Efficacy of indigenous entomopathogenic nematodes (Rhabditida: Heterorhabditidae, Steinernematidae), from Rio Grande do Sul Brazil, against Anastrephafraterculus (Wied.) (Diptera: Tephritidae) in peach orchards

Laboratory, greenhouse, and field experiments were performed with the objective of selecting efficient indigenous strains of entomopathogenic nematodes (EPNs) from Rio Grande do Sul (RS) state, Brazil, for controlling the South American fruit fly, Anastrepha fraterculus (Wied.). Laboratory experiments were conducted in 24 well-plates filled with sterile sand and one insect per well. In greenhouse experiments, plastic trays filled with soil collected from the field were used, while in field experiments, holes were made in soil under the edge of peach tree canopies. Among 19 EPN strains tested, Heterorhabditis bacteriophora Poinar RS88 and Steinernema riobrave Cabanillas, Poinar, & Raulston RS59 resulted in higher A. fraterculus larval (pre-pupal) and pupal mortality, with LD₉₀ of 1630, 457 and 2851, 423 infective juveniles (IJs)/cm², respectively. Greenhouse experiments showed no differences in pupal mortality at 250 and 500 IJs/cm² of either nematode. In the field, H. bacteriophora RS88 and S. riobravae RS59 sprayed individually over natural and artificially infested fruit (250 IJs/cm²) resulted in A. fraterculus larval mortality of 51.3%, 28.1% and 20%, 24.3%, respectively. There was no significant difference in A. fraterculus pupal mortality sprayed with an aqueous suspension of either nematode; however, when using infected insect cadavers, H. bacteriophora RS88 was more efficient than S. riobrave RS59. Our results showed that H. bacteriophora RS88 was more virulent to insect larvae, with an efficient host search inside the infested fruit and control of pupae in the soil after being applied by aqueous suspension or infected cadavers.     

Development Rates in Entomopathogenic Nematodes: Infected Hosts vs. Aqueous Suspension

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.     

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.     

Target Host Finding by Steinernema feltiae and Heterorhabditis bacteriophora in the Presence of a Non-Target Insect Host

The ability of Steinernema feltiae or Heterorhabditis bacteriophora infective juveniles (IJ), when applied to the soil surface, to infect a Galleria mellonella larva at the base of a soil-filled cup (276 cm³) was evaluated in the presence and absence of 100 larvae of a non-target insect, the aphid midge Aphidoletes aphidimyza, near the soil surface. In all four trials with either S. feltiae or H. bacteriophora, A. aphidimyza presence did not affect the number of IJ finding and infecting a G. mellonella larva. Steinernema feltiae and H. bacteriophora IJ movement (as measured by the percentage of IJ aggregating on either side of an experimental arena) in the presence of one or many A. aphidimyza larvae was evaluated in agar- and soil-filled petri dishes, respectively. Infective juvenile movement in the presence of A. aphidimyza did not differ from random, indicating that IJ were not attracted to A. aphidimyza. It is suggested, therefore, that A. aphidimyza does not reduce IJ efficacy when these two forms of biological control agent are present together in a field situation even though it is known that A. aphidimyza is susceptible to IJ of these species.     

Control of Grapholita molesta (Busck, 1916) (Lepidoptera: Tortricidae) with entomopathogenic nematodes (Rhabditida: Heterorhabditidae,Steinernematidae) in peach orchards

Oriental fruit moth Grapholita molesta (Busck, 1916) (Lepidoptera: Tortricidae) is considered a major pest in temperate fruit trees, such as peach and apple. Entomopathogenic nematodes (EPNs) are regarded as viable for pest management control due to their efficiency against tortricid in these trees. The objective of this study was to evaluate the effectiveness of native EPNs from Rio Grande do Sul state against pre-pupae of G. molesta under laboratory and field conditions. In the laboratory, pre-pupae of G. molesta were placed in corrugated cardboard sheets inside glass tubes and exposed to 17 different EPNs strains at concentrations of 6, 12, 24, 48 and 60 IJs/cm² and maintained at 25 °C, 70 ± 10% RH and photophase of 16 h. Insect mortality was recorded 72 h after inoculation of EPNs. Steinernema rarum RS69 and Heterorhabditis bacteriophora RS33 were the most virulent strains and selected for field application (LC₉₅ of 70.5 and 53.8 IJs/cm², respectively). Both strains were highly efficient under field conditions when applied in aqueous suspension directed to larvae on peach tree trunk, causing mortality of 94 and 97.0%, respectively.     

Behavioural and physiological responses of infective juveniles of the entomopathogenic nematode Heterorhabditis to desiccation

Infective juveniles (IJs) of entomopathogenic nematodes (EPNs) are susceptible to a wide variety of environmental factors, including desiccation, which limit their usefulness as biocontrol agents. Although EPNs can be subjected to a gradual loss of water in their natural environment they are not full anhydrobiotes, being able to survive only moderate levels of desiccation at high relative humidities (rh). We investigated the desiccation tolerance of IJs of several Heterorhabditisspecies and strains when exposed to fast and slow desiccation regimes. We also investigated the behavioural and biochemical responses of Heterorhabditis IJs when exposed to 98% rh for 4 days. IJs of H. megidis UK211 (but not IJs of H. indica) aggregate into large clumps when desiccated at high rh, but unlike Steinernema spp., neither H. megidis nor H. indica IJs showed any tendency to coil. Preincubation of H. megidis UK211 IJs at high (98%) rh enhances their ability to survive for 150 min at 57% rh. We show that preincubation of H. megidis and H. indica at 98% rh induces the synthesis of glycerol but not of trehalose, whereas identical preincubation conditions do induce trehalose synthesis in Steinernema carpocapsae and Aphelenchus avenae. The biosynthesis of glycerol rather than trehalose by IJs of two species of Heterorhabditis in response to moderate levels of desiccation indicates that Heterorhabditis is unlikely to have the necessary metabolic responses to desiccation required to enable it to enter into a fully anhydrobiotic state.     

Entomopathogenic nematodes, phoretic Paenibacillus spp., and the use of real time quantitative PCR to explore soil food webs in Florida citrus groves

Quantitative real-time PCR (qPCR) is a powerful tool to detect and quantify species of cryptic organisms such as bacteria, fungi and nematodes from soil samples. As such, qPCR offers new opportunities to study the ecology of soil habitats by providing a single method to characterize communities of diverse organisms from a sample of DNA. Here we describe molecular tools to detect and quantify two bacteria (Paenibacillus nematophilus and Paenibacillus sp.) phoretically associated with entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematodae. We also extend the repertoire of species specific primers and TaqMan® probes for EPNs to include Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema feltiae and Steinernema scapterisci, all widely distributed species used commercially for biological control. Primers and probes were designed from the ITS rDNA region for the EPNs and the 16S rDNA region for the bacteria. Standard curves were established using DNA from pure cultures of EPNs and plasmid DNA from the bacteria. The use of TaqMan probes in qPCR resolved the non-specificity of EPN and some bacterial primer amplifications whereas those for Paenibacillus sp. also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two species that are not phoretically associated with nematodes. The primer-probe sets for EPNs were able to accurately detect three infective juvenile EPNs added to nematodes recovered from soil samples. The molecular set for Paenibacillus sp. detected the bacterium attached to Steinernema diaprepesi suspended in water or added to nematodes recovered from soil samples but its detection decreased markedly in the soil samples, even when a nested PCR protocol was employed. Using qPCR we detected S. scapterisci at low levels in a citrus grove, which suggested natural long-distance spread of this exotic species, which is applied to pastures and golf courses to manage mole crickets (Scapteriscus spp.). Paenibacillus sp. (but not P. nematophilus) was detected in low quantities in the same survey but was unrelated to the spatial pattern of S. diaprepesi. The results of this research validate several new tools for studying the ecology of EPNs and their phoretic bacteria.     

Effect of Application Method on Fitness of Entomopathogenic Nematodes Emerging at Different Times

The entomopathogenic nematode species Steinernema feltiae and Heterorhabditis bacteriophora were compared for survival and infectivity of infective juveniles (IJ) collected with a standard White trap (i.e., emerging from hosts and accumulating in water) and later applied to sand (treatment A) to IJ allowed to emerge from hosts into sand (treatment C). Percentage IJ survival and infectivity was compared between treatments for S. feltiae IJ that emerged between days 1 to 3 and days 4 to 6. For H. bacteriophora, percentage IJ survival and infectivity was compared between treatments only for infective juveniles that emerged between days 4 to 6. For S. feltiae IJ percentage survival and infectivity decreased with time (P ≤ 0.05) and was greater (P ≤ 0.05) for IJ from treatment C than for IJ from treatment A. For H. bacteriophora IJ percentage survival decreased (P ≤ 0.05) and percentage infectivity increased (P ≤ 0.05) with time. While percent survival was higher (P ≤ 0.05) for treatment C than for A, percent infectivity was not different between treatments.     

Virulence and sex-dependent invasion efficiency of entomopathogenic nematodes on developmental stages of Phthorimaea operculella (Lep., Gelechidae)

Virulence and invasion efficiency of the three entomopathogenic nematodes, Heterorhabditis bacteriophora, Steinernema carpocapsae and S. feltiae against the potato tuber moth (PTM), Phthorimaea operculella was evaluated. Also evaluated were the sex ratio of Steinernema spp. and host stages to determine if 1) the developmental stage of the host affects sex ratio of nematodes; 2) infective juveniles (IJs) concentration affects sex ratio in host developmental stages and 3) the establishment of IJs is affected by developmental stages of host. The PTM pre-pupa and pupa were exposed to IJs in filter substrate petri dish bioassays. By increasing the IJs concentrations, the number of established Steinernema spp. in both PTM stages increased and only decreased at the highest concentration. No reduction in established nematode numbers at the highest concentration was observed for H. bacteriophora. Sex ratio of S. carpocapsae in pre-pupa was affected by IJ concentration. PTM was more susceptible to Steinernema spp. than H. bacteriophora. Pre-pupa were more susceptible to S. feltiae but S. carpocapsae recorded as the most virulent EPN on pupa. Invasion efficiencies were similar for Steinernema and considerably higher than for H. bacteriophora. Despite a higher invasion efficiency of Steinernema into pupae, mortality was lower compared to pre-pupa No correlation was recorded between the invasion efficiencies of the EPNs and mortalities of PTM. The results showed that the invasion efficiency is not appropriate criterion to reflect the virulence of studied EPNs. Compared to H. bacteriophora both tested Steinernema spp. were good candidates for further studies as biocontrol agents of PTM.     

Dispersal, Infectivity and Sex Ratio of Early- or Late-Emerging Infective Juveniles of the Entomopathogenic Nematode Steinernema carpocapsae

Differences in activity between infective juveniles (IJ) of the entomopathogenic nematode Steinernema carpocapsae that emerged directly from cadavers onto either a sand or agar substrate compared with those emerging from a cadaver into water and then being placed on the same substrate are known to occur. Differences between S. carpocapsae IJ that emerged directly from a cadaver vs. those that emerged from a cadaver and held in water were further elucidated. Dispersed and non-dispersed IJ from a cadaver were compared with those held in water between two time periods designated as early- (first two days) or late-emerging IJ (seventh day). A significantly greater proportion of early-emerging IJ from the cadaver treatment dispersed, compared with late-emerging IJ from a cadaver or either group of emerging IJ held in aqueous suspension. Moreover, IJ from cadavers were more infectious than those from the aqueous suspensions, and IJ that dispersed were less infectious than those that did not disperse. IJ that emerged early were mostly males, whereas those that emerged late were mostly females. For the non-dispersed IJ, most that emerged early were males, and those that emerged later were females, but among dispersing IJ, there was no difference in sex ratio between early- and late-emerging nematodes.     

Entomopathogenic nematodes induce components of systemic resistance in plants: Biochemical and molecular evidence

Antagonism between entomopathogenic nematodes (EPNs) and plant-parasitic nematodes (PPNs) has been documented over the past two decades but its mechanism and ecological significance remain elusive. We investigated the effects of Steinernema carpocapsae and its symbiotic bacterium, Xenorhabdus nematophila applied to the potting medium on pyrogallol peroxidase (P-peroxidase), guaiacol peroxidase (G-peroxidase) and catalase activities in Hosta sp. and Arabidopsis thaliana leaves as components of induced systemic resistance. We found that P-peroxidase activity was significantly higher in the leaves from hosta plants treated with S. carpocapsae infective juveniles (IJs) and S. carpocapsae infected insect cadavers than in the leaves from the control plants 2 weeks after treatment. The G-peroxidase activity was significantly higher in S. carpocapsae infected cadaver and X. nematophila treatments 10 and 15 days after treatment (DAT) and in S. carpocapsae IJs treatment 5 and 15 DAT. The catalase activity in hosta leaves was significantly higher in S. carpocapsae infected cadaver and X. nematophilus treatments compared with the control 5 and 15 DAT and in S. carpocapsae IJs treatment 5 and 10 DAT. Further, the catalase activity in A. thaliana leaves was significantly higher in S. carpocapsae IJs treatment than in the control 7 DAT. We also determined the effects of S. carpocapsae infected cadavers and S. carpocapsae IJs on PR1-gene expression in transgenic A. thaliana leaves through GUS (β-glucuronidase) activity assay and found that the PR1-gene was expressed in leaves from all treatments except the control. Thus, we conclude that the EPNs and their symbiotic bacteria can induce systemic resistance in plants which may explain the elusive antagonistic effect of EPNs on PPNs.     

Anhydrobiotic potential and long-term storage of entomopathogenic nematodes (Rhabditida: Steinernematidae)

Anhydrobiosis is considered to be an important means of achieving storage stability of entomopathogenic nematodes that are used in biological control. This study explored the effects of anhydrobiosis on longevity and infectivity of infective juveniles (IJs) of three species of entomopathogenic nematodes Steinernema carpocapsae, Steinernema feltiae, and Steinernema riobrave at 5 and 25 degrees C. Anhydrobiosis was induced in water-dispersible granules (WG) at 0.966-0.971 water activity and 25 degrees C following a 7-day preconditioning of IJs at 5 degrees C in tap water. Survival and infectivity of the desiccated (anhydrobiotic) IJs was compared with non-desiccated IJs stored in water for different periods. Anhydrobiosis increased longevity of S. carpocapsae IJs by 3 months and of S. riobrave by 1 month in WG at 25 degrees C as compared with IJs stored in water. However, desiccation decreased S. feltiae longevity at 25 degrees C and of all three species at 5 degrees C. These results demonstrate a shelf-life of 5 months for S. carpocapsae at 25 degrees C and 9 months at 5 degrees C in WG with over 90% IJ survival. For S. feltiae, over 90% survival occurred only for 2 months at 25 degrees C and 5 months at 5 degrees C in WG. Steinernema riobrave had over 90% survival only for 1 month at 25 degrees C and the survival dropped below 85% within 1 month at 5 degrees C. Induction of anhydrobiosis in WG resulted in 85, 79 and 76% reduction in oxygen consumption by S. carpocapsae, S. feltiae, and S. riobrave IJs, respectively. Differences in IJ longevity among three species in water at 25 degrees C were related both to the initial lipid content and the rate of lipid utilisation, but not at 5 degrees C. The one-on-one infection bioassays indicated that desiccation had no negative effect on the infectivity of any of the nematode species suggesting no harmful effect on the IJs and/or their symbiotic bacteria. The species differences in IJ longevity and desiccation survival at different temperatures are discussed in relation to their foraging strategy and temperature adaptation.     

Laboratory and semi-field evaluation of three entomopathogenic nematode species on two non-target insect predators,Coccinella septumpunctata (Coleoptera: Coccinellidae) and Chrysoperla carnea (Neuroptera: Chrysopidae)

Abstract

Biocontrol potential of the entomopathogenic nematodes (EPNs) on the second-instar larvae of the non-target insect predators, Coccinella septumpunctata and Chrysoperla carnea as compared to Spodoptera littoralis (Boisd.) was evaluated. The pathogenicity of EPNs, namely, Heterorhabditis bacteriophora, Steinernema feltiae and Steinernema carpocapsae at concentrations 100, 200, 400, 800 and 1600 IJs/cup) were tested at 2, 4 and 6 days’ post-inoculation. Laboratory results showed significant differences among the mortality rates of different tested larvae, for each concentration at different time intervals. H. bacteriophora induced the highest mortality followed by S. carpocapsae treatment. However, S. feltiae was found to be more safety on predators as it causes less mortality at 6 days of treatment. The values of half lethal concentrations (LC₅₀) were 614.06, 3797.43 and 676.47 IJs/cup for C. Carnea and 390.60, 1209.88 and 503.65 IJs/cup for C. septumpunctata treated by H. bacteriophora, S. feltiae and S. carpocapsae, respectively. In semi-field experiments, there were non-significant differences among mortality of each predator indicated at concentrations of the different EPNs after 2 days or 6 days’ post-inoculation. The study revealed a lethal pathogenic effect of EPNs against insect pests but caused low mortality on the non-target ones.     

Effects of host desiccation on development, survival, and infectivity of entomopathogenic nematode Steinernema carpocapsae

This study investigates the effect of host desiccation on entomopathogenic nematode (EPN) development, emergence, infectivity, and cross-protection against secondary environmental stress. Galleria mellonella hosts infected with the EPN Steinernema carpocapsae A10 were allowed to dehydrate in an environmental chamber for up to 56 days at 23 degrees C achieving a weight loss of approximately 86% by day 44 post-infection. Host carcasses were rehydrated on water-saturated filter paper in White traps to collect emergent infective juveniles (IJ) at specific time intervals. Populations were counted with an apparent peak coinciding with desiccated hosts rehydrated at 24-day post-infection. Desiccation-stressed IJ populations from each time interval were tested for infectivity, and cross-resistance to secondary temperature and pH stresses and were found to have significant increases in both infectivity and protection from extremes of temperature and pH compared with controls. Total aqueous soluble protein profiles from control and desiccation-stressed IJs were analyzed using 10% SDS Laemmli gels. Several novel proteins were over-expressed in EPN from hosts subjected to desiccation suggesting the induction and expression of stress response genes.     

Pathogenicity of Two Species of Entomopathogenic Nematodes Against the Greenhouse Whitefly,Trialeurodes vaporariorum (Hemiptera: Aleyrodidae), in Laboratory and Greenhouse Experiments

The greenhouse whitefly Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) is a polyphagous pest in greenhouse crops. The efficacy of two entomopathogenic nematodes (EPN), Steinernema feltiae and Heterorhabditis bacteriophora, as biological control agents against T. vaporariorum was evaluated using two model crops typical of vegetable greenhouse productions: cucumber and pepper. Laboratory tests evaluated adults and second nymphal instars for pest susceptibility to different EPN species at different concentrations of infective juveniles (IJ; 0, 25, 50, 100, 150, 200, and 250 IJ per cm²); subsequent greenhouse trials against second nymphal instars on cucumber and pepper plants evaluated more natural conditions. Concentrations were applied in combination with Triton X-100 (0.1% v/v), an adjuvant for increasing nematode activity. In laboratory studies, both life stages were susceptible to infection by the two nematode species, but S. feltiae recorded a lower LC₅₀ than H. bacteriophora for both insect stages. Similarly, in greenhouse experiments, S. feltiae required lower concentrations of IJ than H. bacteriophora to reach the same mortality in nymphs. In greenhouse trials, a significant difference was observed in the triple interaction among nematode species × concentration × plant. Furthermore, the highest mortality rate of the second nymphal instars of the T. vaporariorum was obtained from the application of S. feltiae concentrated to 250 IJ/cm² on cucumber (49 ± 1.23%). The general mortality caused by nematodes was significantly higher in cucumber than in pepper. These promising results support further investigation for the optimization of the best EPN species/concentration in combination with insecticides or adjuvants to reach a profitable control of this greenhouse pest.     

Cryopreservation of Steinernema carpocapsae and Heterorhabditis bacteriophora

A method for the cryopreservation of third-stage infective juveniles (IJ) of Steinernema carpocapsae and Heterorhabiditis bacteriophora was developed. Cryoprotection was achieved by incubating the nematodes in 22% glycerol (S. carpocapsae) or 14% glycerol (H. bacteriophora) for 24 hours, followed by 70% methanol at 0 C for 10 minutes. The viability of S. carpocapsae frozen in liquid nitrogen as 20 μl volumes spread over cover slip glass was > 80%. Survival of H. bacteriophora frozen on glass varied from 10 to 60% but was improved to > 80% by replacing the glass with filter paper. Cryopreservation and storage of 1-ml aliqots of S. carpocapsae IJ resulted in > 50% survival after 8 months; pathogenicity was retained and normal in vitro development took place. Trehalose and glycerol levels increased and glycogen levels decreased during incubation of S. carpocapsae IJ in glycerol. Normal levels of trehalose, glycerol and glycogen were restored during post freezing rehydration.     

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.     

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).     

Sex ratios and sex-biased infection behaviour in the entomopathogenic nematode genus Steinernema

In experimentally infected insects, the sex ratio of first generation nematodes of five species of Steinernema was female-biased (male proportion 0.35-0.47). There was a similar female bias when the worms developed in vitro (0.37-0.44), indicating that the bias in these species is not due to a lower rate of infection by male infective juveniles (IJs). Experimental conditions influenced the proportion of males establishing in insects, indicating that male and female IJs differ in their behaviour. However, there was no evidence that males are the colonising sex in any species, contrary to what has previously been proposed. Time of emergence from the host in which the nematodes had developed influenced sex ratios in experimental infections. In three species (Steinernema longicaudum, Steinernema glaseri and Steinernema kraussei), early emerged nematodes had a higher proportion of males than those that emerged later, with the reverse trend for Steinernema carpocapsae and Steinernema feltiae. In a more detailed in vitro study of S. longicaudum, the proportion of males was similar whether or not the nematodes passed through the developmentally arrested IJ stage, indicating that the female bias is not due to failure of males to exit this stage. The sex ratio in vitro was independent of survival rate from juvenile to adult, and was female-biased even when all juveniles developed, indicating that the bias is not explained by failure of males to develop to adults. The female-biased sex ratio characteristic of Steinernema populations appears to be present from at least the early juvenile stage. We hypothesise that the observed female bias is the population optimal sex ratio, a response to cycles of local mate competition experienced by nematodes reproducing within insect hosts interspersed with periods of outbreeding with less closely related worms following dispersal.