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Nematodes

(Rhabditida: Steinernematidae & Heterorhabditidae)

By David I. Shapiro-Ilan, USDA-ARS, SEFTNRL, Byron, GA &
Randy Gaugler, Department of Entomology, Rutgers University, New Brunswick New Jersey 

Nematodes are simple roundworms. Colorless, unsegmented, and lacking appendages, nematodes may be free-living, predaceous, or parasitic. Many of the parasitic species cause important diseases of plants, animals, and humans. Other species are beneficial in attacking insect pests, mostly sterilizing or otherwise debilitating their hosts. A very few cause insect death but these species tend to be difficult (e.g., tetradomatids) or expensive (e.g. mermithids) to mass produce, have narrow host specificity against pests of minor economic importance, possess modest virulence (e.g., sphaeruliids) or are otherwise poorly suited to exploit for pest control purposes. The only insect-parasitic nematodes possessing an optimal balance of biological control attributes are entomopathogenic or insecticidal nematodes in the genera Steinernema and Heterorhabditis. These multi-cellular metazoans occupy a biocontrol middle ground between microbial pathogens and predators/parasitoids, and are invariably lumped with pathogens, presumably because of their symbiotic relationship with bacteria.

Entomopathogenic nematodes are extraordinarily lethal to many important insect pests, yet are safe for plants and animals. This high degree of safety means that unlike chemicals, or even Bacillus thuringiensis, nematode applications do not require masks or other safety equipment; and re-entry time, residues, groundwater contamination, chemical trespass, and pollinators are not issues. Most biologicals require days or weeks to kill, yet nematodes, working with their symbiotic bacteria, can kill insects within 24-48 hours. Dozens of different insect pests are susceptible to infection, yet no adverse effects have been shown against beneficial insects or other nontargets in field studies (Georgis et al., 1991; Akhurst and Smith, 2002). Nematodes are amenable to mass production and do not require specialized application equipment as they are compatible with standard agrochemical equipment, including various sprayers (e.g., backpack, pressurized, mist, electrostatic, fan, and aerial) and irrigation systems.

Hundreds of researchers representing more than forty countries are working to develop nematodes as biological insecticides. Nematodes have been marketed on every continent except Antarctica for control of insect pests in high-value horticulture, agriculture, and home and garden niche markets.

Life Cycle

Steinernematids and heterorhabditids have similar life histories. The non-feeding, developmentally arrested infective juvenile seeks out insect hosts and initiates infections. When a host has been located, the nematodes penetrate into the insect body cavity, usually via natural body openings (mouth, anus, spiracles) or areas of thin cuticle. Once in the body cavity, a symbiotic bacterium (Xenorhabdus for steinernematids, Photorhabdus for heterorhabditids) is released from the nematode gut, which multiplies rapidly and causes rapid insect death. The nematodes feed upon the bacteria and liquefying host, and mature into adults. Steinernematid infective juveniles may become males or females, where as heterorhabditids develop into self-fertilizing hermaphrodites although subsequent generations within a host produce males and females as well.

The life cycle is completed in a few days, and hundreds of thousands of new infective juveniles emerge in search of fresh hosts.  Thus, entomopathogenic nematodes are a nematode-bacterium complex. The nematode may appear as little more than a biological syringe for its bacterial partner, yet the relationship between these organisms is one of classic mutualism. Nematode growth and reproduction depend upon conditions established in the host cadaver by the bacterium. The bacterium further contributes anti-immune proteins to assist the nematode in overcoming host defenses, and anti-microbials that suppress colonization of the cadaver by competing secondary invaders. Conversely, the bacterium lacks invasive powers and is dependent upon the nematode to locate and penetrate suitable hosts. 

Production and Storage Technology

Entomopathogenic nematodes are mass produced for use as biopesticides using in vivo or in vitro methods (Shapiro-Ilan and Gaugler 2002). In vivo production (culture in live insect hosts) requires a low level of technology, has low startup costs, and resulting nematode quality is generally high, yet cost efficiency is low. The approach can be considered ideal for small markets. In vivo production may be improved through innovations in mechanization and streamlining. A novel alternative approach to in vivo methodology is production and application of nematodes in infected host cadavers; the cadavers (with nematodes developing inside) are distributed directly to the target site and pest suppression is subsequently achieved by the infective juveniles that emerge. In vitro solid culture, i.e., growing the nematodes on crumbled polyurethane foam, offers an intermediate level of technology and costs. In vitro liquid culture is the most cost- efficient production method but requires the largest startup capital. Liquid culture may be improved through progress in media development, nematode recovery, and bioreactor design. A variety of formulations have been developed to facilitate nematode storage and application including activated charcoal, alginate and polyacrylamide gels, baits, clay, paste, peat, polyurethane sponge, vermiculite, and water-dispersible granules. Depending on the formulation and nematode species, successful storage under refrigeration ranges from one to seven months. Optimum storage temperature for formulated nematodes varies according to species; generally, steinernematids tend to store best at 4-8 °C whereas heterorhabditids persist better at 10-15 °C.

Relative Effectiveness and Application Parameters

Growers will not adopt biological agents that do not provide efficacy comparable with standard chemical insecticides. Technological advances in nematode production, formulation, quality control, application timing and delivery, and particularly in selecting optimal target habitats and target pests, have narrowed the efficacy gap between chemical and nematode agents. Nematodes have consequently demonstrated efficacy in a number of agricultural and horticultural market segments. 

Entomopathogenic nematodes are remarkably versatile in being useful against many soil and cryptic insect pests in diverse cropping systems, yet are clearly underutilized. Like other biological control agents, nematodes are constrained by being living organisms that require specific conditions to be effective. Thus, desiccation or ultraviolet light rapidly inactivates insecticidal nematodes; chemical insecticides are less constrained. Similarly, nematodes are effective within a narrower temperature range (generally between 20 °C and 30 °C) than chemicals, and are more impacted by suboptimal soil type, thatch depth, and irrigation frequency (Georgis and Gaugler, 1991; Shapiro-Ilan et al., 2006). Nematode-based insecticides may be inactivated if stored in hot vehicles, cannot be left in spray tanks for long periods, and are incompatible with several agricultural chemicals. Chemicals also have problems (e.g., mammalian toxicity, resistance, groundwater pollution, etc.) but a large knowledge base has been developed to support their use. Accelerated implementation of nematodes into IPM systems will require users to be more knowledgeable about how to use them effectively.

Therefore, based on the nematodes’ biology, applications should be made in a manner that avoids direct sunlight, e.g., early morning or evening applications are often preferable. Soil in the treated area should be kept moist for at least two weeks after applications. Application to aboveground target areas is difficult due to the nematode’s sensitivity to desiccation and UV radiation; however, some success against certain above-ground targets has been achieved and recently approaches have been enhanced by improved formulations (e.g., Shapiro-Ilan et al., 2010). In all cases, the nematodes must be applied at a rate that is sufficient to kill the target pest; generally, 250,000 infective juveniles per m2 of treated area is required (though in some cases an increased or slightly decreased rate may be suitable) (Shapiro-Ilan et al., 2002). Additionally, it is important to match the appropriate nematode species to the particular pest that is being targeted (see the table below for species effectiveness).

Appearance

Nematodes are formulated and applied as infective juveniles, the only free-living and therefore environmentally tolerant stage. Infective juveniles range from 0.4 to 1.5 mm in length and can be observed with a hand lens or microscope after separation from formulation materials. Disturbed nematodes move actively, however sedentary ambusher species (e.g. Steinernema carpocapsae, S. scapterisci) in water soon revert to a characteristic "J"-shaped resting position. Low temperature or oxygen levels will inhibit movement of even active cruiser species (e.g., S. glaseri, Heterorhabditis bacteriophora). In short, lack of movement is not always a sign of mortality; nematodes may have to be stimulated (e.g., probes, acetic acid, gentle heat) to move before assessing viability. Good quality nematodes tend to possess high lipid levels that provide a dense appearance, whereas nearly transparent nematodes are often active but possess low powers of infection. 

Insects killed by most steinernematid nematodes become brown or tan, whereas insects killed by heterorhabditids become red and the tissues assume a gummy consistency. A dim luminescence given off by insects freshly killed by heterorhabditids is a foolproof diagnostic for this genus (the symbiotic bacteria provide the luminescence). Black cadavers with associated putrefaction indicate that the host was not killed by entomopathogenic species. Nematodes found within such cadavers tend to be free-living soil saprophages. 

Habitat

Steinernematid and heterorhabditid nematodes are exclusively soil organisms. They are ubiquitous, having been isolated from every inhabited continent from a wide range of ecologically diverse soil habitats including cultivated fields, forests, grasslands, deserts, and even ocean beaches. When surveyed, entomopathogenic nematodes are recovered from 2% to 45% of sites sampled (Hominick, 2002).

Pests Attacked

Because the symbiotic bacterium kills insects so quickly, there is no intimate host-parasite relationship as is characteristic for other insect-parasitic nematodes. Consequently, entomopathogenic nematodes are lethal to an extraordinarily broad range of insect pests in the laboratory. Field host range is considerably more restricted, with some species being quite narrow in host specificity. Nonetheless, when considered as a group of nearly 80 species, entomopathogenic nematodes are useful against a large number of insect pests (Grewal et al., 2005). Additionally, entomopathogenic nematodes have been marketed for control of certain plant parasitic nematodes, though efficacy has been variable depending on species (Lewis and Grewal, 2005). A list of many of the insect pests that are commercially targeted with entomopathogenic nematodes is provided in the table below. As field research progresses and improved insect-nematode matches are made, this list is certain to expand.

USE OF NEMATODES AS BIOLOGICAL INSECTICIDES

* At least one scientific study reported 75% suppression of these pests using the nematodes indicated in field or greenhouse experiments. Subsequent/other studies may reveal other nematodes that are virulent to these pests. Nematodes species used are abbreviated as follows: Hb=Heterorhabditis bacteriophora, Hd = H. downesi, Hi = H. indica, Hm= H. marelata, Hmeg = H. megidis, Hz = H. zealandica, Sc=Steinernema carpocapsae, Sf=S. feltiae, Sg=S. glaseri, Sk = S. kushidai, Sr=S. riobrave, Sscap=S. scapterisci, Ss = S. scarabaei.
** Efficacy of various pest species within this group varies among nematode species.

Steinernema carpocapsae: This species is the most studied of all entomopathogenic nematodes. Important attributes include ease of mass production and ability to formulate in a partially desiccated state that provides several months of room-temperature shelf-life. S. carpocapsae is particularly effective against lepidopterous larvae, including various webworms, cutworms, armyworms, girdlers, some weevils, and wood-borers. This species is a classic sit-and-wait or "ambush" forager, standing on its tail in an upright position near the soil surface and attaching to passing hosts. Consequently, S. carpocapsae is especially effective when applied against highly mobile surface-adapted insects (though some below-ground insects are also controlled by this nematode). S. carpocapsae is also highly responsive to carbon dioxide once a host has been contacted, thus the spiracles are a key portal of host entry. It is most effective at temperatures ranging from 22 to 28°C. 

Steinernema feltiae: S. feltiae is especially effective against immature dipterous insects, including mushroom flies, fungus gnats, and tipulids as well some lepidopterous larvae. This nematode is unique in maintaining infectivity at soil temperatures as low as 10°C. S. feltiae has an intermediate foraging strategy between the ambush and cruiser type.

Steinernema glaseri: One of the largest entomopathogenic nematode species at twice the length but eight times the volume of S. carpocapsae infective juveniles, S. glaseri is especially effective against coleopterous larvae, particularly scarabs. This species is a cruise forager, neither nictating nor attaching well to passing hosts, but highly mobile and responsive to long-range host volatiles. Thus, this nematode is best adapted to parasitize hosts possessing low mobility and residing within the soil profile. Field trials, particularly in Japan, have shown that S. glaseri can provide control of several scarab species. Large size, however, reduces yield, making this species significantly more expensive to produce than other species. A tendency to occasionally "lose" its bacterial symbiote is bothersome. Moreover, the highly active and robust infective juveniles are difficult to contain within formulations that rely on partial nematode dehydration. In short, additional technological advances are needed before this nematode is likely to see substantial use. 

Steinernema kushidai: Only isolated so far from Japan and only known to parasitize scarab larvae, S. kushidai has been commercialized and marketed primarily in Asia.

Steinernema riobrave: This novel and highly pathogenic species was originally isolated from the Rio Grande Valley of Texas, but has since been also been isolated in other areas, e.g., in the southwestern USA. Its effective host range runs across multiple insect orders. This versatility is likely due in part to its ability to exploit aspects of both ambusher and cruiser means of finding hosts. Trials have demonstrated its effectiveness against corn earworm, mole crickets, and plum curculio. Steinernema riobrave has also been highly effective in suppressing citrus root weevils (e.g., Diaprepes abbreviates and Pachnaeus species). This nematode is active across a range of temperatures; it is effective at killing insects at soil temperatures above 35°C, and can also infect at 15 °C. Persistence is excellent even under semi-arid conditions, a feature no doubt enhanced by the uniquely high lipid levels found in infective juveniles. Its small size provides high yields whether using in vivo (up to 375,000 infective juveniles per wax moth larvae) or in vitro methods.

Steinernema scapterisci: The only entomopathogenic nematode to be used in a classical biological control program, S. scapterisci was isolated from Uruguay and first released in Florida in 1985 to suppress an introduced pest, mole crickets. The nematode become established and presently contributes to control. Steinernema scapterisci is highly specific to mole crickets. Its ambusher approach to finding insects is ideally suited to the turfgrass tunneling habits of its host. Commercially available since 1993, this nematode is also sold as a biological insecticide, where its excellent ability to persist and provide long-term control contributes to overall efficacy.

Heterorhabditis bacteriophora: Among the most economically important entomopathogenic nematodes, H. bacteriophora possesses considerable versatility, attacking lepidopterous and coleopterous insect larvae, among other insects. This cruiser species appears quite useful against root weevils, particularly black vine weevil where it has provided consistently excellent results in containerized soil. A warm temperature nematode, H. bacteriophora shows reduced efficacy when soil drops below 20°C.

Heterorhabditis indica: First discovered in India, this nematode is now known to be ubiquitous. Heterorhabditis indica is considered to be a heat tolerant nematode (infecting insects at 30 °C or higher). The nematode produces high yields in vivo and in vitro, but shelf life is generally shorter than most other nematode species.

Heterorhabditis megidis: First isolated in Ohio, this nematode is commercially available and marketed especially in western Europe for control of black vine weevil and various other soil insects. Heterorhabditis megidis is considered to be a cold tolerant nematode because it can effectively infect insects at temperatures below 15 °C.

Conservation

Conservation strategies are poorly developed and largely limited to avoiding applications onto sites where the nematodes are ill-adapted; for example, where immediate mortality is likely (e.g., exposed foliage) or where they are completely ineffective (e.g., aquatic habitats) (Lewis et al., 1998). Minimizing deleterious effects of the aboveground environment with a post-application rinse that washes infective juveniles into the soil is also a useful approach to increasing persistence and efficacy. Native populations are highly prevalent, but, other than scattered reports of epizootics, their impact on host populations is generally not well documented (Stuart et al., 2006). This is largely attributable to the cryptic nature of soil insects. Consequently, research and guidelines for conserving native entomopathogenic nematodes are in need of advancement. 

Compatibility

Infective juveniles are compatible with most but not all agricultural chemicals under field conditions. Compatibility has been tested with well over 100 different chemical pesticides. Entomopathogenic nematodes are compatible (e.g., may be tank-mixed) with most chemical herbicides and fungicides as well as many insecticides (such as bacterial or fungal products) (Koppenhöfer and Grewal, 2005). In fact, in some cases, combinations of chemical agents with nematodes results in synergistic levels of insect mortality. Some chemicals to be used with care or avoided include aldicarb, carbofuran, diazinon, dodine, methomyl, and various nematicides. However, specific interactions can vary based on the nematode and host species and application rates. Furthermore, even when a specific chemical pesticide is not deemed compatible, use of both agents (chemical and nematode) can be implemented by waiting an appropriate interval between applications (e.g., 1 – 2 weeks). Prior to use, compatibility and potential for tank-mixing should be based on manufacturer recommendations. Similarly, entomopathogenic nematodes are also compatible with many though not all biopesticides (Koppenhöfer and Grewal, 2005); interactions range from antagonism to additivity or synergy depending on the specific combination of control agents, target pest, and rates and timing of application. Nematodes are generally compatible with chemical fertilizers as well as composted manure though fresh manure can be detrimental.

Commercial Availability

Of the nearly eighty steinernematid and heterorhabditid nematodes identified to date, at least twelve species have been commercialized. A list of some nematode producers and suppliers is provided below; the list emphasizes U.S. suppliers. Comparison-shopping is recommended as prices vary greatly among suppliers. Additionally, caution is again advised with regard to application rates. One billion nematodes per acre (250,000 per m2) is the rule-of-thumb against most soil insects (containerized and greenhouse soils tend to be treated at higher rates). A final caveat is that, just as one must select the appropriate insecticide to control a target insect, so must one choose the appropriate nematode species or strain. Ask suppliers about field tests supporting their recommended matching of insect target and nematode.

SOME COMMERCIAL PRODUCERS/SUPPLIERS*

References

Akhurst, R. and K. Smith. 2002. Regulation and safety. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI, New York, NY, pp. 311-332.

Georgis, R. and R. Gaugler. 1991. Predictability in biological control using entomopathogenic nematodes. Journal of Economic Entomology. [Forum] 84: 713-20.

Georgis, R., H. Kaya, and R. Gaugler. 1991. Effect of steinernematid and heterorhabditid nematodes on nontarget arthropods. Environmental Entomology 20: 815-22.

Grewal, P. S., R-U, Ehlers, and D. I. Shapiro-Ilan. 2005. Nematodes as Biocontrol Agents. CABI, New York, NY.

Hominick, W. M. 2002. Biogeography. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI, New York, NY, pp. 115-143.

Koppenhöfer, A. M. and P. S. Grewal. 2005. Compatibility and interactions with agrochemicals and other biocontrol agents. In: Nematodes as Biocontrol Agents. CABI, New York, NY, pp. 363-381.

Lewis, E., J. Campbell, and R. Gaugler. 1998. A conservation approach to using entomopathogenic nematodes in turf and landscapes. In: Barbosa, P. (Ed.), Perspectives on the Conservation of Natural Enemies of Pest Species, Academic Press, New York, pp. 235-254.

Lewis, E.E. and P. S. Grewal. 2005. Interactions with plant parasitic nematodes. In: Grewal, P.S., Ehlers, R.-U., and Shapiro-Ilan, D.I. (Eds.), Nematodes as Biocontrol Agents. CABI, New York, NY., pp. 349-362.
Shapiro-Ilan D. I. and R. Gaugler. 2002. Production technology for entomopathogenic nematodes and their bacterial symbionts. Journal of Industrial Microbiology and Biotechnology 28: 137-146.

Shapiro-Ilan, D. I., D. H. Gouge, and A. M. Koppenhöfer. 2002. Factors affecting commercial success: case studies in cotton, turf and citrus. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI, New York, NY, pp. 333-356.

Shapiro-Ilan, D.I., D. H. Gouge, S. J. Piggott, and J. Patterson Fife. 2006. Application technology and environmental considerations for use of entomopathogenic nematodes in biological control. Biological Control 38: 124-133.

Shapiro-Ilan, D. I., T. E. Cottrell, R. F. Mizell, D. L. Horton, B. Behle, and C. Dunlap. 2010. Efficacy of Steinernema carpocapsae for control of the lesser peachtree borer, Synanthedon pictipes: Improved aboveground suppression with a novel gel application. Biological Control 54, 23–28.

Stuart, R. J., M. E. Barbercheck, P. S. Grewal, R.A.J. Taylor, and C. W. Hoy. 2006. Population biology of entomopathogenic nematodes: Concepts, issues, and models. Biological Control 38: 80-102.

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