Optimization of Inoculation for In Vivo Production of Entomopathogenic Nematodes

Entomopathogenic nematodes are potent biopesticides that can be mass-produced by in vitro or in vivo methods. For in vivo production, consistently high infection rates are critical to efficiency of the process. Our objective was to optimize in vivo inoculation of Steinernema carpocapsae and Heterorhabditis bacteriophora in Galleria mellonella and Tenebrio molitor by determining effects of inoculation method, nematode concentration, and host density. We found immersing hosts in a nematode suspension to be approximately four times more efficient in time than pipeting inoculum onto the hosts. The number of hosts exhibiting signs of nematode infection increased with nematode concentration and decreased with host density per unit area. This is the first report indicating an effect of host density on inoculation efficiency. We did not detect an effect of nematode inoculum concentration on nematode yield per host or per gram of host. Yield was affected by host density in one of the four nematode-host combinations (S. carpocapsae and T. molitor). We conclude that optimization of inoculation parameters is a necessary component of developing an in vivo production system for entomopathogenic nematodes.     

Pathogenicity and Host Specificity of Entomopathogenic Nematodes

The mechanisms of infection and pathogenicity of Steinernematidae and Heterorhabditidae in insect hosts are discussed as factors influencing the host specificity of these nematodes. The invasion and evasion of host defences are important steps in the pathogenic process. The ability of the nematode to penetrate into the insect haemocoel, achieved by the release of proteolytic enzymes, is one specific factor. Another specific factor in the nematode-insect relationship is the ability of the nematode to evade insect defences through failure to be recognized and/or by destruction of insect antibacterial factors. Toxins and extracellular enzymes are important virulence factors released by these nematodes, apparently exhibiting a specific activity against certain insect hosts.     

Evolution of Host Search Strategies in Entomopathogenic Nematodes

There is interspecific variation in infective juvenile behavior within the entomopathogenic nematode genus Steinernema. This variation is consistent with use of different foraging strategies along a continuum between ambush and cruise foraging. To address questions about the evolution of foraging strategy, behavioral and morphological characters were mapped onto a phylogeny of Steinernema. Three species, all in the same clade, were classified as ambushers based on standing bout duration and host-finding ability. One clade of six species were all cruisers based on both host-finding and lack of standing behavior. All species in the ambusher clade had a high rate of jumping, all species in the cruiser clade had no jumping, and most intermediate foragers exhibited some level of jumping. Response to volatile and contact host cues was variable, even within a foraging strategy. Infective juveniles in the ambusher clade were all in the smallest size category, species in the cruiser clade were in the largest size categories, and intermediate foragers tended to be more intermediate in size. We hypothesize that the ancestral Steinernema species was an intermediate forager and that ambush and cruise foraging both evolved at least once in the genus.     

Survival Mechanisms of Entomopathogenic Nematodes

Entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) are isolated from a wide variety of ecosystems ranging from sub-Arctic to arid and tropical climates. It is expected that natural populations of these nematodes will be pre-adapted to specific ecological conditions in their environments. However, little is known about the survival strategies of entomopathogenic nematodes. This review focuses on the main survival mechanisms involved in the long-term persistence of nematodes: extreme temperature tolerance (cold and heat), desiccation tolerance, osmotic tolerance, pesticide resistance and limitations of aeration. Physiological and biochemical mechanisms for survival are discussed. The review addresses the implications of the survival strategies on the establishment of exotic and indigenous steinernematids and heterorhabditids.     

Post-application Persistence of Entomopathogenic Nematodes

Five phases can be distinguished in the post-application persistence of entomopathogenic nematodes and each phase is associated with a specific set of mortality factors. Pre-application factors associated with production, storage and transport conditions determine the survival rate and quality of nematodes at the time of application. The phase of tank mixing and application with a sprayer, hose or other equipment does not usually cause mortality as nematode dauer juveniles are quite tolerant of shear forces. The most critical periods for survival are the first few minutes and hours directly after application. High losses, in the order of 40-80%, often occur during this phase. Ultraviolet radiation and dehydration are probably the most important mortality factors. The remaining nematodes settle in the soil and their numbers gradually decrease at levels of 5-10% per day. Predation, infection by antagonists, depletion of energy and desiccation are probably the main mortality factors during this period. In most cases, after 2-6 weeks less than 1% of the applied population is still alive. Through recycling in host insects, nematodes may then persist for years at these levels. Thus, the pattern is a rapid decline in the first few days followed by a moderate decline over the next 2-6 weeks and then a long period of recycling at a low level. Some nematode species that normally occur in warmer climatic zones can also persist in colder climates. Major side-effects of applications of entomopathogenic nematodes are not likely to occur as the population density decreases to background levels within days or weeks after application. Furthermore, there is little or no migration of the nematodes to neighbouring fields. The relatively short period of persistence of entomopathogenic nematodes and the necessity of their populations to recycle frequently in hosts in order to survive make it unlikely that they could have major effects on non-target organisms. Their selectivity and beneficial traits as biological control agents outweigh the small risks of causing unwanted environmental disturbance in non-target populations.     

Effects of Entomopathogenic Nematodes on Control of a Mushroom-infesting Sciarid Fly and on Mushroom Production

The entomopathogenic nematodes Steinernema feltiae (Biosys strain #27) and Heterorhabditis heliothidis were evaluated for the larval control of a mushroom-infesting sciarid, Lycoriella mali, and for the effects of these nematodes on mushroom (Agaricus bisporus) production. In a series of small-scale mushroom crops, infective-stage H. heliothidis and S. feltiae were applied to the mushroom casing surface in the irrigation water or incorporated into the casing material at densities ranging from 28 to 1120 and 11 to 1120 nematodes cm-2 of casing surface respectively. The mortality of L. mali larvae ranged from 52 to 100% for H. heliothidis and 38 to 100% for S. feltiae. Both nematode species reduced mycelial coverage on the casing surface at primordia initiation. Neither mushroom strain (off-white or white hybrid) or method of application (incorporation into or irrigation onto the casing surface) altered the effect on mycelial coverage. The nematodes's negative effect on mycelial growth confounded the benefit of fly control. At high nematode densities (up to 1120 nematodes cm-2), damage-free mushroom yields for the first week of harvest were less than those from the untreated control. However, at lower nematode densities, at or below 140 cm-2, the nematodes had less effect on mushroom growth, and consequently, damage-free mushroom yields for the first week of harvest were frequently greater than those from the untreated control. In the absence of flies, the first-week mushroom yield generally declined with increasing nematode densities for both white and off-white mushroom hybrids. After 4 weeks of harvest, accumulated mushroom yields had nearly recovered from the earlier decline.     

An Edible-to-insects Calcium Alginate Gel as a Carrier for Entomopathogenic Nematodes

A carrier for entomopathogenic nematodes based on an edible-to-insects calcium alginate gel was developed. The alginate system was produced by external setting through an interaction between an aqueous sodium alginate mixture and calcium ions under acidic conditions. Sodium hexa-metaphosphate was used to control gel formation. Yeast extract used in the gel as a phagostimulant for Spodoptera littoralis larvae improved the insect's relative consumption rate and digestibility. The nematodes in the gel effectively controlled the larvae in a 24-h leaf bioassay, although nematode survival in the gel was ~ 50%. Gels subjected to 31% relative humidity (RH) prior to larval feeding became desiccated and were inedible to insects. However, gels at 61% RH supported larval feeding, although the water loss from the gel due to evaporation from 200-400-mg gel cubes at this humidity exceeded 50%. The gel might be a useful delivery system for nematodes against insects infesting the plant canopy in greenhouses.     

Long-Term Persistence of Yersinia pseudotuberculosis in Entomopathogenic Nematodes

Entomopathogenic nematodes (EPNs) are small worms whose ecological behaviour consists to invade, kill insects and feed on their cadavers thanks to a species-specific symbiotic bacterium belonging to any of the genera Xenorhabdus or Photorhabdus hosted in the gastro-intestinal tract of EPNs. The symbiont provides a number of biological functions that are essential for its EPN host including the production of entomotoxins, of enzymes able to degrade the insect constitutive macromolecules and of antimicrobial compounds able to prevent the growth of competitors in the insect cadaver. The question addressed in this study was to investigate whether a mammalian pathogen taxonomically related to Xenorhabdus was able to substitute for or “hijack” the symbiotic relationship associating Xenorhabdus and Steinernema EPNs. To deal with this question, a laboratory experimental model was developed consisting in Galleria mellonella insect larvae, Steinernema EPNs with or without their natural Xenorhabdus symbiont and Yersinia pseudotuberculosis brought artificially either in the gut of EPNs or in the haemocoel of the insect larva prior to infection. The developed model demonstrated the capacity of EPNs to act as an efficient reservoir ensuring exponential multiplication, maintenance and dissemination of Y. pseudotuberculosis.     

Quality Assessment of Commercially Produced Entomopathogenic Nematodes

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.     

Genetic Enhancement of Nematicide Resistance in Entomopathogenic Nematodes

Entomopathogenic nematodes are highly effective bioinsecticides . Their efficacy may be reduced due to the various pesticides they encounter in the soil . These include insecticides as well as nematicides used against plant - parasitic nematodes . The purpose of this study was to examine the feasibility of genetic selection as a means of enhancing resistance of the entomopathogenic nematode Heterorhabditis bacteriophora strain HP88 to the nematicides: Fenamiphos (an organophosphate) , Oxamyl (a carbamate) and Avermectin (a biological product) . Estimates of heritability ( h 2) of resistance to the three nematicides were obtained from analysis of inbred lines derived from the base population . The heritability estimate for Fenamiphos was h 2 = 0 . 31 , for Oxamyl h 2 = 0 . 71 and for Avermectin h 2 = 0 . 46 . Five rounds of selection were performed . Thereafter , each line was divided into two: for one subline selection continued for six additional rounds . The other subline was reared without selection for the six additional rounds . After the eleventh round , resistance to the nematicides was examined as were several traits relevant to biocontrol efficacy including virulence , heat tolerance and reproduction potential . Selection resulted in an 8 - 9 - fold increase in resistance to Fenamiphos and Avermectin and a 70 - fold increase in resistance to Oxamyl . The enhanced resistance Oxamyl and Avermectin , and to a lesser extent to Fenamiphos , was stable and continued after selection was relaxed . No deterioration in traits relevant to biocontrol efficacy was observed in the selected lines as compared with the base population . The selected lines displayed enhanced cross - resistance towards some , but not all , of the nematicides tested . These results demonstrate that genetic selection can be used to enhance resistance of entomopatho genic nematodes to certain environmental stresses . The selected lines will be useful bioinsecti cides in the context of integrated pest management .     

Entomopathogenic Nematodes as a Model System for Advancing the Frontiers of Ecology

Entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematidae have a mutualistic–symbiotic association with enteric γ-Proteobacteria (Steinernema–Xenorhabdus and Heterorhabditis–Photorhabdus), which confer high virulence against insects. EPNs have been studied intensively because of their role as a natural mortality factor for soil-dwelling arthropods and their potential as biological control agents for belowground insect pests. For many decades, research on EPNs focused on the taxonomy, phylogeny, biogeography, genetics, physiology, biochemistry and ecology, as well as commercial production and application technologies. More recently, EPNs and their bacterial symbionts are being viewed as a model system for advancing research in other disciplines such as soil ecology, symbiosis and evolutionary biology. Integration of existing information, particularly the accumulating information on their biology, into increasingly detailed population models is critical to improving our ability to exploit and manage EPNs as a biological control agent and to understand ecological processes in a changing world. Here, we summarize some recent advances in phylogeny, systematics, biogeography, community ecology and population dynamics models of EPNs, and describe how this research is advancing frontiers in ecology.     

Compatibility of Soil Amendments with Entomopathogenic Nematodes

The impact of inorganic and organic fertilizers on the infectivity, reproduction, and population dynamics of entomopathogenic nematodes was investigated. Prolonged (10- to 20-day) laboratory exposure to high inorganic fertilizer concentrations inhibited nematode infectivity and reproduction, whereas short (1-day) exposures increased infectivity. Heterorhabditis bacteriophora was more sensitive to adverse effects than were two species of Steinernema. In field studies, organic manure resulted in increased densities of a native population of Steinernema feltiae, whereas NPK fertilizer suppressed nematode densities regardless of manure applications. Inorganic fertilizers are likely to be compatible with nematodes in tank mixes and should not reduce the effectiveness of nematodes used for short-term control as biological insecticides, but may interfere with attempts to use nematodes as inoculative agents for long-term control. Organic manure used as fertilizer may encourage nematode establishment and recycling.     

Entomopathogenic Nematodes for Control of Insect Pests Above and Below Ground with Comments on Commercial Production

Entomopathogenic nematodes (EPNs) have been utilized in classical, conservation, and augmentative biological control programs. The vast majority of applied research has focused on their potential as inundatively applied augmentative biological control agents. Extensive research over the past three decades has demonstrated both their successes and failures for control of insect pests of crops, ornamental plants, trees and lawn and turf. In this paper we present highlights of their development for control of insect pests above and below ground. The target insects include those from foliar, soil surface, cryptic and subterranean habitats. Advances in mass-production and formulation technology of EPNs, the discovery of numerous efficacious isolates/strains, and the desirability of reducing pesticide usage have resulted in a surge of commercial use and development of EPNs. Commercially produced EPNs are currently in use for control of scarab larvae in lawns and turf, fungus gnats in mushroom production, invasive mole crickets in lawn and turf, black vine weevil in nursery plants, and Diaprepes root weevil in citrus in addition to other pest insects. However, demonstrated successful control of several other insects, often has not lead to capture of a significant share of the pesticide market for these pests.     

Impact of Entomopathogenic Nematodes on Non-target Hosts

Biological pest control has been thought to be ecologically safe for many years. More recently, it has been questioned whether entomopathogens and beneficial arthropods or nematodes truly have no impact on non-target species. Only a few studies deal with the action of entomopathogenic nematodes on non-target animals, although a broad spectrum of species has been tested in the laboratory. Entomopathogenic nematodes do not affect vertebrates under natural conditions. Mortality caused by the release of entomopathogenic nematodes among non-target arthropod populations can occur, but will only be temporary, will be spatially restricted and will affect only part of a population. In plots treated with entomopathogenic nematodes, the impact on the non-target fauna proved to be negligible. The possible impact of introduced exotic nematode species is discussed and regulatory measures for the release are proposed.     

In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the family Enterobacteriaceae. Xenorhabdus bacteria are associated with steinernematids nematodes while Photorhabdus are symbionts of heterorhabditids. Together nematodes and bacteria form a potent insecticidal complex that kills a wide range of insect species in an intimate and specific partnership. Herein, we demonstrate in vivo and in vitro techniques commonly used in the rearing of these nematodes under laboratory conditions. Furthermore, these techniques represent key steps for the successful establishment of EPN cultures and also form the basis for other bioassays that utilize these organisms for research. The production of aposymbiotic (symbiont–free) nematodes is often critical for an in-depth and multifaceted approach to the study of symbiosis. This protocol does not require the addition of antibiotics and can be accomplished in a short amount of time with standard laboratory equipment. Nematodes produced in this manner are relatively robust, although their survivorship in storage may vary depending on the species used. The techniques detailed in this presentation correspond to those described by various authors and refined by P. Stock’s Laboratory, University of Arizona (Tucson, AZ, USA). These techniques are distinct from the body of techniques that are used in the mass production of these organisms for pest management purposes.     

Entomopathogenic Nematodes Are Not an Alternative to Fenamiphos for Management of Plant-Parasitic Nematodes on Golf Courses in Florida

With the cancellation of fenamiphos in the near future, alternative nematode management tactics for plant-parasitic nematodes (PPN) on golf courses need to be identified. The use of entomopathogenic nematodes (EPN) has been suggested as one possible alternative. This paper presents the results of 10 experiments evaluating the efficacy of EPN at managing PPN on turfgrasses and improving turf performance. These experiments were conducted at various locations throughout Florida over the course of a decade. In different experiments, different EPN species were tested against different species of PPN. Separate experiments evaluated multiple rates and applications of EPN, compared different EPN species, and compared single EPN species against multiple species of PPN. In a few trials, EPN were associated with reductions in certain plant-parasite species, but in other trials were associated with increases. In most trials, EPN had no effect on plant parasites. Because EPN were so inconsistent in their results, we conclude that EPN are not acceptable alternatives to fenamiphos by most turf managers in Florida at this time.     

Entomopathogenic Nematodes, a Case Study: Introduction of Steinernema scapterisci in Florida

Steinernema scapterisci Nguyen & Smart (Rhabditida: Steinernematidae) was established in Florida in 1985 for the control of mole crickets, Scapteriscus spp. Infected hosts were collected in sound traps 23 km from the nearest release, indicating long-distance dispersal and area-wide establishment. In a subsequent pasture study, the nematode dispersed, on average, 60 m in 20 months; dispersal in some pastures was 150 m in 1 year. Establishment was not as successful on golf-courses; however, pest populations were reduced 27% in areas where the nematode persisted. Inoculative applications were successful at 10 of 29 sites in Florida, where sound traps attracted flying Scapteriscus to relatively small numbers of S. scapterisci infective juveniles. The differences in the susceptibility to the nematode for mole cricket life stages and species were determined in laboratory and field trials. The nematode became commercially available in 1993; commercial applications facilitate the establishment of S. scapterisci in many areas of the state.     

Lateral Movement of the Entomopathogenic Nematodes Steinernema glaseri and Heterorhabditis bacteriophora in Sand at Different Temperatures in Response to Host Seeking

Laboratory studies were conducted to determine the lateral movement of Steinernema glaseri and Heterorhabditis bacteriophora in sand at 15, 20, 25 and 30°C in response to Galleria mellonella larvae. Lateral movement was assessed in 42.5 ×5 cm PVC tubes, constructed from 17 individual sections, with G. mellonella larvae placed on one end and the nematodes in the center. The proportion of the nematodes moving towards or away from the larvae at different temperatures was quantified at 8 h intervals. Although both species are reported to be cruisers, only S. glaseri responded to the host cues. The movement of the infective juveniles of both species increased significantly as temperature rose. The extraction efficiency of both species decreased at all temperatures with time.     

Curative Control of the Peachtree Borer Using Entomopathogenic Nematodes

The peachtree borer, Synanthedon exitiosa (Say 1823), is a major pest of stone fruit trees in North America. Current management relies upon preventative control using broad-spectrum chemical insecticides, primarily chlorpyrifos, applied in the late summer or early fall. However, due to missed applications, poor application timing, or other factors, high levels of S. exitiosa infestation may still occur and persist through the following spring. Curative treatments applied in the spring to established infestations would limit damage to the tree and prevent the next generation of S. exitiosa from emerging within the orchard. However, such curative measures for control of S. exitiosa do not exist. Our objective was to measure the efficacy of the entomopathogenic nematode, Steinernema carpocapsae, as a curative control for existing infestations of S. exitiosa. In peach orchards, spring applications of S. carpocapsae (obtained from a commercial source) were made to infested trees and compared with chlorpyrifos and a water-only control in 2014 and 2015. Additionally, types of spray equipment were compared: nematodes were applied via boom sprayer, handgun, or trunk sprayer. To control for effects of application method or nematode source, in vivo laboratory-grown S. carpocapsae, applied using a watering can, was also included. Treatment effects were assessed 39 d (2014) or 19 d (2015) later by measuring percentage of trees still infested, and also number of surviving S. exitiosa larvae per tree. Results indicated that S. carpocapsae provided significant curative control (e.g., >80% corrected control for the handgun application). In contrast, chlorpyrifos failed to reduce S. exitiosa infestations or number of surviving larvae. In most comparisons, no effect of nematode application method was detected; in one assessment, only the handgun and watering can methods reduced infestation. In conclusion, our study indicates that S. carpocapsae may be used as an effective curative measure for S. exitiosa infestations.     

Field Application of Entomopathogenic Nematodes for Control of Delia radicum in Collards

Control of Delia radicum (cabbage maggot) in field collards (Brassica oleracea) was compared after one or two applications of entomopathogenic nematodes, Steinernema carpocapsae (All strain) and Heterorhabditis bacterophora (HP88 strain), a single application of granular chlorpyrifos, and a water-only treatment. Nematodes were applied with a sprayer during the egg stage of first-generation D. radicum, and chlorpyrifos was hand placed around collard stems during the same period. A second nematode application was made 10 days later. Chlorpyrifos treatment resulted in fewer puparia per plant, less root damage and higher yield than all other treatments, including the control. Collard yield from nematode-treated beds did not differ from controls. These data indicate that, under these field conditions, the species or strains of entomopathogenic nematodes tested did not reduce the number of active cabbage maggots, nor did they prevent collard root damage.