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Ecological considerations in the biological control of soil-inhabiting insects with entomopathogenic nematodes
Agriculture, Ecosystems and Environment, 24 (1988) 351-360
351
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Ecological Considerations in the Biological Control of SoU-inhabiting Insects with Entomopathogenic Nematodes* RANDY GAUGLER
D~artment~Entomology, Ru~e~ Unive~i~ NewBru~wickNJ(~S.A.)
ABSTRACT Gaugler, R,, 1988. Ecological considerations in the biological control of soil-inhabiting insects with entomopathogenic nematodes. Agric. Ecosystems Environ., 24: 351-360. Entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae are undergoing intense scrutiny as biological alternatives to chemicals for suppression of soil-inhabiting insects. Although naturally adapted to the soil, these nematodes are currently not as predictable or effective as chemical agents. Efforts to close or narrow this efficacy gap have been directed toward technological solutions, with striking advances being made recently in nematode mass rearing, shipping, storage, formulation and genetic improvement. Our understanding of entomopathogenic nematode soil ecology has not kept pace. In particular., we are unable to predict nematode behavior in the soil, biotic interactions with soil antagonists remain virtually unstudied, and the abiotic conditions optimal for survival and mobility are insufficiently understood. There is a compelling need for a standardized protocol that would permit cause and effect relationships to be established for successful and unsuccessful field trials. Fundamental to the applied use of entomopathogenic nematodes is gaining insight into nematode interactions with environmental parameters that determine the likelihood and outcome of nematode-insect encounters.
INTRODUCTION
Records of nematode diseases in insects extend back for more than 350 years (Aldrovandi, 1623; from Poinar, 1975), yet efforts to use nematodes for the biological control of pests were not begun until the 1930s (Glaser, 1932), well after attempts with parasitoids, predators and microbial pathogens. Research has increased almost exponentially in recent years and entomopathogenic nematology is emerging as one of the most innovative and dynamic areas in biological control. Nearly all of this work is directed toward investigations of two families: Steinernematidae and Heterorhabditidae, nematodes characterized *New Jersey Agricultural Experiment Station, Publication No. F-08251-01-87.
0167-8809/88/$03.50
© 1988 Elsevier Science Publishers B.V.
352 by their mutualistic association with bacteria in the genus Xenorhabdus. Infective nematode stages, finding an insect host, penetrate quickly to the host's body cavity and release the bacterium from their intestinal lumen. The bacterium multiplies rapidly in the host hemolymph, causing a lethal bacteremia within 24-48 h. The nematodes feed upon the bacterial cells and liquefying host tissues, mature, mate and produce progeny which emerge from the cadaver as infective-stage juveniles in search of new hosts. Although infectives are able to kill hosts in the absence of their bacterial partner, albeit more slowly, reproduction is reduced Significantly without essential nutrients supplied by Xenorhabdus. The bacterium benefits from the host-finding abilities of the motile infectives and nematode destruction of the host immune proteins (Gotz et al., 1981). The intense interest in steinernematid and heterorhabditid nematodes is a reflection of their impressive potential for biological control. Most significantly, because the associated bacteria kill hosts so quickly, the nematodes have not had to adapt to a specific host life cycle, and are able to parasitize hundreds of insect pests. This spectrum of activity is more characteristic of a chemical than biological insecticide, making the nematodes attractive subjects for commercial development. Despite their broad host range and high virulence for insects, extensive testing has demonstrated a complete lack of mammalian pathogenicity (Gaugler and Boush, 1979; Poinar et al., 1982) and the U.S. Environmental Protection Agency has subsequently exempted steinernematid and heterorhabditid nematodes from registration and regulation requirements (Gorsuch, 1982). Mass production is accomplished easily with existing technology (Bedding, 1984), and there is strong promise that liquid fermentation in bubble columns (Georgis, 1987) may enable nematodes to be economically competitive with many chemicals. A breakthrough in inducing infectives to enter anhydrobiosis (Georgis, 1987) may have resolved the questions of shelf life which plague most biocontrol agents. Finally, infective stages possess chemoreceptors (LeBeck, 1979) and mobility, enabling infective stages to seek out even well-hidden insects. No other biological control agents offer a comparable list of attributes. FIELD EVALUATIONS Despite these advantages, field evaluations of efficacy have produced mixed results. In early trials, billions of Steinernema ( = Neoaplectana ) glaseri ( Steiner) nematodes were produced on artificial media and released into the soil as part of a massive biological control effort against Japanese beetle populations in New Jersey (Glaser and Farrell, 1935; Girth et al., 1940). These tests gave encouraging results initially, but ultimately were unsuccessful, presumably because the investigators were unaware of the nematode's symbiotic partner, and eliminated the bacterium by incorporating antimicrobials into the rearing me-
353 dia. There is also evidence suggesting that "normal" third-stage juveniles were sometimes released (Girth et al., 1940) rather than ensheathed and infective third-stage dauer juveniles. Subsequent tests were suspended for 20 years until S. feltiae Filipjev ( = N . carpocapsae Weiser) was isolated, loosing a flood of evaluations that have made this parasite among the most extensively tested of all biological-control agents. Applications against foliage-feeding insects were heavily emphasized, initially because of the economic impact of these pests and the ease of conducting field trials in aboveground systems. The sensitivity of infective stages on exposed surfaces to inactivation by desiccation (Kamionek et al., 1974), solar radiation (Gaugler and Boush, 1978) and temperature (Molyneux, 1985), however, left foliage treatments heavily dependent upon prevailing weather conditions, producing highly erratic results. The soil, by comparison, offers buffering against the physical environment, is the natural reservoir of steinernematid and heterorhabditid nematodes, and plays host to more than 90% of all insect pests at some point in their life cycle. These obvious advantages have resulted in a gradual but nearly complete shift of effort to the soil. Although progress has been slow, which is a measure of the difficulty of working in soil ecosystems, a number of successful field trials have been conducted against soil-inhabiting insects. The most favorable results have been obtained against root weevils in containerized soils, where Heterorhabditis spp. regularly reduce black vine weevil densities by 90% (Bedding and Miller, 1981; Dolmans, 1983; Stimman et al., 1985). Zimmerman and Simons (1986) conclude that a single application of 50-100 infective nematodes cm -2 of soil can provide consistent and effective control. Excellent control of fire ants has been achieved, with soil drenches of S. feltiae causing colony mortalities of 80% at one million infectives per mound (Poole, 1976) and 88-97% at twice that dosage (Quattlebaum, 1980). Encouraging results have also been obtained in soil treatments against mole crickets (Cobb and Georgis, 1987), wireworms (Kovacs et al., 1980), Colorado potato beetles (Wright et al., 1987), root maggots (van Sloun and Sikora, 1986) and cutworms (Lossbroek and Theuissen, 1985). While instances of successful field trials demonstrate the utility of steinernematid and heterorhabditid nematodes in the soil, it is instructive to consider a single example in more detail. White grubs are currently the subject of an intense reevaluation of nematode control potential because of increasing restrictions on the use of chemical larvicides on turfgrass. The results of these field experiments are generally positive (M. Klein, personal communication, 1987), with H. heliothidis Khan, Brooks and Hirschmann often performing as well as, or better than, chemical treatments against Japanese beetle larvae, giving 74-90% control in one test, although other tests were less impressive at 30-60% control. Steinernema feltiae provided more moderate and erratic control (7-72%); inexplicably, there was no evidence of a dose response. Results with the northern masked chafer, a grub closely related to the Japanese beetle,
354 were particularly puzzling: spring applications of H. heliothidis reduced pest densities by 0-60%, while S. feltiae failed to achieve any level of control; yet these nematodes persisted to cause grub reductions of 84-94% and 11-86% in the summer generation of larvae. Jan Jackson (personal communication, 1987) has also observed wide and unexplained variation in extensive field tests against the corn rootworm. The factors regulating nematode performance in the soil are poorly understood, but sources of variation that probably contribute to inconsistency include application method, nematode strain/species, host stage and defense, and the biotic and abiotic environments. FACTORSINFLUENCINGEFFICACY The greatest single factor reducing the efficacy of aboveground field trials has been poor nematode persistence on exposed foliage. The exceptionally wide distribution of steinernematid and heterorhabditid nematodes, however, suggests a high capacity for persistence in their natural habitat. Threats to survival from the abiotic environment are less severe in the soil where many nematodes can respond to low soil moisture by entering anhydrobiosis (Crowe and Madin, 1975) and there is shelter from solar radiation. Nematodes introduced into the soil are capable of remaining infective for weeks (Edwards and Oswald, 1981; Gray and Johnson, 1983 ) or even months (Harlan et al., 1971 ), more than sufficient time to find hosts and cause infection. Persistence may be a serious limitation to efficacy, primarily when applications are made onto the soil surface, where infectives are exposed to radiation and desiccation unless they quickly migrate into the protective soil. Unfortunately, most infectives begin host-seeking movements slowly, if at all, even when a host is present (R. Gaugler, unpublished data, 1987). Since most applications against soilinhabiting insects have been to the soil surface, the poor migration ability of infectives may be responsible for some of the observed variations in efficacy. Even nematodes that have entered the soil successfully are not immune to inactivation by the physical environment. Inactivation may not be lethal, however, but may be expressed as temporary immobility that precludes host seeking. At low soil moistures, for example, nematodes may not be infective because they lack a sufficient water film for effective movement to their hosts (Wallace, 1958, 1969). Further loss of soil moisture would inactivate the nematodes by inducing anhydrobiosis, whereas a large gain of moisture would restrict host seeking by filling soil pores and eliminating the water-film surface tension nematodes require to push against for locomotion. If movement is optimal when soils are near their field capacity (i.e. most soil pores have been drained) as Wallace (1958) has suggested, there may be a soil moisture "window" when infective steinernematids and heterorhabditids are able to initiate host seeking and cause high rates of infection. Similarly, extremes of temperature can be lethal, while slightly less severe temperatures would merely inhibit mobility,
355 but inactivation would result in either case. Field tests conducted when these environmental windows are closed would meet with limited success. A high host-seeking capability is regarded as the most desirable trait in an effective natural enemy (Doutt and DeBach, 1964). Host seeking by steinernematid and heterorhabditid nematodes is a directed response to host-released carbon dioxide (Gaugler et al., 1980; R. Gaugler and J. Campbell, unpublished data, 1988). This capability, coupled with the ubiquitous nature of these nematodes in the soil, suggests there is considerable selective pressure for soil insects to evolve strategies to avoid parasitism. Thus, the tendency of many quiescent soil insects, especially pupae, to release carbon dioxide in bursts rather than continuously, with an interburst period of as much as 7 h (Chapman, 1982 ), may be in part a coevolutionary response to escape detection and, therefore, avoid nematode parasitism. The spiracles are nearly closed during the interburst period, denying access through a key portal of nematode entry. Other insects move almost constantly through the soil, increasing the difficulty of orientation to potential hosts. Stationary insects should be easy targets, but many species, including root-feeding scarabaeids, construct pupal cells of densely packed soil particles that may present a formidable physical barrier to infection. Soil-inhabiting social insects such as termites should be highly susceptible to nematode infection because they are apparent and predictable, yet they are reported to wall off infected individuals, halting spread of the infection (Fujii, 1975 ). Akhurst ( 1986 ), citing unpublished observations by R. Bedding, lists the defensive mechanisms of scarabaeid larvae to include sieve plates over spiracles, low carbon dioxide output, high defecation rate, and the ability to push nematodes away from their mouths with the anterior legs. Defensive strategies against nematode attack seem skewed toward behavioral counter measures, especially to escape detection or avoid penetration, rather than cellular/humoral responses, phenological separation, or chemical defences. Coevolution has created an equilibrium that prevents steinernematid and heterorhabditid nematodes from overexploiting their hosts. Economic entomologists attempt to break this equilibrium by introducing enormous numbers of infectives into the soil. Japanese beetle control requires dosages of several billion infectives acre -1 (M. Klein, personal communication, 1987), making it difficult Or impossible for hosts to escape nematode detection and overwhelming other defensive mechanisms. The consistently good results achieved in spot treatments against fire ant mounds, and pests in containerized soils, are largely a result of the massive nematode dosages that can be delivered to small restricted areas. Striking differences in efficacy exist between steinernematid and heterorhabditid nematodes, and their various species and strains. Heterorhabditis heliothidis, for example, is effective in field trials against Japanese beetles, but provides extremely poor results when mole crickets are challenged (Cobb and Georgis, 1987), whereas S. feltiae is effective against mole crickets and pro-
356 vides modest control of Japanese beetle grubs. The reasons why one nematode is more efficacious than another against a particular insect remain unclear, but are certainly related, in part, to differences in behavior. Thus, the highly studied H. heliothidis may provide better results against most soil insects than S. feltiae because of its superior host-seeking abilities (R. Gaugler and Campbell, unpublished data). Steinernema feltiae seems to show a preference for soil near the surface (Reed and Came, 1967; Moyle and Kaya, 1981), and, therefore, may be best adapted to attack insects like mole crickets, which feed at the soillitter interface (Walker, 1984). Characterizing nematode strains/species and matching their habits to the activity zones of target insects would aid in achieving increased effectiveness. The method used to introduce nematodes into the soil is certain to influence their effectiveness. Optimal application strategies remain to be determined, and the most used method is likely to be the same as for chemical insecticides, i.e. soil surface sprays. This broadcast approach is quick, easy and gives excellent coverage, but leaves nematodes not immediately entering the soil exposed to inactivation by solar radiation. Since only a small proportion of infectives have good migration abilities most surface-applied infectives may have little impact on pest populations. Even the earliest workers were aware that subsurface applications provided better results than surface-applied nematodes (Glaser and Farrell, 1935 ). Infective stages should be introduced directly into the soil by injection or burying whenever possible, with emphasis on placing the nematodes in the pest feeding zone. This procedure is often not practical, so the best compromise is to make surface applications in the evening followed by irrigation to work the nematodes into the soil, thereby increasing both survival and mobility. In the case of spot treatments, such as applications against termite mounds, drenches can be an effective means of getting the nematodes off the soil surface quickly. Infectives can also be disseminated by field release of laboratory-infected hosts that enter the soil (Akhurst, 1986), or in protective alginate capsules (Kaya and Nelsen, 1985). An innovative formulation approach to preserving field stability being developed by Biosys, Inc., involves mixing desiccated (i.e. anhydrobiotic) nematodes together with a bait attractive to insects feeding in the duff (e.g. mole crickets); ingested nematodes rehydrate in the insect gut to cause lethal infections (Georgis, 1987). CONCLUSIONS Growing dissatisfaction with chemical insecticides used in the soil, especially concern for safety, resistance and increasing microbial degradation, is serving as a strong impetus for the development of alternative control measures. Steinernematid and heterorhabditid nematodes have emerged as the strongest alternative biocontrol candidates, possibly because they occupy a middle ground between predators/parasitoids and pathogens that endows them
357 with a unique combination of attributes. For example, they share a capacity for host-seeking and an exemption from government registration requirements for predators/parasitoids, yet can be mass-reared and stored on a scale only feasible for some pathogens. Unfortunately, these organisms also share a limitation common to all biocontrol agents: control that is variable and unpredictable. Although significant, this flaw is not ruinous, since chemicals also provide unreliable levels of control in the soil (33-99% in M. Klein's white grub tests). With the exception of spot treatments, however, steinernematid and heterorhabditid nematodes generally give less consistent results and lower levels of control than recommended chemical insecticides. Substantial efforts are being directed toward developing new technologies that will close the efficacy gap between chemicals and nematodes. Continued innovation may be expected in formulation technology as it becomes recognized that nematode formulations need not parallel those of chemicals. Similarly, there is further need for application systems specifically designed for use with nematodes. Rapid progress in mass production technology may provide indirect improvements in consistency by permitting dosage levels that are now economically unfeasible. Genetic selection is a worthwhile approach to enhancing nematode effectiveness (Gaugler, 1986, 1987), and improvements of up to 15-fold in S. feltiae host-seeking capability have been achieved in the laboratory (R. Gaugler, unpublished data, 1988). Emphasis should be placed concurrently on devising quick screening procedures for identifying genetically superior new strains. Impressive technological strides have already been made with entomopathogenic nematodes (Georgis, 1987), improvements that will certainly contribute to increased efficacy. But technology cannot resolve all the limitations; the real key to effective use of nematodes will be integrating technology with knowledge of steinernematid and heterorhabditid ecology. Improvements in application technology, for example, are inadequate without detailed information on optimal timing, placement and post-treatment means of maximizing nematode persistence and mobility. In short, we need a fuller understanding of nematode interactions with environmental factors that may determine the outcome of nematode-insect encounters. We lack definitive information on the fate of nematodes introduced into the soil, on factors regulating their population dynamics, on the optimal conditions for epizootic initiation and on the ecological barriers to infection. The most obvious information void concerns nematode interactions with the biotic soil environment. The soil is an intensely colonized habitat, and while antagonism doubtlessly occurs, the influence of the soil fauna on nematode releases remains virtually unstudied. Even for moisture, arguably the most carefully considered abiotic parameter, there is insufficient information to estimate the soil tension needed for infection to
358
occur, yet predictable control depends upon the ability to predict nematode behavior in the soil. These questions are not amenable to technological solutions. A particularly useful approach for gaining insight into steinernematid and heterorhabditid soil ecology would be to explore critically the differences between successful and unsuccessful field trials. Most soil applications have been empirical studies, with surprisingly little regard shown for ascertaining factors which limit effectiveness. Soil parameters are often not adequately monitored, precluding the establishment of cause and effect relationships. The reasons for trial success or failure are likely to continue to remain obscure unless test protocols are devised that provide a degree of standardization. At a minimum, the following test parameters should be reported for soil surface applications: time and method of application, air and soil temperatures, cloud cover, thatch depth, soil type, soil moisture, pest density, pest stage, pest activity zone, post-treatment irrigation and precipitation. Such protocols would not address biotic interactions, but would generate field data on the impact of the physical environment. This would help to reduce the incidence of failures by providing guidelines on where, when and how entomopathogenic nematodes might be most advantageously used, or alternatively, how the environment might best be manipulated to encourage parasitism. Ultimately, understanding steinernematid and heterorhabditid soil ecology is fundamental to understanding how to enhance their use. REFERENCES
Akhurst, R.J., 1986. Controlling insects in soil with entomogenous nematodes. In: R.A. Samson, J.V. Vlak and D. Peters (Editors), Fundamental and Applied Aspects of Invertebrate Pathology. 4th I.C.I.P., Wageningen, The Netherlands, p. 265-267. Aldrovandi, U., 1623. De Animalibus Insectis. Frankfort: 271. Bedding, R.A., 1984. Large scale production, storage and transport of the insect-parasitic nematodes Neoaplectana spp. and Heterorhabditis spp. Ann. Appl. Biol., 104: 117-120. Bedding, R.A. and Miller, L.A., 1981. Disinfesting blackcurrant cuttings of Synanthedon tipuliformis, using the insect parasitic nematode, Neoaplectana bibionis. Environ. Entomol., 10: 449-453. Chapman, R.F., 1982. The Insects: Structure and Function. Harvard University Press, Cambridge, MA, 3rd ed., 919 pp. Cobb, P. and Georgis, R., 1987. Mole cricket damage control on Bermudagrass turf with entomogenous nematodes. (Unpublished manuscript), 6 pp. Crowe, J.H. and Madin, K.A.C., 1975. Anhydrobiosis in nematodes: evaporative water loss and survival. J. Exp. Zool., 193: 323-334. Dolmans, N.G.M., 1983. Biological control of the black vine weevil (Otiorhynchus sulcatus) with a nematode (Heterorhabditis spp.). Meded. Fac. Landouwwet. Rijksuniv. Gent, 48: 417-420. Doutt, R.L. and DeBach, P., 1964. Some biological concepts and questions. In: P. DeBach (Editor), Biological Control of Insect Pests and Weeds. Reinhold, New York, NY, pp. 118-142. Edwards, C,A. and Oswald, J., 1981. Control of soil-inhabiting arthropods with Neoaplectana carpocapsae. Proceedings 1981 British Crop Protection Conference, pp. 467-473.
359 Fujii, J.K., 1975. Effects of an entomogenous nematode, Neoaplectana carpocapsae Weiser, on the Formosan subterranean termite, Coptotermes formosanus Shiraki, with ecological and biological studies on C. formosanus. Ph.D. dissertation. University of Hawaii, Honolulu, 179 pp. Gaugler, R., 1986. Feasibility of genetically improving steinernematid and heterorhabditid nematodes. In: R.A. Samson, J.V. Vlak and D, Peters (Editors), Fundamental and Applied Aspects of Invertebrate Pathology. 4th I.C.I.P., Wageningen, The Netherlands, pp. 300-303. Gaugler, R., 1987. Entomogenous nematodes and their prospects for genetic improvement. In: K. Maramorosch (Editor), Biotechnological Advances in Invertebrate Pathology and Cell Culture. Academic Press, New York, NY, pp. 457-484. Gaugler, R. and Boush, G.M., 1978. Effects of ultraviolet radiation and sunlight on the entomogenous nematode, Neoaplectana carpocapsae. J. Invertebr. Pathol., 32: 291-296. Gaugler, R. and Boush, G.M., 1979. Nonsusceptibility of rats to the entomogenous nematode, Neoaplectana carpocapsae. Environ. Entomol., 8: 658-660. Gaugler, R., LeBeck, L., Nakagaki, B. and Boush, G.M., 1980. Orientation of the entomogenous nematode Neoaplectana carpocapsae to carbon dioxide. Environ. Entomol., 9: 649-652. Georgis, R., 1987. Nematodes for biological control of urban insects. Division of Environmental Chemistry of the American Chemical Society Meeting, 30 August-4 September, New Orleans, 6 pp. (Extended abstract). Girth, H.B., McCoy, E.E. and Glaser, R.W., 1940. Field experiments with a nematode parasite of the Japanese beetle. N. J. Dept. Agric. Circ., No. 37, 21 pp. Glaser, R.W., 1932. Studies on Neoaplectana glaseri, a nematode parasite of the Japanese beetle, (PopiUiajaponica). N. J. Dept. Agric. Circ., No. 211, 34 pp. Glaser, R.W. and Farrell, C.C., 1935. Field experiments with the Japanese beetle and its nematode parasite. J. N. Y. Entomol. Soc., 18: 345-371. Gorsuch, A.M., 1982. Regulations for the enforcement of the Federal Insecticide, Fungicide, and Rodenticide Act exemption from regulation of certain biological control agents. Fed. Regist., 47: 23928-23930. Gotz, P., Boman, P. and Boman, H.G., 1981. Interactions between insect immunity and an insectpathogenic nematode with symbiotic bacteria. Proc. R. Soc. Lond. Set. B, 212: 333-350. Gray, P.A. and Johnson, D.T., 1983. Survival of the nematode Neoaplectana carpocapsae in relation to soil temperature, moisture and time. J. Georgia Entomol. Soc., 18: 454-460. Harlan, D.P., Dutky, S.R., Padgett, G.R., Mitchell, J.A., Shaw, Z.A. and Bartlett, F.J., 1971. Parasitism of Neoaplectana dutkyi in white-fringed beetle larvae. J. Nematol., 3: 280-283. Kamionek, M., Maslana, I. and Sandner, H., 1974. The survival of invasive larvae of Neoaplectana carpocapsae Weiser in a waterless environment under various conditions of temperature and humidity. Zesz. Probl. Postepow Nauk Roln., 154: 409-412. Kaya, H.K. and Nelsen, C.E., 1985. Encapsulation of steinernematid and heterorhabditid nematodes with calcium alginate: a new approach for insect control and other applications. Environ. Entomol., 14: 572-574. Kovacs, A., Dese6, K.V., Poinar, G. and De Leonardis, A., 1980. Prove di lotta contro insetti con applicazione di nematodi entomogeni. Atti G. Fitopathol., 1:449-456 (English summary). LeBeck, L.M., 1979. A scanning and transmission electron microscopic study of the entomogenous nematode Neoaplectana carpocapsae, with emphasis on surface sensory organs. M.S. thesis, University of Wisconsin, 88 pp. Lossbroek, T.G. and Theuissen, J., 1985. The entomogenous nematode Neoaplectana bibionis as a biological control agent ofAgriotis segetum in lettuce. Entomol. Exp. Appl., 39: 261-264. Molyneux, A.S., 1985. Survival of infective juveniles of Heterorhabditis spp., and Steinernema spp. (Nematoda: Rhabditida) at various temperatures and their subsequent infectivity for insects. Rev. Nematol., 8: 165-170.
360 Moyle, P.L. and Kaya, H.K., 1981. Dispersal and infectivity of the entomogenous nematode, Neoaplectana carpocapsae Weiser (Rhabditida: Steinernematidae), in sand. J. Nematol., 13: 294-300. Poinar, G.O., Jr., 1975. Entomogenous Nematodes. E.J. Brill, Leiden, The Netherlands, 317 pp. Poinar, G.O., Jr., Thomas, G.M., Presser, S.B. and Hardy, J.L., 1982. Inoculation of entomogenous nematodes, Neoaplectana and Heterorhabditis and their associated bacteria, Xenorhabdus spp. into chicks and mice. Environ. Entomol., 11: 137-138. Poole, M.A., 1976. Survey and control efficacy of endoparasites of Solenopsis richteri and S. invicta in Mississippi. Ph.D. dissertation, Mississippi State University, 83 pp. Quattlebaum, E.C., 1980. Evaluation of fungal and nematode pathogens to control the red imported fire ant, Solenopsis invicta. Ph.D. dissertation, Clemson University, 54 pp. Reed, E.M. and Carne, P.B., 1967. The suitability of a nematode (DD-136) for the control of some pasture insects. J. Invertebr. Pathol., 9: 196-204. Stimman, M.W., Kaya, H.K., Burlando, T.M. and Studdert, J.P., 1985. Black vine weevil management in nursery plants. Calif. Agric., 39: 25-26. Van Sloun, P. and Sikora, R.A., 1986. Control ofAgrotis segetum and Delia brassicae with species of Steinernema Heterorhabditis. In: R.A. Samson, J.V. Vlak and D. Peters (Editors), Fundamental and Applied Aspects of Invertebrate Pathology. 4th I.C.I.P., Wageningen, The Netherlands, p. 318. Walker, T.J. (Editor), 1984. Mole crickets in Florida. F1. Agric. Exp. Stn. Bull., No. 845, 54 pp. Wallace, H.R., 1958. I. The influence of pore size and moisture content of the soil on the migration of larvae of the beet eelworm, Heterodera schachtii Schmidt. Ann. Appl. Biol., 46: 74-85. Wallace, H.R., 1969. The influence of a non-ionic detergent on the movement of larvae of Meloidogynejavanica. Nematologica, 15: 107-114. Wright, R.J., Agudelo-Silva, F. and Georgis, R., 1987. Soil applications of steinernematid and heterorhabditid nematodes for control of Colorado potato beetles, Leptinotarsa decemlineata (Say). J. Nematol., 19: 201-206. Zimmerman, G. and Simons, W.R., 1986. Experiences with biological control of the black vine weevil, Otiorhynchus sulcatus (F.). In: R.A. Samson, J.V. Vlak and D. Peters (Editors), Fundamental and Applied Aspects of Invertebrate Pathology. 4th I.C.I.P., Wageningen, The Netherlands, pp. 529-533.
351
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Ecological Considerations in the Biological Control of SoU-inhabiting Insects with Entomopathogenic Nematodes* RANDY GAUGLER
D~artment~Entomology, Ru~e~ Unive~i~ NewBru~wickNJ(~S.A.)
ABSTRACT Gaugler, R,, 1988. Ecological considerations in the biological control of soil-inhabiting insects with entomopathogenic nematodes. Agric. Ecosystems Environ., 24: 351-360. Entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae are undergoing intense scrutiny as biological alternatives to chemicals for suppression of soil-inhabiting insects. Although naturally adapted to the soil, these nematodes are currently not as predictable or effective as chemical agents. Efforts to close or narrow this efficacy gap have been directed toward technological solutions, with striking advances being made recently in nematode mass rearing, shipping, storage, formulation and genetic improvement. Our understanding of entomopathogenic nematode soil ecology has not kept pace. In particular., we are unable to predict nematode behavior in the soil, biotic interactions with soil antagonists remain virtually unstudied, and the abiotic conditions optimal for survival and mobility are insufficiently understood. There is a compelling need for a standardized protocol that would permit cause and effect relationships to be established for successful and unsuccessful field trials. Fundamental to the applied use of entomopathogenic nematodes is gaining insight into nematode interactions with environmental parameters that determine the likelihood and outcome of nematode-insect encounters.
INTRODUCTION
Records of nematode diseases in insects extend back for more than 350 years (Aldrovandi, 1623; from Poinar, 1975), yet efforts to use nematodes for the biological control of pests were not begun until the 1930s (Glaser, 1932), well after attempts with parasitoids, predators and microbial pathogens. Research has increased almost exponentially in recent years and entomopathogenic nematology is emerging as one of the most innovative and dynamic areas in biological control. Nearly all of this work is directed toward investigations of two families: Steinernematidae and Heterorhabditidae, nematodes characterized *New Jersey Agricultural Experiment Station, Publication No. F-08251-01-87.
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© 1988 Elsevier Science Publishers B.V.
352 by their mutualistic association with bacteria in the genus Xenorhabdus. Infective nematode stages, finding an insect host, penetrate quickly to the host's body cavity and release the bacterium from their intestinal lumen. The bacterium multiplies rapidly in the host hemolymph, causing a lethal bacteremia within 24-48 h. The nematodes feed upon the bacterial cells and liquefying host tissues, mature, mate and produce progeny which emerge from the cadaver as infective-stage juveniles in search of new hosts. Although infectives are able to kill hosts in the absence of their bacterial partner, albeit more slowly, reproduction is reduced Significantly without essential nutrients supplied by Xenorhabdus. The bacterium benefits from the host-finding abilities of the motile infectives and nematode destruction of the host immune proteins (Gotz et al., 1981). The intense interest in steinernematid and heterorhabditid nematodes is a reflection of their impressive potential for biological control. Most significantly, because the associated bacteria kill hosts so quickly, the nematodes have not had to adapt to a specific host life cycle, and are able to parasitize hundreds of insect pests. This spectrum of activity is more characteristic of a chemical than biological insecticide, making the nematodes attractive subjects for commercial development. Despite their broad host range and high virulence for insects, extensive testing has demonstrated a complete lack of mammalian pathogenicity (Gaugler and Boush, 1979; Poinar et al., 1982) and the U.S. Environmental Protection Agency has subsequently exempted steinernematid and heterorhabditid nematodes from registration and regulation requirements (Gorsuch, 1982). Mass production is accomplished easily with existing technology (Bedding, 1984), and there is strong promise that liquid fermentation in bubble columns (Georgis, 1987) may enable nematodes to be economically competitive with many chemicals. A breakthrough in inducing infectives to enter anhydrobiosis (Georgis, 1987) may have resolved the questions of shelf life which plague most biocontrol agents. Finally, infective stages possess chemoreceptors (LeBeck, 1979) and mobility, enabling infective stages to seek out even well-hidden insects. No other biological control agents offer a comparable list of attributes. FIELD EVALUATIONS Despite these advantages, field evaluations of efficacy have produced mixed results. In early trials, billions of Steinernema ( = Neoaplectana ) glaseri ( Steiner) nematodes were produced on artificial media and released into the soil as part of a massive biological control effort against Japanese beetle populations in New Jersey (Glaser and Farrell, 1935; Girth et al., 1940). These tests gave encouraging results initially, but ultimately were unsuccessful, presumably because the investigators were unaware of the nematode's symbiotic partner, and eliminated the bacterium by incorporating antimicrobials into the rearing me-
353 dia. There is also evidence suggesting that "normal" third-stage juveniles were sometimes released (Girth et al., 1940) rather than ensheathed and infective third-stage dauer juveniles. Subsequent tests were suspended for 20 years until S. feltiae Filipjev ( = N . carpocapsae Weiser) was isolated, loosing a flood of evaluations that have made this parasite among the most extensively tested of all biological-control agents. Applications against foliage-feeding insects were heavily emphasized, initially because of the economic impact of these pests and the ease of conducting field trials in aboveground systems. The sensitivity of infective stages on exposed surfaces to inactivation by desiccation (Kamionek et al., 1974), solar radiation (Gaugler and Boush, 1978) and temperature (Molyneux, 1985), however, left foliage treatments heavily dependent upon prevailing weather conditions, producing highly erratic results. The soil, by comparison, offers buffering against the physical environment, is the natural reservoir of steinernematid and heterorhabditid nematodes, and plays host to more than 90% of all insect pests at some point in their life cycle. These obvious advantages have resulted in a gradual but nearly complete shift of effort to the soil. Although progress has been slow, which is a measure of the difficulty of working in soil ecosystems, a number of successful field trials have been conducted against soil-inhabiting insects. The most favorable results have been obtained against root weevils in containerized soils, where Heterorhabditis spp. regularly reduce black vine weevil densities by 90% (Bedding and Miller, 1981; Dolmans, 1983; Stimman et al., 1985). Zimmerman and Simons (1986) conclude that a single application of 50-100 infective nematodes cm -2 of soil can provide consistent and effective control. Excellent control of fire ants has been achieved, with soil drenches of S. feltiae causing colony mortalities of 80% at one million infectives per mound (Poole, 1976) and 88-97% at twice that dosage (Quattlebaum, 1980). Encouraging results have also been obtained in soil treatments against mole crickets (Cobb and Georgis, 1987), wireworms (Kovacs et al., 1980), Colorado potato beetles (Wright et al., 1987), root maggots (van Sloun and Sikora, 1986) and cutworms (Lossbroek and Theuissen, 1985). While instances of successful field trials demonstrate the utility of steinernematid and heterorhabditid nematodes in the soil, it is instructive to consider a single example in more detail. White grubs are currently the subject of an intense reevaluation of nematode control potential because of increasing restrictions on the use of chemical larvicides on turfgrass. The results of these field experiments are generally positive (M. Klein, personal communication, 1987), with H. heliothidis Khan, Brooks and Hirschmann often performing as well as, or better than, chemical treatments against Japanese beetle larvae, giving 74-90% control in one test, although other tests were less impressive at 30-60% control. Steinernema feltiae provided more moderate and erratic control (7-72%); inexplicably, there was no evidence of a dose response. Results with the northern masked chafer, a grub closely related to the Japanese beetle,
354 were particularly puzzling: spring applications of H. heliothidis reduced pest densities by 0-60%, while S. feltiae failed to achieve any level of control; yet these nematodes persisted to cause grub reductions of 84-94% and 11-86% in the summer generation of larvae. Jan Jackson (personal communication, 1987) has also observed wide and unexplained variation in extensive field tests against the corn rootworm. The factors regulating nematode performance in the soil are poorly understood, but sources of variation that probably contribute to inconsistency include application method, nematode strain/species, host stage and defense, and the biotic and abiotic environments. FACTORSINFLUENCINGEFFICACY The greatest single factor reducing the efficacy of aboveground field trials has been poor nematode persistence on exposed foliage. The exceptionally wide distribution of steinernematid and heterorhabditid nematodes, however, suggests a high capacity for persistence in their natural habitat. Threats to survival from the abiotic environment are less severe in the soil where many nematodes can respond to low soil moisture by entering anhydrobiosis (Crowe and Madin, 1975) and there is shelter from solar radiation. Nematodes introduced into the soil are capable of remaining infective for weeks (Edwards and Oswald, 1981; Gray and Johnson, 1983 ) or even months (Harlan et al., 1971 ), more than sufficient time to find hosts and cause infection. Persistence may be a serious limitation to efficacy, primarily when applications are made onto the soil surface, where infectives are exposed to radiation and desiccation unless they quickly migrate into the protective soil. Unfortunately, most infectives begin host-seeking movements slowly, if at all, even when a host is present (R. Gaugler, unpublished data, 1987). Since most applications against soilinhabiting insects have been to the soil surface, the poor migration ability of infectives may be responsible for some of the observed variations in efficacy. Even nematodes that have entered the soil successfully are not immune to inactivation by the physical environment. Inactivation may not be lethal, however, but may be expressed as temporary immobility that precludes host seeking. At low soil moistures, for example, nematodes may not be infective because they lack a sufficient water film for effective movement to their hosts (Wallace, 1958, 1969). Further loss of soil moisture would inactivate the nematodes by inducing anhydrobiosis, whereas a large gain of moisture would restrict host seeking by filling soil pores and eliminating the water-film surface tension nematodes require to push against for locomotion. If movement is optimal when soils are near their field capacity (i.e. most soil pores have been drained) as Wallace (1958) has suggested, there may be a soil moisture "window" when infective steinernematids and heterorhabditids are able to initiate host seeking and cause high rates of infection. Similarly, extremes of temperature can be lethal, while slightly less severe temperatures would merely inhibit mobility,
355 but inactivation would result in either case. Field tests conducted when these environmental windows are closed would meet with limited success. A high host-seeking capability is regarded as the most desirable trait in an effective natural enemy (Doutt and DeBach, 1964). Host seeking by steinernematid and heterorhabditid nematodes is a directed response to host-released carbon dioxide (Gaugler et al., 1980; R. Gaugler and J. Campbell, unpublished data, 1988). This capability, coupled with the ubiquitous nature of these nematodes in the soil, suggests there is considerable selective pressure for soil insects to evolve strategies to avoid parasitism. Thus, the tendency of many quiescent soil insects, especially pupae, to release carbon dioxide in bursts rather than continuously, with an interburst period of as much as 7 h (Chapman, 1982 ), may be in part a coevolutionary response to escape detection and, therefore, avoid nematode parasitism. The spiracles are nearly closed during the interburst period, denying access through a key portal of nematode entry. Other insects move almost constantly through the soil, increasing the difficulty of orientation to potential hosts. Stationary insects should be easy targets, but many species, including root-feeding scarabaeids, construct pupal cells of densely packed soil particles that may present a formidable physical barrier to infection. Soil-inhabiting social insects such as termites should be highly susceptible to nematode infection because they are apparent and predictable, yet they are reported to wall off infected individuals, halting spread of the infection (Fujii, 1975 ). Akhurst ( 1986 ), citing unpublished observations by R. Bedding, lists the defensive mechanisms of scarabaeid larvae to include sieve plates over spiracles, low carbon dioxide output, high defecation rate, and the ability to push nematodes away from their mouths with the anterior legs. Defensive strategies against nematode attack seem skewed toward behavioral counter measures, especially to escape detection or avoid penetration, rather than cellular/humoral responses, phenological separation, or chemical defences. Coevolution has created an equilibrium that prevents steinernematid and heterorhabditid nematodes from overexploiting their hosts. Economic entomologists attempt to break this equilibrium by introducing enormous numbers of infectives into the soil. Japanese beetle control requires dosages of several billion infectives acre -1 (M. Klein, personal communication, 1987), making it difficult Or impossible for hosts to escape nematode detection and overwhelming other defensive mechanisms. The consistently good results achieved in spot treatments against fire ant mounds, and pests in containerized soils, are largely a result of the massive nematode dosages that can be delivered to small restricted areas. Striking differences in efficacy exist between steinernematid and heterorhabditid nematodes, and their various species and strains. Heterorhabditis heliothidis, for example, is effective in field trials against Japanese beetles, but provides extremely poor results when mole crickets are challenged (Cobb and Georgis, 1987), whereas S. feltiae is effective against mole crickets and pro-
356 vides modest control of Japanese beetle grubs. The reasons why one nematode is more efficacious than another against a particular insect remain unclear, but are certainly related, in part, to differences in behavior. Thus, the highly studied H. heliothidis may provide better results against most soil insects than S. feltiae because of its superior host-seeking abilities (R. Gaugler and Campbell, unpublished data). Steinernema feltiae seems to show a preference for soil near the surface (Reed and Came, 1967; Moyle and Kaya, 1981), and, therefore, may be best adapted to attack insects like mole crickets, which feed at the soillitter interface (Walker, 1984). Characterizing nematode strains/species and matching their habits to the activity zones of target insects would aid in achieving increased effectiveness. The method used to introduce nematodes into the soil is certain to influence their effectiveness. Optimal application strategies remain to be determined, and the most used method is likely to be the same as for chemical insecticides, i.e. soil surface sprays. This broadcast approach is quick, easy and gives excellent coverage, but leaves nematodes not immediately entering the soil exposed to inactivation by solar radiation. Since only a small proportion of infectives have good migration abilities most surface-applied infectives may have little impact on pest populations. Even the earliest workers were aware that subsurface applications provided better results than surface-applied nematodes (Glaser and Farrell, 1935 ). Infective stages should be introduced directly into the soil by injection or burying whenever possible, with emphasis on placing the nematodes in the pest feeding zone. This procedure is often not practical, so the best compromise is to make surface applications in the evening followed by irrigation to work the nematodes into the soil, thereby increasing both survival and mobility. In the case of spot treatments, such as applications against termite mounds, drenches can be an effective means of getting the nematodes off the soil surface quickly. Infectives can also be disseminated by field release of laboratory-infected hosts that enter the soil (Akhurst, 1986), or in protective alginate capsules (Kaya and Nelsen, 1985). An innovative formulation approach to preserving field stability being developed by Biosys, Inc., involves mixing desiccated (i.e. anhydrobiotic) nematodes together with a bait attractive to insects feeding in the duff (e.g. mole crickets); ingested nematodes rehydrate in the insect gut to cause lethal infections (Georgis, 1987). CONCLUSIONS Growing dissatisfaction with chemical insecticides used in the soil, especially concern for safety, resistance and increasing microbial degradation, is serving as a strong impetus for the development of alternative control measures. Steinernematid and heterorhabditid nematodes have emerged as the strongest alternative biocontrol candidates, possibly because they occupy a middle ground between predators/parasitoids and pathogens that endows them
357 with a unique combination of attributes. For example, they share a capacity for host-seeking and an exemption from government registration requirements for predators/parasitoids, yet can be mass-reared and stored on a scale only feasible for some pathogens. Unfortunately, these organisms also share a limitation common to all biocontrol agents: control that is variable and unpredictable. Although significant, this flaw is not ruinous, since chemicals also provide unreliable levels of control in the soil (33-99% in M. Klein's white grub tests). With the exception of spot treatments, however, steinernematid and heterorhabditid nematodes generally give less consistent results and lower levels of control than recommended chemical insecticides. Substantial efforts are being directed toward developing new technologies that will close the efficacy gap between chemicals and nematodes. Continued innovation may be expected in formulation technology as it becomes recognized that nematode formulations need not parallel those of chemicals. Similarly, there is further need for application systems specifically designed for use with nematodes. Rapid progress in mass production technology may provide indirect improvements in consistency by permitting dosage levels that are now economically unfeasible. Genetic selection is a worthwhile approach to enhancing nematode effectiveness (Gaugler, 1986, 1987), and improvements of up to 15-fold in S. feltiae host-seeking capability have been achieved in the laboratory (R. Gaugler, unpublished data, 1988). Emphasis should be placed concurrently on devising quick screening procedures for identifying genetically superior new strains. Impressive technological strides have already been made with entomopathogenic nematodes (Georgis, 1987), improvements that will certainly contribute to increased efficacy. But technology cannot resolve all the limitations; the real key to effective use of nematodes will be integrating technology with knowledge of steinernematid and heterorhabditid ecology. Improvements in application technology, for example, are inadequate without detailed information on optimal timing, placement and post-treatment means of maximizing nematode persistence and mobility. In short, we need a fuller understanding of nematode interactions with environmental factors that may determine the outcome of nematode-insect encounters. We lack definitive information on the fate of nematodes introduced into the soil, on factors regulating their population dynamics, on the optimal conditions for epizootic initiation and on the ecological barriers to infection. The most obvious information void concerns nematode interactions with the biotic soil environment. The soil is an intensely colonized habitat, and while antagonism doubtlessly occurs, the influence of the soil fauna on nematode releases remains virtually unstudied. Even for moisture, arguably the most carefully considered abiotic parameter, there is insufficient information to estimate the soil tension needed for infection to
358
occur, yet predictable control depends upon the ability to predict nematode behavior in the soil. These questions are not amenable to technological solutions. A particularly useful approach for gaining insight into steinernematid and heterorhabditid soil ecology would be to explore critically the differences between successful and unsuccessful field trials. Most soil applications have been empirical studies, with surprisingly little regard shown for ascertaining factors which limit effectiveness. Soil parameters are often not adequately monitored, precluding the establishment of cause and effect relationships. The reasons for trial success or failure are likely to continue to remain obscure unless test protocols are devised that provide a degree of standardization. At a minimum, the following test parameters should be reported for soil surface applications: time and method of application, air and soil temperatures, cloud cover, thatch depth, soil type, soil moisture, pest density, pest stage, pest activity zone, post-treatment irrigation and precipitation. Such protocols would not address biotic interactions, but would generate field data on the impact of the physical environment. This would help to reduce the incidence of failures by providing guidelines on where, when and how entomopathogenic nematodes might be most advantageously used, or alternatively, how the environment might best be manipulated to encourage parasitism. Ultimately, understanding steinernematid and heterorhabditid soil ecology is fundamental to understanding how to enhance their use. REFERENCES
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360 Moyle, P.L. and Kaya, H.K., 1981. Dispersal and infectivity of the entomogenous nematode, Neoaplectana carpocapsae Weiser (Rhabditida: Steinernematidae), in sand. J. Nematol., 13: 294-300. Poinar, G.O., Jr., 1975. Entomogenous Nematodes. E.J. Brill, Leiden, The Netherlands, 317 pp. Poinar, G.O., Jr., Thomas, G.M., Presser, S.B. and Hardy, J.L., 1982. Inoculation of entomogenous nematodes, Neoaplectana and Heterorhabditis and their associated bacteria, Xenorhabdus spp. into chicks and mice. Environ. Entomol., 11: 137-138. Poole, M.A., 1976. Survey and control efficacy of endoparasites of Solenopsis richteri and S. invicta in Mississippi. Ph.D. dissertation, Mississippi State University, 83 pp. Quattlebaum, E.C., 1980. Evaluation of fungal and nematode pathogens to control the red imported fire ant, Solenopsis invicta. Ph.D. dissertation, Clemson University, 54 pp. Reed, E.M. and Carne, P.B., 1967. The suitability of a nematode (DD-136) for the control of some pasture insects. J. Invertebr. Pathol., 9: 196-204. Stimman, M.W., Kaya, H.K., Burlando, T.M. and Studdert, J.P., 1985. Black vine weevil management in nursery plants. Calif. Agric., 39: 25-26. Van Sloun, P. and Sikora, R.A., 1986. Control ofAgrotis segetum and Delia brassicae with species of Steinernema Heterorhabditis. In: R.A. Samson, J.V. Vlak and D. Peters (Editors), Fundamental and Applied Aspects of Invertebrate Pathology. 4th I.C.I.P., Wageningen, The Netherlands, p. 318. Walker, T.J. (Editor), 1984. Mole crickets in Florida. F1. Agric. Exp. Stn. Bull., No. 845, 54 pp. Wallace, H.R., 1958. I. The influence of pore size and moisture content of the soil on the migration of larvae of the beet eelworm, Heterodera schachtii Schmidt. Ann. Appl. Biol., 46: 74-85. Wallace, H.R., 1969. The influence of a non-ionic detergent on the movement of larvae of Meloidogynejavanica. Nematologica, 15: 107-114. Wright, R.J., Agudelo-Silva, F. and Georgis, R., 1987. Soil applications of steinernematid and heterorhabditid nematodes for control of Colorado potato beetles, Leptinotarsa decemlineata (Say). J. Nematol., 19: 201-206. Zimmerman, G. and Simons, W.R., 1986. Experiences with biological control of the black vine weevil, Otiorhynchus sulcatus (F.). In: R.A. Samson, J.V. Vlak and D. Peters (Editors), Fundamental and Applied Aspects of Invertebrate Pathology. 4th I.C.I.P., Wageningen, The Netherlands, pp. 529-533.