Phased infectivity in Heterorhabditis megidis: the effects of infection density in the parental host and filial generation

Entomopathogenic nematodes can develop through two or more generations in the cadavers of killed insect hosts. Non-feeding infective juveniles from each generation emerge and may spend prolonged periods searching for a new host. The infectivity of the infective juveniles of Heterorhabditis megidis varies with time after emergence and may not reach a maximum until several weeks have passed. 'Phased infectivity' hypotheses propose that this pattern is adaptive, tending to reduce competition in new hosts. Here we provide further evidence that infectivity is phased in H. megidis. In addition, we show that the basic pattern is modified by infection density in the parental host and by filial generation. Two general patterns were observed: first, infective juveniles that developed under the least crowded conditions (F(1) infective juveniles produced in hosts infected with 16 parent nematodes) reached maximum infectivity after only 15 days, compared to 27 or 39 days for infective juveniles that developed under more crowded conditions (F(1) produced in hosts infected with 103 or 424 parent nematodes or F(2) infective juveniles). Second, infective juveniles had lower infectivity overall when produced under the most crowded conditions (F(2) versus F(1); highest versus lowest infection density). We propose that while lower overall infectivity is a necessary consequence of limited resource availability during infective juvenile development, the difference in the timing of peak infectivity reflects a shift in the fitness gains associated with being maximally infective either 'early' or 'late'. F(1) infective juveniles emerge several days before F(2) infective juveniles, and we suggest that filial generation and infection density in the parental host function as indicators of the potential risk of competition within new hosts.     

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.     

Susceptibility of the peachtree borer, Synanthedon exitiosa, to Steinernema carpocapsae and Steinernema riobrave in laboratory and field trials

The nematode Steinernema carpocapsae (All) strain was significantly more effective against peachtree borer larvae (Synanthedon exitiosa [Lepidoptera: Sesiidae]) than Steinernema riobrave (7-12) strain in field and laboratory experiments. Eighty-eight percent control of peachtree borer larvae was obtained with S. carpocapsae in the field trial when applied at 3 x 10(5) infective juveniles per tree, and 92% mortality was obtained in the lab assay using 50 infective juveniles per larva.     

To complete their life cycle, pathogenic nematode-bacteria complexes deter scavengers from feeding on their host cadaver

The life cycle of commercially used molluscicidal rhabditid nematodes Phasmarhabditis hermaphrodita and entomopathogenic steinernematid nematodes is similar: infective stages carry symbiotic bacteria, which kill their host. Nematodes complete their life cycle feeding on the proliferating symbiont and the host tissue. After 1-2 weeks, new infective stages carrying the bacteria leave the host cadaver in search of new hosts. The removal of invertebrate cadavers by scavengers is extremely fast and represents a severe threat to the developing nematodes.Two-choice trials were used to assess prey choice of the generalist predator/scavenger Pterostichus melanarius (Coleoptera: Carabidae) between Deroceras reticulatum (Mollusca: Agriolimacidae) slugs or wax moth Galleria mellonella (Lepidoptera: Pyralidae) larvae killed by infection of P. hermaphrodita/Steinernema affine and control killed by freezing. We demonstrate that the presence of either of the two nematodes tested deters the beetles from consuming infected cadavers. As P. hermaprodita cannot infect an insect host, we hypothesise the deterrent effect being an evolutionary adaptation of the nematode/bacteria complex rather than the ability of the beetles to avoid potentially infective 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.     

Parastrongyloides trichosuri, a nematode parasite of mammals that is uniquely suited to genetic analysis

Commonly studied nematode parasites have not proven amenable to simple genetic analyses and this has significantly reduced the available research options. We introduce here a nematode parasite of mammals, Parastrongyloides trichosuri, which has features uniquely suited for genetic analysis. This parasite has the capacity to undergo multiple reproductive cycles as a free-living worm and thereby amplify the numbers of its infective L3s in faeces. Culture conditions are presented that permit facile laboratory maintenance of this worm for >90 free-living life cycles (to date) without the need for re-entry into a permissive host. Even after long maintenance as a free-living worm, culture conditions can be manipulated to favour development of infective L3 worms, which remain able to successfully infect their marsupial hosts. The switch to infective L3 development is triggered by a secreted factor contained in culture medium conditioned by multiple generations of free-living worm culture. It is simple to perform single pair crosses with P. trichosuri to carry out Mendelian genetics in the laboratory and this has been done multiple times with sibling pairs to generate highly inbred lines. Lines of worms can readily be cryopreserved and recovered. Over 7000 expressed sequence tags have been produced from cDNAs at different life cycle stages and used to identify single nucleotide polymorphisms and microsatellites as genetic markers. Free-living worms live only a few days on average while the patency of parasitic infections can last for several months. Since we show this is not the result of re-infection, we conclude that parasitic worms have a lifespan capacity at least 20-30 times longer than their free-living counterparts. We discuss how it should be possible to exploit these unique features of P. trichosuri as a model for future studies that explore the genetic basis of longevity and parasitism.     

Infectivity and reproductive potential of the Oswego strain of Heterorhabditis bacteriophora associated with life stages of the clover root curculio,Sitona hispidulus

The infectivity and reproductive potential of the entomopathogenic nematode Heterorhabditis bacteriophora (Oswego strain), at different concentrations, was studied. Seventy to 80.0% mortality to late instar larvae of the clover root curculio, Sitona hispidulus, and 40.0-76.0% mortality to pupae, was observed at concentrations of 15-100 infective juveniles. There were no significant differences in mortality among nematode concentrations. LC(50) levels of 4.0 and 21.4 nematodes were determined for clover root curculio larvae and pupae, respectively. Nematodes did not cause significant mortality to adult or first instar clover root curculio. H. bacteriophora was able to complete its development and reproduce in 74.0-95.0% of clover root curculio late instar larvae and pupae. Reproductive potential in curculio larvae and pupae ranged from 0 to 7040 infective juveniles per host. Larvae exposed to 100 nematodes had a reproductive potential significantly higher than in those larvae exposed to 15 and 50 nematodes. Reproductive potential in pupae decreased with an increased nematode dose, indicating potential crowding effects. Host larval and pupal mass were positively correlated with nematode progeny production.     

Dynamics of carbon dioxide release from insects infected with entomopathogenic nematodes

The quality of an insect as a host to an entomopathogenic nematode infective juvenile depends in part on whether or not the insect is already infected and on the stage of that infection. Previous research has shown that nematode response to hosts can change after infection and that, for uninfected hosts, CO(2) can be an important cue used by infective stage juveniles during attraction. We hypothesized that CO(2) production from an insect changes after it is infected, and that these changes could influence nematode infection decisions. Changes in CO(2) released by two insect species (Galleria mellonella and Tenebrio molitor) after infection by one of four nematode species (Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri, or Steinernema riobrave) were measured. Measurements were taken every 2h from time of initial exposure to nematodes up to 224 h after infection. Dead (freeze-killed) and live uninfected insects were used as controls. Infected G. mellonella showed two distinct peaks of CO(2) production: one between 20 and 30 h and the other between 70 and 115 h after exposure to the nematodes. Peaks were up to two times higher than levels produced by uninfected insects. Infected T. molitor showed only one peak between 25 and 50h. We found differences in peak height and timing among nematode and insect species combinations. The influence of these changes in CO(2) production on IJ attraction and infection behavior remains to be determined.     

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.     

Susceptibility of the boll weevil to Steinernema riobrave and other entomopathogenic nematodes

The susceptibility of the boll weevil (BW), Anthonomus grandis Boheman, to Steinernema riobrave and other nematode species in petri dishes, soil (Hidalgo sandy clay loam), and cotton bolls and squares was investigated. Third instar weevils were susceptible to entomopathogenic nematode (EN) species and strains in petri dish bioassays at 30 degrees C. Lower LC(50)'s occurred with S. riobrave TX- 355 (2 nematodes per weevil), S. glaseri NC (3), Heterorhabditis indicus HOM-1 (5), and H. bacteriophora HbL (7) than H. bacteriophora IN (13), S. riobrave TX (14), and H. bacteriophora HP88 (21). When infective juveniles (IJs) of S. riobrave were applied to weevils on filter paper at 25 degrees C, the LC(50) of S. riobrave TX for first, second, and third instars, pupae, and 1-day-old and 10-days-old adult weevils were 4, 5, 4, 12, 13, and 11IJs per weevil, respectively. The mean time to death, from lowest to highest concentration, for the first instar (2.07 and 1.27days) and second instar (2.55 and 1.39days) weevils were faster than older weevil stages. But, at concentrations of 50 and 100IJs/weevil, the mean time to death for the third instar, pupa and adult weevils were similar (1.84 and 2.67days). One hundred percent weevil mortality (all weevil stages) occurred 3days after exposure to 100IJs per weevil. Invasion efficiency rankings for nematode concentration were inconsistent and changed with weevil stage from 15 to 100% when weevils were exposed to 100 and 1IJs/weevil, respectively. However, there was a consistent relationship between male:female nematode sex ratio (1:1.6) and nematode concentration in all infected weevil stages. Nematode production per weevil cadaver increased with increased nematode concentrations. The overall mean yield of nematodes per weevil was 7680IJs. In potted soil experiments (30 degrees C), nematode concentration and soil moisture greatly influenced the nematode efficacy. At the most effective concentrations of 200,000 and 400,000IJs/m(2) in buried bolls or squares, higher insect mortalities resulted in pots with 20% soil moisture either in bolls (94 and 97% parasitism) or squares (92 and 100% parasitism) than those of 10% soil moisture in bolls (44 and 58% parasitism) or squares (0 and 13% parasitism). Similar results were obtained when nematodes were sprayed on the bolls and squares on the soil surface. This paper presents the first data on the efficacy of S. riobrave against the boll weevil, establishes the potential of EN to control the BW inside abscised squares and bolls that lay on the ground or buried in the soil.     

Neosteinernema longicurvicauda n. gen., n. sp. (Rhabditida: Steinernematidae), a Parasite of the Termite Reticuldermes flavipes (Koller)

A nematode isolated from the termite Reticulitermes flavipes (Koller) was identified and described as a new genus and species, Neosteinernema longicurvicauda. Primary distinguishing characters, by contrast to members of the genus Steinernema, were females having prominent phasmids, a curved tail longer than the body width at the anus, a spiral shape in juvenile-bearing females, and juveniles becoming infective-stage juveniles before emerging from the female; males having prominent phasmids, a digitate tail tip, a characteristic shape of the spicules (foot-shaped with a hump on the dorsal side), and 13-14 pairs of genital papillae, with eight pairs preanal; and infective juveniles having prominent phasmids and a filiform curved tail as long as the esophagus. Adult nematodes are found outside the termite cadaver. Diagnosis of the family Steinernematidae was emended to accommodate the new species.     

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.     

Potential negative effects on biological control by Sancassania polyphyllae (Acari: Acaridae) on an entomopathogenic nematode species

Sancassania polyphyllae (Acari: Acaridae) is associated with larvae of the white grub, Polyphylla fullo (Coleoptera: Scarabaeidae), and will feed on the infective juveniles of entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae which are important biological control agents of soil insect pests. We conducted laboratory studies to determine the potential negative effects this mite species might have on biological control of soil insect pests. Our objectives in this study were to (1) determine the response of S. polyphyllae adult mites to a nematode-killed insects on agar, (2) evaluate the predation by mites on Steinernema feltiae infective juveniles from nematode-killed insects on agar and in soil, and (3) assess predation efficiency of the mite on the infective juveniles in the soil. On agar, we found (1) significantly more adult female mites near or on a nematode-killed Ceratitis capitata (Diptera: Tephritidae) larva than near or on the freeze-killed larva or a bamboo mimic suggesting that a chemical or an odor from the nematode-killed larva attracted the mites, and (2) 10 mites consumed 96% of infective juveniles that emerged from an insect cadaver. In soil with a nematode-killed insect, the average number of infective juveniles recovered was <30 when mites were present, whereas the average number of infective juveniles recovered was >375 when the mites were absent. When the infective juveniles alone were placed in different depths in relation to the mites in the soil column for 4 and 10 days, S. polyphyllae was not as efficient at finding the infective juveniles when they were separated from each other in the soil lending support to the idea that the mites were cueing in on the cadaver as a food resource. Our data suggest that emerging infective juveniles from an insect cadaver in the soil in the presence of S. polyphyllae can adversely affect biological control because of nematode consumption by the mites.     

Nematode Population and Community Dynamics in Soybean-Wheat Cropping and Tillage Regimes

The nematode community structures of various soybean-wheat regimes and of a single-cropped, conventionally tilled soybean regime were studied at two sites in Tennessee. Each of the 100 nematode species identified in the study was placed in one of five trophic groups, the most diverse being plant parasites (31 species), followed by Dorylaimida (26 species), bacterivores (23 species), fungivores (15 species), and predators (5 species). No significant differences in overall diversity and dominance among treatments and trophic groups were found. Densities of Heterodera glycines Ichinohe infective juveniles were significantly higher in single-cropped, conventionally tilled soybeans in July. When data were subjected to ordination analysis, it was shown that plant-parasitic nematode communities produced an aggregation of conventionally tilled, single-cropped soybean plots when compared to all double-cropped treatments. Ordination of overall nematode communities yielded similar results.     

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.     

Simple In Vivo Production and Storage Methods for Steinernema carpocapsae Infective Juveniles

Methods are described for standardized in vivo production, rapid harvest, and storage, in a concentrated form, of infective juveniles of the entomopathogenic nematode, Steinernema carpocapsae Mexican strain Kapow selection. Nematodes were stored in nematode wool configurations, consisting of mats of intertwined infective juveniles. Freshly harvested nematodes are readily available in adequate quantities for laboratory and small-scale field evaluations as well as cottage industry production.