Biological Control Potential of Neoaplectanid Nematodes ~ Randy Gaugler 2 Abstract: The neoaplcctanids are among the most studied of all entomogenous nematodes. Because these nematodes kill their insect hosts, they are regarded as having excellent potential as biological control agents. While the host specificity of most entontogenous nematodes tends to limit their potential usefulness, the broad host range and high virulence of neoaplectanids make them attractive candidates for industrial development. Also, recent development of economical mass rearing procedures appears to make production on a commercial basis feasible. Infective stages may be stored for ),ears trader various laboratory condilions. Although entomogenous nematodes, as parasites, are exempt from govermnent registration requirements, the mutualistic association of neoaplectanid nematodes with a bacterium will likely necessitate a detailed safety evaluation. Studies conducted to date indicate a lack of pathogenicity to mammals. Field trial success has been limited by the intolerance of infective stages to mffavorable environmental conditions, particularly low moisture. Applications against pests on exposed plant foliage have been especially disappointing. More encouraging anti consistent results have been obtained in more favorable environments, including soil and aquatic habitats, but the most promising treatment sites ntay be cryptic habitats where infective stages are shehered from environmental extremes. Cryptic habitats also exploit the ability of neoaplectanids to actively seek out hosts in recessed places where conventional insecticide applications are intpractical. Key words: Neo- aplectana bibionis, Neoaplectana carpocapsae, Neoapleetana glaseri, entomogenous nematodes.

Although nematodes are most evident as control attempts: N. glaseri, N. bibionis, injurious parasites of man, animals, and and N. carpocapsae. First reported in 1954, plants, those species which attack inverte- N. carpocapsae has been studied more ex- brates can play a significant role in limiting tensively than any other neoaplectauid. populations of agriculturally and medically Conseqttently, most of our knowledge con- important insect pests. Nematode-insect in- cerning the genus has been derived from teractions may range from phoresy to obli- this single species. gate endoparasitism and inchlde host death, There is little doubt that neoaplectanids sterility, reduced fecundity, delayed devel- show great potential as pest control agents, opment, or aberrant behavior. The majority hut several difficulties remain to be fully of these interactions are harmless or mildly resolved. This review will examine both the debilitative; comparatively few entomogen- prontise and the problems. ous nematodes cause host mortality. Among those which do kill their hosts are members BIOLOGY of the genus Neoaplectana. Reviewers have consistently noted that these nematodes Tile ensheathed third-stage juvenile is show great promise for use as biological con- the neoaplectanid invasive form which lo- trol agents of insect pests (25,57,76). Despite cates new hosts and initiates infections. this acclaim, the neoaplectanids have yet to Host finding by these infective-stage juve- realize their potential. niles is an active process occurring in re- As many as 20 species have been de- sponse to chemical stimuli provided by the scribed within the genus Neoaplectana; host. NeoapIectana carpocapsae, for ex- however, Poinar (59) considers all but ample, forms aggregations adjacent to in- seven of these either as strains of earlier sect larvae (63,68) and fecal components described species, as belonging in other (69) and is capable of directed orientation genera, or as being insufficiently described. up a gradient of carbon dioxide (18), a Only three species have been used in insect compound emitted by insect spiracles. When a suitable host is located, infective juveniles Received for publication 12 December 1980. enter via natural body openings (i.e., ~Symposium paper presented at the annual meeting of moltth, anus, or spiracles), exsheath, and the Society of Nematologists, New Orleans, Louisiana, Au- gust 1980. Published by permission of the Director, New penetrate into the hemocoel. Here the York State Museum, State Education Department, Journal nematodes release an associated bacterium Series No. 318. USenior Scientist, Biological Survey, New York State from their intestinal lumen into the hemo- Museum, State Education Department, Albany, NY 12230. lymph. The bacterium rapidly multiplies Continuing financial support from the National Institutes of Health (AI-15605) is gratefully acknowledged. and kills the host by septicemia. The nema-

241 242 ]ournal o[ Nematology, Volume 13, No. 3, ]uly 1981 lodes feed upon the bacteria and degraded frequently been illustrated in tests with N. host tissue, mature, mate, and produce two carpocapsae, where initial laboratory suc- or three generations which emerge as in- cesses could not be duplicated in the field fective-stage juveniles in search of new hosts. (32,39,65,66,79). The relationship between the nematode and its associated bacterium, Xenorhabdus SAFETY (= Achromobacter) nematophilus in the case of N. carpocapsae (75), is one of classic Insect control agents being considered mutualism. The bacterium requires the for wide application must have their safety nematode for protection, transport, and established. Entomogenous nematodes are penetration into the host hemocoel, while regarded as parasites rather than microbial the nematode is dependent upon the bac- pathogens by the U. S. Environmental Pro- terium for supplying nutrients essential for tection Agency (EPA), and as such are not successful reproduction. The bacterium also subject to government registration (53). establishes suitable conditions for nema- Tile specific bacterium associated with neo- tode development by repressing cadaver aplectanid nematodes is, however, a mic- colonization by other bacteria, thereby al- robe and plays a major role in the infection lowing the nematode to complete its life process. Therefore, it is anticipated that cycle without cadaver putrefaction. Tiffs registration of the nematode-bacterium bactericidal effect may be the result of de- complex will be required by EPA. fective bacteriophages produced by the Although EPA guidelines for safety test- bacterium (61). The only other genera of ing of neoaplectanids are unavailable, some nematodes known to be mutualistically as- xnammalian pathogenicity tests have been sociated wth bacteria, Heterorhabditis (62) conducted. No adverse effects were observed and Steinernema (51), are also parasites of when high dosages of infective-stage juve- insects. niles of N. carpocapsae were fed to mice (70) and rats (57). In per os and intra- HOST RANGE peritoneal inoculations (17), infective stages did not cause detectable injury in rats, nor The intense interest in Neoaplectana did the nematodes survive: juveniles admin- spp. as biological control agents is largely a istered per os were recovered dead in the function of their unusually broad host feces, and intraperitoneally-injected juve- range. Nearly 250 insect species from 10 niles were encapsulated by rat peritoneal orders are reported to serve as hosts for N. macrophages. Kaya (35) has shown that carpocapsae (59); the host range of other homoiothermic parasitism is unlikely be- neoaplectanids is probably no less exten- cause growth of both the nematode and sive. The basis for this lack of host specific- bacterium is inhibited at temperatures ity is tile associated bacterium, which kills above 30 C. Thus, safety considerations the host so quickly that the nematodes are should not prevent the use of neoaplect- not hampered by a defense reaction or re- anids as agents of pest control. quired to adapt to the host life cycle. Even in those few insects where invasion elicits PROPAGATION a strong host reaction, encapsulation seldom protects the insect since the bacterium is not An essential requirement in the utiliza- inactivated (81). Apparently, neoaplect- tion of ally biological control agent is the anids kill any insect they penetrate. capability of propagating large numbers of Despite the impressive host range attrib- infective stages at an acceptable cost. Be- uted to neoaplectanids, it is extremely un- cause neoaplectanids can be easily mass pro- likely that so many insects serve as hosts in duced using either in vivo or in vitro meth- nature. Most species described as susceptible ods, they have been widely available for were exposed in laboratory petri dishes field use. Dutky et al. (13) used larvae of the under optimal conditions where host-para- greater wax moth, Galleria melIonella, to site contact was assured. In nature, host propagate the DD-136 strain of N. carpo- range may be restricted by ecological and capsae in vivo and achieved yields of up to behavioral barriers (67). This point has 200,000 infective juveniles per host. A1- Neoaplectanids and Biocontrol: Gaugler 243 though the in vivo technique is simple and dominates, Bedding (3) has established that in wide usage, it is economically unsuited his culture media should be inocuIated to large-scale rearing. Efficient mass produc- with the primary form bacteria prior to tion for commercial use or large-scale field nematode introduction. trials is best accomplished through in vitro Neoaplectanid infective stages may be techniques. Neoaplectanids can be reared stored for prolonged periods (5 yr) without on a wide range of materials, reflecting the loss of infectivity when kept at low tempera- ability of the associated bacterium to con- tures (7 C) in an aerated aqueous suspen- vert proteinaceous substances into suitable sion (13), or when stored in water shallow media for nematode development (2,59). enough to allow oxygen diffusion (42). High An artificial media, dextrose-veal infusion survival is also reported when juveniles are agar, was first used for propagation of N. held on moist filter paper at 3 C (29). An glaseri in 1931 (21); however, the yields alternative approach to aqueous storage (400-800 nematodes/cm -° of culture area) may be storage in paraffin oils, which hold were inadequate for field tests. Subse- tip to 15 times as much oxygen as water (2). quently, yeast-fermented potatoes (46) and ground veal pulp (45) were developed as ENVIRONMENTAL SENSITIVITY culture media with progressively greater The intolerance of neoaplectanid nema- yields. A dog food based medium devised todes to environmental extremes is regarded by House et al. (28) was used on a com- as the key factor limiting their effectiveness mercial scale to produce the DD-136 strain as insect control agents. This sensitivity sev- of N. carpocapsae at a cost of $1 (1971) per erely restricts their use as short-term control million infective juveniles (56). Hara et al. agents (biological insecticides) and pre- (26) later refined this method, stressing cludes their use for long-term control (col- monoxenicity, to produce 125 million nema- onization) in many habitats. todes/wk from 100 (log food-agar petri There is considerable evidence showing dishes at a cost of $0.28 (1980) per million. that moisture is the most important en- Abandoning petri dish methods as un- vironmental factor affecting N. carpocapsae economical, Bedding (2) developed and field persistence. Aqueous spray applica- patented a rearing system which provided tions of infective stages onto exposed sur- a large surface area to volume ratio, rearing faces quickly result in juvenile desiccation. the nematode-bacterium complex on Aspen Neoaplectanids sprayed on plant foliage in wood wool coated with homogenized the field may survive for less than an hour chicken heart. In a subsequent improvement (79), and even at 85% relative humidity, (3), shredded plastic foam was soaked in 98% mortality has occurred after 102 hr pork kidney-beef fat homogenate and (33). Simons and Poinar (72) have indicated placed in 500-ml flasks. This method was that foliar applications are ineffective be- suitable for rearing several species of neo- cause evaporation is rapid and, therefore, aplectanid and heterorhabditid nematodes, lethal. They concluded that juvenile sur- with an average yield of 38 million N. vival depended on gradual desiccation and carpocapsae juveniles per flask, at a cost of suggested that soil application was, there- less than $0.02 (1980) per million. One tech- fore, more practical than application on nician can produce 16 million nematodes/ foliage. In support of this conclusion, wk with this technique (3). Moore (48) reported that infective stages Akhurst (1) has determined that there survived 90 min on an exposed leaf surface, are two distinct forms of the bacterium but up to 24 d in a quart of slowly dried nmtualistically associated with neoaplect- soil. anid and heterorhabditid nematodes. The Infective stages are sensitive to tempera- primary form, found in infective-stage juve- ture extremes as well. Their activity de- niles, is initially produced, but is later dis- creases at temperatures exceeding 32 C, and placed by the secondary form. Noting that 100% juvenile mortality occurs after 16 h nematode reproduction is optimal when of exposure at 37 C (71). At low tempera- only the primary form is present, and di- tures (< 15 C), juvenile ability to induce minished when the secondary form pre- host mortality is reduced (Gaugler, un- 244 Journal of Nematology, Volume 13, No. 3, July 1981 published data), although high rates of host population of N. glaseri was reisolated, that mortality have been achieved at tempera- tile long suspected presence of the associated tures as low as 11 C (47) and the infective bacterium was finally confirmed (60). In- nematodes will survive freezing (71). The terest in N. glaseri declined abruptly with optimal temperature for N. carpocapsae the development in the 1940s of milky dis- growth and reproduction is 25 C, with no ease (Bacillus popillae) as an effective con- development occurring at l0 C or above trol agent of the Japanese beetle. However, 3~ c (~5). tile rediscovery of xenic populations of N. Solar radiation, usually regarded as the glaseri should revive interest in this nema- environmental factor most detrimental to tode's use against insect pests in the soil. microbial insecticides (31), also adversely Neoaplectana bibionis was originally affects neoaplectanid nematodes. Exposure considered to be a parasite of questionable of N. carpocapsae juveniles to 60 min of pathogenicity (5) and has only recently direct natural sunlight reduced their patho- found favor with biological control work- genicity to G. mellonella larvae by 95%, ers. In one of the most notable demonstra- while nematode development, reproduc- tions of the biocontrol potential of neo- tion, and pathogenicity were severely im- aplectanids, Bedding and Miller (4) applied paired following 3.5 min of irradiation with N. bibionis to 50,000 blackcurrant cane short (254 nm) ultraviolet light (15). cuttings and obtained nearly total suppres- Other physical environmental param- sion of currant borer larvae. They con- eters (e.g., pH, photoperiod, oxygen, salin- cluded that this nematode could econom- ity) are unlikely to be serious limiting fac- ically disinfest blackcurrant cuttings on a tors in most target habitats. Elements of colnmercial basis. the biotic environment have been almost Most attempts to control field popula- ignored as factors affecting the survival of tions of insects with nematodes have used neoaplectanids, even though nematodes are strains of N. carpocapsae. Poinar (59) lists known to have many natural enemies (44). 34 field trials conducted up to 1978; at least The mermithid nematode R. culicivorax, 10 additional trials have also been reported for example, is attacked by numerous pred- recently (8,10,12,14,20,37,39,43,64,82), for a ators (54,55) and at least one pathogen (73). minimum of 44 trials in 12 countries. These tests have yielded inconsistent results, pro- FIELD TRIALS ducing nearly as many failures as successes, The first attempts to control insects but poor choice of target habitat would ap- with nematodes used N. glaseri against soil- pear to account for many of the failures. inhabiting pests in the ]930s. Billions of in- For example, attempts to control foliage- fective stages were applied in these tests, feeding insects have been generally dis- primarily in the eastern United States couraging, with frequent reports of low against the Japanese beetle, Popillia mortality, insignificant population reduc- ~aponica. Initial results were encouraging, tion, or inadequate crop protection. Interest with high grub mortality and nematode in such applications has consequently establishment in the test plots (22). Control dwindled. Only 6% (I/17) of the field tests was not obtained in subsequent applica- reported in the last decade have been foliar tions, although establishment was some- applications, compared to 70% (9/13) dur- times recorded (23,24,30). Noting that early ing the 10-yr period following the nema- workers were unaware of the nematode's tode's discovery. Not coincidently, the in- association with a symbiotic bacterium, cidence of successful trials also increased. Poinar (59) found it remarkable that es- The use of neoaplectanids appears more tablishment occurred at all, since the nema- promising against soil-inhabiting insects. todes were cultured on media containing Soil applications have reduced field popula- antimicrobial agents. These agents prob- tions of wireworms (37), root maggots (9, ably eliminated the associated bacterium, 80), and root weevils (8,27,74), although resulting in reduced nematode develop- population reductions have not always re- ment, reproduction, and pathogenicity. It sulted in adequate control. In contrast to was not until 1977, when a natural field foliar applications, where posttreatment Neoaplectanids and Biocontrol: Gaugler 245 survival has generally been brief, viable spray application; a larger test the subse- juveniles have been recovered from soil qttent year resulted in a 55% reduction in treatment plots 2 (39) to 16 (27) months the pest population and a 34% reduction after application. A possible limitation to in almond damage. Moore (49) demon- neoaplectanid use in the soil is that nema- strated the nematode's ability to enter and tode movement may be restricted during survive in bark beetle tunnels, recording periods of low soil moisture (39). Under 44~/~ mortality of brood and adults follow- favorable moisture conditions, infective- ing spray applications on pine bark. In tests stage juveniles of N. carpocapsae can mi- against the codling moth, Carpocapsa grate in soil up to 14 cm laterally and 12 pomonella, on apple trees, Dutky (11) cm vertically to cause host infection (50). sprayed infective-stage N. carpocapsae juve- The aquatic environment provides niles onto tree trunks to produce more than nearly optimal physical conditions for neo- 60% mortality among larvae seeking co- aplectanid survival but remains virtually cooning sites in bark crevices. He found unexploited as a site for field trials. Briand that tire nematodes coukt tolerate even ex- and Welch (6) briefly noted that applica- tended periods of drought under the pro- tions of N. carpocapsae in mosquito pools tective canopy of leaves. reduced larval density and adult emergence, although establishment did not result. In PERSPECTIVE a stream trial against blackfly larvae, Gaug- The ultimate objective of most neo- ler and Molloy (20) obtained 50% mortal- aplectanid research is to make these para- ity of late instar Simulium vittatum despite sites widely available for effective pest low stream temperatures (9-12 C), but, controI. Tile most logical step toward the again, nematode establishment did not oc- accomplishment of this goal would be com- cur. Tire effective use of N. carpocapsae mercial production. Commercialization against blackflies is restricted to late instar seems justified, since neoaplectanids possess populations, because early instars are re- nearly all the attributes of an ideal biolog- sistant and mid instars only moderately sus- ical control agent (Table 1). However, ceptible (19). several problems remain to be resolved. The most encouraging trials have been The broad host range of neoaplectanids conducted against insects in protective, is certainly a desirable attribute. Industry cryptic habitats, where the nematode is is often reluctant to invest in a control sheltered from environmental extremes. agent attacking only one or two insect pests, Such applications are attractive, not only because this results in a limited market. On because survival is enhanced, but also be- the other band, a broad host range may cause they capitalize on the nematode's also be a liability since nontarget inverte- ability to seek out concealed pests, a cap- brates, including beneficial insects, could be ability not shared by chemical insecticides. adversely affected. Although Kaya and Lindegren et al. (43) reported total sup- Hotchkin (36) emphasize that even a detri- pression of first-instar carpenter worms in- mental affect on beneficials by N. carpo- festing fig trees after treatment with the capsae should not preclude the nematode's Mexican strain of N. carpocapsae. These insect pests are nearly invulnerable to con- Table 1. Anributes and liabilities of neoaplect- ventional control methods because of their anid nematodes as biological control agents of in- cryptic habitat (moist, frass-filled galleries sects. in the tree heartwood). Similarly, larvae of the navel orangeworm, Arayelois transitella, Attributes Liabilities a serious pest in almond orchards, are Broad host range Broad host range shielded from most insecticides by inhabit- Safety Registration ing the almond interior. Lindegren et al. Laboratory storage Commercial storage (40), noting that the moist almond interior High efficacy Field persistence also provides a higldy favorable environ- Ease of production Ease of application ment for nematode survival, achieved up to Power of search 100% mortality of these insects in a 1976 246 Journal of Nematology, Volume 13, No. 3, July J981 development as a biological control agent, tectant (16) spray additives, but the value this potential problem has received scant of these chemicals under field conditions attention and requires a full assessment has been inadequately evaluated. Another based upon the results of both field and approach is nematode application only in laboratory study. selected habitats which provide shelter and All available evidence indicates that moisture, and thus the greatest potential neoaplectanid nematodes are specific for [or their survival; e.g., soil, aquatic environ- arthropods and not hazardous to man and merit, and cryptic habitats. Soil is an espe- other vertebrates. Nevertheless, establishing cially attractive site because, as the natural their safety to the satisfaction of govern- reservoir for neoaplectanids, it offers the ment regulatory agencies remains an ob- possibility of establishment and recycling. stacle to neoaplectanid development. Colonization of other habitats seems un- Laboratory storage of infective-stage ju- likely; continued pest control in these sites veniles is readily accomplished, but existing would require repeated inundative applica- procedures appear unsuitable for commer- tions. Thus, neoaplectanids should be con- cial purposes, where viability must be pre- sidered as a "specialized tool" (78) and not served inexpensively, often under sub- for use against all pests in all habitats. optimal conditions, tyophilization or a re- Previous field testing has been restricted lated technique would seem ideal, but such to small plot treatments because of the rela- methods are unlikely to be developed soon. tively inefficient methods used to mass pro- Only Bedding's (2) work with paraffin oils duce neoaplectanids. Advances with in vitro seems to offer immediate promise, althongh culture now allow large-scale testing at a detailed information on this method is un- cost approaching that of many conventional available. Perhaps problems in storage and insecticides. It is hoped this breakthrough shipping could be avoided if the nematodes will induce industry to seriously reconsider were produced locally by "cottage indus- commercial production. In the meantime, tries" for immediate application. since the greatest cost associated with pro- Field applications frequently have shown duction is labor (3,26), the use of neoaplect- that neoaplectanids possess high efficacy for anids would seem especially appealing in controlling pest populations when applied developing countries with inexpensive under favorable conditions. Several success- labor. ful attempts have been made to further in- Another desirable neoaplectanid charac- crease efficacy, including the use of infective teristic is high virulence. Whereas many stages in conjunction with chemical (64) microbial agents have only a slow debilita- and microbial (34,38) agents. In novel ex- rive action, neoaplectanid infection causes periments to exploit the behavioral charac- host mortality within 24-48 h, thus limiting teristics of N. carpocapsae, Burman and Pye pest damage. Such rapid host death might (7) suggest that infective stages might be be expected to restrict dispersion, but conditioned to migrate toward the same soil "rapid spread" of N. glaseri by infected temperature level as the target pest. En- beetles has been reported (24). hanced efficacy might also result if efforts Application technology should not be an were directed toward determining which impediment to biological control with neo- species or strains are most effective against aplectanids. Infective stages are resistant to a particular pest. For example, N. bibionis most agricultural chemicals (3) and may be was shown to be far superior to N. carpo- applied with conventional high-pressure capsae and H. heliothidis in finding and spray equipment, either by ground or air, killing currant borers (4), while N. glaseri without loss of viability (11,41). is regarded as best suited for use against In contrast to chemical and microbial soil insects (58). agents, which must rely on chance contact, Instances of low field efficacy have usu- neoaplectanid nematodes can actively search ally been attributed to poor nematode field for hosts, permitting their use against even persistence. Efforts aimed at extending per- well-hidden target pests. Bedding and Miller sistence have focused on the development of (4) reported striking behavioral differences evaporetardant (2,52,77) and photopro- between neoaplectanid species in their abil- Neoaplectanids and Biocontroh Gaugler 247 ity to locate and parasitize insects inhabit- Hylobius abietus (Coleoptera, Curculionidae). Ann. ing relatively inaccessible environments. Entomol. Fennici 45:88. 9. Cheng, H. H., and G. E. Bucher. 1972. Field Additional research is needed concerning comparison of the neoaplectanid nematode DD 136 neoaplectanid behavior if we are to snccess- with diazinon for control of Hylemya spp. on to- fully exploit sttch differences. Selection for bacco. J. Econ. Entomol. 65:1761-1763. strains with even greater searching capabil- 10. Drooz, A. T. 1960. The larch sawfly: Its biol- ogy and control. USDA Tech. Bull. No. 1212. ities also deserves investigation. 11. Dutky S. R. 1959. Insect microbiology. Adv. Appl. Microbiol. 1:175-200. CONCLUSIONS 12. Dutky, S. R. 1967. An appraisal of the DD-136 nematode for the control of insect populations and Neoaplectanid nematodes have been some biochemical aspects of its host-parasite rela- tionships. Proc. Joint us-Japan Seminar on Micro- evaluated as biological control agents for bial Control of Insect Pests, Fukuoka, pp. 139-140. more than 50 yr, yet none are in general use. 13. Dutky, S. R., J. V. Thompson, and G. E. Still, tremendous progress has been made in Cantwell. 1964. A technique for the mass propaga- the last 5 yr and interest in their use has lion of the DD-136 nematode. J. Insect Pathol. 6: 417-422. accelerated, as indicated by the recent crea- 14. Finney, J. R., and C. Walker. 1977. The DD- tion of the Neoaplectana Newsletter (G. O. 136 strain of Neoaplectana sp. as a potential control Poinar, Jr., ed.). In the United States alone, agent for the European ehn bark beetle, Scolytus at least eight states support neoaplectanid scolytus. J. Invertebr. Pathol. 29:7-9. research, and worldwide their control po- 15. Gaugler, R., and G. M. Boush. 1978. Effects of ultraviolet radiation and sunlight on the en- tential is being examined in Argentina, tomogenous nematode, Neoaplectana carpocapsae. Australia, Canada, China, England, France, J. lnvertebr. Pathol. 32:291-296. Italy, New Zealand, and the Soviet Union. 16. Gaugler, R., and G. M. Boush. 1979. Labora- In light of this gathering momentum, it tory tests on ultraviolet protectants of an entomo- genous nematode. Environ. Entomol. 8:810-813. seems likely that some of the potential of 17. Gaugler, R., and G. M. Boush. 1979. Non- these nematodes for pest suppression will be susceptibility of rats to the entomogenous nematode, realized during the 1980s. Neoaplectana carpocapsae. Environ. Entomol. 8: 658-660. 18. Gaugler, R., L. LeBeck, B. Nakagaki, and LITERATURE CITED G. M. Boush. 1980. Orientation of the entomogenous nematode Neoaplectana carpocapsae to carbon di- 1. Akhurst, R. J. 1981. Morphological and func- oxide. Environ. Entomol. 9:649-652. tional dimorphism in Xenorhabdus spp., bacteria 19. Gangler, R., and D. Molloy. 1981. Instar symbiotically associated with the insect pathogenic susceptibility of Simulium vittatum (Diptera: nematodes Neoaplectana and Heterorhabditis. J. Simuliidae) to the entomogenous nematode, Neo- Gen. Microbiol., in press. aplectana carpocapsae. J. Nematol. 13:1-5. 2. Bedding, R. A. 1976. New methods increase 20. Gaugler, R., and D. Molloy. 1981. Field the feasibility of using Neoaplectana spp. (Nema- evaluation of the entomogenous nematode, Neo- toda) for the control of insect pests. Pp. 250-254 in aplectana carpocapsae as a biological control agent Proc. Int. Colloq. Invertebr. Pathol., Kingston. of black flies (Diptera: Simuliidae). Mosquito News, 3. Bedding. R. A. 1981. Low cost, in vitro mass in press. production of Neoaplectana and Heterorhabditis 21. Glaser, R. W. 1931. The cultivation of a species, (Nematoda) for field control of insect pests. nematode parasite of an insect. Science 73:614. Nematologica, in press. 22. Glaser, R. W. 1932. Studies on Neoaplectana 4. Bedding, R. A., and L. A. Miller. 1981. Disin- glaseri, a nematode parasite of the Japanese beetle festing blackcurrant cuttings of Synanthedon tipuli- (l'opillia japon'ca). N. J. Dept. Agric. Circ. 211: forints using the insect parasitic nematode, Neo- 3-34. aplectana bibionis. Environ. Entomol., in press. 23. Glaser, R. W., and C. C. Farrell. 1935. Field 5. Bovien, P. 1937. Some types of association be- experiments with the Japanese beetle and its nema- tween nematodes and insects. Viden. Meddel. Dansk tode parasite. J. N. Y. Entomol. Soc. 43:345-371. Naturhist. Foren 101:1-114. 24. Glaser, R. W., E. E. McCoy, and H. B. Girth. 6. Briand, L. J., and H. E. Welch. 1963. Use of 1940. 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