European earwig (Forficula auricularia) as a novel host for the entomopathogenic nematode Steinernema carpocapsae

The natural history of many entomopathogenic nematode species remains unknown, despite their wide commercial availability as biological control agents. The ambushing entomopathogenic nematode, Steinernema carpocapsae, and the introduced European earwig, Forficula auricularia, forage on the soil surface. Since they likely encounter one another in nature, we hypothesized that earwigs are susceptible to nematode infection. In the laboratory, the LC₅₀ for F. auricularia was 226 S. carpocapsae/earwig and the reproductive potential was 123.5 infective juvenile nematodes/mg tissue. This susceptibility depended on host body size with significantly higher mortality rates seen in larger earwigs. In a study of host recognition behavior, S. carpocapsae infective juveniles responded to earwig cuticle as strongly as they did to Galleria mellonella cuticle. We also found that earwigs exposed to S. carpocapsae cleaned and scratched their front, middle and back legs significantly more than controls. Coupled with previous field data, these findings lead us to suggest that F. auricularia may be a potential host for S. carpocapsae.     

Attraction of entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora to the red palm weevil (Rhynchophorus ferrugineus)

The red palm weevil (RPW), Rhynchophorus ferrugineus, is a serious pest of date palms. Its larvae bore deep into the trunk disrupt the vascular tissues and kill the infested trees. Behavioral features of entomopathogenic nematodes (EPNs), reflected by attraction and distribution patterns, are fundamental aspect in determining their parasitic ability and potential management of RPW. We studied the attraction behavior of the EPNs Steinernema carpocapsae and Heterorhabditis bacteriophora to the RPW under simulated natural conditions in tubes to evaluate their infective potential. In all experiments, a certain proportion of infective juveniles (IJs) (16–20%) stayed near the inoculated site and a major proportion (38–48%) was attracted to the host end. Both H. bacteriophora and S. carpocapsae were efficient crawlers, climbing up and descending when locating their insect host. They were efficiently attracted to the various larval sizes and stages of the RPW life cycle. Host localization by ascending movement was more prominent in S. carpocapsae than in H. bacteriophora. In general, H. bacteriophora is classified as a cruiser forager and S. carpocapsae as an ambusher. However, in this study, we discovered a higher percentage of cruiser foragers among S. carpocapsae IJs. They dispersed much faster and their cruising behavior was prominent characteristic in controlling the cryptic RPW concealed in organic habitats.     

Overwintering behavior of the entomopathogenic nematodes Steinernema scarabaei and Heterorhabditis bacteriophora and their white grub hosts

Entomopathogenic nematodes (EPNs) are obligate pathogens known to naturally persist in many habitats. Because survival is a fundamental component of persistence, we investigated whether vertical movement and other avoidance behaviors (i.e., in‐host survival and latent infection), previously speculated as viable survival mechanisms, are exhibited during the cooler months in a temperate turfgrass habitat. The vertical distribution of populations of two EPN species, Steinernema scarabaei Stock & Koppenhöfer (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), and two important hosts of these EPN species, the white grub species Popillia japonica Newman and Anomala orientalis Waterhouse (both Coleoptera: Scarabaeidae), were regularly monitored in turf plots from October through April in two consecutive years. Entomopathogenic nematode vertical distribution showed limited changes for H. bacteriophora but none for S. scarabaei. Recovery of H. bacteriophora showed a strong and consistent decline at 0–4 cm depth in the 1st year and a weaker decline at 4–10 cm in the 1st year and at 0–4 cm in the 2nd year. Due to high variability in the data, it was not possible to determine whether the decline in the upper soil layers was due to downward migration or attrition of infective juvenile nematodes. The decline occurred mostly during the first half of the season before the soil froze to any significant extent. The vertical distribution of both white grub species changed with temperature during fall and spring, but not during winter. Overwintering infective juveniles were only recovered in the soil. There was no evidence for successful in‐host survival or latent infection by the nematodes in endemic white grub populations.     

Field efficacy of the entomopathogenic nematode Steinernema feltiae against the Mediterranean flat-headed rootborer Capnodis tenebrionis

The Mediterranean flat-headed rootborer, Capnodis tenebrionis (L.) (Coleoptera: Buprestidae), is an economically important pest of stone fruit and seed fruit in Mediterranean areas. The potential control of the entomopathogenic nematode Steinernema feltiae (Filipjev) (strain Bpa), isolated from a dead C. tenebrionis larva, was tested in a cherry tree orchard in Ullastrell, Barcelona (Spain). Nematode infective juveniles (IJs) were applied by drench and injection. In both the treatments, a rate of 1 million IJs was applied per tree every week during 4 or 8 weeks, with a total dose of 4 × 10⁶ IJs/tree and 8 × 10⁶ IJs/tree. Number, stage and localization of insects in each tree trunk were recorded. In both the experiments, S. feltiae significantly reduced the population of C. tenebrionis providing control ranging from 88.3% to 97%. No significant differences were recorded between the different treatments. Persistence of nematodes was recorded until 6 weeks after application. Results indicate that the application of S. feltiae (Bpa) provides adequate control of C. tenebrionis in cherry trees.     

Effect of soil type on infectivity and persistence of the entomopathogenic nematodes Steinernema scarabaei, Steinernema glaseri, Heterorhabditis zealandica, and Heterorhabditis bacteriophora

We tested the effect of soil type on the performance of the entomopathogenic pathogenic nematodes Steinernema scarabaei, Steinernema glaseri, Heterorhabditis zealandica, and Heterorhabditis bacteriophora. Soil types used were loamy sand, sandy loam, loam, silt loam, clay loam, acidic sand, and a highly organic potting mix. Infectivity was tested by exposing third-instar Anomala orientalis or Popillia japonica to nematodes in laboratory and greenhouse experiments and determining nematode establishment in the larvae and larval mortality. Infectivity of H. bacteriophora and H. zealandica was the highest in potting mix, did not differ among loamy sand and the loams, and was the lowest in acidic sand. Infectivity of S. glaseri was significantly lower in acidic sand than in loamy sand in a laboratory experiment but not in a greenhouse experiment, and did not differ among the other soils. Infectivity of S. scarabaei was lower in silt loam and clay loam than in loamy sand in a greenhouse experiment but not in a laboratory experiment, but was the lowest in acidic sand and potting mix. Persistence was determined in laboratory experiments by baiting nematode-inoculated soil with Galleria mellonella larvae. Persistence of both Heterorhabditis spp. and S. glaseri was the shortest in potting mix and showed no clear differences among the other substrates. Persistence of S. scarabaei was high in all substrates and its recovery declined significantly over time only in clay loam. In conclusion, generalizations on nematode performance in different soil types have to be done carefully as the effect of soil parameters including soil texture, pH, and organic matter may vary with nematode species.     

Susceptibility and behavioral responses of the dampwood termite Zootermopsis angusticollis to the entomopathogenic nematode Steinernema carpocapsae

Termites exploit microbially rich resources such as decayed wood and soil that are colonized by potentially pathogenic and parasitic fungi, bacteria, viruses, and nematodes. In colonies composed of thousands of individuals, the risk of infection among nestmates is significant, and individual and social behavior could involve various adaptations to resist disease and parasitism. Here we show that the dampwood termite Zootermopsis angusticollis (Hagen) exhibits a dosage dependent susceptibility to the soil nematode Steinernema carpocapsae (Weiser) (Mexican strain) and that this social insect significantly alters its behavior in response to this entomopathogenic roundworm. Relative to their baseline behavior, termites exposed to infective juveniles increased the frequency and duration of allogrooming and vibratory displays as well as two other novel behaviors, abdominal tip-raising and self-scratching. Whereas the first two behaviors likely reflect general adaptations to reduce susceptibility to a variety of pathogens and parasites, the latter behaviors might be specific to nematodes because they have never been observed in Z. angusticollis in any other pathogenic context. Our results support the hypotheses that behavioral responses in termites are important in the control of pathogenic and parasitic microorganisms and that termite susceptibility is socially mediated.     

Enhanced cold tolerance of the entomopathogenic nematode Steinernema feltiae through genetic selection

Cold sensitivity of entomopathogenic nematodes severely restricts their biological control potential in some environments. We selected the SN strain of Steinernema feltiae together with its bacterial symbiont, Xenorhabdus bovenii, for improved cold tolerance by repeated passage through the wax moth Galleria mellonella larvae at 15°C. Nematode virulence (total insect mortality and speed of kill) and establishment (initiation of nematode development following penetration) were evaluated after six (= 12–24 generations) and 12 passages (= 24–36 generations). Cold selection enhanced nematode virulence at the cooler temperatures. Virulence measured as total insect-mortality at 8°C improved by 5.3- and 6.6-fold after six and 12 passages, respectively. Only small improvements (1.2–1.5-fold) were observed in speed of kill. Nematode establishment improved at all temperatures after 12 passages; the highest increase of 9-fold was observed at 8°C. Our results lend support to the hypotheses that functional traits along a continuously distributed environmental variable are genetically correlated and that the area under the fitness function is not always constant. Trade-offs in percentage mortality and speed of kill by the selected nematodes were observed at the warmer extreme after six passages of selection only. The implications of rapid changes in thermal sensitivity for economic mass-production of nematodes are discussed.     

Ecological characterization of Steinernema anatoliense (Rhabditida: Steinernematidae)

Our study describes the basic ecological characteristics of the entomopathogenic nematode Steinernema anatoliense including its response to temperature, moisture, and host range. The effect of temperature and soil moisture on the infection of Galleria mellonella larvae by S. anatoliense was determined. The temperature range for infectivity was greater than that for development. The optimal temperature for infection and development was 25 degrees C. Although S. anatoliense infected the hosts at 10 degrees C, no reproduction occurred at this temperature. This nematode species that was isolated from a cold region of Turkey exhibited warm-adapted temperature characteristics. Optimum water content of the soil for S. anatoliense to infect the host was 10%.     

Comparative study of the entomopathogenic nematode, Steinernema carpocapsae, reared on mutant and wild-type Xenorhabdus nematophila

The symbiotic interaction between Steinernema carpocapsae and Xenorhabdus nematophila was investigated by comparing the reproduction, morphology, longevity, behavior, and efficacy of the infective juvenile (IJ) from nematodes reared on mutant or wild-type bacterium. Nematodes reared on the mutant X. nematophila HGB151, in which an insertion of the bacterial gene, rpoS, eliminates the retention of the bacterium in the intestinal vesicle of the nematode, produced IJs without their symbiotic bacterium. Nematodes reared on the wild-type bacterium (HGB007) produced IJs with their symbiotic bacterium. One or the other bacterial strain injected into Galleria mellonella larvae followed by exposing the larvae to IJs that were initially symbiotic bacterium free produced progeny IJs with or without their Xenorhabdus-symbiotic bacterium. The two bacterial strains were not significantly different in their effect on IJ production, sex ratio, or IJ morphology. IJ longevity in storage was not influenced by the presence or absence of the bacterial symbiont at 5 and 15 °C, but IJs without their bacterium had greater longevity than IJs with their bacterium at 25 and 30 °C, suggesting that there was a negative cost to the nematode for maintaining the bacterial symbiont at these temperatures. IJs with or without their symbiotic bacterium were equally infectious to Spodoptera exigua larvae in laboratory and greenhouse and across a range of soil moistures, but the absence of the bacterial symbiont inhibited nematodes from producing IJ progeny within the host cadavers. In some situations, such as where no establishment of an alien entomopathogenic nematode is desired in the environment, the use of S. carpocapsae IJs without their symbiotic bacterium may be used to control some soil insect pests.     

Hybridisation and genetic selection for improving desiccation tolerance of the entomopathogenic nematode Steinernema feltiae

Shelf life of biological control products based on the entomopathogenic nematode Steinernema feltiae is rather limited. In order to prolong shelf life, the metabolism of nematodes during storage must be reduced, either by low temperature or by means of desiccation of the third stage dauer juveniles (DJs). Tolerance to desiccation is limited in S. feltiae. Their tolerance can be increased by an adaptation to moderate desiccation conditions. The objective of this study was to screen for tolerant strains among wild type populations of S. feltiae, hybridise most tolerant strains and further improve desiccation tolerance by subjection of the hybrid strain to genetic selection. Dehydrating conditions, measured as water activity (a _w-values), were produced by treating DJs with different concentrations of the polymer polyethylene glycol 600. Significant variation was recorded among 24 S. feltiae strains. The mean tolerated water activity survived by 50% of the population (WA₅₀) ranged from 0.78 to 0.93 when nematodes were not adapted to desiccation stress and from 0.66 to 0.88 when preadapted to desiccation stress. The six most desiccation tolerant strains of non-adapted and adapted nematode populations were crossed. Preadapted tolerance of hybrids was superior to the tolerance of parental strains, whereas non-adapted tolerance was not increased. The most tolerant hybrid had a WA₅₀ when preadapted of 0.67 and 0.86 when not adapted. The tolerance was lost after few reproductive cycles in the insect Galleria mellonella but was recovered again after six selection cycles with exposure to increasing stress conditions. Virulence and reproduction potential was not negatively affected by the selection. Stabilisation of the selection progress will be a major challenge to enable commercial exploitation of the genetic improvement.     

Interactions between isolates of the entomopathogenic fungus Metarhizium anisopliae and the entomopathogenic nematode Heterorhabditis bacteriophora JPM4 during infection of the sugar cane borer Diatraea saccharalis (Lepidoptera: Pyralidae)

Interactions between the nematode Heterorhabditis bacteriophora isolate JPM4 and the fungus Metarhizium anisopliae, isolates LPP45 and LPP39, were studied during dual infections of Diatraea saccharalis. Mortality, production of infective juveniles (IJs) and production of conidia were evaluated. A positive effect was demonstrated for host mortality in duel infections of JPM4 and LPP39, causing 100% mortality with LT₅₀ and LT₉₅ values of 1.8 and 2.8 days, respectively. Higher values were seen when using the nematode or fungi individually. However, a combination of JPM4 + LPP39 caused a significant reduction in IJ production. The results show that faster time to death, a moderately virulent fungal isolate could be combined with the nematode, however at the expense of IJ production.     

Efficacy of entomopathogenic bacteria for control of Musca domestica

The aim of this study was to evaluate the larvicidal activity, and sub lethal effects of entomopathogenic bacteria Brevibacillus laterosporus, Bacillus thuringiensis var. israelensis, B. thuringiensis var. kurstaki, and a commercial formulation of Bacillus sphaericus on Musca domestica. Bacterial suspensions were prepared in different concentrations and added to the diet of newly-hatched larvae which were monitored until the adult stage. The larvae were susceptible to the B. laterosporus, B. thuringiensis var. israelensis, and B. thuringiensis var. kurstaki bacteria in varied concentration levels. These bacteria have larvicidal and sub lethal effects on the development of flies, reducing both adult size, and impairing the reproductive performance of the species.     

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.     

In-vitro liquid culture of the entomopathogenic nematode,Steinernema colombiense,in orbitally shaken flasks

Steinernema colombiense, an entomopathogenic nematode species (EPN) was grown in two types of orbitally shaken flasks at 130?rpm and 28°C, containing 10 or 20?mL, respectively of a complex culture medium with an initial EPN-concentration of 1,000 Infective Juveniles (IJ)/mL. At the 10th day, the EPN-concentration was 58,771 individuals/mL with 87% of them in the IJ stage. No significant differences were found between the EPN growth kinetics in both types of flasks. The nematode-population growth was modelled by a re-parameterized Gompertz equation of three-parameters with best-fit values of 3.8 days for the lag time, 33.8 day^-1 for the maximum growth rate, and 57.3 (dimensionless) for the maximum asymptotic growth.     

Enhanced biological control potential of the entomopathogenic nematode,Steinernema carpocapsae,applied with a protective gel formulation

Entomopathogenic nematodes (EPNs) are adapted to subterranean environments, and are prone to damage by UV light and desiccation. EPNs, Steinernema carpocapsae, combined with a protective gel, and anti-UV ingredients, have potential for above-ground pest management. We (1) ascertained whether the gel could provide protection to EPNs at low concentrations when applied in direct sun, (2) determined if other ingredients added to the gel increased efficacy, and (3) quantified retention and survival of EPNs applied to foliage with the gel. EPNs in 1% protective gel caused higher host mortality (60%) than other treatments (2–37%). UV protection provided by titanium dioxide (TD) and octyl methoxycinnamate with 1% protective gel solution was tested outdoors; these formulations resulted in higher host mortality (43% and 25%) than other treatments (2–7%). After 8?h in the greenhouse, 0.25% protective gel solution had the highest percentage of live EPNs on leaves. The gel at low concentration protects EPNs, and addition of TD enhanced the protective properties of the formulation. It is important to continually improve options for different growing systems and insect pest behaviour. TD added to a low-concentration formulation of the protective gel makes this application technique more viable for growers to use.     

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

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

Phenotypic characterization of the Xenorhabdus bacterial symbiont of a Texas strain of the entomopathogenic nematode Steinernema riobrave, and characterization of the Xenorhabdus bovienii bacterial symbiont of a Newfoundland strain of Steinernema feltiae

Two bacterial symbionts of entomopathogenic nematodes, one of which originated from Texas, U.S.A., and the other from Newfoundland, Canada, were characterized phenotypically. These strains belonged to the genus Xenorhabdus. The Newfoundland (NF) strain was shown to be X. bovienii but the Texas (TX) strain was not identified at the species level. Four additional cultures of Xenorhabdus were included in the study. These were a strain of X. bovienii (Ume?), which was from a nematode of European origin, and strains of X. nematophilus, X. beddingii, and X. poinarii. The tests used in this study indicated identical properties for the NF (North American) and Ume? (European) strains of X. bovienii. These could be differentiated from the other strains studied by their failure to grow at 34 degrees C and resistance to low concentrations of a mixture of amoxilline and clavulanic acid. The Xenorhabdus TX strain could be differentiated from the other strains studied by its failure to grow at 10 degrees C. Of the tests done, approximately 30 were useful in distinguishing between the strains and species studied.     

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.