Real-time PCR as an effective technique to assess the impact of phoresy by Paenibacillus sp. bacteria on Steinernema diaprepesi nematodes in nature

Quantitative real‐time PCR (qPCR) is a powerful tool to study species of cryptic organisms in complex food webs. This technique was recently developed to detect and quantify several species of entomopathogenic nematodes (EPNs), which are widely used for biological control of insects, and some natural enemies of EPNs such as nematophagous fungi and the phoretic bacteria Paenibacillus sp. and Paenibacillus nematophilus. A drawback to the use of primers and TaqMan probes designed for Paenibacillus sp. is that the qPCR also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two closely related species that are not phoretically associated with EPNs. Here, we report that the detection of Paenibacillus sp. DNA in nematode samples was two orders of magnitude greater (P < 0.001) when the bacterium was added to soil together with its EPN species‐specific host Steinernema diaprepesi than when it was added concomitantly with other EPNs or with species of bacterial‐feeding nematodes. Just 6% of samples detected trace amounts of P. thiaminolyticus and P. popilliae exposed to the same experimental conditions. Thus, although the molecular assay detects Paenibacillus spp. DNA in nonphoretic associations, the levels are essentially background compared to the detection of Paenibacillus sp. in association with its nematode host.     

Bionomics of a Phoretic Association Between Paenibacillus sp. and the Entomopathogenic Nematode Steinernema diaprepesi

Spores of an unidentified bacterium were discovered adhering to cuticles of third-stage infective juvenile (IJ) Steinernema diaprepesi endemic in a central Florida citrus orchard. The spores were cup-shaped, 5 to 6 mm in length, and contained a central endospore. Based on 16S rDNA gene sequencing, the bacterium is closely related to the insect pathogens Paenibacillus popilliae and P. lentimorbus. However, unlike the latter bacteria, the Paenibacillus sp. is non-fastidious and grew readily on several standard media. The bacterium did not attach to cuticles of several entomopathogenic or plant-parasitic nematodes tested, suggesting host specificity to S. diaprepesi. Attachment of Paenibacillus sp. to the third-stage cuticle of S. diaprepesi differed from Paenibacillus spp. associated with heterorhabditid entomopathogenic nematodes, which attach to the IJ sheath (second-stage cuticle). The inability to detect endospores within the body of S. diaprepesi indicates that the bacterial association with the nematode is phoretic. The Paenibacillus sp. showed limited virulence to Diaprepes abbreviatus, requiring inoculation of larvae with 108 spores to achieve death of the insect and reproduction of the bacterium. The effect of the bacterium on the nematode population biology was studied in 25-cm-long vertical sand columns. A single D. abbreviatus larva was confined below 15-cm depth, and the soil surface was inoculated with either spore-free or spore-encumbered IJ nematodes. After 7 days, the proportion of IJ below 5-cm depth was seven-fold greater for spore-free IJ than for spore-encumbered nematodes. Mortality of D. abbreviatus larvae was 72% greater (P <= 0.01) for spore-free compared to spore-encumbered S. diaprepesi. More than 5 times as many progeny IJs (P <= 0.01) were produced by spore-free compared to spore-encumbered nematodes. These data suggest that the bacterium is a component of the D. abbreviatus food web with some potential to regulate a natural enemy of the insect.     

New citriculture system suppresses native and augmented entomopathogenic nematodes

Since its first detection in 2005, the bacterial disease huanglongbing (HLB or citrus greening) has emerged as a critical threat to the citrus industry in Florida. An “Advanced Production System” (APS) could mitigate the impact of HLB by bringing citrus trees into production more quickly and economically than conventional citriculture methods. However, unlike conventional practices, APS fertigates plants daily, thereby changing the soil properties in ways that might impact soil biota. We tested the hypothesis that changes to soil properties caused by APS would affect the abundance of native entomopathogenic nematodes (EPNs) and/or the survival of augmented EPNs. The densities of organisms at different trophic levels were measured by real-time qPCR in three experiments conducted in an ongoing field experiment. Target organisms included 6 entompathogenic nematodes, 5 nematophagous fungi (NF) and a phoretic bacterium, Paenibacillus sp. Soil properties, free-living nematodes and citrus fibrous roots were also measured. Compared to soil under conventional citriculture (CC), APS increased soil pH and Mg content, while reducing the electrical conductivity, and content of K, Mn and Fe. The naturally occurring EPN Steinernema diaprepesi was 5 times less abundant in APS plots where these nematodes were more heavily encumbered by the phoretic bacterium Paenibacillus sp., which limits the foraging success of EPNs. In general, when EPNs were augmented in either treatment, fewer Steinernema riobrave than Heterorhabditis indica were recovered and recovery of both species declined rapidly over time. As seen with native S. diaprepesi, fewer augmented S. riobrave were recovered from APS plots in two of the three experiments, whereas the management system did not affect the recovery of H. indica. More of some endoparasitic and trapping NF were recovered from soil augmented with S. riobrave than with H. indica. However, variation in the responses of NF to the management systems suggests that these NF species were not primarily responsible for the steinernematid responses to APS. Although APS has the potential to reduce EPN populations and exacerbate herbivory by subterranean pests such as the root weevil Diaprepes abbreviatus, additional study of the physical causes of this effect may reveal ways to avoid the problem.     

Variations in Immune Response of Popillia japonica and Acheta domesticus to Heterorhabditis bacteriophora and Steinernema Species

The infectivities of Steinernema carpocapsae, S. glaseri, S. scapterisci, and Heterorhabditis bacteriophora to Japanese beetle larvae, Popillia japonica, and house cricket adults, Acheta domesticus, were compared using external exposure and hemocoelic injection. Only H. bacteriophora and S. glaseri caused high P. japonica mortality after external exposure. When nematodes were injected, P. japonica had a strong encapsulation and melanization response to all species except S. glaseri. Heterorhabditis bacteriophora and S. carpocapsae were able to overcome the immune response, but S. scapterisci was not. All species except S. scapterisci were able to kill and reproduce within the host. Only S. scapterisci and S. carpocapsae caused A. domesticus mortality after external exposure. When nematodes were injected, A. domesticus had a strong immune response to all species except S. scapterisci. Steinernema carpocapsae effectively overcame the strong immune response and caused high host mortality, but S. glaseri and H. bacteriophora did not. Steinernema scapterisci caused high host mortality and reproduced, S. glaseri and H. bacteriophora caused low host mortality but only S. glaseri reproduced, and S. carpocapsae was able to kill the host but reproduced poorly. Most (ca. 90%) of the S. carpocapsae in the hemocoel of P. japonica became encapsulated and melanized within 8 hours postinjection. The symbiotic bacterium, Xenorhabduf nematophilus, was often released before this encapsulation and melanization.     

Growth and Virulence of Steinernema glaseri Influenced by Different Subspecies of Xenorhabdus nematophilus

Three Xenorhabdus nematophilus subspecies influenced Steinernema glaseri growth profiles and growth rates, but this was not necessarily because of different bacterial growth rates. Virulence of dauer nematodes in larval Galleria mellonella varied with the number of dauers retaining bacteria and the bacterial subspecies. Virulence was least for dauers grown on X. nematophilus subsp. bovienii because of the lack of retained bacteria. Virulence was subsequently restored by culturing these nematodes on X. nematophilus subsp. poinari.     

Natural occurrence of entomopathogenic nematodes in Liaoning (Northeast China)

Entomopathogenic nematodes (EPNs) can provide effective biological control of pest. In order to contribute to knowledge on these organisms for regional biological control programs, we studied EPN distribution and ecological requirements in Liaoning Province, Northeast China. One hundred and forty-nine soil samples were taken from 36 locations. EPNs were recovered from 22 of the 36 locations (61.11%). Forty-four samples contained steinernematids (89.80%) and 5 samples contained heterorhabditids (10.20%). EPN recovery varied among the different soil and habitat type. Most EPNs were isolated from sandy loam, and most of the samples containing EPNs were collected from woodland and fruit crop habitats. The morphological characters of infective juveniles were used for preliminary species diagnosis. We preliminarily identified 15 species of Steinernematidae (Steinernema litorale, Steinernema silvaticum, Steinernema feltiae, Steinernema bicornutum, Steinernema robustispiculum, Steinernema affine, Steinernema riobrave, Steinernema yirgalemense, Steinernema kushidai, Steinernema scapterisci, Steinernema carpocapsae, Steinernema ritteri, Steinernema tami, Steinernema rarum and Steinernema sasonense) and 4 species of Heterorhabditidae (Heterorhabditis megidis, Heterorhabditis zealandica, Heterorhabditis brevicaudis and Heterorhabditis bajardi).     

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).     

Effect of insect cadaver desiccation and soil water potential during rehydration on entomopathogenic nematode (Rhabditida: Steinernematidae and Heterorhabditidae) production and virulence

We examined the influence of insect cadaver desiccation on the virulence and production of entomopathogenic nematodes (EPNs), common natural enemies of many soil-dwelling insects. EPNs are often used in biological control, and we investigated the feasibility of applying EPNs within desiccated insect cadavers. Desiccation studies were conducted using the factitious host, Galleria mellonella (Lepidoptera: Pyralidae, wax moth larvae) and three EPN species (Heterorhabditis bacteriophora ‘HB1’, Steinernema carpocapsae ‘All’, and Steinernema riobrave). Weights of individual insect cadavers were tracked daily during the desiccation process, and cohorts were placed into emergence traps when average mass losses reached 50%, 60%, and 70% levels. We tracked the proportion of insect cadavers producing infective juveniles (IJs), the number and virulence of IJs produced from desiccated insect cadavers, and the influence of soil water potentials on IJ production of desiccated insect cadavers. We observed apparent differences in the desiccation rate of the insect cadavers among the three species, as well as apparent differences among the three species in both the proportion of insect cadavers producing IJs and IJ production per insect cadaver. Exposure of desiccated insect cadavers to water potentials greater than −2.75 kPa stimulated IJ emergence. Among the nematode species examined, H. bacteriophora exhibited lower proportions of desiccated insect cadavers producing IJs than the other two species. Desiccation significantly reduced the number of IJs produced from insect cadavers. At the 60% mass loss level, however, desiccated insect cadavers from each of the three species successfully produced IJs when exposed to moist sand, suggesting that insect cadaver desiccation may be a useful approach for biological control of soil insect pests.     

Steinernema scapterisci n. sp. (Rhabditida: Steinernematidae)

Steinernema scapterisci n. sp., isolated in Uruguay from the mole cricket Scapteriscus vicinus, can be distinguished from other members in the genus by the presence of prominent cheilorhabdions, an elliptically shaped structure associated with the excretory duct, and a double-flapped epitygma in the first-generation female. The spicules of the male are pointed, tapering smoothly to a small terminus, and the shaft (calomus) is long, bearing a sheath. The gubernaculum has a long, upward-bent anterior part. The ratio of head to excretory pore divided by tail length of the third-stage juvenile is greater for S. scapterisci n. sp. than for S. carpocapsae. Steinernema scapterisci n. sp. did not hybridize with S. carpocapsae strain Breton. In laboratory tests, S. scapterisci n. sp. killed 10% or less of non-orthopteran insects, including the wax moth larva, a universal host for other species of Steinernema.     

Pathogenic effect of entomopathogenic nematode-bacterium complexes on terrestrial isopods

In this study, we evaluated the effect of entomopathogenic nematodes (EPNs) Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora, symbiotically associated with bacteria of the genera Xenorhabdus or Photorhabdus, on the survival of eight terrestrial isopod species. The EPN species S. carpocapsae and H. bacteriophora reduced the survival of six isopod species while S. feltiae reduced survival for two species. Two terrestrial isopod species tested (Armadillidium vulgare and Armadillo officinalis) were found not to be affected by treatment with EPNs while the six other isopod species showed survival reduction with at least one EPN species. By using aposymbiotic S. carpocapsae (i.e. without Xenorhabdus symbionts), we showed that nematodes can be isopod pathogens on their own. Nevertheless, symbiotic nematodes were more pathogenic for isopods than aposymbiotic ones showing that bacteria acted synergistically with their nematodes to kill isopods. By direct injection of entomopathogenic bacteria into isopod hemolymph, we showed that bacteria had a pathogenic effect on terrestrial isopods even if they appeared unable to multiply within isopod hemolymphs. A developmental study of EPNs in isopods showed that two of them (S. carpocapsae and H. bacteriophora) were able to develop while S. feltiae could not. No EPN species were able to produce offspring emerging from isopods. We conclude that EPN and their bacteria can be pathogens for terrestrial isopods but that such hosts represent a reproductive dead-end for them. Thus, terrestrial isopods appear not to be alternative hosts for EPN populations maintained in the absence of insects.     

Wide interguild relationships among entomopathogenic and free-living nematodes in soil as measured by real time qPCR

Entomopathogenic nematodes (EPNs) are promising biological control agents of soil-dwelling insect pests of many crops. These nematodes are ubiquitous in both natural and agricultural areas. Their efficacy against arthropods is affected directly and indirectly by food webs and edaphic conditions. It has long been suggested that a greater understanding of EPN ecology is needed to achieve consistent biological control by these nematodes and the development of molecular tools is helping to overcome obstacles to the study of cryptic organisms and complex interactions. Here we extend the repertoire of molecular tools to characterize soil food webs by describing primers/probe set to quantify certain free-living, bactivorous nematodes (FLBNs) that interact with EPNs in soil. Three FLBN isolates were recovered from soil baited with insect larvae. Morphological and molecular characterization confirmed their identities as Acrobeloides maximum (RT-1-R15C and RT-2-R25A) and Rhabditis rainai (PT-R14B). Laboratory experiments demonstrated the ability of these FLBNs to interfere with the development of Steinernema diaprepesi, Steinernema riobrave and Heterorhabditis indica parasitizing the weevil Diaprepes abbreviatus (P < 0.001), perhaps due to resource competition. A molecular probe was developed for the strongest competitor, A. maximum. We selected the highly conserved SSU rDNA sequence to design the primers/probe, because these sequences are more abundantly available for free-living nematodes than ITS sequences that can likely provide better taxonomic resolution. Our molecular probe can identify organisms that share ?98% similarity at this locus. The use of this molecular probe to characterize soil communities from samples of nematode DNA collected within a citrus orchard revealed positive correlations (P < 0.01) between Acrobeloides-group nematodes and total numbers of EPNs (S. diaprepesi, H. indica and Heterorhabditis zealandica) as well as a complex of nematophagous fungi comprising Catenaria sp. and Monachrosporium gephyropagum that are natural enemies of EPNs. These relationships can be broadly interpreted as supporting Linford’s hypothesis, i.e., decomposition of organic matter (here, insect cadavers) greatly increases bactivorous nematodes and their natural enemies.     

Does scavenging extend the host range of entomopathogenic nematodes (Nematoda: Steinernematidae)?

Living and freeze-killed natural and laboratory hosts, with different susceptibility to entomopathogenic nematodes, were exposed to the larvae of Steinernema affine and Steinernema kraussei in two different experimental arenas (Eppendorf tubes, Petri dishes), and the success of the colonisation and eventual progeny production were observed. Both nematodes were able to colonise both living and dead larvae of Galleria mellonella (Lepidoptera) and adult Blatella germanica (Blattodea) even though the progeny production in dead hosts was lower on average. Living carabid beetles, Poecilus cupreus, and elaterid larvae (Coleoptera) were resistant to the infection, however, both nematodes were able to colonise and multiply in several dead P. cupreus and in a majority of dead elaterid larvae. By scavenging, EPNs can utilise cadavers of insects that are naturally resistant to EPN infection, and so broaden their host range.     

Use of real-time PCR to discriminate parasitic and saprophagous behaviour by nematophagous fungi

Entomopathogenic nematodes (EPNs) are important pathogens of soilborne insects and are sometimes developed commercially to manage insect pests. Numerous nematophagous fungal species (NF) prey on nematodes and are thought to be important in regulating natural or introduced EPN populations. However, nematophagy by these fungi in nature cannot be inferred using existing methods to estimate their abundance in soil because many of these fungi are saprophytes, resorting to parasitism primarily when certain nutrients are limiting. Therefore, we developed an assay to quantify NF DNA in samples of nematodes. Species-specific primers and TaqMan probes were designed from the ITS rDNA regions of Arthrobotrys dactyloides, Arthrobotrys oligospora, Arthrobotrys musiformis, Gamsylella gephyropagum and Catenaria sp. When tested against 23 non-target fungi, the TaqMan real-time PCR assay provided sensitive and target-specific quantification over a linear range. The amount of A. dactyloides or Catenaria sp. DNA in 20 infected nematodes, measured by real-time PCR, differed between fungal species (P=0.001), but not between experiments (P>0.05). However, estimates of relative NF parasitism using a bioassay with 20 nematodes infected by either species, differed greatly (P<0.001) depending on whether the fungi were alone or combined in the samples used in the assay. Tests done to simulate detection of NF DNA in environmental samples showed that, for all species, background genomic DNA and/or soil contaminants reduced the quantity of DNA detected. Nested PCR was ineffective for increasing the detection of NF in environmental samples. Indeed, real-time PCR detected higher amounts of NF DNA than did nested PCR. The spatial patterns of NF parasitism in a citrus orchard were derived using real-time PCR and samples of nematodes extracted from soil. The parasitism by Catenaria sp. was positively related to the abundance of both heterorhabditid and steinernematid EPNs. The possible significance of the associations is ambiguous because NF attack a broad range of nematode taxa whereas EPNs are a small minority of the total nematode population in a soil sample. These studies demonstrate the potential of real-time PCR to study the role of NF parasitism in soil food webs.     

Distribution of the entomopathogenic nematodes from La Rioja (Northern Spain)

Entomopathogenic nematodes (EPNs) distribution in natural areas and crop field edges in La Rioja (Northern Spain) has been studied taking into account environmental and physical-chemical soil factors. Five hundred soil samples from 100 sites of the most representative habitats were assayed for the presence of EPNs. The occurrence of EPNs statistically fitted to a negative binomial distribution, which pointed out that the natural distribution of these nematodes in La Rioja was in aggregates. There were no statistical differences (p < or = 0.05) in the abundance of EPNs to environmental and physical-chemical variables, although, there were statistical differences in the altitude, annual mean air temperature and rainfall, potential vegetation series and moisture percentage recovery frequency. Twenty-seven samples from 14 sites were positive for EPNs. From these samples, twenty isolates were identified to a species level and fifteen strains were selected: 11 Steinernema feltiae, two S. carpocapsae and two S. kraussei strains. S. kraussei was isolated from humid soils of cool and high altitude habitats and S. carpocapsae was found to occur in heavy soils of dry and temperate habitats. S. feltiae was the most common species with a wide range of altitude, temperature, rainfall, pH and soil moisture, although this species preferred sandy soils. The virulence of nematode strains were assessed using G. mellonella as insect host, recording the larval mortality percentage and the time to insect die, as well as the number of infective juveniles produced to evaluate the reproductive potential and the time tooks to leave the insect cadaver to determinate the infection cycle length. The ecological trends and biological results are discussed in relationship with their future use as biological control.     

Competition Between Entomopathogenic and Free-Living Bactivorous Nematodes in Larvae of the Weevil Diaprepes abbreviatus

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.     

Substrate modulation, group effects and the behavioral responses of entomopathogenic nematodes to nematophagous fungi

Laboratory experiments were conducted on the behavioral responses of five species of entomopathogenic nematodes (EPNs; Steinernema diaprepesi, Steinernema sp. glaseri-group, Steinernema riobrave, Heterorhabditis zealandica, Heterorhabditis indica) to three species of nematophagous fungi (NF; trapping fungus Arthrobotrys gephyropaga; endoparasites Myzocytium sp., Catenaria sp.). We hypothesized that EPN responses to NF and their putative semiochemicals might reflect the relative susceptibility of EPNs to particular NF species. EPN responses to “activated” NF (i.e., induced to form traps or sporangia by previous interactions with nematodes) versus controls of non-activated NF or heat-killed EPNs were compared in choice experiments on water agar in Petri dishes (dia = 9 cm) and in horizontal sand columns (8 cm L × 2.7 cm dia). On agar, all EPN species were attracted to all activated NF species except for S. riobrave, which was neutral. In sand, all EPN species were repelled by activated Arthrobotrys but attracted to activated Myzocytium and Catenaria, except H. indica (neutral to Myzocytium) and Steinernema sp. (neutral to Catenaria). EPN behavioral responses appeared unrelated to relative susceptibility to NF except that H. indica exhibited low susceptibility and a neutral response to Myzocytium in sand whereas the remaining EPNs were highly susceptible and attracted. These results indicate potential complexity (i.e., mixed responses, aggregation or group movement) and species specificity in the responses of EPNs to NF, demonstrate that results on agar can differ markedly from those in sand, and underline the potential importance of utilizing natural substrates to properly assess the role of semiochemicals in nematode-fungus interactions.     

Modifying orchard planting sites conserves entomopathogenic nematodes,reduces weevil herbivory and increases citrus tree growth,survival and fruit yield

In Florida, a root weevil pest of citrus, Diaprepes abbreviatus, is more damaging and attains higher population density in some orchards on fine textured, poorly drained “flatwoods” soils than in those on the deep, coarse sandy soils of the central ridge. Previous research revealed that sentinel weevil larvae were killed by indigenous entomopathogenic nematodes (EPNs) at significantly higher rates in an orchard on the central ridge, compared to one in the flatwoods. We hypothesized that filling tree planting holes in a flatwoods orchard with sandy soil from the central ridge would provide a more suitable habitat for EPNs, thereby reducing weevil numbers and root herbivory. Fifty trees were planted in oversized planting holes filled with coarse sand and 50 trees were planted in native soil in a split plot design where whole plots were species of introduced EPNs and split plots were soil type. Each of Steinernema diaprepesi, Steinernema riobrave, Heterorhabditis indica, Heterorhabditis zealandica, or no EPNs were introduced into the rhizospheres in 10 plots of each soil type. During four years, EPN numbers in soil samples and the relative abundance of seven species of nematophagous fungi associated with nematodes were measured three times using real-time PCR. The efficacy of EPNs against sentinel weevil larvae was also measured three times by burying caged weevils in situ. EPN species richness (P = 0.001) and diversity (P = 0.01) were always higher in sand than native soil. Soil type had no effect on numbers of EPNs in samples, but EPNs were detected more frequently (P = 0.01) in plots of sandy soil than native soil in 2011. Two nematophagous fungi species, Paecilomyces lilacinus and Catenaria sp. were significantly more abundant in nematode samples from sandy soil on all three sampling dates. Efficacy of EPNs against weevil larvae was greater in sandy soil inoculated with S. diaprepesi (P = 0.03) in June 2010 and in all treatments in sandy soil in May 2011 (P = 0.03). Sixty-eight percent more adult weevils (P = 0.01) were trapped emerging from native soil during two years than from sandy soil. By May 2011, the cumulative number of weevils emerging from each plot was inversely related (P = 0.01) to the numbers of EPNs detected in plots and to EPN efficacy against sentinels. Three trees in sandy soil died as a result of root herbivory compared to 21 trees in native soil. Surviving trees in sandy soil had trunk diameters that were 60% larger (P = 0.001) and produced 85% more fruit (P = 0.001) than those in native soil. Although it is not possible to characterize all of the mechanisms by which the two soil treatments affected weevils and trees, substitution of sand for native soil was an effective means of conserving EPNs and shows promise as a cultural practice to manage D. abbreviatus in flatwoods citrus orchards with a history of weevil damage to trees.     

Liquid Culture of the Entomogenous Nematode Steinernema feltiae with Its Bacterial Symbiont

The insect-parasitic nematode, Steinernema feltiae Filipjev strain 42, was reared in liquid culture along with its bacterial symbiont, Xenorhabdus nematophilus Thomas &Poinar. First-stage juveniles developed into reproducing adults in a maintenance salts medium containing resuspended Xenorhabdus cells and the yeast Kluyveromyces marxianus (Hansen) van der Walt or cholesterol. Cultures with media depths greater than 4 mm required aeration. Nematode populations increased as bacterial density increased. An optimal culture system was obtained when the bacteria and nematodes developed in a semidefined medium containing tryptic soy, yeast extract, and cholesterol and were incubated on a rotary shaker at 25 ± 1 C. Under these conditions, up to 86% of the final population were infective juveniles.     

Foraging Ants as Scavengers on Entomopathogenic Nematode-Killed Insects

Ants were the most apparent invertebrate scavengers observed foraging on entomopathogenic nematode-killed insects (i.e., insect cadavers containing entomopathogenic nematodes and their symbiotic bacteria) in the present study. Workers of the Argentine ant,Linepithema humile(Mayr), scavenged nematode-killed insects on the surface and those buried 2 cm below the soil surface. Ant workers scavenged significantly more steinernematid-killed (60–85%) than heterorhabditid-killed (10–20%) insects. More 4-day-postinfected cadavers (hosts died within 48 h after exposure to nematodes) were scavenged than 10-day-postinfected cadavers. Ten-day-postinfected hosts contained live infective juvenile nematodes therefore ants may serve as phoretic agents. Other ant species, includingVeromessor andrei(Mayr),Pheidole vistanaForel,Formica pacificaFrancoeur, andMonomoriom ergatogynaWheeler, also scavenged nematode-killed insects. These ant species removed or destroyed about 45% of the steinernematid-killed insects. These results suggest that survival of steinernematid nematodes may be more significantly impacted by invertebrate scavengers, especially ants, than that of heterorhabditid nematodes, and placement of steinernematid-killed insects in the field for biological control may be an ineffective release strategy. Because entomopathogenic nematodes kill insects with the help of symbiotic bacteria, we tested the role of these bacterial species in deterring invertebrate scavengers by injecting bacteria (without nematodes) into insects and placing the cadavers in the field. None of the insects killed by the symbiotic bacterium,Photorhabdus luminescens(Thomas and Poinar) fromHeterorhabditis bacteriophoraPoinar, were scavanged, whereas 70% of the insects killed by the symbiotic bacterium,Xenorhabdus nematophilus(Poinar and Thomas) fromSteinernema carpocapsae(Weiser), and 90% of the insects killed byBacillus thuringiensisBerliner were scavenged by the Argentine ant. We conclude thatP. luminescensis responsible for preventing ants from foraging on heterorhabditid-killed hosts.     

Steinernema feltiae Intraspecific Variability: Infection Dynamics and Sex-Ratio

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.