Technical Bulletin 218 - Nematode Infections General Features Taxonomic Status Mermithidae General Features Steinernematidae Hosts and Geographical Distribution Life Cycle Heterorhabditidae Hosts and Geographical Distribution Bacteria Associated with Steinernematidae and Heterorhabditidae Control of Helicoverpa (=Heliothis) zea and Heliothis virescens with Nematodes References Nematodes associated parasitically with insects have received increased attention as biological control agents in recent years. They have been found associated with most of the insect orders. There are more than 3100 natural associations between insects and nematodes involving 11 orders of nematodes and 19 orders of insects. The association may range from a phoretic relationship to obligate entomoparasitism and include host death, sterility, reduced fecundity, delayed development, or aberrant behavior. Some that are associated with one host and its special ecology are highly specialized and difficult to propagate on an artificial medium (some terrestrial mermithids). Other less specialized forms have wide host ranges and can be mass produced on artificial media and be used at the present time for control of agricultural pests (Steinernematidae). General Features Nematodes can be arranged into several groups depending on the method of feeding. This includes 1) free-living, 2) predatory, 3) plant-parasites, 4) vertebrate-parasites, and 5) parasites of invertebrates. Insect-parasitic nematodes must meet two criteria to be considered as a candidate for biological control: 1) they must attack insects that are considered pests to man, and 2) they must either kill, sterilize, or hamper the development of the insect. Taxonomic Status The phylum Nematoda is divided into two classes: Adenophorea and Secernentia (syn. Phasmidia), both of which contain important insect-parasitic nematodes. In the class Adenophorea, parasitic nematodes have been found in the family Mermithidae and in the class Secernentea in the families Steinernematidae and Heterorhabditidae. Three genera of nematodes are known, at this time, as containing parasites of heliothines; they are Hexamermis sp., Steinernema spp., and Heterorhabditis spp. Mermithidae Figure 1. Helicoverpa zea parasitized by Hexamermis sp. and Galleria mellonella larva parasitized by Steinernema carpocapsae (= Neoplectana=DD-136 strain of S. carpocapsae). Pre-adult Hexamermis sp. emerging from H. zea larva. Immature forms of a Hexamermis sp. family Mermithidae were isolated from both Helicoverpa (=Heliothis) zea and H. virescens larvae collected in cotton fields in Mississippi and were identified by Gerard M. Thomas (University of California, Berkeley) as members of this group of nematodes. The immature juvenile stages of some mermithids were 10 cm in length. In order to obtain adult forms, the nematodes should be kept in soil for one to two months. However, our attempts to rear it to the adult stage failed (only adults can be identified to species). In the chapter on lepidopterans, Wouts (1984), in a group of unidentified Hexamermis species, listed numerous species of Noctuidae, including H. armigera as hosts of this mermid. In the literature, a large number of lepidopterans have been observed as hosts of Hexamermis species and other unidentified mermithids (Wouts, 1984). He speculated that, in the majority of cases, the parasite involved was H. trunctata. General Features Mermithids are parasites of invertebrates. In most cases, the newly emerged infective second stage nematode directly penetrates the cuticle of its host. In the body cavity, the mermithid absorbs food directly through its cuticle. Inside the host, mermithids can grow up to 50 cm in length. The pre-adults of most mermithids emerge from the host to mature in the soil to a free-living nonfeeding adult, mate, and produce progeny. It is generally presumed that mermithid parasites of insects can significantly reduce their host population and thus are excellent candidates for biological control of insects. Steinernematidae An infective juvenile of S. carpocapsae. S. carpocapsae juveniles emerging from G. mellonella larva (photograph by Dr. S. R. Dutky). Hosts and Geographical Distribution Steinernematids are parasites of invertebrates (including H. zea and H. Virescens). In 1929, R. W. Glaser isolated Steinernema (= Neoaplectana) glaseri from Japanese beetle (Popilla japanica) and cultured it on artificial media (Glaser, 1931 and 1932). Steinernema carpocapsae may kill its hosts in 48 hours and has a very wide host range. A number of Steinernema species have been described from natural infections of insects, and all have a mutualistic association with several species of bacteria in the genus Xenorhabdus (Enterobacteriaceae). Infective juveniles are resistant to desiccation and can survive in damp soil for several months. In 0.1% formalin at 5 °C, they may survive for several years (Wouts, 1984). Steinernema chresima (syn. S. carpocapsae) was found naturally infecting corn earworms, H. armigera. Steinernema carpocapsae has been isolated from seven different species of insects (natural infection) and has been experimentally transmitted to more than 200 different insect species (Poinar, 1979). Life Cycle (Tanada and Kaya, 1993) The infective juveniles (third-stage nematode) are ingested by the host and enter the hemocoel by penetrating directly through the midgut. The nematode may also enter the host through the spiracles and penetrate the tracheae. In the host hemocoel, the nematodes release bacteria, which kill the host by septicemia within 48 hours. The nematodes feed on the Xenorhabdus bacteria and the host tissues. The nematodes develop rapidly to the adult stage, mate, and produce eggs. As soluble nutrients in the cadavers are depleted, the progeny of the second and third generations develop into infective juveniles, which exit from the cadaver and seek new hosts. In the laboratory, infective juveniles exit from the host 8-14 days after infection. Matrical endotoky occurs in older adult female nematodes. The eggs hatch within the body, and the juveniles feed on the body contents of the mother (Tanada and Kaya, 1993). In Steinernema, the juveniles develop into dioecious adults. The relationship between the nematode and the bacterium is symbiotic. Xenorhabdus spp. are symbiotically associated with the family Steinernematidae (Thomas and Poinar, 1979). The bacterium benefits because the nematode acts as a vector for the bacterium. The nematode benefits because the bacterium causes septicemia, which causes death of the insect, and the host tissues are changed into nutrients suitable for the nematode to use as a food source (Kaya and Gaugler, 1993). The nematode can kill its host without its associated bacterium but is unable to reproduce, and the bacterium cannot invade the hemocoel of its host without the nematode (Tanada and Kaya, 1993). Heterohabditidae Members of the genus Heterorhabditis are obligate parasites of insects that share with the steinernematids the specialized character of carrying bacteria. The type species of the genus is H. bacteriophora (Poinar, 1979). Heterorhabditids are similar to steinernematids in general life cycle and gross morphology (Wouts, 1984). The major difference between the two families is in the reproductive strategies. Heterorhabditid adults resulting from the infective juveniles are hermaphrodites; therefore, only one juvenile is needed to enter the host for progeny production (Tanada and Kaya, 1993). The larvae and pupae of infected hosts usually turn dark brick-red upon death. Four to five days after death of the insects, the nematodes reach mature size and become giant hermaphrodite females. Eggs laid by the hermaphrodites produce juveniles that develop into males and females within the body of the host. Hosts and Geographical Distribution The original host of H. bacteriophora was Heliothis punctigera, but Milstead and Poinar (1978) reported that representatives of four orders (Coleoptera, Diptera, Lepidoptera, and Orthoptera) can serve as hosts for H. bacteriphora. Heterorhabditis bacteriophora was isolated from diseased larvae of H. punctigera in Brecon, South Australia (Poinar, 1975). Heterorhabditis heliothidis was collected from prepupae and pupae of H. zea in North Carolina. Tanada and Kaya (1993) reported H. heliothidis as a synonym for H. bacteriophora. The genus Heterorhabditis is represented by several species with a worldwide distribution (Poinar, 1979). A Heterorhabditis sp. (Nematode 41088) was collected from white-fringed beetles at Saucier, Mississippi in 1941. This nematode was then recorded from 66 insect species from Louisiana, Alabama, Florida, Georgia, North Carolina, and Mississippi (Poinar, 1979). Bacteria Associated with Steinernematidae and Heterorhabditidae The bacteria associated with these families are in the genera Xenorhabdus and Photorhabdus. Xenorhabdus nematophilus, associated with Steinernema species, is the type species of the genus (Thomas and Poinar, 1979). A new genus Photorhabdus (syn. Xenorhabdus luminescens), with the description of the type species, Photorhabdus luminescens was proposed by Boemare et al. (1993). Photorhabdus luminescens is the bacterium associated with Heterorhabditis species. Both species of nematodes carry their specific mutualistic bacteria in their intestines. The infective juveniles enter their insect host and release the bacteria into the host's hemolymph within 5 hours of invasion. In general, the bacteria are highly virulent when delivered to the host's hemocoel. Several Steinernema species have been shown to be pathogenic for insects in the absence of their Xenorhabdus symbiont. Apparently, both nematodes and bacteria produce numerous toxins. However, the significance of multiple-toxin production by the bacterium/nematode complexes has not been established (Ankhurst and Dunphy, 1993). Control of H. zea and H. virescens with Nematodes Some members of the families Steinernematidae and Heterorhabditidae are regarded as being excellent biological control agents for a number of insect pests in soil and cryptic habitats (Gaugler and Kaya, 1990; Kaya and Gaugler, 1993). Bell (1995) reported a reduction in H. virescens adult emergence from soil under cotton plants treated with S. riobravis. The number of moths emerging from treated soils was reduced by an average of 66% compared to the untreated controls. Purcell et al. (1992) used S. carpocapsae to control H. zea on corn plants with some success. Bong and Sikorowski (1983) tested the effectiveness of S. carpocapsae in controlling H. zea larvae on corn ears. The nematodes were suspended in a water solution containing 0.01% Triton X-100 surfactant and applied to the corn silk with a pressurized hand sprayer. They reported 88% control with 4 x 104 nematodes/ml in early June. Kaya (1993) stated that natural habitat of steinernematid and heterorhabditid nematodes is soil, and for the foreseeable future, the major application will likely be as biological control agents against soil insects. Various preparations of nematodes available commercially in the United States at this time are formulations based on S. riobravis (against mole crickets and plant-parasitic nematodes in turfgrass), based on S. feltiae against mushroom flies and fungus gnats, and based on S. carpocapsae against a wide range of soil insects in grass and ornamentals. References Akhurst, R. J., and G. B. Dunphy. 1993. Tripartite interactions between symbiotically associated entomopathogenic bacteria, nematodes, and their insect hosts, pp. 1-23. In N. E. Beckage, S. N. Thompson, and B. A. Federici (eds.), Parasites and pathogens of insects, Vol. 2: pathogens. Academic Press, New York, NY. Bell, M. R. 1995. Effects of an entomopathogenic nematode and nuclear polyhedrosis virus on emergence of Heliothis virescens (Lepidoptera: Noctuidae). J. Entomol. Sci. 30: 243-250. Boemare, N. E., R. J. Akhurst, and R. G. Mourant. 1993. DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus Photorhabdus gen. nov. International Journal of Systemic Bacteriology 43: 249-255. Bong, C. F. J., and P. P. Sikorowski. 1983. Use of DD136 strain of Neoaplectana carpocapsae Weiser (Rhabditida: Steinernematidae) for control of corn earworm (Lepidoptera: Noctuidae). J. Econ. Entomol. 76: 590-593. Glaser, R. W. 1931. The cultivation of a nematode parasite of an insect. Science 73: 614. Glaser, R. W. 1932. Studies on Neoaplectana glaseri, a nematode parasite of the Japanese beetle (Popillia japonica). N. J. Dep. Agric. Circ. No. 211. Gaugler, R., and H. K. Kaya. 1990. Entomopathogenic nematodes in biological control. CRC Press, Inc., Boca Raton, FL. Kaya, H. K. 1993. Contemporary issues in biological control with entomopathogenic nematodes. Extension bulletin No. 375. University of California, Davis, CA. Kaya, H. K., and R. Gaugler 1993. Entomopathogenic nematodes. Ann. Rev. Entomol. 38: 181-206. Milstead, J. E., and G. O. Poinar, Jr. 1978. A new entomogenous nematode for pest management systems. Calif. Agric. 32: 12. Poinar, G. O., Jr. 1975. Description and biology of a new insect parasitic rhabditoid, Heterorhabditis bacteriophora n.gen., n. sp. (Rhabditida: Heterorhabditidae n. family). Nematologica 21: 463-470. Poinar, G. O., Jr. 1979. Nematodes for biological control of insects. CRC Press, Boca Raton, FL. Purcell, M., M. W. Johnson, L. M. LeBeck, and A. H. Hara. 1992. Biological control of Helicoverpa zea (Lepidoptera: Noctuidae) with Steinernema carpocapsae (Rhabditida: Steinernematidae) in corn used as a trap crop. Environ. Entomol. 21: 1441-1447. Thomas, G. M., and G. O. Poinar, Jr. 1979. Xenorhabdus gen. nov., a genus of entomopathogenic nematophilic bacteria of the family Enterobacteriaceae. Int. J. Syst. Bact. 29: 352-360. Tanada, Y., and H. K. Kaya. 1993. Insect Pathology. Academic Press, New York, NY. Wouts, W. M. 1984. Nematode parasites of lepidopterans, pp. 655-696. In W. R. Nickle (ed.) Plant and insect nematodes. Marcel Dekker, Inc., New York, NY.