriophora Poinar (1743 strain); and H. indica Poinar, Karunakar and David (HAM-64 strain). Activity of all strains was determined at four different nematode densities in Petri dishes and in concrete containers. A significant nematode density effect was detected for all nematode species tested. Overall, Heterorhabditis bacteriophora, S. siamkayai, and S. pakistanense were among those that showed the highest virulence to pulse beetle larvae and adults. For all nematode species, the last larval stage of the pulse beetle seems to be more susceptible than the adult. LC(50) values in Petri dish and concrete containers were 14-340 IJs/larvae and 41-441 IJs/larvae, respectively, and 59-1376 IJs/adult and 170-684/adult, respectively.     

Distribution of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) in natural habitats in California, USA

A total of 270 soil samples from 30 different habitats in 10 geographic regions of California were evaluated for the presence of rhabditid entomopathogenic nematodes. Nematodes were isolated from 26.3% of the samples. The recovered isolates were identified as Steinernema carpocapsae, S. feltiae, S. kraussei, S. longicaudum, S. oregonense, Heterorhabditis marelatus and H.bacteriophora. Among the steinernematids, S. kraussei and S. feltiae were the most commonly encountered species, generally occurring in acidic soils high in organic matter. Among the heterorhabditids, H. bacteriophora was isolated along the southern coast, whereas H. marelatus was recovered along the northern coast of California. Steinernematids were recovered from coniferous forests, oak woodlands and grasslands whereas heterorhabditids were isolated from coastal marshes.     

Diversity and distribution of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) in Turkey

The diversity and distribution of entomopathogenic nematodes in thefamilies Steinernematidae and Heterorhabditidae were assessed throughout anextensive soil survey in Turkey during 1999 and 2000. Entomopathogenic nematodeswere recovered from six out of seven regions sampled, with 22 positive sites(2%) out of 1080 sites sampled. A single nematode isolate was recovered at eachof the positive sites, of which 15 were steinernematid isolates and seven wereheterorhabditid isolates representing a total of four species. Based onmorphometric and molecular data, the nematode species were identified asHeterorhabditis bacteriophora, Steinernemafeltiae, S. affine, andSteinernema n. sp. The most common species was S.feltiae, which was isolated from 10 sites in six regions, followed byH. bacteriophora from seven sites in five regions,S. affine from four sites in two regions, andSteinernema n. sp. from one site. Heterorhabditisbacteriophora and S. feltiae have been found inmany parts of the world, whereas S. affine, so far, hasonly been recovered in Europe until our survey. Steinernemaaffine was isolated from the European (Marmara) as well as theAsiatic region (Middle Anatolia) of Turkey. A new undescribedSteinernema sp. was isolated from the most eastern region(East Anatolia) of Turkey. Soils of the positive sites were classified as sandy,sandy loam, or loam (68.2%) and sandy–clay–loam or clay loam (31.8%) and the pHranged from 5.6 to 7.9. The habitats from which the entomopathogenic nematodeswere isolated were broadly classified as disturbed (59.1%), which includedagricultural fields and poplar planted for lumber and wind breaks, andundisturbed (40.9%), which included pine forest, grassland, marsh and reed sites.Steinernema feltiae, S. affine, andH. bacteriophora were recovered from both disturbed andundisturbed habitats. The new Steinernema sp. was recoveredfrom grassland. Our survey showed that these nematodes occur widely throughoutTurkey, but at a frequency below that reported for other parts of the world.     

Host cadavers protect entomopathogenic nematodes during freezing

The entomopathogenic nematodes Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema glaseri, and Steinernema feltiae were exposed to freezing while inside their hosts. Survival was assessed by observing live and dead nematodes inside cadavers and by counting the infective juveniles (IJs) that emerged after freezing. We (1) measured the effects of 24h of freezing at different times throughout the course of an infection, (2) determined the duration of freezing entomopathogenic nematodes could survive, (3) determined species differences in freezing survival. Highest stage-specific survival was IJs for S. carpocapsae, and adults for H. bacteriophora. When cadavers were frozen two or three days after infection, few IJs emerged from them. Freezing between five and seven days after infection had no negative effect on IJ production. No decrease in IJ production was measured for H. bacteriophora after freezing. H. bacteriophora also showed improved survival inside versus outside their host when exposed to freezing.     

Molecular and morphological characterization of heterorhabditid entomopathogenic nematodes from the tropical rainforest in Brazil

Despite massive losses of primary forest, the Amazonian rainforest remains an extremely rich source of biodiversity. In recent years, entomopathogenic nematodes (EPNs) have been isolated from soil in various parts of the world and used successfully as biological control agents against numerous insect pests. Therefore, a sampling in the rainforest of Monte Negro, Rond?nia, Brazil was conducted with the aim of discovering new strains and/or species of EPNs for future development as biological control agents. From 156 soil samples taken at nine collecting sites, 19 isolates were obtained, all of them belonging to the genus Heterorhabditis. Four strains were subjected to detailed morphological and molecular evaluation. Based on morphometrics and internal transcribed spacer (ITS) sequence data, the strains LPP1, LPP2 and LPP4 were identified as Heterorhabditis indica, whereas LPP7 was considered Heterorhabditis baujardi. Comparative analysis of the ITS1 sequence of H. indica and H. baujardi isolates showed a polymorphic site for the restriction enzyme Tth 111 that could be used to distinguish the two species. Consequently, strains LPP1, LPP2, LPP3, LPP4, and LPP9 were identified as H. indica, whereas LPP5, LPP7, LPP8 and LPP10 were identified as H. baujardi.     

Effectiveness of different species of entomopathogenic nematodes for biocontrol of the Mediterranean flatheaded rootborer, Capnodis tenebrionis (Linné) (Coleoptera: Buprestidae) in potted peach tree

The susceptibility of larvae of the Mediterranean flatheaded rootborer (Capnodis tenebrionis) to 13 isolates of entomopathogenic nematodes was examined using GF-677 potted trees (peachxalmond hybrid) as the host plant. The nematode strains tested included nine Steinernema feltiae, one S. affine, one S. carpocapsae and two Heterorhabditis bacteriophora. Nematodes showed the ability to locate and kill larvae of C. tenebrionis just after they enter into the roots of the tree. S. feltiae strains provided an efficacy ranging from 79.68% to 88.24%. H. bacteriophora strains resulted in control of 71.66-76.47%. S. carpocapsae (B14) and S. affine (Gspe3) caused lower control of C. tenebrionis larvae (62.03% and 34.76%, respectively). The influence of foraging strategy and the use of autochthonous nematodes to control C. tenebrionis larvae inside the roots is discussed.     

Application technology for the entomopathogenic nematodes Heterorhabditis indica and Steinernema sp. (Rhabditida: Heterorhabditidae and Steinernematidae) to control Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) in corn

The effects of different application technologies were evaluated on the concentration, viability, and efficiency of infective juveniles of the nematodes Heterorhabditis indica Poinar, Karunakar & David and Steinernema sp. (IBCB-n6) to control Spodoptera frugiperda Smith on corn plants. Two hundred and eighty infective juveniles of Steinernema sp. were required to kill 100% third-instar fall armyworms in petri dishes, as compared to 400 infective juveniles of the H. indica nematode to obtain 75% fall armyworm control. It is possible to spray entomopathogenic nematodes without significant loss in their concentration and viability, with equipment that produces electrical charges to the spraying mix, and with those using hydraulic and rotary nozzle tips. The concentrations of infective juveniles of H. indica and Steinernema sp. nematodes were reduced by 28% and 53%, respectively, when hydraulic spraying nozzles that require 100-mesh filtrating elements were used. Tensoactive agents of the organosilicone and ethoxylate groups did not affect the viability of infective juveniles of Steinernema sp. juveniles. Spraying corn plants (V6 growth stage) with up to 288 million infective juveniles of Steinernema sp. per hectare, diluted in the spraying mix up to 800 L ha-1, with 0.01% ethoxylate tensoactive agent, or at the same volume followed by artificial rain (6 mm water depth) was not sufficient to control S. frugiperda in a controlled environment.     

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.     

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

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

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.     

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).     

Steinernema aciari sp. n. (Nematoda: Steinernematidae), a new entomopathogenic nematode from Guangdong, China

A new species of entomopathogenic nematode, Steinernema aciari sp. n. was described. It was recovered from a soil sample collected from Haimen town, Shantou district in the eastern coast of Guangdong province, the People's Republic of China during a survey for entomopathogenic nematodes. S. aciari sp. n. belongs to the Steinernema glaseri group. It can be separated from all described Steinernema species by the combined morphological and morphometrical characters of various stages of the nematodes. For male, the new species can be recognized by spicule length (86+/-6.3 microm); spicule tip blunt with a hook-like structure; gubernaculum with a short and Y-shaped cuneus and corpus well-separated posteriorly. For infective juvenile, the combination of the following characters: body length (1113+/-68 microm), distance from anterior end to excretory pore (95+/-3.7 microm), tail length (78+/-5.2 microm), and E % (123+/-7) can be used to differentiate the new species from other nematodes. For female, the tail (conoid with a long mamillate terminus and a distinct postanal swelling) and vulva (slightly protruding from body surface with conspicuous double flapped epiptygma) shapes can be used as diagnostic characters for the new species. The new species can also be distinguished from other Steinernema species by DNA sequences of either a partial 28S rDNA or the internal transcribed spacer regions of rDNA, and from the close related species S. glaseri, Steinernema longicaudum CWL05, and Steinernema guangdongense by cross-breeding test.     

Ecological characterization of entomopathogenic nematodes isolated in stone fruit orchard soils of Mediterranean areas

Entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae were isolated from stone-fruit orchards in two Mediterranean regions of Spain. A total of 630 soil samples (210 sites) from Catalonia and 90 soil samples (30 sites) from Murcia were evaluated resulting in 5.2% and 20% of the soils testing positive for nematodes, respectively. Ten steinernematid isolates and three heterorhabditid isolates were recovered using the Galleria mellonella baiting method. Based on morphometric data, molecular data, and cross-breeding experiments the nematode species were identified as Steinernemafeltiae and Heterorhabditis bacteriophora. Environmental tolerance to heat, desiccation and hypoxia, the effect of temperature on infectivity and reproduction and nematode migration in sand columns were compared among isolates and one Steinernema carpocapsae strain. Results showed differences among species and a great variability within species. Beneficial traits for each strain were added up to identify a superior candidate to control Mediterranean flat-headed rootborer, Capnodis tenebrionis. When all analyzed factors were considered, three S. feltiae isolates (Bpa, Sor and M116) obtained the best scores, and when hypoxia was removed, two of the strains (Bpa and Sor) continued ranking superior to other strains.     

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.     

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。