Studies on the Insecticidal Action of Nereistoxin, 4-N, N-dimethylamino- 1, 2-dithiolane I. Insecticidal Properties1 By Michihiko SAKAI Laboratory of Insecticide, the Research Laboratories, Takeda Chemical Industries Ltd., Ichijoji, Kyoto Some chemical, pharmacological and tox- of nereistoxin will be worthwhile to de- icological studies on a toxic substance velop new insecticides. in a marine annelid, Lumbriconereis hetero- The present paper publishes the results poda MARENZ, have been conducted, since of toxicological experiments with nereistox- it is well-known that the dead body of in on various species of insects and mites. annelid is toxic to flies, ants etc. This Those expereiments were so planned as substance was first isolated by NITTA (1934), to obtain more detailed informations of and named •gNereistoxin•h. Its chemical the insecticidal properties of nereistoxin. structure was determined as 4-N, N-di- methylamino-1, 2-dithiolane (I) by OKAICHI MATERIALS AND METHODS and HASHIMOTO (1962a), and its synthesis Synthetic nereistoxin hydrogen oxalate was accomplished by HAGIWARA et al. (1962). was used throughout the experiments. This material, prepared by the procedure (I) of HAGIWARAet al. (1962), was supplied by the Synthetic Department, the Research NITTA (1941) concluded from the results Laboratories, Takeda Chem. Ind. Ltd., Osa- of his pharmacological experiments, that ka. nereistoxin is a neuroactive substance Diluted solutions of test compounds were which affects the activities of motor prepared with acetone or water. Since nerves, but not those of sensory nerves nereistoxin hydrogen oxalate was not easi- in mammals. The toxicity to several insect ly soluble in acetone directly, the stock species was investigated by OKAICHI and solution prepared with acetone-water mix- HASHIMOTO (1962b). They found that, in ture (1:1) was diluted with acetone and spite of somewhat lower toxicity compared used as a acetone solution. To make a with DDT, parathion, etc., treated insects water solution of water-insoluble com- were knocked down rapidly, and some of pounds such as parathion, DDT, etc., a ten the knocked down insects revived in a per cent emulsifiable concentrate was pre- certain period. pared dissolving the compounds in the The interesting facts are that nereistoxin mixture of Tween-20 and acetone (1:4) is regarded to be neuroactive, and that it as a stock solution. belongs to a new compound possesing in- Experiments were so conducted as to secticidal action, which is distinguishable compare the effect of nereistoxin with from the insecticides of today. According- those of some commercial insecticides on ly, as OKAICHI and HASHIMOTO(1962b) sug- houseflies (Musca domestica L., Lab-em-7- gested, the chemical and insect toxicologi- em strain), German cockroaches (Blattella cal studies of the analogous compounds germanica L.), rice stem borers (Chilo sup- 1A part of this report was presented at the Annual Meeting of Japanese Society of Applied Entomology and Zoology (April 1964, Tokyo) (Received for publication, August 17, 1964) 324 December 1964 SAKAI: Insecticidal action of nereistoxin 325 pressalis WALKER), azuki bean weevils (Cal- Acetone solutions were topically applied losobruchus chinensis L.), tobacco cut worms on the back of thoraces of flies, using a (Prodenia litura FABRICIUS), common cab- micrometer-driven syringe. For the test by bage worms (Pieris rapae L.), diamond- injection, insecticidal solutions were pre- back moths (Plutella maculipennis CURTIS), pared by dissolving the insecticides into striped flea beetles (Phyllotreta vittata FAB- 0.9 per cent sodium chloride solution. Flies RICIUS), soy bean aphids (Aphis glycines were injected into their thoraces with a MATSUMURA), mulberry mealy bugs (Pseudo- fine stainless steel injection needle attach- coccus comstocki KUWANA) and spider mites ed to a micrometer-driven syringe. The (Tetranychus kanzawai KISHIDA). House- treated flies were kept in a wire-mesh flies, cockroaches, bean weevils, tobacco cage and fed with a mixture of milk and cut worms, cabbage worms and diamond- sugar. Thirty flies were treated for each back moths were reared in a room at of the dosages. the temperature of 25•}1•Ž. Soy bean Tests with German cockroaches by topi- aphids and mites were also laboratory cal application and injection were done in colonies reared in a greenhouse on seed- the same way as performed with houseflies lings of soy bean and kidney bean, re- except the treated cockroaches were fed spectively. Although the other insects with water. Oral administration of insec- were captured out-of-doors, they were kept ticidal solution was done as follows: after under the temperature of 25•}1•Ž for the insects were anaesthetized with carbon several days prior to the conduction of dioxide, they were fixed with adhesive the tests. tape ventral side up on a glass plate, For testing with houseflies, three to five then quantified droplets of insecticidal day's old male adults were used by four solution from a micrometer-driven syringe different testing methods. One was a bait were applied to the mouth parts of the test; i.e. ten flies were introduced into a cockroaches. In this manner, the insects a wire-mesh cage (9cm in diameter and swallowed the solution comparatively rap- 15cm deep) in which a cotton pad soaked idly. The treated cockroaches were trans- in the insecticidal solution was placed. ferred into glass pots and fed with water. The insecticidal solution was prepared by In these tests, ten males were used for a dissolving nereistoxin in 5 per cent sugar single dosage. solution. Three cages were prepared for Rice stem borer larvae employed in the a single concentration. topical application test were in the dor- In the second test, one ml. of acetone mant stage. They were collected from solution of insecticide was pipetted onto heavily infested rice plants in the suburb a bottom of a Petri dish (9cm in diameter). of Kusatsu City, Shiga Prefecture, in After evaporation of the solvent under November, 1963, and used for tests in gentle air stream, the dishes were covered January, 1964. They were kept in a re- with sheets of drawing paper on which ten frigerator up to 7 days before the tests flies (anaesthetized with carbon dioxide) were conducted, then transferred into 25•} were covered with a top side of dishes. 1•Ž. The larvae were treated topically on At the time of complete revival of flies the back of abdomen with a droplet of from anaesthesia, the paper sheets were acetone solution of test substance. Twenty drawn off; thus the flies were immedi- larvae were treated with each of the ately introduced into the dishes. To ob- dosages. tain the information on the revival symp- The possibility of killing rice stem borers toms, knocked down flies were transferred in the stems of rice plant was examined into uncontaminated Petri dishes. by spraying artificially infested rice plants The tests by topical application and in- (Kin-maze, one month after transplanting) jection were conducted with houseflies. with insecticidal solutions. Four days 326 Japanese Journal of Applied Entomology and Zoology Vol 8, No.4 after the egg-masses placed on the leaves used for the others. hatched out, i.e. the larvae bored into Adult soy bean aphids were transferred the stems, the plants were set on a turn onto soy bean seedlings three days before table, and sprayed with 30ml of insecti- the spray of insecticides. The plants cidal solution per pot with a paint sprayer were sprayed with insecticides dissolved from the distance of 80cm. Five days in water containing the wetting agent after spraying, the survival rates were (0.02 per cent). determined cutting open the stems. The In the test with the spider mites, the insecticidal solution for spray was pre- toxicity to adult females and ovicidal ac- pared by dissolving the test substances in tion were investigated. To test the toxicity water containing 0.02 per cent of a wet- to the adults, twenty females were trans- ting agent (Dine, manufactured by Takeda ferred to bean leaves which were placed Chem. Ind.). on wetted filterpaper in open Petri dishes. Male azuki bean weevils were introduced The mites were sprayed with a vertical into a Petri dish which was treated on its sprayer, and the mortalities were deter- bottom surface with a deposit from ace- mined after 2 days. To observe ovicidal tone solution containing insecticide. Thir- action, kidney bean leaves on which eggs ty weevils were introduced per dish. In (1•`2 days old) of the mites were ovipo- another series of test with the weevils, sited were used. The leaves were sprayed the insecticidal solution was prepared by with the test solutions, then the hatching dissolving insecticides in water containing rates of twenty eggs were determined 0.02 per cent of the wetting agent. Imme- seven days after the spray. In these tests, diately afterward the insects were wrap- three Petri dishes were sprayed with a ped in gauze and dipped into the test solu- single insecticidal solution. tion for ten seconds; then they were trans- Mulberry mealy bugs were exposed to ferred onto a sheet of filter-paper to re- the deposits of test substances in Petri move excess solution. The treated insects dishes. The deposits were produced as in were kept in a Petri idsh, and the mor- the same way as performed in the test of talities were determined. houseflies or azuki bean weevils. Ten in- In the tests with tobacco cut worms sects were employed for a single Petri dish. (second instar larvae), cabbage worms All experiments, except the spray test (third instar larvae), diamond-back moths of rice stem borers and the aphids, were (immature larvae), striped flea beetles conducted in a room at the temperature (adults) and daikon leaf beetles (immature of 25•}1•Ž. larvae), they were fed with pieces of leaf RESULTS AND DISCUSSION of their host plants, which were treated with insecticides.