Possible Commercial Formulations of Insect- Parasitic Nematodes *

Possible Commercial Formulations of Insect- Parasitic Nematodes * W. R. NICKLE, Nematology Laboratory, Plant Protection Institute, Science and Education Administration, Agricultural Research, Beltsville Agricultural Research Center, U.S. Department of Agriculture, Beltsville, Maryland 20705 Summary At least two experimental nematode biological insecticide formulations are currently being produced in small quantities around the world. These formulations are efficacious and people are willing to pay money for them. Advantages for the use of these parasites can be: nonpolluting, self-perpetuating, safe, and not harmful to beneficial organisms. The disadvantages are: relatively short shelf-life, acceptability, moisture, price, and dispersal techniques. One of the preparations is a nematode that vectors a bacterium. It can be produced at the rate of 100 x lo6 infective nematodes! 2 liter container at a cost of 2 cents/106. It can kill over 1000 species of insects. The other product kills over 60 species of mosquitoes and has been sold as mermithid nematode eggs in moist sand. This mosquito parasite can be established in the new site and self-perpetuate to suppress mosquito populations. It kills the mosquito before pupating. Increased research activity, using these parasites in the last ten years, has stimulated interest in these organisms for pest insect control. INTRODUCTION Insect parasitic nematodes are roundworms, 0.5-150 mm in length, which have evolved to parasitize a wide variety of insects. They live in the body cavities of insects and debilitate, sterilize, or cause the death of their hosts. Some make large exit holes and come out, thus behaving more like parasitic Hymenoptera than pesticides. Some vector a bacterium causing septicemia and death of the hosts. They are multicellular animals as opposed to the viruses and bacteria that are the more well-known biological control organisms. However, in some pest insects found in the soil, water, or on plants growing near the ground, we can find high parasitism by nematodes. * Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. Biotechnology and Bioengineering, Vol. XXII, Pp. 1407-1414 (1980) @ 1980 John Wiley & Sons, Inc. 00063592/8O/oO22-1407$01.oO

1408 NICKLE Examples of pest insects commonly attacked by nematodes include: black flies, mosquitoes, chirononfids, rootworms, grubs, caterpillars, grasshoppers, and bark beetles. Most of the research on insect nematodes has been done by nematologists and entomologists and the field is therefore lacking in product formulation technology. Problems involving carriers, preservatives, packaging, shelf-life, and storage are more easily understood by people familiar with such technology and help is needed in applying these areas of expertise for production of nematodes as microbial agents. Two nematode parasites of insects have been mass reared to the stage at which commercial production is anticipated. The modes of action and rearing procedures are different for these two candidate microbial insecticides and so they are considered separately. MOSQUITO MERMITHID PARASITE This nematode, Romanomermis culicivorax (Reesimermis nielsen;), was one of a group of nematodes found in mosquitoes in Louisiana by Petersen et al.,* who submitted the specimens to this author for identification. This author separated them into five distinct species5 Nickle and Hogger3 described the infective stage that Petersen was able to manipulate into the mosquito mass-rearing pans (Fig. I), leading to the development of an efficient in vivo production plant for this para~ite.~ Although it is mass reared on Culex pipiens quinquifusciarus, it can kill over 60 species of mosquitoes in several genera. The mode of action is not unlike that of a hymenopterous parasitoid; it simply makes a large fatal exit hole in the mosquito wiggler (Fig. 2). The parasitized mosquitoes do not pupate. Fortunately, larval mosquitoes are not the injurious stages but the reduction in the potential populations of the pest adult mosquitoes by this parasite makes this type of host more suitable for nematode suppression. A culture of 1-2 x lo6 nematode eggs which will hatch into infective-stage nematodes when placed into a mosquito-breeding area is a product that is well worth a price of 5-10 dollars. The possibility that the parasite will establish in the pond and self-perpetuate is an added attraction for the user. Three companies are currently experimentally producing this parasite in the United States (US). The mosquito nematode has been dispersed by Chapman and Petersen and others to scientists all over the world for research purposes and field trials. So far, no adverse affects have been noted. The Fairfax Biological

INSECT-PARASITIC NEMATODES 1409 Fig. 1. Rearing apparatus for the in vivo culture of 100 g nematode pai.asites from 300000 mosquito larvae. Laboratory produced and sold this nematode as Skeeter Doom in 1976 and 1977. Nutrilite Corporation will produce a product of this mosquito nematode in 1980 in California and the Sandoz Corporation of Florida has a pilot study for the possible commercial production of this product. The Italian company SIAPA has plans to market the mosquito mermithid. The Indian government is producing a native mosquito mermithid similar to this one at the Vector Control Research Center at Pondicherry. They have distributed cultures throughout India in the mosquito areas. The nematode, unlike a lady bird beetle which often flies to another area, is not self-dispersing in nature. This parasite kills the last-stage mosquito larva before it pupates and goes to the bottom of the pond where it remains to produce eggs that hatch, producing

1410 NICKLE Fig. 2. Culex pipiens mosquito larva with fatal nematode parasitism in thorax. infective stages that parasitize other young mosquito larvae in the same pond. It does not enter the adult stage of the mosquito and therefore is not widely dispersed in nature. So it is necessary to add the parasite to the mosquito-breeding area, making the product more desirable. Finney5 has produced female mosquito mermithid nematodes in vitvo using Grace's insect tissue culture media along with fetal calf serum. The nematodes lacked some structures and they will be tested to see if they can produce eggs. Efficacy in the field has been tested extensively by Petersen' and high percentages of parasitism are often attained. Work in Maryland

INSECT-PARASITIC NEMATODES 141 1 with this Louisiana parasite' showed that it probably could establish and be effective in suppressing populations of summertime mosquitoes in that state, thus extending the useful range of the parasite. The advantages of this type of microbial agent for control of mosquitoes are nonpolluting, nonpoisonous, safe, not harmful to beneficial organisms, resistance to malathion, and often self-perpetuating. Also, the Environmental Protection Agency (EPA) in 1976 ruled that this nematode would be classified as a parasite and not a pesticide. 'The disadvantages are: relatively short shelf-life of two to three months, acceptability, only freshwater mosquito hosts, price, and dispersal techniques. Lack of patentability discourages larger companies from producing it because if and when the product is perceived to be useful, competing companies could undercut the price. The production of this parasite is currently labor-intensive, rendering it relatively expensive to produce. Neoaplectana carpocapsae (DD- 136) This nematode was first found by Dutky in the early 1950s from codling moth in the US and later Weiser8 described a nematode as Neoaplectana carpocapsae from fixed material from Czechoslovakia. He decided it was the same species as the culture of DD-136 that Dutky sent him. Dutky's original culture is the one that has been widely distributed and we now know that it will kill over 1000 species of insects. In vivo culture methods developed by Dutky are explained in detail in Refs. 9 and 10. The technique involves the mass rearing on the greater wax moth larvae and also the aseptic maintenance of the nematode on cooked pork kidney. The most industrious efforts in mass rearing of this nematode have been reported by Bedding." He produced 100 x lo6 infectivestage nematodes in a 2 liter container at a cost of 2 cents/106 on homogenized chicken heart on Aspen wood wool. He also made an important contribution to the commercialization of this nematode by helping to solve the desiccation problem by adding 0.1% paraffin oil/wax medium. Recently Lindegren et a].'' described a technique of rearing N. carpocapsae on adult navel orangeworms, Amyelois transitella (Walker). About 6 X lo6 infective-stage nematodes can be stored without aeration at 6 C in Petri dishes. Dutky and Weiser have distributed cultures of this nematode to

1412 NICKLE scientists all over the world since the early 1950s. So far, no adverse effects have been uncovered. Nutrilite Corporation produced this nematode briefly a few years ago. A French company, RENO, will produce it commercially next year. Also, the Italian company, Fig. 3. Wax moth larva with N. carpocapsae DD-136 nematodes in the body cavity.

INSECT-PARASITIC NEMATODES 1413 SIAPA will produce it shortly after receiving some favorable data from experimental tests. The mode of action requires the infective-stage nematode larva to enter the body cavity of the host insect, usually by oral ingestion. However, they can actively enter through the anus or spiracles. After entering the body cavity of the insect, they release a bacterium, which they vector in their esophagus into the insect's hemolymph. The bacteria build up and cause a septicemia and death of the host insect. The nematodes feed on the bacteria in the cadaver and produce young (Fig. 3). This author has sprayed infective stages on potatoes and beans for control of the larvae of Colorado potato beetles and Mexican bean beetles and the insects stopped feeding in 36 hr and died. The advantages of using this nematode to control pest insects are nonpolluting, nonpoisonous, relatively safe, large host range, relatively quick kill, and resistance to many pesticides. The disadvantages are moisture requirements, acceptability, price, dispersal techniques, shelf-life, and it could possibly vector other bacteria that may be noxious to man or plants. DISCUSSION AND CONCLUSIONS We have come a long way in accumulating basic information and technology showing that some insect parasitic nematodes can kill pest insects. We need more work on shelf-life, formulations, and patent protection. The lack of patent protection keeps larger companies out of the production of biologicals because if the product becomes commercially successful, competing companies might enter the market and produce the formulation at reduced prices. References 1. J. J. Petersen, H. C. Chapman, and D. B. Woodward, Mosquiro News, 28(3), 346 ( 1968). 2. W. R. Nickle, J. Nemurol., 4, 113 (1972). 3. W. R. Nickle and C. H. Hogger, Proc. Helminrhol. Soc. Wush.. 41(2), 173 (1974). 4. J. J. Petersen and 0. R. Willis, Mosquiro News, 32, 226 (1972). 5. J. R. Finney, in Proceedings of the First Inrernutionul Colloquiuin on Invertebrute Parhology (Kingston, Ontario,!976), pp. 225-226. 6. J. J. Petersen, Exp. Purusirol., 33, 239 (1973). 7. W. R. Nickle, Proc. Helrninrhol. Soc. Wush., 46(1), 21 (1979). 8. J. Weiser, Vestn. CesL. Spol. Zool., 19, 44 (1955). 9. S. R. Dutky, J. V. Thompson, and G. E. Cantwell, J. Insecr Puthol., 6, 417 (1964).

1414 NICKLE 10. W. R. Nickle, Insect Diseases (Marcel Dekker, New York, 1974), Chap. 7, pp. 327-376. 11. R. A. Bedding, in Proceedings of the First International Colloquium on Invertebrate Pathology (Kingston, Ontario, 1976), pp. 250-254. 12. J. E. Lindegren, D. F. Hoffmann, S. S. Collier, and R. D. Fries, USDA Advances in Agricultural lechnology (USDA, Washington, D.C., 1979), No. AATw-3, p. 5. Accepted for Publication November 2, 1979