Search

Summary Background Dietary salts sodium (Na+), potassium (K+), magnesium (Mg2+), and calcium (Ca2+) are important in metabolic diseases. Yet, we do not have sufficient understanding on the salts global molecular network in these diseases. In this systematic review we have pooled information to identify the general effect of salts on obesity, insulin resistance and hypertension. Aims To assess the roles of salts in metabolic disorders by focusing on their individual effect and the network effect among these salts. Methods We searched articles in PubMed, EMBASE and Google Scholar. We selected original laboratory research, systematic reviews, clinical trials, observational studies and epidemiological data that focused on dietary salts and followed the preferred reporting items for systematic review in designing the present systematic review. Results From the initial search of 2898 studies we selected a total of 199 articles that met our inclusion criteria and data extraction. Alterations in metabolic pathways associated with the sensitivity of sodium, potassium, magnesium and calcium may lead to obesity, hypertension, and insulin resistance. We found that the results of most laboratory research, animal studies and clinical trials are coherent but some research outcome are either inconsistent or inconclusive. Conclusion Important of salts in metabolic disorder is evident. In order to assess the effects of dietary salts in metablic diseases, environmental factors, dietary habits, physical activity, and the microbiome, should be considered in any study. Although interest in this area of research continues to grow, the challenge is to integrate the action of these salts in metabolic syndrom.

To understand the infection strategies of the mosquito parasitic mermithid nematodes Romanomermis iyengari (Welch, 1964) and Strelkovimermis spiculatus (Poinar and Camino, 1986), we have investigated the foraging behaviors of the pre-parasites of both species. Romanomermis iyengari with body length of 0.99 ± 0.071 mm (twice as S. spiculatus 0.55 ± 0.01 mm) swims faster (2.76 ± 0.13 cm/min) than S. spiculatus (1.61 ± 0.09 cm/min). The nematode pre-parasites of R. iyengari showed negative phototaxis in responding to natural light, which caused them to migrate to shade where mosquito larvae tend to aggregate. This behavior helped the nematode to gain significantly higher parasitism when shade was present (37.7 ± 2.74%) than no shade (11.3 ± 0.95%). In contrast, S. spiculatus pre-parasites had no response to natural light and therefore, shade contributed no parasitism gain to this species. The host searching pattern data indicated that the two species used different strategies for host searching. The pre-parasites of R. iyengari tended to do long range search and responded to host aggregation habitat but slowed down and do more localized search when host present. However, the species of S. spiculatus , tended to do localized movement when hosts were absent regardless shade. Once host larvae present in nearby location, the nematodes responded to the host cue and moved faster toward their hosts. The active foraging behavior of R. iyengari may suggest that the species is more suitable for mosquito control in clear and moving water such as streams. However, the passive stay and wait behavior of S. spiculatus might be more suitable against mosquitoes in small sized water bodies such as temporarily flooded or stagnant ponds.

The mosquito-parasitic nematode, Strelkovimermis spiculatus (Mermithidae: Nematoda) emerges from hosts and aggregates to form mating clusters characterized by intense male-male competition for females. Successful males deposit an adhesive copulatory plug over the female vulva after insemination. In choice experiments, males strongly preferred virgin females, whereas plugged females were ignored. Males made no attempt to displace copulatory plugs deposited by previous males. Plugged females repelled males without the need for physical contact. The observed chemical repellency was independent of females, since excised plugs alone showed the same negative male response. Removal of the plug shortly after mating reduced fecundity by 90%, presumably due to spermatids leaking from the vulva. The plug as a nutritional gift hypothesis was rejected because there was no post-mating reduction in plug size that would have indicated absorption. Despite being a male adaption to sperm competition, we conclude that the copulatory plug serves the fitness needs of both males and females in multiple roles that include paternity preservation, reduced male harassment, improved male search for mates, increased fecundity, and sperm retention.

Background Lipid metabolism dysfunction leading to excess fat deposits (obesity) may cause tumor (cancer) development. Both obesity and cancer are the epicenter of important medical issues. Lipid metabolism and cell death/proliferation are controlled by biochemical and molecular pathways involving many proteins, and organelles; alteration in these pathways leads to fat accumulation or tumor growth. Mammalian Kruppel-like factors, KLFs play key roles in both lipid metabolism and tumor development. Scope of review Substantial epidemiological and clinical studies have established strong association of obesity with a number of human cancers. However, we need more experimental verification to determine the exact role of this metabolic alteration in the context of tumor development. A clear understanding of molecules, pathways and the mechanisms involved in lipid metabolism and cell death/proliferation will have important implications in pathogenesis, and prevention of these diseases. Major conclusion The regulatory role of KLFs, in both cell death/proliferation and lipid metabolism suggests a common regulation of both processes. This provides an excellent model for delivering a precise understanding of the mechanisms linking altered expression of KLFs to obesity and tumor development. General significance Currently, mouse and rats are the models of choice for investigating disease mechanisms and pharmacological therapies but a genetic model is needed for a thorough examination of KLF function in vivo during the development of an organism. The worm Caenorhabditis elegans is an ideal model to study the connectivity between lipid metabolism and cell death/proliferation.

a b s t r a c t Suppression of Aedes albopictus populations is a substantial challenge for mosquito control programs globally because juveniles of this species are found in numerous kinds of domestic artificial containers that are difficult to detect, access, and eliminate. We conducted a multi-year assessment of the effect of different interventions to control Ae. albopictus near the northernmost geographic boundary of the species in temperate North America and deployed an array of BG-Sentinel traps for adult surveillance. Here we present the results of a comparative examination of adult sex ratios in urban and suburban areas, shifts in sex ratios after control interventions, and a discussion of the critical drivers of population dynamics of Ae. albopictus in our area. We collected significantly more male mosquitoes in urban as compared to suburban areas in June through September, but not in May (p < 0.001). The higher number of male mosquitoes in urban areas could be attributed to a higher number of larval habitats within a closer proximity of the surveillance traps and the lower flight dispersal of males. Following application of adulticides in urban areas, Ae. albopictus male populations were reduced by 88% on average, which was higher than the 69% reduction in female populations. The higher reduction of male mosquitoes could be attributed to the smaller body mass of the males and their higher susceptibility to adulticides. The results of this study are directly relevant to the development of suitable control strategies that depend on manipulation of males, such as the sterile insect technique. The results could also be used to refine mosquito abatement by providing more accurate methods to determine the need and timing of vector

a b s t r a c t Autodissemination of insecticides is a novel strategy for mosquito management. We tested if contami- nated Aedes albopictus (Skuse) mosquitoes from a small area treated with commercial formulations (79 gm a.i. pyriproxyfen/ha) using conventional techniques, would disseminate pyriproxyfen over a wider area. Pyriproxyfen showed LC50 = 0.012 ppb for Ae. albopictus. Direct treatment and autodissemination effi- cacy was measured as a pupal mortality by conducting Ae. albopictus larval bioassay. A tire pile (n = 100 tires) treated by backpack sprayer as a point-source treatment showed higher pupal mortality in 2010 (60.8% for week 0-6) than in 2011 (38.3% for week 0-6). The sentinel containers placed for autodissem- ination in four compass directions out to 200-400 m from the tire pile showed 15.8% pupal mortality (week 1-6) in the first year, and 1.4% pupal mortality in the second year. No significant difference was detected among the distances and direction for pupal mortality. In area-wide treatments, vegetation was sprayed in checkerboard pattern (3.7% of 105 ha) using backpack sprayer in 2010 and in strips (24.8% of 94 ha) using truck-mounted ultra-low volume (ULV) sprayer in 2011. In both years, the area-wide direct treatment efficacy was lower (30.3% during 2010 and 5.3% in 2011) than point-source treatments. Autodissemination in area-wide plots was higher in 2010 (10.3%) than 2011 (2.9%). However, area-wide treatments were ineffective on field populations of Ae. albopictus as monitored by using BGS traps. We found accumulation of pyriproxyfen in the week 6 autodissemination containers in both experiments. The differences in autodissemination in 2010 and 2011 can be attributed to higher rainfall in the second year that may have eroded the pyriproxyfen from treatment surfaces and sentinel containers. Our study shows that ULV surface treatments of conventional formulation do not work for autodissemination. The effectiveness of pyriproxyfen in autodissemination may be improved by developing specific formulations to treat vegetation and tires that can load high doses on mosquitoes.

Mating aggregations in the mosquito parasitic nematode, Strelkovimermis spiculatus, were investigated in the laboratory. Female postparasites, through their attraction of males and, remarkably, other females, drive the formation of mating clusters. Clusters may grow in size by merging with other individual or clusters. Female molting to the adult stage and reproductive success are enhanced in larger clusters. Male mating behavior is initiated when the female begins to molt to the adult stage by shedding dual juvenile cuticles posteriorly. Males coil their tail around the adult cuticle, migrating progressively along the female in intimate synchrony with the molting cuticle until the vulva is exposed and mating can occur. The first arriving male is assured of access to a virgin female, as his intermediate location between the vulva and subsequently arriving males blocks these competitors. Males deposit an adhesive gelatinous copulatory plug into and over the vulva before departing the female. Fecundity was greater in larger mating clusters, but this was a function of a greater rate of molting which is a prerequisite for mating. Males compete for virgin females by emerging and molting to the adult stage earlier than females. Mating aggregations have previously only been examined in snakes, but these studies have tended to be observational as snakes offer a challenging system for study. The relatively easy to culture and manipulate mermithid system may offer a model for experimental studies of male-male competition, protandry, copulatory plugs and female choice in mating clusters.

a b s t r a c t Interspecific variations in the susceptibility of freshly and embryonated eggs of Aedes albopictus, Ae. aegypti, Ae. atropalpus and Culex pipiens were tested against three classes of insect growth regulators (IGRs) including ecdysone agonist (azadirachtin), chitin synthesis inhibitor (diflubenzuron) and juvenile hormone analog (pyriproxyfen) at 0.001, 0.01, 0.1 and 1.0 ppm concentrations. Egg hatching inhibition was dose dependent, the highest being at 1.0 ppm concentration for freshly laid eggs of Ae. albopictus (pyriproxyfen: 80.6%, azadirachtin: 42.9% and diflubenzuron: 35.8%). Aedes aegypti showed lower egg hatching inhibition when exposed to pyriproxyfen (47.3%), azadirachtin (15.7%) and diflubenzuron (25.5%). Freshly laid eggs of Cx. pipiens were most susceptible to diflubenzuron. Aedes atropalpus eggs were tolerant to all three classes of IGRs. Embryonated eggs of Ae. albopictus, Ae. aegypti, Ae. atropalpus and Cx. pipiens were resistant to pyriproxyfen, azadirachtin and diflubenzuron than freshly laid eggs. The median desiccation time (DT50) of Ae. atropalpus eggs was maximum (5.1 h) as compared to Ae. aegypti (4.9 h), Ae. albopictus (3.9 h) or Cx. pipiens (1.7 h) eggs. Insignificant relationship between the rates of desiccation and egg hatching inhibition suggests other factors than physical providing eggs the ability to tolerate exposures to various IGRs. Egg hatching inhibition was due to the alteration in embryonic development caused by IGRs. Changes in the egg shell morphology and abnormal egg hatching from the side of the egg wall instead of operculum, was observed at higher concentrations of diflubenzuron. Morphological and physiological variations in eggs may be the key factor to influence the ovicidal efficacy of IGRs. The present data provide a base line for the improvement of the ovicidal efficacy of the insecticide and its formulation.

Lipid metabolism is coordinately regulated through signaling networks that integrate biochemical pathways of fat assimilation, mobilization and utilization. Excessive diversion of fat for storage is a key risk factor for many fat-related human diseases. Dietary lipids are absorbed from the intestines and transported to various organs and tissues to provide energy and maintain lipid homeostasis. In humans, disparity between triglycerides (TG) synthesis and removal, via mitochondrial β-oxidation and VLDL (very low density lipoprotein) secretion, causes excessive TG accumulation in the liver. The mutation in Caenorhabditis elegans KLF-3 leads to high TG accumulation in the worm's intestine. Our previous data suggested that klf-3 regulates lipid metabolism by promoting fatty acid β-oxidation. Depletion of cholesterol in the diet has no effect on fat deposition in klf-3 (ok1975) mutants. Addition of vitamin D in the diet, however, increases fat levels in klf-3 worms. This suggests that excess vitamin D may be lowering the rate of fatty acid β-oxidation, with the eventual increase in fat accumulation. We also demonstrate that mutation in klf-3 reduces expression of C. elegans dsc-4 and/or vit genes, the orthologs of mammalian microsomal triglyceride transfer protein and apolipoprotein B, respectively. Both microsomal triglyceride transfer protein and apolipoprotein B are essential for mammalian lipoprotein assembly and transport, and mutation in both dsc-4 (qm182) and vit-5 (ok3239) results in high fat accumulation in worm intestine. Genetic interactions between klf-3 and dsc-4, as well as vit-5 genes, suggest that klf-3 may have an important role in regulating lipid assembly and secretion.

In vertebrates, adipose tissue stores energy in the form of fat. Fat storage is tightly controlled by and dynamically balanced with energy expenditure under physiological settings; the perturbation of fat in either excess (obese) or deficit (lipodystrophy) has devastating pathologic consequences in the fueling of homeostasis and organismal fitness. The process by which fat storage is coordinated through positive and negative feedback signals is still poorly understood. To address potential mechanisms underlying fat storage we study a Caenorhabditis elegans Krüppel-like transcription factor, Ce-klf-3 and demonstrate that klf-3 is a hitherto unrecognized key regulator of fat metabolism in C. elegans. The Ce-klf-3 is highly expressed during larval development and predominantly present in intestine: the site of fat digestion, absorption, storage, and utilization. We found a strong positive correlation between klf-3 expression and fat deposition in a worm's intestine. Significantly, a klf-3 (ok1975) loss-of-function mutation, characterized by the deletion of a 1658-bp sequence spanning the 3' end of exon 2 through to the 5' end of exon 3 of klf-3, enhanced fat deposition in the intestine and caused severe defects in worm reproduction. Although klf-3 mutants seemed very similar to wild type worms in appearance and life span, 70% of mutants became semi-sterile, each producing 40-50 viable progenies, and the remaining 30% were rendered completely sterile toward adulthood. Notably, both mutant types displayed extensive deposition of fat in the intestine. Our study also demonstrates that klf-3 is critical for maintaining normal fatty acid composition by regulating genes involved in a fatty acid desaturation pathway. Strikingly, klf-3 mutant animals with impaired fatty acid beta-oxidation pathway genes resulted in fat accumulation in the mutant worm. We present the first clear in vivo evidence supporting essential regulatory roles of KLF-3 in fat storage in C. elegans and shed light on the human equivalent in disease-gene association.

Reproduction of entomopathogenic nematodes requires that they escape recognition by a host’s immune system or that they have mechanisms to escape encapsulation and melanization. We investigated the immune responses of larvae for the greater wax moth (Galleria mellonella), tobacco hornworm (Manduca sexta), Japanese beetle (Popillia japonica), northern masked chafer (Cyclocephala borealis), oriental beetle (Exomala orientalis) and adult house crickets (Acheta domesticus), challenged with infective juveniles from different species and strains of entomopathogenic nematodes. The in vivo immune responses of hosts were correlated with nematode specificity and survival found by infection assays. In P. japonica, 45% of injected infective juveniles from Steinernema glaseri NC strain survived; whereas the hemocytes from the beetle strongly encapsulated and melanized the Heterorhabditis bacteriophora HP88 strain, S. glaseri FL strain, Steinernema scarabaei and Steinernema feltiae. Overall, H. bacteriophora was intensively melanized in resistant insect species (E. orientalis, P. japonica and C. borealis) and had the least ability to escape the host immune response. Steinernema glaseri NC strain suppressed the immune responses in susceptible hosts (M. sexta, E. orientalis and P. japonica), whereas S. glaseri FL strain was less successful. Using an in vitro assay, we found that hemocytes from G. mellonella, P. japonica, M. sexta and A. domestica recognized both nematode species quickly. However, many S. glaseri in M. sexta and H. bacteriophora in G. mellonella escaped from hemocyte encapsulation by 24 h. These data indicate that, while host recognition underlies some of the differences between resistant and susceptible host species, escape from encapsulation following recognition can also allow successful infection. Co-injected surface-coat proteins from S. glaseri did not protect H. bacteriophora in M. sexta but did protect H. bacteriophora in E. orientalis larva; therefore, surface coat proteins do not universally convey host susceptibility. Comparisons of surface coat proteins by native and SDS–PAGE demonstrated different protein compositions between H. bacteriophora and S. glaseri and between the two strains of S. glaseri.

Entomopathogenic nematode in vivo production depends on a high level of successful infection during the inoculation process. Some host–nematode combinations do not provide sufficient or consistent infection levels and are, therefore, not suitable for in vivo production. Our objective was to determine whether infection levels could be improved through host or nematode exposure to physical and chemical stressors. Elevated stress may compromise host insect defenses, thus increasing its vulnerability to nematode infection. Specifically, we investigated the effects of stressors on Heterorhabditis bacteriophora infection of Tenebrio molitor larva. The stressors included temperature extremes, insecticidal oils and metal cations. Most chemical stressor–nematode combinations caused high host mortality and low infectivity rates. However, infective juvenile stimulation by the cation Mn2+ did enhance infectivity compared to the control. Dry heat (40 °C for 30 min) and hot water (60–70 °C for 1 s) treatments increased the host’s susceptibility to H. bacteriophora infection with respect to unstressed controls, and the yields and virulence of infective juveniles produced in hosts stressed by these treatments were not affected. Thus, host stress methods can be used to successfully enhance infection levels for in vivo production of H. bacteriophora.

Serial culturing of organisms used for biological pest suppression often leads to detrimental genetic changes and loss of utility. We established that genetically homozygous inbred lines can deter beneficial trait decline in the entomopathogenic nematode, Heterorhabditis bacteriophora . Three inbred lines and the foundation population were serially cultured in the insect host, Galleria mellonella . Trait stability was evaluated by comparing serially cultured with non-cultured populations. Laboratory data indicated that serial culture of the foundation population (16 passages) resulted in more than a 30% loss in traits deemed beneficial for biological pest suppression i.e., virulence to an insect host ( Diaprepes abbreviatus ), reproductive capacity, heat tolerance (at 38 °C), and host-seeking ability. In contrast, the three inbred lines were impervious to decline in all beneficial traits. A greenhouse test targeting D. abbreviatus provided additional evidence that the biocontrol efficacy of the inbred lines remained stable during serial culture. Our results indicate that genetic factors played an important role in trait change, and creation of inbred lines may be a useful technique for maintaining beneficial traits.

Scarab beetle larvae are widespread insect pests that can be controlled by the entomopathogenic nematode Heterorhabditis bacteriophora. The nematode is applied as an inundative biological control agent, which means that large numbers ( ≈2.5×10 9 ha −1 ) are applied uniformly to crops. Nematode persistence in these systems tends to be poor. Many theoretical mechanisms have been proposed whereby spatial structure can promote population persistence. We hypothesized that nematode persistence could be enhanced by applying nematodes in spatial distributions other than the typical uniform distribution. We applied H. bacteriophora ( 2.5×10 9 ha −1 ) to 3×3 m plots of turf in three spatial patterns: (1) uniform distribution; (2) one central circular patch (diameter 1.12 m); and (3) nine individual patches (diameter 0.38 m). Nematode persistence and spatial distribution were monitored over 1 year. In all treatments, nematode numbers declined to similar levels. Over time, the nematodes applied in patches moved from their initial application sites and became more evenly distributed, whereas the distribution of nematodes in plots with uniform application became patchier as nematodes died. No significant differences were observed in nematode numbers or spatial pattern from week 20 until the end of the experiment. Grub densities were measured at the end of the experiment. Nematodes that had been applied uniformly or in nine patches significantly reduced grub numbers, but the one-patch application did not.

1. Entomopathogenic nematodes penetrate and kill Galleria mellonella within 48 h at optimal temperatures. 2. Low temperature induces infection latency, preventing host death until optimal conditions resume. 3. Infected Galleria survived 25 days at 5°C. On transfer to 25°C, 100% and 12.5% of Steinernema carpocapsae and Steinernema riobravis infected larvae died within 72 h. 4. Infective juvenile penetration decreased with decreasing temperature; declining from 49.7 and 49.3 nematodes/host at 25°C to 6.3 and 0.25 nematodes/host at 5°C for S. carpocapsae and S. riobravis , respectively. 5. Latent infection occurs, albeit infrequently, due to low host penetration at low temperature.

As the only biocontrol agent available for many soil insects, entomopathogenic nematodes should be poised for wider use, as regulatory scrutiny in the USA continues to restrict or ban important chemical insecticides. One component of nematode production in the USA is a cottage industry of low volume producers using in vivo technology, based on a method devised in 1927: the White trap. We report the first scalable system for in vivo nematode mass production. Unlike the White trap, there is no requirement for nematode migration to a water reservoir. The LOTEK system of tools and procedures provides process technology for low-cost, high-efficiency mass production. The system consists of: (1) perforated holding trays to secure insect hosts during inoculation, conditioning (synchronizing nematode emergence), and harvesting, (2) an automated, self-cleaning harvester with misting nozzles that trigger infective juvenile emergence and rinse the nematodes through the holding trays to a central bulk storage tank, and (3) a continuous deflection separator for washing and concentrating nematodes. The harvester collects 97% of Heterorhabditis bacteriophora Poinar that emerged from Galleria mellonella (L.) cadavers in 48 h. The separator removes 97.5% of the wastewater in three passes, while nematode concentration increased 81-fold. The rearing system offers an increase in efficiency relative to the conventional White trap method with reduced labor and space.

Several entomopathogenic nematode species are currently under evaluation for mass production and field efficacy for biological control of insect pests. However, quality and quantity of in vitro -produced entomopathogenic nematodes vary considerably, depending on media, temperature, and production method. In addition, nematode production should be cost effective. We investigated nematode yield, production time, total lipid content, and fatty acid composition of Heterorhabditis bacteriophora produced in artificial media supplemented with different lipid sources. Lipid source significantly affected lipid quantity and quality in H. bacteriophora. Media supplemented with extractable insect lipids produced yields 1.9 times higher than did beef fat- or lard-supplemented media. Moreover, the developmental rate in media supplemented with host lipids was 1.7 times faster than that in media supplemented with beef fat or lard. Nematodes grown in media supplemented with insect lipids accumulated significantly higher lipid proportion per dry biomass than those grown in media supplemented with other lipid sources. H. bacteriophora produced in media supplemented with insect lipids, olive oil, or canola oil had similar fatty acid patterns, with oleic (18:1) acid as the major lipid fatty acid. Media supplemented with other lipid sources produced nematodes with fatty acid patterns different from those of media supplemented with insect lipids. We recommend addition of fatty acid mixtures that resemble natural host lipids for mass-producing entomopathogenic nematodes. This could provide nematode quality similar to in vivo -produced nematodes and could improve yield.

In previous greenhouse studies, the insecticide imidacloprid and the entomopathogenic nematode Heterorhabditis bacteriophora Poinar interacted synergistically against third instars of the masked chafers Cyclocephala hirta LeConte and C. pasadenae Casey (Coleoptera: Scarabaeidae). We tested this interaction for two additional nematode species and three additional scarab species under field conditions. In greenhouse tests, H. bacteriophora and Steinernema glaseri (Steiner) interacted synergistically against third instars of the Japanese beetle, Popillia japonica Newman, the oriental beetle, Exomala orientalis Waterhouse, and the masked chafers Cyclocephala borealis Arrow, C. pasadenae, and C. hirta. The degree of interaction varied with nematode species. The strongest synergism occurred between imidacloprid and S. glaseri. Synergism between imidacloprid and H. bacteriophora was weaker and the interaction was not always significant. Combinations of imidacloprid and S. kushidai Mamiya only resulted in additive mortality. The synergistic interaction was also observed in field trials but the results were more variable than those under greenhouse conditions. The combination of nematodes and imidacloprid could be used for curative treatments of white grub infestations, especially against scarab species that are less susceptible to nematodes and/or imidacloprid. This combination has a low environmental impact and high compatibility with natural biological control of turfgrass insects. The possible roles of these combinations in augmentative control approaches are discussed.

The quality of the most widely commercialized entomopathogenic nematodes, Steinernema carpocapsae (Weiser) and Heterorhabditis bacteriophora Poinar, was assessed from 30 shipments from six United States suppliers mailed to three locations. These suppliers comprise a cottage industry aimed in large part at a mail-order market. Most companies were accessible and reliably shipped pure populations of the correct species on time, in sturdy containers, and often with superb accompanying instructions. Nematodes were received in satisfactory condition with acceptable levels of viability. Consistency, however, was a problem, with each supplier having one or more weak spots to bolster. Most shipments did not contain the expected nematode quantity, and one shipment had no nematodes. Pathogenicity of several products against Galleria mellonella (L.) larvae was not equivalent to our controls. H. bacteriophora was not always available when ordered. A few products contained mixed populations of both nematode species. Application rate recommendations provided by some suppliers appeared unsound. We conclude that (1) the entomopathogenic nematode cottage industry lacks rigorous quality control, (2) self-regulation is problematic without feedback on quality, and (3) consumers are rarely able to provide this feedback. Improved reliability by the nematode industry will most likely be achieved via industry-generated agreement on standards for quality.

The lipid, sterol, and fatty acid compositions of Heterorhabditis bacteriophora infective juveniles reared by in vivo and in vitro methods (Galleria mellonella, Popillia japonica, solid and liquid culture) were compared. The greatest accumulation of lipids per dry weight was achieved by growing nematodes in P. japonica and solid culture (54.7 and 60.3%, respectively). Nematodes produced by liquid fermentation accumulated lower levels of sterols (2%) per total lipids compared with other production methods. However, the level of polar lipids was higher in nematodes produced in artificial media. Fractionation of the polar lipids revealed that an increased level of phosphatidylglycerol accompanied production in liquid culture. This was not observed for nematodes produced in solid culture in which the major polar lipid fractions were phosphatidylcholine and phosphatidylethanolamine. Lipids from nematodes produced in artificial media had more linoleic (C18: 2) fatty acids, whereas lipids from in vivo cultures had more stearic (C18: 0) fatty acids. The ratio of saturated to unsaturated fatty acid composition decreased in cultures produced in vitro. Artificial media composition should be adjusted to meet the nutritional composition of a natural host. Media for H. bacteriophora culture should be modified to boost deficiencies in sterols (e.g., liver extract) and oleic acid (e.g., olive oil). r 1999 Academic Press

A strain of the entomopathogenic nematode,Heterorhabditis bacteriophora,was transformed by the addition of a heat-shock protein gene (hsp70A) from the free-living nematodeCaenorhabditis elegans.Laboratory experiments with wild-type and transgenic nematodes were done at 16, 25, 30, and 37°C to investigate infectivity, reproductive capacity, and survival in water. Heat-shocked transgenic nematodes (ones subjected to a heat shock that is lethal to wild-type nematodes) were included in experiments to determine if this treatment caused sublethal damage. We found no significant differences in the ability of wild-type, transgenic, and heat-shocked-transgenic nematodes to infect or to reproduce in last-instarGalleria mellonellalarvae or to survive at these temperatures. We compared the ability of wild-type and transgenic nematodes to infect and kill 11 species of invertebrates, representing groups known to be susceptible and nonsusceptible to wild-type nematodes. Transformation caused no detectable change in virulence of nematodes to any test invertebrates. Transgenic and wild-type nematodes were also injected interperitoneally into mice and transgenic nematodes were fed by injection to mice without mouse mortality or changes in growth rate. We conclude that genetic engineering has provided a precise method to alter the heat shock tolerance ofH. bacteriophorawithout affecting other important life history characteristics and that the transgenic nematode is unlikely to pose any threat to the environment if released on a wide scale.

The host immune response is a key obstacle to entomopathogenic nematodes in making the transition from the free-living state to parasitism. The entomopathogenic nematode Steinernema glaseri has evolved mechanisms to evade immune encapsulation in larvae of the Japanese beetle, Popillia japonica. Host intrahemocoelic injection tests show that live axenic nematodes of S. glaseri not only avoid host melanotic encapsulation but also protect dead nematodes injected after the live ones. This result indicates that the nematodes release anti-immune factor(s). We extracted the nematode surface coat proteins and found that at least one protein (SCP3a) from the S. glaseri surface coat can suppress the host immune system. This suppression protects unrelated nematode species from encapsulation and latex beads from phagocytosis. We conclude that S. glaseri uses an anti-immune protein to defeat the host immune system, thereby protecting itself from encapsulation. Presumably its symbiotic bacteria are similarly protected from phagocytosis.

Bacteria in the genera Photorhabdus and Xenorhabdus are highly pathogenic to insects and are symbiotically associated with nematodes in the genera Heterorhabditis and Steinernema , respectively. We compared extracellular protease production and resistance to antibiotics (chloramphenicol, erythromycin, neomycin and tetracycline) for 32 isolates of P. luminescens and 11 isolates of X. poinarii taken from nematodes isolated from soil in southern New Jersey. P. luminescens produced greater amounts of protease, and was more resistant to erythromycin and less resistant to neomycin than X. poinarii . No interspecific differences in resistance to chloramphenicol or tetracycline were detected. Variability within species was not related to the site, habitat or soil core from which the isolates were obtained, and was not associated with variation in the color of the host cadaver for P. luminescens . Resistance to erythromycin was positively correlated with resistance to both neomycin and tetracycline for P. luminescens , but was negatively correlated with resistance to neomycin for X. poinarii . Antibiotic resistance profiles and extracellular protease production might be useful characteristics for distinguishing among species and strains of these bacteria, probably have ecological significance with respect to intra- and interspecific competition within host cadavers, and could have implications for the utility of these organisms for biological control.

In laboratory assays, male infective juveniles of some species of entomopathogenic nematodes in the genus Steinernema differ from females in their tendency to disperse and in their responses to the volatile cues emitted by parasitized and nonparasitized insects. These differences suggest that male infective juveniles might locate and establish in insect hosts before females, and that infection by males might render hosts suitable for nematode development and more attractive to females: "the male colonization hypothesis." We tested this hypothesis in laboratory experiments in which larvae of the greater wax moth, Galleria mellonella (L.), were exposed to infective juveniles of S. glaseri (Steiner) (NC strain) in sand columns for various periods of time. The hosts were dissected to determine the sex ratio of the adult nematodes that became established. We found that infective juveniles entered hosts over periods of up to at least 14 h, and that sex ratios varied among experiments. However, there were no temporal differences in colonization by males and females during the infection process: the proportion of males in host cadavers was not related to exposure time or to the total number of nematodes in host cadavers. This result is inconsistent with the male colonization hypothesis. We conclude that differential colonization of hosts over time by males and females either does not occur in S. glaseri, or occurs only rarely or under conditions differing from ours, or occurs to such a small extent that it is difficult to detect. In S. glaseri, male infective juveniles tend to emerge from host cadavers before females. This might give males an advantage over females in locating new hosts following a natural emergence. Nonetheless, to date, males have not been shown to colonize hosts before females in any steinernematid species. Copyright 1998 Academic Press.

Entomopathogenic nematodes Steinernema riobravis, S. carpocapsae and S. glaseri survived prolonged exposure to freezing at −4°C. LT50 values were 2.1, 1.8 and 0.6 days respectively. Steinernema riobravis and S. carpocapsae were still pathogenic after 6 days of freezing and S. glaseri showed poor pathogenicity over 4 days of freezing at −4°C. Incubation in 20% glycerol for 48 h prior to freezing at −20°C enhanced survival and pathogenicity of S. carpocapsae. Survival increased to 30 days; nematodes were still pathogenic after 24 days of exposure.

1. 1. The entomopathogenic nematode Steinernema riobravis grows and reproduces over a wide temperature range (15, 20, 25, and 30°C) in the insect host Galleria mellonella . 2. 2. The lipid content of S. riobravis varied in amount and composition. PE and PC were the two major constitutive classes of polar lipids whereas triglycerols were the major constitutive classes of neutral lipids. Lipid content of nematodes grown at 15°C was marginally lower than at other growth temperatures. Nematodes accumulated higher proportions of saturated fatty acids when grown at high temperature (30°C). 3. 3. The ability of S. riobravis to adjust its lipid and fatty acid composition in response to growth temperature could contribute to thermal tolerance. We recommend culturing S. riobravis at 25–30°C. At these temperatures more lipids are accumulated which may improve the physiological quality of S. riobravis compared to nematodes produced at lower temperatures (15–20°C). In addition, the growth and development cycle at high growth temperature in the insect cadaver is shorter compared to low temperature, which reduces the cost of mass production if applied in fermentation.

A strain of the entomopathogenic nematodeHeterorhabditis bacteriophora,genetically enhanced for thermotolerance by introduction of a heat-shock protein gene from the free-living nematodeCaenorhabditis elegans,was released in turfgrass field microplots in the spring, summer, and fall of 1996. As predicted, transgenic and wildtype strains did not differ in their ability to persist. We document the regulatory procedures at the federal, state, university, and local levels needed before field release, none of which posed any significant difficulties. Our risk assessment study supports the regulatory view that the transgenic nematode strain is an unlikely environmental threat. Subsequent regulatory reviews in the United States appear likely to continue to be decided on a case-by-case basis according to organism phenotype rather than the techniques used to generate them. This is the first report of a nonmicrobial, genetically engineered insect natural enemy being released into the environment.

1. 1. All organisms tested respond to a sudden increase of temperature by synthesizing heat-shock proteins, which helps organisms to survive high temperature. A correlation with increased thermotolerance and production of major 70 kDa protein has been observed in many organisms. 2. 2. Many studies have been designed on a large number of animal species to assess their adaptation to different thermal environments. Genetic analysis of the hsp 70 gene in entomopathogenic nematodes inhabiting different environments may provide insight into the physiological roles of hsps in these nematodes and could be useful for ecological studies. 3. 3. To assess variation among species of entomopathogenic nematodes for thermotolerance, we initiated a search for the molecular organization of heat-inducible hsp 70 genes in these nematodes. Five Heterorhabditis species/isolates with different temperature optima for survival and one warm-adapted species of Steinernema were tested 4. 4. PCR and RFLP analyses of hsp 70 in Heterorhabditis species and S. scapterisci demonstrated a putative homology with the Caenorhabditis elegans hsp 70 A gene, thus indicating evolutionary conserved nature among different nematode species. 5. 5. RFLPs with the hsp 70 A gene probe revealed different banding patterns for Heterorhabditis species and isolates. 6. 6. This is the first report on the identification of any hsp 70 gene in entomopathogenic nematodes. 7. 7. Our observation establishes a unique correlation between geographical distribution and polymorphisms for hsp 70 A gene in these nematodes.

Entomopathogenic nematodes (Rhabditida: Heterorhabditidae, Steinernematidae) are lethal insect parasites that have a symbiotic association with bacteria in the family Enterobacteriaceae. We evaluated the changes in thermal sensitivity of two nematodes Heterorhabditis bacteriophora and Steinernema anomali. The nematodes were genetically selected, together with their respective symbiotic bacteria, at nematodes' reproductive thermal niche breadth extremes, 15deg; and 30deg;C, by repeated passage through larvae of the wax moth Galleria mellonella. Nematode virulence (rate of insect mortality), establishment, and reproduction were evaluated after 12 passages (equal to 24-36 generations). Direct and correlated responses of the selected strains were compared with the ancestral strains, maintained at 25deg;C, at the selection and novel temperatures. The thermal limits for virulence and establishment were extended in both species and at both selection regimes. The thermal reproductive niche breadth of H. bacteriophora was extended from 15deg;-30deg;C to 12deg;-32deg;C after selection at either 15deg; or 30deg;C, but that of S. anomali remained unchanged. Virulence of S. anomali was enhanced across the entire thermal niche breadth, but H. bacteriophora improved in one portion of the thermal niche and declined in another portion. Enhanced virulence of S. anomali was most likely due to the improvements in growth rate of the symbiotic bacteria, Xenorhabdus sp. Establishment of both species and reproduction of H. bacteriophora improved at most temperatures. S. anomali reproduction showed no direct response to temperature selection, but decrements and/or improvements were evident at novel temperatures. Our results demonstrate that the temperature-specific virulence and thermal niche breadth of entomopathogenic nematodes are malleable. Adaptation to either cold or warm temperature enhanced fitness in both cold and warm environments. S. anomali adapted to novel temperature by enhancing virulence, whereas H. bacteriophora improved fecundity. Trade-offs in fitness were rarely observed.

1. 1. Nematodes survive subzero temperatures using either a freeze-avoiding or freezing-tolerant strategy. Steinernema anomali, S. feltiae, and Heterorhabditis bacteriophora were all found to be freezing tolerant. 2. 2. The lower lethal temperatures were −22, −19 and −14°C for S. feltiae, H. bacteriophora and S. anomali, respectively. 3. 3. Survival after prolonged freezing at −4°C was 6, 5 and 3 days for S. feltiae, H. bacteriophora and S. anomali, respectively. 4. 4. Acclimation to lower temperatures increased freezing tolerance. The freezing tolerance of Heterorhabditis bacteriophora increased under a stepwise acclimation regime; S. feltiae acclimated better under a direct acclimation regime.

Understanding the ability of natural populations of predators, parasitoids, and pathogens to suppress prey populations is important in determining their potential as biological control agents. We measured the seasonal population dynamics of endemic entomopathogenic nematodes ( Steinernema carpocapsae and Heterorhabditis bacteriphora ) in turfgrass and their effect on populations of Japanese beetle, Popillia japonica, larvae and mobile arthropods associated with the soil surface and thatch layer. Both nematode species were recovered in central New Jersey in samples collected from April to December. S. carpocapsae tended to be more prevalent in the plots (32.6 and 37.1% of sections) than H. bacteriophora (8.3 and 0.3% of sections). However, when only the positive sections were analyzed H. bacteriophora had a higher density (63.6 ± 49.3 nematodes/cm 2 ) than S. carpocapsae (17.0 ± 4.9 nematodes/cm 2 ). Sections with H. bacteriophora had significantly lower P. japonica densities compared to sections without nematodes (58.5 ± 20.2 compared to 112.7 ± 9.2 larvae/m 2 ). S. carpocapsae did not have a measurable impact on P. japonica populations. Mobile surface arthropods, as measured by pitfall catches, were higher in H. bacteriophora positive sections than in sections without nematodes. This difference was due to higher catches of Araneae and Lithobiomorpha. Mobile surface arthropod populations did not differ between sections with and without S. carpocapsae . The scope of H. bacteriophora′s impact on host populations is likely to be limited because of the nematodes patchy distribution.

We report the first successful transformation of an entomopathogenic nematode. Foreign genes were introduced in Heterorhabditis bacteriophora HP88 by microinjection using vectors carrying the Caenorhabditis elegans genes coding for the roller phenotype and 16-kDa heat shock protein (hsp16) gene. A translational fusion made by inserting lacZ in frame into hsp16 was expressed in the body musculature, hypodermis, and pharyngeal muscles. The transcription of the hsp16/lacZ transgenes resulted in the rapid synthesis of detectable levels of beta-galactosidase. This research opens new avenues for genetic modification of entomopathogenic nematodes.

Defense mechanisms of Japanese beetle larvae, Popillia japonica, and their effectiveness against entomopathogenic nematodes were investigated in the laboratory. Behavioral grooming by P. japonica larvae was effective in reducing invasion of both Steinernema glaseri and Heterorhabditis bacteriophora. Forty percent of H. bacteriophora infective juveniles were inactivated or killed in the host gut fluid within 8 hr, whereas 11% of S. glaseri were killed. Host melanotic encapsulation, induced by injection of 20 H. bacteriophora infective juveniles, caused 75% nematode mortality. S. glaseri was less susceptible to the host immune response, which caused only 12% mortality of injected nematodes. In a sand column assay, S. glaseri was found in the host hemocoel 8 hr earlier than H. bacteriophora. However, Photorhabdus luminescens, the bacterium of H. bacteriophora, was detected 16 hr earlier in the host's hemolymph than S. glaseri's bacterium, Xenorhabdus poinarii. Despite the differences in effectiveness of host defenses, S. glaseri and H. bacteriophora caused similar host mortality in the sand column assay. We demonstrated that the two nematode species use different strategies for invasion of P. japonica larvae. S. glaseri, which tolerated P. japonica gut fluid and avoided the host immune response, has a high initial rate of penetration and survival; H. bacteriophora, which exhibited poor tolerance to the host gut fluid and induced a strong host immune response, releases its bacteria during or soon after penetration to kill the host.

The entomopathogenic nematode Steinernema glaseri enters into the host through natural openings and penetrates through the internal walls before entering into the hemocoel. In the present study we found that in addition to mechanical means used for penetration, entomopathogenic nematodes release proteolytic enzymes. When nematodes were treated with protease inhibitors and injected into Galleria mellonella gut, the percentage mortality of G. mellonella was reduced to 17 ± 2 for iodoacetamide and 30 ± 4 for 1,10-phenanthroline-treated nematodes compared to 75 ± 3 for control after 18 hr post anal injection. Treatment of the nematodes with combinations of protease inhibitors and protein synthesis inhibitors did not result in a synergistic effect on G. mellonella mortality. Nematode penetration of G. mellonella gut after their treatment with iodoacetamide and 1,10-phenanthroline was reduced to 13.4 ± 1.1 and 15.6 ± 1.9, respectively, after 12 hr postinjection through the anus. The antibiotic chloramphenicol inhibited the symbiotic bacteria Xenorhabdus poinarii cells and reduced the average viable number retained/100 nematodes to 40 ± 30. Cycloheximide delayed G. mellonella mortality but not gut penetration. This suggests that it might have some toxic effect on the nematodes.

We demonstrate a relationship between the foraging strategies of entomopathogenic nematodes and the pattern of emergence of male.and female infective juveniles from their host. We hypothesize that protandry as a mating strategy may be most adaptive in situations where cruising search is employed to find hosts. Alternatively, when attachment to a host is more passive, as in ambush foraging, a more constantly female-biased sex ratio should be maintained. Cruise-foraging infective juvenile Steinernema glaseri that develop into adult males emerge from host cadavers before those that develop into females and are immediately sensitive to volatile host cues. Females that emerge later are less attracted to host volatiles than are males during the first week of the infective stage. However, recently emerged females are strongly attracted to parasitized hosts. Infective juvenile Steinernema carpocapsae showed the opposite trend, with significantly more females emerging on the first day than the later harvest. In most protandrous mating systems, males take the risks of colonizing a new resource. The benefit derived by the early males is to have prolonged access to females inside the new host. Females penetrate into the safer parasitized hosts to find males. Their chances of prereproductive death are decreased in two ways; males prepare the host by breaking down defenses, and males make it possible for the females to mate immediately when they reach adulthood. Reported variability between harvests of S. glaseri may be partially attributed to protandry.

Larvae of the Japanese beetle, Popillia japonica , respond to attack by infective juveniles of the entomopathogenic nematode Heterorhabditis bacteriophora with aggressive and evasive behaviors. Brushing with the legs and rubbing with the abrasive raster were key aggressive behaviors associated with nematode attack in petri dish assays. Remarkably, contact with even a single live nematode triggered behavioral countermeasures directed at eliminating the attacker. Unrestrained P. japonica free to display aggressive behaviors removed more than 60% of attacking nematodes from their cuticle and experienced significantly less parasitism than larvae restrained from displaying defensive behaviors (13% or less of the nematodes removed). Unrestrained larvae were also capable of killing 20.6 ± 4.7% of the attacking nematodes. Observations of aggressive behavior in soil assays supported our petri dish results. Moreover, larvae were observed to respond to the presence of nematodes with evasive behavior. These defense behaviors are so generalized it appears unlikely that they evolved as a specific scarab evolutionary response to selection pressure from entomopathogenic nematodes, but rather to any threat or irritant.

The distribution and abundance of entomopathogenic nematodes within and among sites were investigated by taking soil samples along transects at several locations in southwestern New Jersey and by assaying for nematodes using the Galleria -bait method. Entomopathogenic nematodes were found at 12 of 13 sites (92.3%) and in 72 of 600 soil samples (12.0%). Heterorhabditids (probably Heterorhabditis bacteriophora ) were found in 60 samples from 12 sites. Steinernematids were rarer, with Steinernema glaseri being found in 8 samples from 3 sites, S. feltiae (=bibionis) in 4 samples from 2 sites, and S. carpocapsae in 2 samples from 2 sites. Heterorhabditids differed significantly in abundance among sites with from 0 to 47.5% of samples from individual sites proving positive. Overall, heterorhabditids were equally abundant in turf and weedy habitats but significantly less abundant in closed-canopy forest where no positive records were obtained. Within sites, heterorhabditids were aggregated, with individual soil samples being much more likely to contain sufficient nematodes to infect multiple Galleria larvae than predicted based on the total number of positive soil samples and a random distribution. However, this patchiness occurred on a relatively small scale since the occurrences of nematodes in successive soil samples taken at 4-m intervals along transects were statistically independent of one another. Spatial heterogeneity is probably a fundamental aspect of the population biology of these species and has important ramifications for their population dynamics, population genetics, and community structure.

Grubs of the scarabaeid Cyclocephala hirta infected with the milky disease bacterium, Bacillus popilliae (Bp), were more susceptible to the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema glaseri than healthy grubs. Increased susceptibility was due to greater ease of nematode penetration through the midguts of Bp-infected insects and occurred after > 10 days exposure to B. popilliae spores. By that time, the bacterium was proliferating rapidly in the insect hemolymph and possibly stressed the insect by physiological starvation. Attraction of the nematodes to the insects and cuticular penetration by the nematodes were ruled out as possible reasons for the enhanced susceptibility of Bp-infected grubs. In greenhouse pot trials, S. glaseri killed more Bp-infected grubs than healthy grubs, whereas no differences were noted with H. bacteriophora. Thus, to achieve the best grub control in the presence of B. popilliae, application of S. glaseri is recommended. The compatibility of B. popilliae and entomopathogenic nematodes in the field is ensured by the existence of a diseased insect class with a low level of susceptibility to the nematode, the presence of B. popilliae in grubs not interfering with nematode progeny production, and the ability of B. popilliae spores to survive invading nematodes and their associated bacteria.

A strain of Steinernema carpocapsae selectively bred in the laboratory for improved host-finding of scarab larvae did not provide enhanced field efficacy of Japanese beetle larvae, Popillia japonica. Selected (S20) and wildtype (All) strains reduced larval populations by 25-30%, compared with 70% for the chemical insecticide. Similar results were obtained in laboratory exposures conducted in soil-filled pots against the scarab Maladera matrida . The selected S20 strain was, nevertheless, superior to the All and HP88 strains at locating larvae. S20 nematodes, which are known to have enhanced chemosensitivity to carbon dioxide, were observed to aggregate heavily at or near the spiracles. Scarab spiracles are covered with sieve plates and, therefore, are not a portal of nematode entry. Despite selection for greater response to host cues, S20 persisted in nictating, standing on their tails, even when a host was present. The unfavorable field results usually obtained with S. carpocapsae against scarab larvae have been attributed to low powers of host search. Our results indicate that even when larvae are located S. carpocapsae is poorly adapted to cause infection. Genetic improvement cannot he expected to overcome poor adaptation. Future selection efforts with nematodes should begin with species adapted for the target host.

Density-dependent factors within a host can have an important influence on the population dynamics of parasites. We compared the effects of increasing entomopathogenic nematode ( Steinernema carpocapsae and Heterorhabditis bacteriophora ) density within a Galleria mellonella larva. Although the number of invading nematodes increased with increasing dose, percentage penetration declined. This density-dependent penetration was not sufficient to prevent the detrimental effects of overcrowding. Although both S. carpocapsae and H. bacteriophora had optimal survival at a density of approximately 100 nematodes per host, H. bacteriophora was better able to tolerate higher densities. Fecundity, measured as female size and infective juvenile production, declined at densities above and below 100 nematodes per host. The longest infective juveniles were produced at the lowest nematode densities, indicating a tradeoff between size and number of progeny. Effects of high density appear to result from competition for limited nutrients within the host. Effects of density should be taken into account in the laboratory culture and field release of entomopathogenic nematodes.

Penetration of infective juveniles of Steinernema glaseri and Steinernema carpocapsae into Japanese beetle larvae, Popillia japonica , was compared using external exposure and injection methods. Steinernematids entered the grubs through the mouth and anus, but not through the spiracles or cuticle. For all treatments, S. glaseri had higher levels of penetration than S. carpocapsae . Penetration of the gut by orally injected nematodes began 4-6 hr postinoculation. All regions of the gut were penetrated, with more than 82% of all penetrating nematodes invading the host hemocoel via the midgut. Gastric cecae were important sites for nematode penetration. Penetration sites of anally injected nematodes were the rectal sac of the hindgut. Penetration rates of isolated guts and that in vivo did not differ. Defecation by P. japonica was rapid, with orally injected nematodes appearing in the feces in 4-8 hr. Our results indicate that compared with S. glaseri, S. carpocapsae is poorly adapted to parasitize Japanese beetle larvae.

Infectivity of the entomopathogenic nematode Steinernema scapterisci to a range of insect species was evaluated using the standard petri dish/filter paper assay. The nematodes infected and reproduced in insects in the orders Coleoptera, Diptera, Lepidoptera, and Orthoptera, but some insects were resistant. Resistent insects were the coleopteran larva Leptinotarsa texana, the dermapteran adult earwig Forficula auricularia, the orthopteran adult Periplaneta americana, and the lepidopteran pupae Spodoptera exigua. S. scapterisci from two geographical areas (Argentina and Uruguay) infected and reproduced equally in the last-instar wax moth Galleria mellonella . The nematodes were more pathogenic to G. mellonella larvae than to the adult house cricket Acheta domesticus with the LC50 (median lethal concentration) for A. domesticus being 38 times higher than that for G. mellonella. The host finding tactic of S. scapterisci appears to be similar to that of Steinernema carpocapsae wherein a large proportion of infective juveniles ambush hosts. In a sand-based assay, temperature affected the infectivity (penetration ability and insect mortality) of both S. scapterisci and S. carpocapsae, S. scapterisci being more infective at higher temperatures than S. carpocapsae. The optimum temperature for nematode penetration and establishment in G. mellonella larvae was 24°C for S. carpocapsae and 32°C for S. scapterisci. Temperature also affected the rate of G. mellonella mortality by the nematodes; compared with S. carpocapsae, S. scapterisci killed hosts slowly. In water, S. scapterisci survived and retained infectivity during the 16-week test period; however, percentage survival was greater at 10 and 25°C than at 4°C. The greater infectivity, development, reproduction, and storage stability of S. scapterisci at relatively higher temperatures indicate their probable adaptation to warm climates. It is concluded that the wide experimental host range, characteristic of entomopathogenic nematodes, also holds true for S. scapterisci.

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" (

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.

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.

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.

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.

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.