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Infective-stage juveniles of Steinernema carpocapsae entered the Liriomyza trifolii leafmine via the oviposition puncture created by the female fly during egg laying, or through any unnatural tear in the mine surface. Nematodes could not enter mines by penetrating the intact leaf cuticle. Upon contact with the host larva, nematodes were 12 times more likely to infect the insect via the anus than the mouth. Spiracles, both larval and puparial, were not entry routes because of their small size and morphology. Susceptible stages of L. trifolii to S. carpocopsae included all larval stages, the prepuparium, and the "early puparium" (

Two species of entomopathogenic nematodes, an ambush forager (Steinernema carpocapsae) and a cruising forager (S. glaseri), were assayed for their responses to volatiles associated with Galleria mellonella (Insecta: Lepidoptera). The responses of entomopathogenic nematodes to six host-associated volatile treatments were assayed. Live G. mellonella, live G. mellonella with the cuticular hydrocarbons removed, live G. mellonella with carbon dioxide removed, dead G. mellonella, dead G. mellonella with the cuticular hydrocarbons removed, and volatiles from S. exiqua feces were assayed in a Y-tube choice aparatus. Steinernema glaseri were attracted only to treatments producing carbon dioxide. Steinernema carpocapsae showed no significant response to any treatments tested. To assess the directionality of the response toward live G. mellonella, net average movement per nematode after 5, 10, and 20 min was measured for both nematode species. The assay method was based on the "quadrant plate" bioassay, with a 9 cm Petri dish containing 2% agar. Steinernema carpocapsae showed little directionality of response, whereas S. glaseri was attracted to carbon dioxide. We conclude that carbon dioxide attracts S. glaseri to areas likely to hold hosts. Ambush foragers are less influenced by cues associated with hosts or their habitat.

Control of Japanese beetle ( Popillia Japonica Newman) larvae by Heterorhabditis Bacteriophora Poinar (HP88 strain and a New Jersey strain, NJ-2) and Steinemema glaseri Steiner (NC strain and a New Jersey strain, NJ-43) showed that all strains reduced larval populations to a level comparable with that achieved by use of bendiocarb. Nematode strains and species did not differ significantly in mean level of population reductions ( H. bacteriophora HP88 and NJ-2, 51.0 and 71.6%; S. glaseri NC and NJ-43, 50.4 and 70.1%, respectively). However, variation in the level of population reduction provided by NJ-43 was less than that by the NC strain. After a 3-wk test period, nematode persistence and downward migration in the field did not differ among species and strains. In a laboratory bioassay, New Jersey strains provided higher levels of larval mortality compared with strains cultured in the laboratory for at least a decade. A new method used to count viable bacteria retained by individual infective juveniles showed that NJ-43 not only retained more bacteria than the NC strain (19.5 ± 0.2 compared with 4.9 ± 0.06 bacteria per infective juvenile) but also that the proportion of nematodes retaining bacteria was higher (77% compared with 55%). Bacterial retention by infective juveniles did not differ significantly between H. bacteriophora strains.

[Entomopathogenic nematodes (Heterorhabditidae and Steinernematidae) are insect parasites that appear to use both cruising and ambushing search strategies. Cruising species Heterorhabditis bacteriophora, Steinernema feltiae, and S. glaseri spent more time crawling (88.8, 80.3, and 92.5% of observation period, respectively) and therefore traveled farther (22.4, 18.6, and 24.3 mm, respectively) and searched a larger area (18.3, 13.7, and 26.6 mm2, respectively) than ambushing species. Ambusher species S. carpocapsae and S. scapterisci traveled shorter distances (4.8 and 4.7 mm, respectively) and searched smaller areas (3.0 and 2.5 mm2, respectively) because they spent most of the observation period nictating (77.5 and 78.2%, respectively). Nictating infective juveniles raise greater than 95% of their body off the substrate and maintain a straight posture. A nictating species S. carpocapsae was up to 43 times as effective at finding mobile insect hosts compared to a non-nictating species H. bacteriophora. When unable to nictate host-finding of mobile insects declined from 35.7±7.19 to 3.9±1.11 infective juveniles per host. Nictation, by reducing the surface tension forces holding the nematode to the substrate, can increase the nematodes ability to attach to passing insects. S. carpocapsae also tended to search more effectively along a surface than through a matrix. Although entomopathogenic nematodes have a broad potential host range, differences in search behavior may result in a narrower actual host range. Ambushers being more specialized for mobile insects on the soil surface and cruisers for more sedentary and/or subterranean insects., Entomopathogenic nematodes (Heterorhabditidae and Steinernematidae) are insect parasites that appear to use both cruising and ambushing search strategies. Cruising species Heterorhabditis bacteriophora, Steinernema feltiae, and S. glaseri spent more time crawling (88.8, 80.3, and 92.5% of observation period, respectively) and therefore traveled farther (22.4, 18.6, and 24.3 mm, respectively) and searched a larger area (18.3, 13.7, and 26.6 mm2, respectively) than ambushing species. Ambusher species S. carpocapsae and S. scapterisci traveled shorter distances (4.8 and 4.7 mm, respectively) and searched smaller areas (3.0 and 2.5 mm2, respectively) because they spent most of the observation period nictating (77.5 and 78.2%, respectively). Nictating infective juveniles raise greater than 95% of their body off the substrate and maintain a straight posture. A nictating species S. carpocapsae was up to 43 times as effective at finding mobile insect hosts compared to a non-nictating species H. bacteriophora. When unable to nictate host-finding of mobile insects declined from 35.7±7.19 to 3.9±1.11 infective juveniles per host. Nictation, by reducing the surface tension forces holding the nematode to the substrate, can increase the nematodes ability to attach to passing insects. S. carpocapsae also tended to search more effectively along a surface than through a matrix. Although entomopathogenic nematodes have a broad potential host range, differences in search behavior may result in a narrower actual host range. Ambushers being more specialized for mobile insects on the soil surface and cruisers for more sedentary and/or subterranean insects.]

Competitive interactions may facilitate or repel invaders into new communities, and these interactions may depend on other environmental conditions such as the presence of pesticides. Malathion is widely used in controlling agricultural pests and mosquitoes worldwide. Small amounts of malathion, previously considered inconsequential, may in fact increase in lethality when combined with biotic stressors in aquatic systems. We tested whether low concentrations of malathion (0.11 ppm) that are often detected in aquatic systems, affect competition between two invasive mosquito species Aedes albopictus (Skuse) and Aedes japonicus Theobald. There were no survivors of Ae. japonicus larvae in malathion. There was a significant negative effect of Ae. japonicus density on Ae. albopictus survival, but this effect was absent in the presence of malathion. There was also a moderate negative effect of Ae. japonicus density on Ae. albopictus female size, but this effect was absent in the presence of malathion. These findings indicate that pesticide-mediated alterations in competition and species-specific differences in susceptibility to pesticides could play a role in enhancing invasive potential of Ae. albopictus.

The nematode Steinernema carpocapsae infects and kills many pest insects in agro-ecosystems and is commonly used in biocontrol of these pests. Growth of the nematodes prior to distribution for biocontrol commonly results in deterioration of traits that are essential for nematode persistence in field applications. To better understand the mechanisms underlying trait deterioration of the efficacy of natural parasitism in entomopathogenic nematodes, we explored the maintenance of fitness related traits including reproductive capacity, heat tolerance, virulence to insects and ‘tail standing’ (formerly called nictation) among laboratory-cultured lines derived from natural, randomly mating populations of S. carpocapsae. Laboratory cultured nematode lines with fitness-related trait values below wild-type levels regained wild-type levels of reproductive and heat tolerance traits when outcrossed with a non-deteriorated line, while virulence and ‘tail standing’ did not deteriorate in our experiments. Crossbreeding two trait-deteriorated lines with each other also resulted in restoration of trait means to wild-type levels in most crossbred lines. Our results implicate inbreeding depression as the primary cause of trait deterioration in the laboratory cultured S. carpocapsae. We further suggest the possibility of creating inbred lines purged of deleterious alleles as founders in commercial nematode growth.

BACKGROUND: Aedes (Stegomyia) albopictus (Skuse), the Asian tiger mosquito, is an introduced invasive species in the United States that is responsible for a significant proportion of service requests to local mosquito control programs. This containerutilizing mosquito is refractory to standard mosquito abatement measures in the United States. This study is part of a USDA-ARS project to develop an area-wide management strategy for Ae. albopictus. The goal was to identify three study sites, similar in socioeconomic parameters, geography and Ae. albopictus abundance, in urban and suburban areas in Mercer and Monmouth counties in New Jersey. Prior service requests and light trap counts and also detailed county maps were used to chose nine preliminary sites (four in Mercer and five in Monmouth) where weekly surveillance forAe.albopictus was performed throughout the 2008 active season. RESULTS: Although outliers were detected, socioeconomic variables in the study sites within each county were fairly consistent. Ae.albopictus abundance was associated with poverty levels and had the highest maxima in Mercer, although average mosquito abundance was similar in urban Mercer and suburban Monmouth. CONCLUSION: Three study sites in each county were identified for future studies. The summer-long surveillance also revealed socioeconomic variables critical for the development of integrated mosquito management. c � 2011 Society of Chemical Industry

Billions of nematodes were released from 1939 to 1942 throughout the state of New Jersey (563 sites) in an effort to colonize the entomopathogenic species Steinernema glaseri for biological control of the Japanese beetle, Popillia japonica. Because of the onset of World War 11 and the postwar development of chlorinated hydrocarbon insecticides, little effort was expended to evaluate the outcome of these introductions. We evaluated this colonization program by collecting soil samples in 1991 from 304 geographically and ecologically diverse sites across New Jersey. The soil samples were assayed for entomopathogenic nematodes using the Galleria bait method. Overall, 66 (21.7%) soil samples were positive for entomopathogenic nematodes: 24 steinernematids and 42 heterorhabditids. The most common species isolated was Heterorhabditis bacteriophora (38 isolates), followed by S. glaseri (14), S. carpocapsae (4), S. feltiae (4), Heterorhabditis spp. (4), and Steinernema spp. (2). S. glaseri was recovered only from the southernmost third of the state. We conclude that the colonization effort initiated more than 50 years ago was unsuccessful. The reasons remain uncertain, but intolerance of S. glaseri to temperate climates is one likely explanation. That is, southern New Jersey appears to represent the northernmost range of this neotropical species. Moreover, early workers were unaware of the nematode's mutualistically associated bacterium, Xenorhabdus poinarii, which plays important roles in killing insect hosts and in nematode reproductive potential. We show that the bacterium is inhibited by antimicrobial compounds used by these workers during mass rearing, so it is probable that only the nematode portion of the nematode-bacterium complex was released.

Males of the horse flies Tabanus nigrovittatus Macquart and Tabanus conterminus (Walker) engage in daily periods of hovering and mating activity; the time period when hovering takes place is species-specific and strongly temperature-dependent. T. nigrovittatus males exhibit two distinct daily hovering periods, which occur over different temperature ranges. Thoracic temperatures of hovering males were elevated 10–12°C and were independent of air temperature; temperatures of early-hovering and late-hovering T. nigrovittatus males were significantly different. We found no evidence of endothermy in perching males; preflight warmup apparently was strictly ectothermic. Female T. nigrovittatus in forward flight did not thermoregulate as strongly as males. Males of the two groups of T. nigrovittatus did not exhibit significant differences in wing area, wing length, wing load, body mass, rate of convective cooling, or wingbeat frequency that might explain their different flight temperatures. Differences in the internal temperature associated with hovering may represent a prezygotic isolating mechanism to reduce or prevent interaction between T. nigrovittatus and T. conterminus . Alternatively, the bimodal pattern of mating activity may be a consequence of physiological or biochemical limitations on hovering flight.

Infective juveniles of the entomopathogenic nematode Heterorhabditis bacteriophora exposed to medium-wave ultraviolet radiation (302 nm) were inactivated at radiation levels that did not affect Steinernema carpocapsae . Irradiated H. bacteriophora showed a significant delay in progeny emergence at an exposure of 1.5 min, a decline in reproductive capacity at 2 min, and loss of pathogenicity at 4 min. Negative effects were not noted until exposures of at least 6 min for S. carpocapsae . Complete inactivation of H. bacteriophora resulted following 4 min of irradiation, compared with 10 min for S. carpocapsae . The infective juvenile sheath, an additional cuticle retained from the second-stage molt, did not provide either nematode species with significant protection from uv inactivation. The poor uv stability shown by H. bacteriophora is consistent with the tendency of heterorhabditids to be less tolerant of environmental stress than steinernematids. Heterorhabditid nematodes appear to be among the most vulnerable of all entomopathogens to uv inactivation. Their application onto exposed surfaces such as foliage should be discouraged.

Background The success of a biological control agent depends on key traits, particularly reproductive potential, environmental tolerance, and ability to be cultured. These traits can deteriorate rapidly when the biological control agent is reared in culture. Trait deterioration under laboratory conditions has been widely documented in the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora (Hb) but the specific mechanisms behind these genetic processes remain unclear. This research investigates the molecular mechanisms of trait deterioration of two experimental lines of Hb, an inbred line (L5M) and its original parental line (OHB). We generated transcriptional profiles of two experimental lines of Hb, identified the differentially expressed genes (DEGs) and validated their differential expression in the deteriorated line. Results An expression profiling study was performed between experimental lines L5M and OHB of Hb with probes for 15,220 ESTs from the Hb transcriptome. Microarray analysis showed 1,185 DEGs comprising of 469 down- and 716 up-regulated genes in trait deteriorated nematodes. Analysis of the DEGs showed that trait deterioration involves massive changes of the transcripts encoding enzymes involved in metabolism, signal transduction, virulence and longevity. We observed a pattern of reduced expression of enzymes related to primary metabolic processes and induced secondary metabolism. Expression of sixteen DEGs in trait deteriorated nematodes was validated by quantitative reverse transcription-PCR (qRT-PCR) which revealed similar expression kinetics for all the genes tested as shown by microarray. Conclusion As the most closely related major entomopathogen to C. elegans, Hb provides an attractive near-term application for using a model organism to better understand interspecies interactions and to enhance our understanding of the mechanisms underlying trait deterioration in biological control agents. This information could also be used to improve the beneficial traits of biological control agents and better understand fundamental aspects of nematode parasitism and mutualism.

Knowledge of the host-feeding behavior and extent of interactions with human hosts are important in evaluating the role and vector potential of invasive mosquitoes in transmission of native arboviruses. We collected blood-engorged females of the recently established exotic species Aedes japonicus japonicus from sites in New Jersey during 2000 to 2007 and identified the sources of vertebrate blood meals by sequencing portions of the cytochrome b gene of mitochondrial DNA. Over 1/3 (36%, n = 36) of the engorged mosquitoes acquired blood meals from humans. Other mammalian hosts included white-tailed deer (53%), fallow deer (5%), horse (3%), and Virginia opossum (3%). No avian, amphibian, reptilian, or mixed blood meals were identified. Our detection of a comparatively high prevalence of human bloodfeeding in Ae. j. japonicus in association with its local abundance, vector competence, and repeated detection of West Nile virus from field-collected specimens illustrates the potential for this invasive mosquito to serve as a "bridge" vector in transmission of West Nile and other mosquito-borne viruses in North America.

Standard surveillance traps in North America for adult Aedes albopictus (Skuse) (Diptera: Culicidae), an invasive mosquito with public health implications, are currently ineffective. We compared the efficacy of the BG-Sentinel trap (BGS) with and without lures (BG-lure, octenol, and CO2), the Centers for Disease Control and Prevention light trap (CDC) with and without lures, and the gravid trap (GT) for Ae. albopictus collection in two urban sites in New Jersey. The BGS with or without lures collected more Ae. albopictus compared with other trap configurations and was more specific for Ae. albopictus. In Camden County, the BGS with lures collected three times more Ae. albopictus than the CDC (with CO2 only) and five times more than the GT. In Mercer County, BGS with lures collected the most mosquitoes, with 3 times more Ae. albopictus than the CDC with all lures and 50 times more than the GT. The BGS collected more male Ae. albopictus than other traps in both counties, providing further population monitoring. The GT and BGS provided a relative measure of the enzootic activity of West Nile virus in Culex spp. and the potential epidemic activity of WNV in Ae. albopictus. The BGS provides effective chemical and visual cues for host-seeking Ae. albopictus and should be used as a part of existing surveillance programs and new initiatives targeting this mosquito.

Background The entomopathogenic nematode Heterorhabditis bacteriophora and its symbiotic bacterium, Photorhabdus luminescens, are important biological control agents of insect pests. This nematode-bacterium-insect association represents an emerging tripartite model for research on mutualistic and parasitic symbioses. Elucidation of mechanisms underlying these biological processes may serve as a foundation for improving the biological control potential of the nematode-bacterium complex. This large-scale expressed sequence tag (EST) analysis effort enables gene discovery and development of microsatellite markers. These ESTs will also aid in the annotation of the upcoming complete genome sequence of H. bacteriophora. Results A total of 31,485 high quality ESTs were generated from cDNA libraries of the adult H. bacteriophora TTO1 strain. Cluster analysis revealed the presence of 3,051 contigs and 7,835 singletons, representing 10,886 distinct EST sequences. About 72% of the distinct EST sequences had significant matches (E value < 1e-5) to proteins in GenBank's non-redundant (nr) and Wormpep190 databases. We have identified 12 ESTs corresponding to 8 genes potentially involved in RNA interference, 22 ESTs corresponding to 14 genes potentially involved in dauer-related processes, and 51 ESTs corresponding to 27 genes potentially involved in defense and stress responses. Comparison to ESTs and proteins of free-living nematodes led to the identification of 554 parasitic nematode-specific ESTs in H. bacteriophora, among which are those encoding F-box-like/WD-repeat protein theromacin, Bax inhibitor-1-like protein, and PAZ domain containing protein. Gene Ontology terms were assigned to 6,685 of the 10,886 ESTs. A total of 168 microsatellite loci were identified with primers designable for 141 loci. Conclusion A total of 10,886 distinct EST sequences were identified from adult H. bacteriophora cDNA libraries. BLAST searches revealed ESTs potentially involved in parasitism, RNA interference, defense responses, stress responses, and dauer-related processes. The putative microsatellite markers identified in H. bacteriophora ESTs will enable genetic mapping and population genetic studies. These genomic resources provide the material base necessary for genome annotation, microarray development, and in-depth gene functional analysis.

Entomopathogenic nematodes are lethal insect parasites that reproduce exclusively inside their hosts in nature. Infection decisions made by the free-living infective-stage juveniles have an impact on reproductive success, but it is likely that mating decisions are made by adults while inside their host. We investigated sexual communication between male and female adult stages of Steinernema carpocapsae (Rhabditida: Steinernematidae) to assess whether mating is chemically mediated during the adult stage or results from incidental encounters between adults inside the insect host. To assess chemical communication, we measured the behavioral response of adult male S. carpocapsae to several different potential sources of chemical information. Male S. carpocapsae responded to virgin females only and were not influenced by mated conspecific females, conspecific males, or heterospecific females. These results show that species-specific communication takes place between adult entomopathogenic nematodes within the host cadaver just prior to mating.

Entomopathogenic nematodes are used for biological control of insect pests. A method for improved cryopreservation of infective juvenile stage nematodes has been developed using Steinernema carpocapsae and Heterorhabditis bacteriophora. Optimum survival for both species was achieved with 12,000 infective juveniles/ml in glycerol and 7,500/ml in Ringer's solution. For S. carpocapsae, maximum survival also was observed with 60,000 infective juveniles/ml in glycerol and 25,000/ml in Ringer's solution. These concentrations resulted in 100% post-cryopreservation survival of S. carpocapsae and 100% retention of original virulence to Galleria mellonella larvae. This is the first report of achieving 100% survival of an entomopathogenic nematode after preservation in liquid nitrogen. Maximum survival of H. bacteriophora following cryopreservation was 87%.

We describe methodology used for the laboratory colonization of Aedes japonicus japonicus, an exotic mosquito species native to eastern Asia and first collected in New Jersey as larvae in 1999. We created a free mating colony in 2000 that readily bloodfed on restrained bobwhite quail (Colinus virginianus). A larval diet of finely ground Purina Lab Diet© dissolved in dechlorinated water has proven acceptable. This is the first report of Ae. j. japonicus colonization from mosquitoes collected in the United States.

Single and multiple releases of the entomopathogenic nematode, Heterorhabditis bacteriophora Poinar (= H. heliothidis Khan, Brooks, and Hirschmann) (HP88 strain), were evaluated for controlling populations and damage of the sweetpotato weevil, Cylas formicarius (Fabricius), during two consecutive growing seasons in southern Florida. Sweet potato, Ipomoea batatas (L.) Lam., plants were treated with one, two, or three applications of H. bacteriophora in a Krome very gravelly loam soil. Weevil population reduction and damage in nematode-treated sweet potato plots were compared with that in plots treated with monthly applications of chemical insecticides and in nontreated plots. Persistence of nematodes in soil was assessed at various times after release by baiting soil samples with greater wax moth larvae, Galleria mellonella (L.). Applications of H. bacteriophora did not consistently reduce weevil abundance, but did consistently reduce damage to sweet potato storage roots compared with nontreated plants. Weevil densities and damage did not differ between plants treated with one, two, or three applications of H. bacteriophora , suggesting that a single, early season release of this nematode was adequate for managing this weevil. Weevil damage on plants treated with chemical insecticides was intermediate to that on nematodetreated and nontreated plants. H. bacteriophora persisted in soil for over 130 and 250 days after application in the first and second experiment, respectively.

The effect of culture method on the efficacy of entomopathogenic nematodes was assessed by analysis of 511 greenhouse and field trials. In tests against larvae of the Japanese beetle, Popillia japonica Newman, and black vine weevil, Otiorhynchus sulcatus (F.), infective juveniles of Steinernema carpocapsae (Weiser) reared by in vivo, in vitro solid, and in vitro liquid culture methods provided equivalent larval reductions. Heterorhabditis bacteriophora Poinar infective juveniles produced in vivo and on solid media provided equivalent results in trials against Japanese beetle larvae and northern masked chafer, Cyclocephala borealis (Arrow). By contrast, H. bacteriophora produced by liquid culture achieved significantly lower Japanese beetle reductions than those reared in vivo or on solid media. Against the northern masked chafer, liquid culture H. bacteriophora provided significantly less host mortality than solid culture nematodes. Field persistence studies provided further evidence that liquid culture H. bacteriophora were inferior to infective juveniles produced on solid media. The poor performance of liquid culture H. bacteriophora was attributed to low lipid assimilation.

Summary Infective juveniles of the entomopathogenic nematode Steinernema carpocapsae show a low level of locomotory activity that is presumed to limit their usefulness as biological insecticides. A 30 μg ml-1 solution of the carbamate pesticide oxamyl reduced the proportion of nonmobile nematodes by nearly two thirds to 35%, while stimulating a 7.5-fold increase in sinusoidal movement. This increase in activity did not result in a corresponding increase in host-finding. Oxamyl treatment did not enhance infective juvenile pathogenicity to Galleria mellonella larvae. At higher concentrations, oxamyl caused aberrant nematode movement and partial paralysis. Heterorhabditis bacteriophora infective juveniles maintain a high level of locomotory activity. Treatment with 30 μg ml-1 oxamyl increased the proportion of sinusoidal over nonsinusoidal movements, but infective juvenile host-finding and pathogenicity were significantly reduced. Higher rates impaired movement and induced complete paralysis. We conclude that oxamyl is incompatible with S. carpocapsae and H. bacteriophora. The concept of chemically activating infective juveniles to increased locomotory activity and thereby achieving enhanced efficacy is inconsistent with our results.

In an examination of entomopathogenic nematodes used in inundative releases on turfgrass against Japanese beetle larvae, Popillia japonica Newman, 380 treatments from 82 field trials performed from 1984 to 1988 were analyzed using a standard protocol. The results show that most test failures can be explained on the basis of unsuitable nematode strains or environmental conditions. Steinemema carpocapsae (Weiser) appears ill-adapted to parasitize Japanese beetle larvae under any range of conditions. By contrast, the HP88 strain of Heterorhabditis bacteriophora Poinar, produced on solid media, provides control comparable with that by chemical insecticides at the appropriate season (fall), soil temperature (>20°C), soil type (silty clay), irrigation frequency (1-4-d intervals), and thatch depth

The effect of entomopathogenic nematodes on nontarget arthropods in the laboratory, field soils, and a stream were assessed. In the laboratory, adult predators were less susceptible to the nematodes Steinernema carpocapsae (Weiser) (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) than the immature stages. In field tests, entomopathogenic nematodes that had significantly suppressed pest populations ( Popillia japonica Newman, Japanese beetle, Scapteriscus vicinus Scudder, tawny mole cricket, Otiorhynchus sulcatus (F.), black vine weevil, Delia radicum (L.), cabbage maggot, and Diabrotica virgifera virgifera LeConte, western corn rootworm) did not adversely affect the numbers of nontarget soil arthropods in comparison with the untreated control. In contrast, broad-spectrum chemical insecticides (isofenphos, ethoprop, or chlorpyrifos used as chemical checks) significantly reduced or showed a tendency to reduce nontarget arthropod populations. In a stream trial, S. carpocapsae significantly reduced black fly larval populations, but the nontarget insects often increased in the treatment sites. Decreases in nontarget populations were matched by approximately equal or greater reductions in the upstream controls. We conclude that entomopathogenic nematodes do not adversely affect nontarget arthropods when used for short-term control of insect pests.

Infective juveniles of the entomopathogenic nematodes Steinernema carpocapsae (All and Mexican strains) and Heterorhabditis bacteriophora (HB1 and NC1 strains) are ensheathed in a cuticle retained from the previous molt. Exsheathment marks the transition from the free-living to the parasitic life cycle. Chemical and non-chemical exsheathment methods were examined. S. carpocapsae exsheaths more readily than H. bacteriophora. Movement on a substrate increased exsheathment in S. carpocapsae but had no effect on H. bacteriophora. Exposure to 1% sodium hypochlorite for 5 min resulted in nearly 100% sheath removal in both species. Storage increased exsheathment, with greater than 70% exsheathed in both species after 64 days at 7°C. Agitation and carbon dioxide did not stimulate exsheathment. Temperature alone did not stimulate exsheathment, however, increased temperatures accelerated exsheathment during storage. The availability of reliable exsheathment methods will enable analysis of exsheathment behavior and clarification of the role of the sheath in entomopathogenic nematodes.

The G13 strain of Steinernema carpocapsae (Weiser), previously selected for enhanced host-finding of waxmoth larvae, Galleria mellonella L., was subjected to selection for the same trait against scarab larvae. Twenty rounds of selection produced a 3.7-fold increase, from 18.4 to 68.7%, in nematode (S20 strain) location of nondiapausing larvae of the Japanese beetle, Popillia japonica Newman. However, the overall improvement over wild-types (Foundation and All strains), after 33 rounds (13 against G. mellonella and 20 against scarabs) was 153- and 72-fold, respectively. Infective juveniles had difficulty locating diapausing P. japonica larvae, and selection did not result in any enhancement in finding these larvae. Selection for host-finding of scarabs resulted in a 24.6% gain in host-finding of G. mellonella , suggesting selection was for a general feature present at different levels in different hosts. The use of KOH to absorb CO2 resulted in the complete inhibition of S20 strain response to insect hosts, verifying that nematodes have been selected for enhanced sensitivity to CO2. S20 nematodes were significantly less storage stable than the Foundation strain.

Soil temperature, moisture, and relative humidity (RH) effects on the survival and pathogenicity of Steinernema carpocapsae and S. glaseri were tested in the laboratory. Survival and pathogenicity of S. carpocapsae were significantly greater at lower temperatures (5–25°C) than at the highest temperature (35°C). Conversely, S. glaseri survival and pathogenicity were significantly greater at higher temperatures (15–35°C) than at the lowest temperature (5°C). S. carpocapsae and S. glaseri survived best at low soil moistures of 2 and 4%, respectively. After rehydration by bringing up all tested moistures to 16%, both nematode species caused lethal infection of Galleria mellonella larvae. Survival and pathogenicity of these two nematode species decreased as RH decreased from 100 to 25% over the 32-day test period. At 100% RH, S. carpocapsae and S. glaseri survived for 32 days, but as RH was lowered, survival decreased. For example, at 25% RH, S. carpocapsae survived for 2 days and S. glaseri survived for only 4 hr. Pathogenicity was reinitiated after rehydration and showed a trend similar to survival. These differences in survival and pathogenicity may be attributed to the climatic origins or the soil habitats of these two nematode species.

The host-finding response of a strain (G13) of the entomopathogenic nematode, Steinernema carpocapsae, selected for enhanced host-finding was compared with that of two wild-type strains (All and Foundation). The G13 strain showed a significant response to insect hosts up to a distance of 20 cm, compared to 3.5 cm in the wild types. Increasing the exposure period from 1 to 8 hr did not result in a corresponding increase in host-finding for any strain. G13 selection against the wax moth, Galleria mellonella, enhanced host-finding of other insect species as well, but equivalent enhancement was achieved only against the house cricket, Acheta domesticus. Host-finding by G13 infective juveniles was positively correlated with host carbon dioxide production. Removal of host carbon dioxide as a cue resulted in complete inhibition of chemotaxis in all three nematode strains. Our results indicate that the basis for enhanced host-finding in G-13 nematodes was selection for enhanced chemosensitivity to carbon dioxide.

Soils from 351 sites representing ecologically diverse habitats from six Hawaiian Islands (sea level to 4,200 m) were assessed for entomopathogenic nematodes using the Galleria baiting technique. Twenty-four sites (6.8%)were positive for entomopathogenic nematodes. Twenty-two sites (6.3%) were positive for a Heterorhabditis sp. from the islands of Kauai (6), Oahu (5), Maui (4), Molokai (1), and Hawaii (6), and two sites were positive for a Steinernema sp. from Maui. No entomopathogenic nematodes were recovered from soils on the island of Lanai. Heterorhabditids were highly correlated with ocean beaches within 100 m of seashore (0 m elevation). These positive sites had soils containing sand grains from coral and shells with moderately alkaline pH (8.0) and low organic content (12%). The steinernematid isolates came from inland areas in silty clay and silt loam soils with higher organic content (15–35%).

The free-living infective juveniles of entomopathogenic nematodes are ensheathed in the retained cuticle from the previous moult. Desiccation tolerance is essential for persistence in a soil environment and the sheath may have a protective function. We evaluated the role of the sheath in the desiccation tolerance of two species of entomopathogenic nematodes, Steinernema carpocapsae and Heterorhabditis bacteriophora. The sheath of H. bacteriophora was critical for survival at 97% relative humidity with 80.9% of sheathed infective juveniles surviving after 21 d versus 20.0% and 6.3% for exsheathed and chemically desheathed nematodes respectively. The sheath did not influence S. carpocapsae survival after 21 d at 97% relative humidity. In sand assays at 2, 4, 8 and 16% moisture levels, desheathed H. bacteriophora caused significantly less Galleria mellonella mortality than sheathed H. bacteriophora. No differences were found between the three S. carpocapsae treatments or sheathed H. bacteriophora. Our results indicate that the sheath plays an important role in desiccation tolerance of H. bacteriophora but has no effect on tolerance of S. carpocapsae.

Culex territans Walker (Diptera: Culicidae) larvae share nonpolluted freshwater habitats with amphibians, which are bloodmeal hosts of their adults. To examine synchrony of Cx. territans with amphibian species, 10 larval habitat sites were sampled weekly from March to November of 2004. Cx. territans larvae were temporally and spatially associated with the green frog, Rana clamitans Latrielle. We predicted that if the preferred hosts were abundant at low temperatures, Cx. territans might be able to digest bloodmeals at those same temperatures. Using the thermal heat summation model, 192.3 d above 3.9 degrees C were needed to complete the gonotrophic cycle. This is the lowest thermal minimum reported for a Nearctic species of mosquito. Using this model, we calculated that the first larvae of Cx. territans field collected on 6 May 2004 were the progeny of females that bloodfed during the last week of March or first week of April. We conclude that Cx. territans has physiological mechanisms that allow them to take advantage of early season bloodmeal sources.

Surface coat proteins (SCPs) of entomopathogenic nematodes are implicated in the suppression/evasion of host immune responses, which is required for successful host colonization. Steinernema glaseri NC strain SCPs suppressed immune responses in oriental beetle larvae (Exomala orientalis), a susceptible host for S. glaseri, in a dosage-dependent manner, thus protecting Heterorhabditis bacteriophora from being killed in the same host. Melanization of H. bacteriophora decreased from 92 ± 5% in the untreated check to 1 ± 3% when protected by injection of 230 ng of S. glaseri SCPs. As the SCPs dosage increased, freely moving H. bacteriophora increased from 3 ± 4% in the untreated group to 57 ± 15% with an SCPs dose of 940 ng. At 2 h and in the absence of SCPs, 8% and 11% of hemocytes of E. orientalis were stained by propidium iodide and Hoechst, respectively. When exposed to 300 ng/l SCPs, 70% and 96% were stained, respectively. At 6 h, propidium iodide stained 37% and 92% of the hemocytes without and with SCPs, respectively. In contrast, more than 90% of the cells were stained by Hoechst with or without SCPs. As native proteins, two isolated S. glaseri SCPs had an immunosuppressive effect; they were each composed of 38 kDa (PI = 4.6) and 56 kDa (PI = 3.6) subunits. SCP peptides were sequenced using LC-MS/MS and the mass fingerprints obtained with MALDI-TOF-MS; there were no significant matches found in peptide databases, which suggests that the SCPs studied are novel proteins. Twelve cDNA sequences were derived based on short peptides and 7 of them had no significant match against the Caenorhabditis elegans protein database. One of the cDNA matched an unknown C. elegans protein and the remaining 4 cDNAs matched proteins of C. elegans and Brugia malayi.

We compared Heterorhabditis bacteriophora GPS11 expressed sequence tags (ESTs) to the ESTs of animal-parasitic, human-parasitic, plant-parasitic, and free-living nematodes. We identified 127 previously nondescribed ESTs of which 119 had homologs in ESTs and 8 had homologs in proteins of free-living nematodes. These ESTs were assigned putative functions in transcription, signal transduction, cell cycle control, metabolism, information processing, and cellular processes, thereby providing better insight into H. bacteriophora metabolism, sex determination, and signal transduction. We also identified 36 H. bacteriophora ESTs that had significant similarities to ESTs of parasitic nematodes, but not to ESTs or proteins of free-living nematodes species. Among these are the ESTs encoding a centrin, an ankyrin-repeat containing protein, and a nuclear hormone receptor. Our analysis also revealed that parasitic nematode-specific ESTs in this H. bacteriophora data set had more homologs in animal-parasitic nematodes than those parasitizing humans or plants.

The salt marsh horse fly, Tabanus nigrovittatusMacquart, exhibits two nonoverlapping daily periods of hovering and mating activity, which are correlated with different environmental temperatures. Allelic and genotypic frequencies of hovering males collected during the two periods were compared by electrophoresis of three polymorphic enzyme loci. Approximately 26% of early-hovering males possessed a Pgmallozyme that was absent in our sample of late-hovering males. However, based on other allozyme loci, we found no evidence for reproductive isolation between early and late hoverers. All the genetic data are consistent with the hypothesis that the Pgmpolymorphism is associated with behaviorally and physiologically distinct groups of males that, by all other criteria, form a single Mendelian population.

A strain of Steinernema carpocapsae (G-13) selectively bred for improved host-finding was compared against two wild-type strains, the commercially available All strain and the Foundation strain from which the G-13 strain was derived, for changes in fitness. Selection did not affect pathogenicity, mobility, sex ratio, or morphology. However, the G-13 strain showed a gain of fitness with regard to host penetration and reproductive potential and a loss of fitness for storage stability. Acquiring enhanced host-finding abilities does not appear to be correlated with a serious reduction in overall fitness, and the potential of the selected G-13 strain for inundative biological control appears unlikely to be impaired. The possible significance of the three correlated responses to selection are discussed.

Survival and pathogenicity of two entomopathogenic nematodes, Steinernema carpocapsae (= Neoaplectana ) and Steinernema glaseri , were tested in four types of soil: sand, sandy loam, clay loam, and clay over a period of 16 weeks. Significant differences in the survival and pathogenicity of both nematodes occurred in all soil types. S. carpocapsae survival was 44.9, 38.8, 32.9, and 26.7% in sandy loam, sand, clay loam, and clay, respectively, whereas S. glaseri survival was 30.1, 25.9, 22.5, and 19.3% in sand, sandy loam, clay loam, and clay, respectively, at the end of the test. S. glaseri survival was significantly lower than S. carpocapsae in all soil types. Survival for S. carpocapsae was greatest in sandy loam, while survival for S. glaseri was best in sand. Pathogenicity assays with recovered nematodes supported the survival data. Persistence of both species decreased as the proportion of clay increased.

The Asian tiger mosquito, Aedes albopictus, is a highly invasive mosquito species that transmits chikungunya and dengue. This species overwinters as diapausing eggs in temperate climates. Early diapause termination may be a beneficial strategy for winter mosquito control; however, a mechanism to terminate the diapause process using chemicals is not known. We tested the hypothesis that a hormonal imbalance caused by the administration of juvenile hormone analog would terminate egg diapause in A. albopictus. We tested the insect growth regulator pyriproxyfen on all developmental stages to identify a susceptible stage for diapause termination. We found that pyriproxyfen treatment of mosquito eggs terminated embryonic diapause. The highest rates of diapause termination were recorded in newly deposited (78.9%) and fully embryonated (74.7%) eggs at 0.1 and 1 ppm, respectively. Hatching was completed earlier in newly deposited eggs (25-30 days) compared to fully embryonated eggs (71-80 days). The combined mortality from premature diapause termination and ovicidal activity was 98.2% in newly deposited and >98.9% in fully embryonated eggs at 1 ppm. The control diapause eggs did not hatch under diapausing conditions. Pyriproxyfen exposure to larvae, pupae and adults did not prevent the females from ovipositing diapausing eggs. There was no effect of pyriproxyfen on diapausing egg embryonic developmental time. We also observed mortality in diapausing eggs laid by females exposed to pyriproxyfen immediately after blood feeding. There was no mortality in eggs laid by females that survived larval and pupal exposures. In conclusion, diapausing eggs were the more susceptible to pyriproxyfen diapause termination compared to other life stages. This is the first report of diapause termination in A. albopictus with a juvenile hormone analog. We believe our findings will be useful in developing a new control strategy against overwintering mosquito populations.

The entomopathogenic nematode-bacteria complexes Heterorhabditis bacteriophora/Photorhabdus luminescens and Steinernema carpocapsae/Xenorhabdus nematophila are mass produced for use as biological insecticides. Stability of the bacterial partner in culture is essential for maintaining traits important for both biological control and production. Two geographically distinct strains of each bacterial species were isolated from their nematode partners and serially subcultured on in vitro media to assess trait stability. Subculturing resulted in a shift to secondary cell production in one P. luminescens strain and both X. nematophila strains within ten in vitro culture cycles. However, when cell phenotypic variation was controlled in X. nematophila strains by regular selection for primary variants, no trait change was detected in the primary variant after prolonged subculture. When P. luminescens cell phenotypic variation was controlled by selection for primary variants, changes in the primary variant of both strains were noted including reductions in cell and inclusion body size and inclusion body prevalence. Bacterial ability to cause lethal infections following injection into the hemocoel of Tenebrio molitor larvae declined by more than half in primary variants of one P. luminescens strain. Conversely, yield was enhanced, with the subcultured P. luminescens strains showing 53.5 and 75.8% increases in primary cell density. Field adapted traits of primary variant P. luminescens strains tend to deteriorate during in vitro culture as tradeoffs for gains in yield. In vitro producers of the P. luminescens/H. bacteriophora complex must weigh the need for superior bacterial yield against the need to preserve traits important for biological control.

Silicon micromachining has been used to fabricate microprobes for injecting DNA into cells. Arrays of very sharp pyramidal points are etched on a silicon substrate. Pressing these points into a culture of cells, allows biologically active material to cross the cell wall barrier. Using the microprobes, DNA has been injected into plant (tobacco leaves) and animal (nematodes) cells.

Our overall goal is to improve insecticidal nematodes by genetically engineering strains capable of entering an enhanced state of dormancy that provides improved stability. Objectives: 1. Clone and sequence tps-l homologue from Steinernema carpocapsae. (Revised: A failure to isolate the tps gene group from Steinernema precipitated a redirection to identifying other genes involved in insecticidal nematode desiccation process.) 2. Incorporate cloned tps-l gene into S. carpocapsae to obtain overexpression, thereby, enhancing desiccation tolerance. (Revised: Other stress genes in addition to tps-l genes were cloned and efforts at expression in S. carpocapsae were conducted) 3. Characterize the transgenic strains. No other biological control agent offers more impressive attributes than insecticidal nematodes. However, their potential is limited by the bane of nearly all biological control agents: poor stability. This leads to inadequate shelf-life and ultimately reduced field efficacy. Nematode storage is based on desiccation, yet insecticidal species are only capable of partial desiccation termed quiescent anhydrobiosis. Overwhelming evidence has shown that when the disaccharide compound trehalose is elevated in anhydrobiotic organisms such as yeast, plants, and nematodes it enables these organisms the ability to survive environmental stresses i.e., desiccation. Armed with this information our goal was to improve insecticidal nematodes stability by engineering trehalose overexpression.

Entomopathogenic nematodes (genera Steinernema and Heterorhabditis) kill insects with the aid of mutualistic bacteria. The nematode-bacteria complex is mass produced for use as biopesticides using in vivo or in vitro methods, i.e., solid or liquid fermentation. In vivo production (culture in live insect hosts) is low technology, has low startup costs, and resulting nematode quality is high, yet cost efficiency is low. In vitro solid culture, i.e., growing the nematodes and bacteria on crumbled polyurethane foam, offers an intermediate level of technology and costs. In vivo production and solid culture may be improved through innovations in mechanization and streamlining. In vitro liquid culture is the most cost-efficient production method but requires the largest startup capital and nematode quality may be reduced. Liquid culture may be improved through progress in media development, nematode recovery, and bioreactor design. A variety of formulations is available to facilitate nematode storage and application.