mmcmmmu {NVESTIGATEQNS ON THE GNEQN MAGGQT HYLEMYA ANTEQUA {MERE} Thesis For the Degree a! Ph. D. MIClfiGAN S‘E‘ATE UNIVERSITY Husein M. Eimosa 3960 This is to certify that the thesis entitled TOXICOLOGICAL INVESTIGATIONS ON THE ONION MAGGO'I‘, HYLEMYA ANTIQUA (MEIG.). presented by HUSEIN M. ELMOSA has been accepted towards fulfillment of the requirements for PH 0 D 0 degree in ENEMOLEY [awxa Major professor I i Date AUGUST 19, 1960 0-169 LIBRARY Michigan Stan: University TOKICOLOGlCAL INVESTIGATIONS ON THE CKION AnGGOT fiYLEmYA ANTILUA (QEIG.) BY HJSEIR m. ELmOSA A TnESIS Submitted to the School for advanced Graduate Studies of Michigan State University 3r Agriculture and Applied Science in partial fulfillment of the requirenents for the degree of DOCTOR OF PHILOSOPHY Departhent of Entomology August, 1960 5/572 7 0W“ ACKNODLEDGME‘TS The writer wishes to express his sincere appreciation to the Michigan State University Entomology Department for granting him a two-year research assistantShip. The financial assistance made this research possible. Sincere appreciation is also empressed to }rofessor Ray Hutson, head, Department of Entomology, for his suggestions and criticism during the writing of this thesis and for read- ing the manuscript critically. Special thanks go to Professor Herman hing, under whose supervision and guidance this study was made. It was with his constant help and valuable suggestions that this research was completed and to whom it is herewith dedicated. Thanks are also extended to Dr. Gordon Guyer for his many helpful suggestions and assistance which contributed much toward the completion of this study. Acknowledgments are also extended to Dr. Philip Clark, of the Zoology Department, for his valuable suggestions in the statistical analysis and reading the manuscript critically; to Professor Erwin Benne, of the Agricultural Chemistry Department, and Dr. Roland Fischer for reading the manuscript critically. Appreciation is also eXpressed to Mr. Arthur Wells for his help in collecting some of the pupae used in this work, and to Mr. Robert McClanahan for reading parts of the thesis critically. TOXICOLOGlCAL lNVEbTIGnTIONo ON IHE ONION MAGGOT nYLEMYA ANTlan (MEIG.) BY fiUSElN M. ELMOSA AN ABSTRACT for the degree of DOCTOR OF PdILOSOPhY Department of Entomology August, 1960 ABSTRACT The reSpective toxicities of 15 chlorinated hydro- carbon and phOSphate insecticides to the onion maggot adults hylemya antigua (Meig.), were determined by topical application. The insecticides tested were methyl parathion, dimethoate, Diazinon (0,0-diethy1 O-(B-isopropyl-é-methyl- 6-pyrimidinyl) phOSpnorothioate), parathion, Bayer 21/199 (Co-ral)(Q-(B-chloro-e-metnylumbelliferone) 0,0-diethy1 phoSphorothioate),American Cyanamid 18153 (0,0-diethy1 O-B-pyrazinyl phosphorothioate),trithion, American Cyanamid 18706 (0,0-diethyl—S—(N-ethylcarbamoylmethyl) phosphorodi- thioate), Thimet (0,0-diethy1 S-(ethylthio) methyl phos- phorodithioate), ethion, DDT, Guthion (0,0-dimethyl S-(4~oxo«bh-l, 2, S-benzotriazine—3~methy1) phOSphoro— dithioate), endrin, dieldrin and heptachlor. The results snowed that of the insecticides tested methyl parathion, dimethoate and Diazinon were the most toxic to the onion fly. heptachlor, dieldrin, endrin and DDT were the least toxic. n syringe-microburet which was modified for topical application and used to apply the insecticide solutions to the insect is described. Onion maggot adults from michigan, suspected of being resistant to chlorinated hydrocarbon insecticides, were iv compared with a laboratory strain of onion flies for susceptibility to dieldrin. The data confirmed that the flies obtained from michigan were resistant to dieldrin. EXperiments were conducted to determine the effect of different pre-treatment and post-treatment temperatures, various post-treatment relative numidities, age, sex, stage of development and site of application on the toxicity of dieldrin and ethion to the onion maggot adults. ire—treatment temperature did not affect the toxicity of either dieldrin or ethion to the onion maggot adults. Post-treatment temperatures had a profound effect on the toxicity of both dieldrin and ethion to the fly. In general, the higher the temperature the greater the toxicity. Variations in post-treatment relative humidity did not affect the toxicity of either dieldrin or ethion to the onion maggot adults. There was no significant difference in the effect of age on the toxicity of dieldrin and ethion to the onion maggot adults. Although greater kills were obtained with the older flies than the young ones, there was more natural mortality with the older flies. The lesser kills obtained with the younger flies were attributed to vigor resistance. Female flies were significantly more resistant to the action of dieldrin and ethion than the hales. There was V no significant difference in weight between female and male flies. it was suggested that the difference in resistance between female and male flies may be a case of physiological resistance and worth investigating. The sex ratio of the onion fly was 55 per cent males to 45 per cent females. This was not statistically significant from a 1:1 ratio. The onion maggot adults were less resistant to the action of both dieldrin and ethion than the second instar larvae, third instar larvae and pupae. Because at the doses used dieldrin was not effective against the second instar larvae, third instar larvae and/pupae, no comparison could be made between these stages. The order of resistance in the stages of develOpment of the onion maggot to ethion was pupa) third instar larva)»and adult. although there was no significant difference in the effect of the site of application on the toxicity of dieldrin and ethion to the onion maggot adults, greater kills were obtained when ethion was applied to the mesono- tum of individual insects than when applied to the dorsum of the abdomen. ' 'l‘ABLE Oi“ CONTENTS Page 1 COCOOOOOOOOOOOOIOOO0.00...... Introduction............. I" oocoooooocoooooooococoo '3 Review of Literature........... [Viaterials and K’ietilodSOOOOOOOOOOOOOIOOOOOOOOOO0.0.0.0.019 Method of obtaining onion flies..................19 Rearing metllods O O O O O O O O O O O O O O O O O O O O D O O O O C O I O O O O O O 1? TOpical application apparatus....................3$ Temperature contrOl. O O O O O O I O O O O C O O O I O O O O O O O O O O O O O 2 Humidity control..... Method of treating onion flies with the insecticide solutions.........................85 InseCtiCideSOOOIOOCIOOOOOCOOCOOCOOOOOOO.O0.0.0.0087 .0.OOOOOOOOOOOOOOOOOOOOOOI0.25 Experimental Procedures and Results...................2; Experiment 1.0....0..00...OOOOOOOOOOOOOOOOOOOOOOO29 Part I. The reapective toxicities of 15 insecticides to the onion maggot adults..............................39 Part Il.Toxicity of dieldrin to a laboratory strain of onion.uaggot adults...OOOOIOOOOOOOOOOOOOOOOO.0.0.35 Experiment 11. Effect of different pre-treatment. temperatures on the toxicity of both dieldrin and ethion to the onion maggot adults...;...........34 Experiment 111. Effect of different post-treat- ment temperatures on the toxicity of both dieldrin and ethion to the onion maggot adults.......... r as Experiment IV. Effect of various after-treatment relative humidities on the toxicity of ethion or dieldrin to the onion maggot adults........44 Experiment V. Effect of age of the onion maggot adults on the toxicity of dieldrin or ethionOOOOOOOOOOOOOIOOO.00.0.00.47 Experiment VI. Susceptibility of the sexes of onion maggot adults to ethion or dieldrinoooooo0.000000000000010...53 vii page EXperiment VII. Susceptibility of the stages of deveIOpment of onion fly to ethion or dieldrin...............55 Effect of site of applicatiOn on the toxicity of both dieldrin and ethion to onion maggot adults..................... ...67 Experiment VIII. DiscuSSiOllOOOOOOOO00.000.00.000. Summary and Conclusions......... OCOOOOCOOOOOOOOOIOC lippendiXOOOOOOOOOOOOOOOOOOOOOOOO The mathematical procedure in determining the LDSU for lhimet by the prooit analysis method....51 The individual weights in milligrams of onion flies obtained from nichigan, onion flies from Cornell University, male and female nichigan onion flies, second instar larvae, third instar larvae, dlld IguPaBOOOOOOOOOOOOOOI0.00000000000000035 Literature CiteJOOO0.0.0.00...OOIOOOOOOOOOOOOOOOOOOOUgo, Table l. 2. lo. 11. 12. 1:5. 14. LIST OF TaBLES Toxicities of 15 insecticides as determined by topical application to the onion fly.............ol Number of adult onion maggots killed with dieldrin after conditioning for three days at dif.fererlt telnperaturesoooco00000000000000.000000030 Number of adult onion maggots killed with ethion after conditioning for three days at different 0.00.00.057 temperatureSOOO...OOOOOOOOOOOOOOOOOOOO. Number of adult onion maggots killed with ethion when held at different temperatures after treatmentooOOOOOOOIOOOOOOOOOOOOOOOOOOOIOOOOOO0.0.40 humber of adult onion maggots killed with dieldrin when held at different temperatures after treat- 4) 0......OOOOOOOOOOOOIOOOOO... mentOOOOOOIOOOOOOOCOO Number of adult onion maggots killed with ethion when held at different humidities after treatmentaS adult onion maggots killed with dieldrin Number of when held at different humidities after treatmentéfi Number of adult onion maggots of various age groups Killed “'ith dieldriHOOOOOO0.0.0....000000049 Number of adult onion maggots of various age groups killed ‘Uivith ethiOnOOOOOOOOOOOOO0.0.0.0000.50 Per cent mortality in various age groups of the onion maggot adults treated with dieldrin........51 Per cent mortality in various age groups of the onion maggot adults treated with etnion.......... 52 Toxicities of dieldrin and ethion as determined by tOpical application to the onion fly females.. 55 Toxicities of dieldrin and ethion as determined by tOpical application to the onion fly females.. 55 Number of surviving and dead larvae after treat- ment of the second instar larvae of the onion fly 59 with different doses of dieldrin................. ix Table 15. 17. 18. 19. 20. 21. 6 fl ‘\ BU. Page Number of larvae pupated, larvae survived and larvae dead after treating the third instar larvae of the onion fly with different concentrations of dieldrin.......................oO Number of adult onion maggots and number of undeveloped pupae after treatment with different doses of dieldrin................................61 Number 01 adult onion maggots killed when exposed to different concentrations of dieldrin..........02 Number of pupae, larvae of the onion fly dead, larvae survived after treating the third instar larvae with different concentrations of ethion...65 and Number of adults of the onion maggot that emerged and pupae Which failed to develop after treat- Silent Vv'ith et4rlai-On000000000000000000.00000000000000t>4 Number of adult onion maggots killed when exposed to different concentrations of ethion............0 Number of adult onion maggots killed by applying dieldrin to the thoracic mesonotum or dorsum of abdomen OI. the insect000000.00000000000000000000068 Number of adult onion maggots killed by applying ethion to the thoracic mesonotum or dorsum of abdomen of the insect............................09 The individual weights in milligrams of onion flies obtained from Micnigan, onion flies from Cornell University, male and fenale michi an onion flies, second instar larvae, third instar larvae, and pupae..................... 85 00000000... I‘J’l‘iODUCTIO N The onion maggot, hylemya antique (Meig.) is one of the host important pest of onions in the United States. It mas introduced to this country in the first half of harris (1852) reported heavy the nineteenth century. At damage caused by the insect in Massachusetts in 1841. the present time the onion maggot is found in practically every region in the country where onions are grown. ln Michigan, Merrill and hutson (1955) reported that from a total of 52 Species of flies attaching onions, hzlemza antigua caused most of the damage to michigan grown onions. Several methods have been employed to control the onion maggot since its introduction into this country. Calomel was used with excellent results for several years but was eXpensive and phytotoxic. After 1944 with the development of the chlorinated hydrocarbon insecticides, the onion maggot was satisfactorily controlled with these materials. As early as 1957 there were reports by various in— vestigators from different parts of the country that the onion naggot had develOped resistance to the chlorinated In Michigan excellent control hydrocarbon insecticides. had been ootained with the chlorinated hydrocarbon insecti- cides since 1951, out in 1957 and 1958 the same insecticides failed to control the pest (Guyer a hells, 1959) The failure of these insecticides to control this insect in the field in 1958 initiated the study of various facets of the insect's biology as well as the laboratory evalua- tion of various materials. The insecticides tested were topically applied to the insect by a nodified syringe-microburet and the results are described herein. The effect of temperature on the toxicity of a number of insecticides has been reported by previous workers. A brief summary of these reports on the following hacres shows two general trends in the effect of temperature on toxicity. One is an increase in toxicity at the lower temperatures, and the other an increase in toxicity at the higher temperatures. The effect or tehperature on the hortality of insects nay be of considerable practical importance. This was indicated by hoffman and lindquist (1949) when they wrote "From a practical standpoint it appears that house flies are controlled with low dosages of DDT in northern or cool climates. For example, the states of nashington and Oregon have recommended a wettable-powder Spray containing only 0.5 per cent of DDT for residue spraying in barns. Because only 5 to 6 ml. of fluid can be applied on a vertical wood surface before run-off, the dosages used ranged from 15 to 50 mg. of active ingredient per square foot. According to extension workers these dosages have given good control. In comparison, the southern States recommended a dosage of 200 mg. per square foot for satisfactory control." Because relative humidity is closely related to temperature, a study of the effect of various relative humidities on the toxicity of both dieldrin and ethion was considered to be important. In toxicological investigations it is or importance 0 Research on Q. to be able to obtain consistant result insecticides involves two components, the insect and the insecticide; and in order to obtain repeatable results It is well known that both of them must be standardised. in a given Species of insect, large variation in Busvine (1957) listed the sources susceptibility nay occur. of variability in insects under two headings, intrinsic differences and extrinsic differences. Under intrinsic factors wnich affect the susceptibility of insects to insecticides ne listed sex, age and the stage of development. Under extrinsic factors temperature and relative humidity were listed. also tne brief review of literature summari- zed on the following pages revealed that the relative toxicity of insecticides is affected by such factors as age, sex, site of application and the stage of develOpment of the insect. These factors also could be of practical importance in control, because different stages, ages and sexes of the insect may be present at the same time. This is eSpecially true in case of the stage of development of the animal because with any given Species large vari— ations in susceptibility may occur throughout the life cycle. It would seem important for the purpose of practical control that some knowledge be available on the change of resistance with the stage of development in order to establish the correct timing of application. 'uith these points in view investigations on the effect of temperature, relative humidity, sex, age, site of application and the stage of develOpment on the toxicity of insecticides to the onion maggot were undertaken. The insecticides chosen to be tested here dieldrin and ethion because the insect is resistant to the former but not to the latter. This was done with the hOpe that some ideas might be ootained as to the mechanism of resistance of the onion maggot to the chlorinated hydrocarbon insecticides. I. Relative toxicities of insecticides to onion maggots. ‘ Considerable attention nas been devoted to the control of the onion naggot both in North Anerica and Europe. The control aeasures employed prior to 1958 were classified by Wright (1958) into four groups: 1. The use of deterrents to prevent egg laying. 2. Trapping and killing adults by poison baits. 5. The use of larval poisons. 4. Cultural measures. The first category included, among other materials, powdered naphthalene, tar oil-sand mixture, and paraffin emulsions. The disadvantages in the use of all deterrents were the necessity for their constant renewal and when applied over a large area, the adult flies appeared to become accustomed to their presence and suffer little inconvenience. ‘rne use of poisonous baits was not attended with much success and could not be relied upon. Among larval poisons used were various mercury Salts, corrosive sublimate solution, and calomel. Satisfactory control has been obtained by the use of powdered calomel, which could be applied to the seed coat. Cultural operations were of little value alone in controlling heavy infestations but could be important in combating minor attacks. After 1944, with the interest in hydrocarbon insecti- cides, Sparked by DDT, control of the onion maggot appeared to be imminent. mcLeod (1946) tried a total of twenty- nine different treatments, involving a number of materials and combinations, to control the onion naggot. n calomel treatment gave almost perfect control but was considered impractical because of its high cost and the retarded growth of treated plots. BLC and DDT also gave satisfactory control. munro (1947) in North Dakota obtained 90 per cent control of the maggot by means of Spraying onions with 1 ounce of 25 per cent DDT per gallon of water. Maan (1947) using DDT in the powder form as seed treatment, at the rate of 40 grams per 100 grams of seeds, obtained better control than with calomel. Morrison and drovell (1952) reported that aldrin and chlordane gave economical control of the onion maggot in Oregon. Tozloski (1954) working in Massachusetts snowed that 10 per cent DDT and l per cent dieldrin applied to onion seed as dry materials, and agitated to produce an evenly dispersed mixture of seed and insecticides, gave the most effective control of the onion maggot. Shirck (1957) reported that coating the seed with aldrin, dieldrin, heptachlor, or chlordane enulsions gave tne best control on seed crops. Doane a Chapman (19o?) reported that populations of onion maggots wnich were extremely resistant to several Chlorinated hydrocarbon insecticides were present in some localities of Iisconsin. howitt (195:) stated that prior to 1953 the onion fly was readily controlled with aldrin, heptachlor, chlordane, and dieldrin. After 1955 the chlorinated hydrocarbons became less effective. he also reportel that cooperative experinents conducted in Washing- ton, British Columbia, Uregon and Idaho demonstrated that strains of onion nfiSfiOL highly resistant to the chlori- nated hydrocarbons were present at least in certain areas of the lacific Northwest. In MiChigan Drew and Guyer (1956) stated that the onion flies emerging from pupae collected from the field were resistant to chlorinated hydrocarbon insecticides. The increased failure of the chlorinated hydrocarbon insecticides to control this pest prompted the laboratory evaluations reported in the present paper. II. Effect of pre-treatment temperature on the toxicity of insecticides to insects. Relatively little worn has been done on the effect of pre-treatnent temperature on tne toxicity of insecticides to insects. Cotton (1932) reported that confused flour beetles preconditioned at lower temperatures for several days required a higher concentration of ethylene oxide to give 100 per cent nortality. <,uayle (1934) revealed that in fumigation of scale insects higher Kills were obtained when the insects had been conditioned at a lower tenperature. Lindgren a Dicnson (l9al) morning on the fumigation of the purple scale with nydrocyanic acid found that insects pre- anditioned at 5003. were nore easily milled than those pre-conditioned at 750F.; while the latter were xilled more readily than those pre-conditioned at 90°F. Munson (1955) reported that anerican cockroaches were less resistant to the action of DDT when pre-conditioned at 6400. than when they were pre-conditioned at 1700. nunson 23. 31. (1954) found that the American cockroach, Periplaneta americana (L.); the vinegar fly, Drosophila melanogaster Meig.; and the s w—toothed grain beetle were less susceptible to DDT wnen xept at low temperature before treatment than when Kept at high tenperature. Crauford- Benson (1938) showed that temperature before treatment has a definite effect on the toxicity of derris to the foreign grain beetle, Ahasverus advena Haiti. The beetles were less susceptible to derris when reared at 8000. than when Kept upon at .30 (J. 111. Effect of post-treatment temperature on the toxicity of insecticides to insects. Lindquist gg.‘al. (1944) studied the effect of after- treatment temperature on the toxicity of DDT to house flies. flies were exposed at 63°F. in containers treated with DDT and wnen all were down they were divided and transferred to clean cages and Kept at different temperatures. The results showed 100, 92, and as per cent xiii then flies were nept at 7d? so? and lOOOF. respectively. rouse flies were exposed by Lindquist ep.‘al. (1945) to pyrethrum films and xept in recovery cages at 709 8d? 9daand lOOOF. The authors snowed that nortality was higher at higher tem- peratures. harliger (1949) determined the effect of post- treatment temperature on the toxicity of DDT, calcium arsenate, BhC and parathion to the honey bee. When the insects were fed the poison from a micropipette, then placed in containers at 20°, 28°, and 5600. DDT was much more toxic at lower temperatures, calcium arsenate more toxic at higher temperatures. The post-treatment temperature had no influence on the toxicity of BLC or parathion to the honey bee. weaver (l9a9) in an experiment to determine the relative toxicity of certain organic insectiCLdes to the honey bees found that after-treatment temperature has a profound effect on the toxicity of chlordane to the honey .o , bees. At temperatures below 70 C. chlordane was only lO . ,.oq . . . . slightly toXic but at so J. its toxiCity increased greatly. Pradhan (1949) reported that When adult Tribolium castaneum (hbst.) were exposed to a film of DDT for about 24 hours at the same temperature and then kept away from it at different temperatures there was a higher kill at- the lower temperature. Loffman a Lindquist (1949) reported that DDT and nethoxychlor gave faster knockdown of house flies at 7003- than at 9003. These compounds also caused greater mortality at low temperature than at high temperature. The reverse was true with heptachlor, parathion, chlordane, and toxaphene. Guthrie (1950) established the effect of tenperature on toxicity of certain organic insecticides applied topically to the German cockroach. The temperature vas regulated at 14.50, 220, and 52°C. and the treated roaches were subjected to one of the three temperatures for five days. The results snowed that DDL, pyrethrun, and lindane were more toxic at the lower temperatures while the reverse was true with aldrin and dieldrin. Vinson 32-.2i- (1952) tested the effect of poet-treatment temperature on the toxicity of toPicaily applied and injected DDT to the American cock- Both topical and injected doses of DDT roach females. ‘ “' ‘ r T‘ ' ues Save less kill at the higher temperature. ine LDSO val it 150" and 75 to 100 micrograms v. 9 to 5 micrograms were 5 to 10 micrograms o ‘ ° n at 550C. for topically applied on; and o ll at 150 C.and 20 to 25 micrograms at 550 C.for injected DDT. Cressman e3. al.(l9oo)working in California on some factors influencing the effectiveness of parathion against California red scale, found that after treatment tempera— ture had no effect on the toxicity of parathion to the scale. doth gt. 31. (1955) conducted an experiment to determine the amounts of DDT actually absorbed by the house flies at different temperatures. he found that the flies absorbed 64 per cent more of DDT with a lower mor- tality when kept at 900 F. than when neld at 700 E. IV. The effect of post-treatment relative humidity on the toxicity of insecticides to insects. iradhan (1949) studied the effect of relative humidity on the toxicity of insecticides to insects. he reviewed the literature up to 1949 and concluded "a perusal of literature does not help to deduce any general principal regarding the effect of humidity on the toxicity of insecticides. in some cases the rise in relative nunidity has been found to increase the percentage kill and in others to decrease it. There are also cases in Which changes in relative humidity have been found to have no appreciable effect on toxicity." Gains and Dean (1949) investigated the effect of temperature and humidity on the toxicity of certain 18 insecticides to the boll weevil. They reported that high nunidity reduced the toxicity of calcium arsenate, toxapnene, and Chlordane. Collins and hing (l953) studied the effect of hunidity on the toxicity of residual films of DDT to the adult of Tribolium confusum Duv. Their results indicated that with the temperature hept constant at 60° fiLtnere was no significant difference in mortality at 55 per cent and $9 per cent relative humidity. Glyhne— Jones and Edwards (1952) tested the effect of after- treatnent hunidity on toxicity of suspensions of acid DhCC and a solution of the sodiun salt of QNOC. Tharfound that no difference occurred vith the acid DNOC when humidity was changed from 45 - 50 per cent relative humidity to (I) 5 - 90 per cent, but the sodium salt was much more toxic at the higher humidity. V. Effect of age on the susceptibility of insects to insecticides. Simanton a miller (1957) investigated house fly age as a factor in susceptibility to pyrethrun Sprays. They tested 1.5 hour, a hour, 21 hour, 45 hour, a day, a day, 7 day and 10 day age groups. The results showed that very young house flies were more easily paralyzed but less easily Killei than were older flies. Craufurd-Benson (1938) reported on the resistance of adult Ahasverus advena taltl. at different ages to derris. he showed that the insects were less resistant to the insecticides at both very young and increasing ages. theod (1944) experimented on the effect of age of the fruit flies on resistance to nicotine sulphate solution. He found that 5—day old flies were gore susceptible than 6-day old; and the latter were more susceptible than l«day old flies. David & Bracey (lean) investigated the change of resistance of nedes aegypti (Linn.) with age to pyretnruh and DDT. The data indicated that there was a progressive decrease in resistance with age. One-day old insects were more resistant than 5 to 4-day old insects; and the latter vere less susceptible than 4 to 5-day old insects. Collins and King (1953) tested the resistance of 7—day old and 54 to 58- day old fribolium confusum Duv. to residual films of DDT. Their results showed that there was no significant difference in resistance between the two ages with an exposure of 5 hours, but there was highly significant difference with the 6-hour exposure. The mortality has higher with the older beetles. herr (1954) studied the variation with age of adult D, melanogaster meig. in the susceptibility to DDT. he snoted that susceptibility was high in young flies, but rapidly decreased tith age, to a minimum at about 5 days, thereafter increasing rapidly in sales and not significantly in females. he concluded that ”the need for 14 sexing and standardizing age in flies used for toxicological investigations was thus demonstrated.” VI. Effect of sex on the susceptibility of insects to insecticides. Simanton a niller (1937) in a study of house fly age as a factor in susceptibility to pyrethrum sprays showed that the fenale house fly has more resistant than the hale. a dose of pyrethrun wnich gave 100 per cent male hill resulted in ob.6 per cent female mortality. hurray (1937) reported that the female house fly was more resistant to the action of pyrethrins than the male. Gough (1940) working on the toxicity of sulfur dioxide to the bed—bug reported that the nales and the females were equal in susceptibility. David a Dracey (1946) investi- gated the relative resistance of dale and female Aedes aegypti (Linn.) to insecticides. They found that the females were more resistant than the males to pyrethrins, and to a mixture of pyrethrins and DDT. Barber a Schmitt (1948) working on house fly resistance to DDT residue sprays showed that the females were here resistant than the males. henusan (1948) experimented on the toxicity of insecticides administered in various ways to several species of insects. he reported that the difference in susceptibility to insecticides between male and female cockroaches varied 15 greatly depending on the insecticide and the method of testing. There was less sex difference in resistance to an insecticide when the material was injected into the blood stream than when the same material was fed or tested for contact toxicity. All materials tested showed the male to be less resistant than the fenale, with the exception of dichloro ethyl ether where the reverse was true. hacCuaige (1956) deternined the resistance of the desert locust and the African migratory locust to DNC in relation to height, age, and sex. he found no difference in susceptibility of the sexes to DEC. V11. Effect of stage of development on resistance of insects to insecticides. Campbell (1926) compared the susceptibility of the last four instars of silxworm larvae to arsenic. his results snowed that second instar were 3.5 times as susceptible to arsenic as the fifth instar larvae. He concluded that the susceptibility of the silkworm to arsenic decreased during its larVal development. Cotton (1952) working on the relation of the reSpiratory metabolism of insects to their susceptibility to fumigants showed that When test lots of 50 adults, 25 larvae and 25 pupae of T. confusun Duv. were placed in d-liter glass flasxs and fumigated for 3 hours with carbon disulphide, the adults Were Killed with a dose of 125 mg. per liter, larvae lo5 mg. per liter and pupae ole mg. per liter. Lindgren (1955) investigated the order of resistance of different stages of Tribolium conquum Duv. to various funigants and reported that with carbon disulphide the resistance of the egg) pupa) adult) larva (low humidity), the pupa) egg) adult)1arva (high humidity); with chloropicrin, the resistance of the egg) pupa) adult)larva; with ethylene oxide, the resistance of the pupa)'adult) larVa)iegg. Gunderson and Strand (1959) studied the toxicity of hydrogen cyanide, chloropicrin and ethylene oxide to eggs, nymphs, and adults of the bed-bug, Cimex lectularius L. They showed that the eggs of the bed-bug were less resistant to hydrogen cyanide and ethylene oxide than the nymphs and adults. The eggs were nuch more resistant to chloropicrin. DuChanois (1947) studied the toxicity of gamia DhC to the pre-imaginal stages of the house fly. The stages used were the eggs, second instar larva, third instar larva, and pupa. he reported that gamma EEC was not toxic to ova. Second instar larvae were the most susceptible and the pupal stage was considerably more resistant than the larval stages. Cochran (1955) compared last instar male and fenale nymphs with adult females as to their susceptibility to DDT. his findings indicated that there was no difference between adult fenale, male nymphs, or 17 female nymphs of the American cockroacn with respect to DDT susceptibility. Benschoter (1960) investigated the susceptibility of life stages of the Mexican fruit fly to fuhigation with ethylene chlorobromide. He found that the order of susceptibility was larva) adult) egg)pupa. VIII. Effect of loci of application on susceptibility of insects to insecticides. fl O'hane ep..al. (1935) studied the effect of nicotine, pyretnrun, strycninne sulphate, cocoanut oil, fatty acid, white petrolium oil and nerosene applied to different spots of the American cockroach, Oriental cockroach, German cocxroacn and the larvae of the mealworm. They found that the reaction rate to a given dose depended on the location of application. Jilson (1949) investigated the effect of pyrethrum and two of its synergists, piperonyl butoxide and piperonyl cyclonene on house flies and blow flies when applied to different parts of the body. he found that when placed on the top of the tnorax, 0.1 microgran of py- retnrins Knocxed down only 10 per cent of house flies and $5 per cent of blow flies. The sane dose Knocked down 100 per cent of the flies when applied to the mouth parts. Ball and secs (1951) worning on the role of the circulatory and nervous systens in the toxic action of parathion to the American cockroach reported that the site of application was important. ihej found that the closer the site of application to the central nervous system the shorter the xnochdovn tide. Fisher (1952) reported on the importance of the locus of application on the effectiveness of DDT for the house fly. dis results showed that lower mortal- ities were produced as the locus of application moved distally along the legs, and posteriorly from head to abdomen. hashins 22. a1. (1952) reported on sohe factors influencing the contact of chenical and test insect. he found that application of lindane to the tarsi of the house fly was more effective than to the ventral surface of the -tnorax. MacCuaig (1956) studied the resistance of the desert locust and the african nigratory locust to ONC. his findings snoted that the site of application had relatively little effect on the mills obtained, except when the dose was applied to the fenur or wings. 'fiJ" ' nnfixlni; ALB M;-nODE Method of Obtaining flies The flies used in these experiments, with the exception of experiment 1, part 11, were obtained from pupae which had seen collected from infested Michigan onion fields. The pupae were stored at Q30 r.nhen flies were required for testing, pupae were transferred to a tenperature cabinet at 77° Fland adults allowed to emerge. after emergence the flies were held in a small insect cage until they were tanen to the laboratory to be tested. The nethod of obtaining the flies used in experinent 1, part II, will he described under rearing netnods. The food for the flies consisted of honey and br were yeast prepared on a moist slice of bread. The petri dish containing the food was placed in the cage. also Shall droplets of a mixture of honey and brewers yeast were placed on tap of the cage. hater was supplied by cotton wichs inserted through a corK in a small Erlenmeyer flash. Rearing Methods The few references in the literature conCerning suCcessful rearing techniques for the onion fly were summarized oy‘aorhnan (1958). 19 20 In the present research, attempts were nade to rear insecticide-susceptible onion flies obtained from Cornell University, and chlorinated hydrocarbon-resistant onion flies from Michigan, in a basement room at hichigan State University. an air conditioner Kept the temperature in the rearing room between 650 f.and 750.3 at all times. Cages 20 inches long, 10 inches wide, and 10 inches high were used for rearing the onion fly. Che cages were covered with 1d nesh screen with cloth sleeve openings at the front. Light for the rearing cages was supplied by four, forty watt, white fluorescent lamps. The four foot lamps were suspended ld inches above the rearing cages. fhe lights were controlled by a time switch and were in Operation from 8 a.m. until aidhignt, giving in hours light eacn day. at first the food for the adult flies consisted of honey and brewers yeast prepared on a moist slice of bread in a petri disn and placed in the rearing ca; 3. The flies from Cornell University produced eggs satisfactorily with this diet, but the flies from michigan produced very few eggs. The diet was tnen changed to mils (with formaldehyde, two parts per thousand, to retard Spoilage) and honey. To 25 parts of honey was mixed one part of a liquid vitamin concentrate containing vitamins A, D, El, 32, B5, C, E, 21 niacinamide, pantothenic acid, choline chloride, and inositol. milk was supplied to the onion flies in small jars inverted on the screen on the top of the cage with cheese cloth placed over the Jar opening to prevent spillage. fhe honey was placed on top of the cage in small droplets. Satisfactory egg production resulted from this diet with the insecticide-susceptible flies from Cornell University. Chlorinated hydrocarbon-resistant onion flies from Michigan still failed to produce many eggs. This diet was the same one used by Workman (1953) in rearing the onion fly successfully in the greenhouse in Oregon. Small onion bulbs were planted in lo X 10 inch enamel pans containing much soil and placed in the rearing cages. Females oviposited on the onion plants or on the muck. Also halves of large onion bulbs were placed out side dOMn on the soil for the emerging larvae to feed on. it is important to note that the onion flies obtained from nicninan and reared simultaneously by the same methods as the flies from Cornell University, failed to produce eggs satisfactorily. There is no complete explanation for this; however, nosnins and Gordon (1956) stated that the fertility of resistant insects was often so low that Protagation was impossible. *his may have been the case with the chlorinated hydrocarbon—resistant onion flies. flr—e Topical Application Apparatus In topical application of insecticides to individual insects accuracy and ease of operation are most important. Topical application is usually accomplisned by treating individual insects with small volumes of insecticide solutions. Trevan (1932) suggested that small droplets of fluid could be produced by using the pitcned thread of a maCninist's micrometer to drive the plunger of a shall syringe. after Trevan developed the micrometer-syringe, topical application was widely employed in insect toxi- cology, and most of the topical application apparatus in use today emoodies Trevan's principle (Metcalf, 1958). The apparatus used for topical application in this research was a syringe-microburet, nude by the Micro-Metric Instrument Company of Cleveland, Ohio. The syringe-nicroburet (Fig. l) employs a thumbscrew—operated shaft which advances tne stem of the direct—dial micrometer gauge. Unis sten, which is carried through the gauge, advances the plunger of the (Syringe and delivers the insecticide solution through the Syringe. The syringe used in the torn reported 1ere delivers a volume of 0.2 microliter of solution for each unit of displacenent on the dial. Syringes of other sizes eSpecially calibrated for use with this instrument are available froa the manufacturer. 23 SVRIIGE MICROIUREY MODEL N0 532 ALudkaAuumuid CLEVELAND - OHIO Figure 1. The modified syringe-microburet Because this apparatus is intended primarily for microtitration, the manufacturer supplies glass buret tips wnich can be attached to the syringes. hith these glass buret tips attached it was impossible to deliver the small volumes of insecticide solutions necessary for topical application. In order to use this syringe-microburet for topical application, it was necessary to replace the burst tips by Bo-gauge hypodermic needles polisned at the tips with a fine abrasive stone and bent at 90° angle. The adVantage of the modified syringe-microburet is threefold: 1. Most important, volume deliveries are made quickly and accurately, and they are read on a direct-reading dial gauge. 0 The syringes fit interchangeably into a V-block at the delivery end of the burst stand. 5. Storage facilities for the filled syringes are provided on the burst stand. Temperature Control Eour electric incubators rith thermostatic controls were employed. In order to produce temperatures below room temperature it was necessary to place two of the electric incubators in a cold room. One o1 these was 0 a regulated at 600 F.and the other at 75 a. One of the two remaining cabinets was regulated at 770 E.and the . i0, other at 90 .& humidity Control Constant humidities were obtained byfisupersaturated3E salt solutions. Chemically pure salts appropriate for each relative humidity at different temperatures mere used (hodgman, 19a9). They were placed at the bottom of desiccators and enough distilled water was added to cover the surface of the salt. Method of Treating Onion Flies with the Insecticide Solutions In the following eXperiments, individual insects were treated with the respective insecticide solutions after they had been anesthetized with carbon dioxide. The carbon dioxide was obtained as a liquified gas in cylinders under a pressure of about 800 pounds per square inch. The gas was allowed to escape through a pressure reducing valve at a slow rate through a 4 inch Eachner funnel in which the test insects were confined. The modified syringe-microburet was used to apply the insecti- - o . o _ ‘71" 9 cide solutions to the indiVidual insects (rig. w). After treatment the flies were held in pint-size xShould read saturated, with an excess of salt present, here and wherever mentioned in this context later. 26 .1,“ . ., Figure 2. The method by which the toxicant was topically applied to the insects. 27 ice cream cartons which were covered Wit“ cheese cloth to prevent the escape of treated flies. The food for the- treated insects consisted of honey and brewers yeast placed on top of the cheese cloth. Hater was supplied in small droplets added to the ice cream cartons. Insecticides The insecticides used, their purity, and the sources from wnich they were ootained are listed below. The names used here are those approved as of April, 1959 by the Conaittee on Insecticide TerminolOgy of the Entomological Society of anerica. . Per Cent 8 urce InsectiCide Purity 0 Methyl parathion 80 Nutritional Biochemical Corp., Cleveland, Ohio Dimethoate Technical American Cyanamid Co. Stamford, Connecticut Diazinon (0,0—diethyl 0- Technical Geigy Chemical Corp. (2-isoprpyl-4-methy1-6- Mcintosh, alabama pyrinidinyl) phosphoro- tnioate Nutritional Biochemical , - Technical . Parathion Corp., Cleveland, Ohio n - l . q_ n ' ' ” --v Corp. oajer 31/199 (Co—ral) (Ola lechnlcal Hhefldér9 , , chloro-4-methylumbellifer- heW 30r‘:,Ne“ York one) 0,0-diethyl phos- phorothioate) . . Per Cent P lnsectic1de Purity oource American Cyanamid 13155 Technical American Cyananid Co. (0,0-diethyl O-z—pyrazinyl Stanford, Connecticut phosphorothioate) Trithion (S—(cnlorophenylthio)Technical Stauffer dhenical Corp. methyl 0,0—diethyl phos- Richmond, California phorothioate) American Cyananid 18706 (0,0-Tecnnical American Cyananid Co. dinethyl-S-(N—ethyl- Stamford, Connecticut carbamoylmethyl) pnosphorotnioate Tnimet (0,0-diethyl S-(ethylio) American Cyanamid Co. methyl phoSpnorothioate) 'Pechnical Stamford, Connecticut Etnion 'Sechnical Niagara Uhenical Uiv. Food machinery and Chemical Corp., uiiddleport , I‘leVJ YOI‘K DDT Technical Nutritional Viocheuical Corp., Cleveland, Ohio Gutnion (0,0-dimetnyl S— 16.7 Chemagro Corp. (4-oxo-l,2,5ebenzotriazine- New Yorh, N w York o-netnyl) phosphoro- dithioate) Endrin Technical Velsicol Chemical Corp. Chicago, Illinois Dieldrin 85 Nutritional Riochenical Corp., Cleveland, Ohio neptachlor 72 Nutritional Ciochenical Corp;, Cleveland, Ohio armagnmlt i-RUCEDUHEZS “no assume Experiment I Part I This eXperinent was conducted in 1958 to determine the respective toxicities of lo chlorinated hydrocarbon and organic phOSphate insecticides to the onion maggot adults. nll insecticides tested were or technical grade with the exception of methyl parathion, heptachlor, dieldrin, and Guthion (0,0-dinethyl S-(e-oxo-oh-l,2,3- benzotriazine-e—nethyl) phoSphorothioate). These were so per cent, 2 per cent, 85 per cent, and 16.7 per cent pure reSpectively. The L350 in terms of micrograms of insecticides per gran of fly was determined by the probit analysis nethod (Finney 1952). nfl example of the mathematical procedure in determining the nDSO for Thimet by the probit analysis method is shown in the appendix. Flies 2 to 5-days old, obtained as previously described were used for testing. Each toxicant was dissolved in acetone and applied in ninute droplets to the nesonota of individual insects by the syringe-microburet. Cne micro— liter of solution was applied to each fly as described 29 previously. at least four dose levels of each insecticide were used to deternine the LDSO. Four to five replicates of 10 flies each tere used at each dose level. also a control treataent of 10 flies was conducted with each replicate. These controls received acetone only. nortali- ty counts were tanen 24 hours after treatment. The criterion for mortality was the inability of the flies to show active locomotion. The L350 in micrograms of toxicant per gram of fly, 95 per cent confidence limits, and the slopes of the log. dose-probit lines for the 15 insecticides tested are presented in Table l. The hOSt effective insecticides against the onion naggot were netnyl parathion, dimethoate, and Diazinon (0,0-diethy1 U-(Z-iSOpropy1-4 methyl-6- pyrimidinyl) phOSphorotnioate). The least effective were the four chlorinated hydrocarbon insecticides tested. It is to US noted that the sanple of Guthion used was only 16.7 per cent pure and was therefore not comparable to the samples of other naterials. 3y calculation, it day be estinated that a sample containing 85.5 ,er cent Guthion (5 times as pure as used in the laboratory) would nave an MDSO of 4.39 nicrograns per gran of fly, and would be comparable to Trithion \S-(p-chlorophenylthio) methyl 0,0-diethy1 phoSphorodithioate) in toxicity. Table 1 -— Toxicities of 15 insecticides as deternined by topical ayplication to the onion fly. 1D50 in micro- Slope of Insecticide gran per gram 95p confidence Log. dose fly limits probit line Methyl parathion 1.50 0.95—1.50 8.45 Dinethoate 1.52 0.71-2.44 2.79 Diazinon 1.55 0-67—2.71 4.94 iarathion 2.09 1.50-2.90 5.75 Bayer 21/199 (Coral) 2.24 1.74-2.85 5.82 ha. Cyananid 13155 2.24 1.24-4.05 4.76 Trithion 5.72 2.75-6.90 6.05 An. Cyananid 15706 6.92 5.70-2.40 5.95 Thimet 7.06 4.58—11.40 5.50 Ethion 7.41 6.05-9.08 6.01 DDT 11.17 7.16-17.5‘ 4.55 Guthion 21.95 15.1a-29.95 6.41 Endrin 57.95 24.45-55.88 2.98 Dieldrin 542.00 528—205 2.23 heptachlor 2,979.00 2,518-5,524 6.42 In a review of arthropod resistance to chemicals hosKins and Gordon (1956) stated that the L050 is an index of the mean tolerance or the nean resistance of the insects tested. The L050, if obtained with wild unexposed 32 insects gives the tolerance of the species, or if obtained with individuals from an area treated with an insecticide it gives a measure of the resistance that has been develOped. Determination of the 1050 from time to tine will show how the average resistance varies and how it is affected by any chosen procedure, such as continued use of insecticide, substitution of another, or use of none at all. Therefore, the L050 is a measure of what already happened and gives little indication of that nay be eXpected. For the latter purpose the slope of the log.dose-probit line is nuch more informative. The slope of the log.dose-probit line is a measure of the diversity of reaponse or the heterogeneity of the insects toward the toxicant used. In the normal susceptible condition of insects, the slope is great. Jhen resistance begins to develop the slope decreases. This decrease in lepe will continue until resistance tends to reach a plateau, and subseiuently the slope increases. This phenomenon seems to be true with the slopes recorded in table 1 for the insecticides tested. Table l snows large L050 and small slopes for endrin and dieldrin. This indicates that the flies are resistant to the two insecticides, and resistance did not reach its limit. It is eXpected that the onion fly will develOp still greater '4 '4 U u resistance to endrin and dieldrin. 0n the other hand, both tne L050 anu the slope for heptachlor are large. This indicates that the onion fly resistance to heptachlor has reached the limit and no further resistance is expected to develop. Table 1 also shows that both the 1050 and the slope for dimethoate are small. One hay speculate that there is a great heterogeneity in the onion fly with respect to resistance to this insecticide and resistance is apt to develop. It would be interesting to deternine the L050 for the insecticides tested from time to time to show how resistance varies. Besides the L050 the slope of the log. dose-probit line should be reported, because it is more informative. Part 11 The purpose of this experiment was to determine the L050 of dieldrin to an insecticide susceptible strain of onion maggot adult. This L050 would be compared to the L050 for dieldrin obtained in part I of this experiment with samples of resistant onion flies from.Michigan. This experiment was conducted in may, 1960. The onion flies tested were reared from a culture of flies obtained from Cornell University. he far as is Known 14 L. this culture of flies had had no previous contact with insecticides. The flies since 1958 have been reared in the laboratory at michigan State University by the previously described method. The L050 of dieldrin was determined by the method described in Part I of this experiment. The L050 and its 95 per cent confidence limits were 1.58 and 0.80-2.59 micrograms per gram of fly. The slope of the log.dose~probit line is 2.009. Table 1 shows that the L050 with the onion flies obtained from Michigan is 542 micrograas per gram of fly. From this it can be concluded that the samples of onion flies from Michigan are highly resistant to dieldrin. It is important to nOte that the difference in weight between the resistant onion flies from Michigan and the susceptible flies obtained from Cornell University was not significant. The individual weights of 20 flies from each of the two localities are recorded in the appendix. Experiment 11 The following experiment was conducted in 1959 to determine the effect of different pre-treatnent temperatures on the toxicity of both ethion and dieldrin to chlorinated hydrocarbon resistant onion maggot adults. m ()1 Three temperature incubators were employed. One incubator was Kept at 500 Eh,another incuaator at 75 3., and the third cabinet was regulated at 900 F. The relatiVe humidity in the incubators was maintained at 55 per cent. One thousand micrOgrams of dieldrin or 8 dicrograms of ethion per gran of fly were applied to 2 to 5-day old flies obtained by the nethod describei previously. Four replicates of 5 males and 5 females of flies were used at each dose level and temperature. also a control treatment was conducted in conjunction with each replicate. These control flies received acetone only. The flies were conditioned at the Specified temperatures for three days. After conditioning the flies were removed from the tempera- ture cabinets and treated with the toxicant at room temperature. The toxicant was dissolved in acetone and applied to tne aesonota of individual insects in one micro- liter anounts by the method described previously. After treatment the flies were held in pint-size ice cream cartons and transferred to a constant temperature cabinet at '770 3, Food and water were supplied to the treated insects. mortality counts were dade 24 hours after treatnent. The results obtainel are shown in tables 2 and 5. analysis of variance of the data indicated that there was no significant difference in the effect of different pre- treatment tehperatures on the toxicity of dieldrin or ethion to the onion maggot adults. m 03 Table 2 -- Number of adult onion naggots Killed with dieldrin after conditioning for three days at different temperatures. Each lot con- sisted of 10 flies. Ere-treatment temperature Insecticide Rep. 500 F. 750 F. 900 F. Dieldrin a 7 6 6 b 6 6 4 c 5 5 6 d 5 5 5 Mean 5.00 5.50 5.25 Control a l l O b l 0 0 c 0 l 0 d 0 0 O T..1ean —5 _.—5 6' analysis of Variance Source of variation 0.3. 5.8. Jean 8. E. F.95 Temperature 2 1.6 .8 0.19 5.55 Dose 1 105.4 105.4 Interaction 2 .5 .15 18 7.7 .45 Within cells Conclusion: The difference between 60? 75°and 90°F. pre- treatment temperatures on the toxicity of dieldrin to the onion maggot adults is not significant. 57 Table 5 -- humber of adult onion maggots Killed with ethion after conditioning for three days at different temperatures. Each lot consisted of 10 flies. Ere-treatment temperature Insecticide 33p, 60° 75°F. 930 F. Ethion a 5 9 7 b 5 5 5 c 7 7 5 d 8 b 5 mean 7.00 0.75 5.50 Control a O O l b 0 0 O c 0 0 0 d 0 O 0 Mean 0 '0 .25 Analysis of Variance Source of variation 0.F. 5.8. Mean 8. E. F.95 Temperature 2 1.8 0.9 0.87 19.45 Dose 1 240.6 240.6 Interaction 2 5.6 1.8 within cells 18 18.5 1.05 . . . . , . o o . ~ as i _ ConcluSion: The diflerence between 60 , 75 , and 90 .. pre treatment temperatures on the toxicity of ethion to the onion maggot adults is not significant. 8 Experiment III This experiment was conducted in 1959 to determine the effect of different post-treatnent temperatures on the toxicity of both dieldrin and ethion to the chlorinated hydrocarbon resistant onion fly. A dose of 1,000 micrograms of dieldrin or 8 micrograms of ethion per gram of flies was used. Three electric incubators were used in this experiment. One cabinet was regulated at 0033., another at 750F. and the third incubator was nept at 90°F. Two to 5-day old flies were obtained as described previously. Four replicates of 5 females and 5 hales of flies were used at each dose level and temperature. a control treatment was conducted along with eacn replicate. The toxicants were dissolved in acetone and topically applied to the aesonota of individual insects. The flies were treated at room temperature and held in pint—size ice cream cartons. Immediately after treatment the flies were mortality counts transferred to the specified temperature. were made 2a hours after treatment. The number of adult onion maggots killed With dieldrin ‘ ’ ‘ " ~ - o - “ta ‘ n eratures and ethion at the diffeient post treatment te p were recorded in tables a and 5. analySis oi variance of the data in tables a and 5 showed that there were highly signifiCant differences in the effects of post-treatment tenperature on the toxicity of ethion and dieldrin to the onion fly. 40 Table 4 -— Number of adult onion maggots Killed mith ethion when held at dlfierent temperatures after treatment. Eacn lot consisted of 10 flies. Eost-treatnent temperature Insecticide Rep. (Von. 75°F. 90°F. Ethion d O 5 10 b l 5 b c 0 2 10 d 0 5 9 mean .25 5.75 T75- Control a O 0 1 b 1 0 0 c 0 0 0 d l 0 O .uean '_TE 0 .25 analysis of variance source of variation 0.E. 0.8. mean 5. f. 3.99 Temperature 2 69.8 ~a.9 51.7 X 6.01 Dose 1 96.0 95.0 Interaction 2 76.7 58.5 Uithin cells 15 20.0 1.1 ‘ ' ' ‘ \ - ‘ . ; 1t teAperature on Conclusion: The effect of post treatmex ' ' ’ ' " "‘ “ rion fl\ is the toxiCity oi ethion to the 01 y highly significant. 41 Further statistiCal analysis revealed that the three means differ significantly from one another. The deans were compared by the modified Tukey nethod (Snedecor 1956, p. 251). Table 5 -- Lumber of adult onion maggots Killed with dieldrin when neld at different temperatures after treatment. Each lot consisted of 10 flies. Post-treatment temperature Insecticide Rep. 600 E. 75oF. 900p, Dieldrin a g l 10 b 1 5 9 c 0 2 8 d .1. 3 a... nean l 2.35 8.75 Control a 0 O 0 b 0 0 O c 0 0 1 d a a .2... Mean 0 O .75 analysis of variance Source of variation D.E. 5.8. mean 8. E. 3.9, Temperature 2 84.2 41.1 74.7XX 6.01 Dose 1 84.4 84.4 Interaction 2 55.8 27.9 Within cells 18 10.2 .57 Conclusion: The effect of post-treatment temperature on . . . . .‘ _. ‘. , ,1, is the toXiCity oi dieldrin to the orion i y highly significant. ,p. 5 Further statistical analysis revealed that the taree means differ significantly from one another. The means were compared by the modifiel Tuney nethod (Snedecor 1956, p. 251). a4 Salter idem: IV The purpose of this experihent was to determine the effect of various after-treatment relative humidities on the toxicity of ethion or dieldrin to the resistant onion maggot adults. This experihent was performed in 1959. Three desiccators containing supersaturated salt solutions were placed in a tenperature cabinet at 7¢>F. The relative humidities in theSe desiccators tere 55, 55, and 75 per cent. The doses of ethion and dieldrin applied were 8 or 100 micrograms ger gram of fly respectively. Eour replicates of 5 male and 5 female flies were used at each dose level and hunidity. A control treatment was conducted along with each replicate. The modifiel syringe- nicroburet previously described was used to apply the insec- ticide solutions to the nesonota of individual insects. The flies were treated at room temperature and neld in pint- size ice cream cartons. After treatment the flies were Mortality counts transferred to the specified humidity. were nade 24 hours after treatment. The number of adult onion maggots killed when held at different hunidities were recorded in tables 6 and 7. Analysis of Variance of the data in the forementioned tables showed that humidity after treatnent had no significant effect on the toxicity of dieldrin or ethion to the onion maggot adults. Table 6 -- Nunber of adult onion uaggots Killed with ethion when held at different humidities after treat— ment. Eacn lot consisted of 10 flies. lost-treatment relative humidity Insecticide Rep. 55} 55$ 75$ Ethion a 6 7 9 b 3 5 b c 5 5 4 d 5 5 8 Mean 4.25 5.75 6.75 Control a O O 1 D U 0 c 0 O 0 d O O 0 ..ie an 5 6' . :5 analysis of Variance Source of variation D.E. 8.5. Mean 5. F. F.95 hunidity 2 7.59 5.50 2. 5 3.55 Dose 1 151.51 181.51 Interaction 8 5.2 2.03 within cells 15 25 1.59 Conclusion: The effect of tost-treatnent humidity on the toxicity of ethion to the onion naggot is not significant. ("'1 46 Table 7 -— Lumber of adult onion naggots Killed with dieldrin when held at different humidities after treatnent. Each lot consisted of 10 flies. Lost—treatnent relative humidity Insecticide Rep. 55$ 55$ 75} Dieldrin a 6 6 6 b 6 5 8 c 8 8 9 d o 5 7 Mean 6.50 6.25 7.50 Control a O O O b 0 0 O c 1 0 0 d 2 0 0 mean .75 O O analysis of variance Source of Variation D.E. 8.5. Mean 3. F. F.95 humidity 2 0.58 0.29 0.17 19.43 Dose 1 240.66 2&0.65 Interaction 2 4.09 9.05 within cells 15 50.50 1.60 Conclusion; The effect of post—treatment hunidity on the toxicity of dieldrin to the onion naggot is not significant. 47 Experiment V To snow the extent to whicn the age of the onion fly affects nortality, an experiaent was conducted in 1959 in which given doses of ethion or dieldrin were tested against flies fron the sane catch of pupae at Specified intervals after emergence. The age groups tested were 2 — 5 day, 9 — 12 day, and 16 - 19 day old flies. Flies were obtained as described previously. The insecticides were dissolved in acetone and applied to the nesonota of individual insects by the topical application apparatus. flour replicates of 10 males and 10 fenales each were tested at eaCn dose and age group. also, a control treatment with acetone was performed. A dose of 1,000 nicrograns of dieldrin or 5 nicrograhs of ethion was applied per gran of fly. after treatment the flies were held in pint—size ice cream cartons and placed in a temperature caoinet at 770E. hortality counts mere made 34 hours after treatnent. The number of flies killed for each age group are recorded in tables 8 and 9. Also, the data are recorded in tables 10 and 11 after correction for natural mortality by Abbott's formula (Kinney 1952, (‘1 p.‘6). analysis of Variance of data in the preceding taoles before and after correction for natural aortality showed no 48 significant difference in susceptibility of the Various age groups to ethion or dieldrin. Revertneless, ins;ection of tables 8 and 9 revealel that hore kills were obtained with the older flies. Also, there was nore natural mortality with the older insects. The nigh natural mortality in the older group was expected because the onion nawaot adult hale flies reach their pean in natural nortality at 25 days of age ( orknan, 1956). also, the writer observed an increa:e in natural male aortality in the rearing cages of 16 - 19 day old group. Table 5 -~ hunber of adult onion naggots of various age groups hilled with dieldrin. Each lot consisted of 20 flies. . . . age Group micrograms of Dieldrin per gram of fly 3-5 day 9-13 day 16-19 da" 1000 11 11 17 1000 12 14 12 1000 11 9 15 1000 9 10 15 Mean 10.75 11 14.35 Control 1 3 4 Analysis of variance Source of Variation 0.3. 5.5. Mean 8. F. 3.93 Treathent 8 00.5 15.25 5.69 5.14 Replicate 5 5.7 Error 6 ‘24.8 4.13 Conclusion: The difference in susceptibility of the Various age groups tested with dieldrin is not significant. 50 Table 9 —- Kunber of adult onion maggots of various age groups Killed with ethion. Each lot consisted of 20 flies. .. , age Group micrograms of ethion per gram fly 2-5day 9-12 day 16-19 day 8 11 17 10 8 1a 12 14 5 15 16 12 8 8 -- 15 he an 1:735 To 12 . 75 Control 0 2 6 Analysis of variance Source of variation D.F. 6.3. Mean 8. r. 1.95 Treatment 2 24.10 12.05 2.12 4.46 Error 8 45.50 5.69 Conclusion: The difference in susceptibility of the various ave groups tested with ethion is not 0 ‘2 significant. 1 fable 10 -- Per cent nortality in Various age groups of the onion maggot adults treated with dieldrin. Liicrograms of dieldrin Age Group per gran of fly 2-5 day 9-12 day 16-19 day 1000 55 47 81 1000 58 65 50 1000 55 55 56 1000 42 41 69 inean 5f"? :7 .674- Control 5 15 20 Analysis of Variance Source of variation D.F. 8.5. mean 8. E. F.95 freatment 2 6.10 5.05 1.97 5.14 Replicate a 5.00 Error 6 9.24 1.54 Conclusion: The difference in susceptibility of the various age groups tested with dieldrin is not significant. After correction for natural nortality. U? l u - . . l U ‘ o Taole 11 -- :er cent nortality in Various age groups of the onion nagéot adults treated witn etnion. _, . age Group micrograms of ethion per gran of fly 2—5 day 9-12 day l6-19 day 8 55 85 29 8 65 55 57 8 65 78 45 8 4O -- 64 Mean 56.25 72.55 48.25 Control 0 10 50 Analysis of Variance Source of variation D.E. 8.8. Mean 8. F. F 95 Treatment 2 10.00 5.00 2.56 4.46 Error 8 15.62 1.95 Conclusion: The difference in susceptioiiity of the various age groups tested with etnion is not significant. lAfter correction for natural mortality. Enterinent V1 lurpose: To determine the difference in susceptibility of the sexes of onion Jaggot adults to ethion or dieldrin. The flies tested in this experiment in 1359 were obtained as previously described. The insecticide solutions were topically applied to the mesonota of 2 to 5 day old insects. The LD5O of both ethion and dieldrin were determined as described in experiment I. at least four dose levels of each insecticide and four replicates of 10 flies of the same sex were treated to deternine the LD50. After treatnent at room tengerature, the flies were held in pint-size ice crean cartons and transferred to a constant temberature cabinet at 770 E. Mortality counts were made 24 hours after treatment. The LD50 for the male and female of the onion fly in nicrograns of toxicant per gram of fly, 95 yer cent confidence limits, and the slopes of the log. dose-probit lines for ethion and dieldrin are shown in tables 12 and 15. a comparison of data in tables 12 and 15 revealed that the difference between the sexes in the LD50 when eXposed to dieldrin or ethion was significant. The females were less susceptible to the action of the chemicals than the males. It is well Known that the lethality of poison is influenced by the height of the test animal. Ihis suggested the possibility of significant difference in weight between the nale and fenale insects. To find whether or not there was significant difference in weight, 40 females and 40 males were weighed individually. These figures are given in table 25 in the appendix. The average weight of the female insect was 10.92 mg. and that of the male was 10.50 mg. analysis of variance of the weights disclosed that the difference in weight between male and female insects was not significant. After the difference in susceptibility of the sexes of the onion fly to ethion and dieldrin was found to be significant, it was decided to deternine the sex ratio of the fly. nt regular intervals 100 pupae were taken from the stock of field-collected pupae stored at 580 F. These pupae were transferred to a temperature cabinet at 770 F. and allowed to develop. The flies which emerged from each 100 pupae were sexed and counted. In a total of 712 flies counted, there were 52o renales and 589 males. This represented a sex ratio of 55 per cent males to 45 per cent fenales. This is not statistically significant fron a 1:1 ratio. Table 12 - To xicities of dieldrin and ethion as determined by topical application to the onion fly females. 1050 in nicro- Slope of log. Insecticide grams per gran eon confidence dose-probit of fly limits line Dieldrin 804 580-1264 2.95 EtniOn 1105 902’1405 602 Table 13 -- Toxicities of dieldrin and ethion as deternined by topical application to the onion fly males. LD5O in nicro- slope of log. Insecticide grams per gram 95p confidence dose-probit of fly limits line Dieldrin 529.2 165.8-655.8 5.15 BthiOfl 5.90 4.40-7.90 3.90 Experiment V11 Eurpose: To deternine the relationsnip between the stage of development of the onion fly and susceptibility to both dieldrin and ethion. The following stages of developnent of the onion fly mere compared as to their susceptibility to dieldrin: Second instar larvae, third instar larvae, pupae and adults. Third instar larvae, pupae, and adults were compared as to their susceptibility to ethion. The insecticides were dissolved in acetone and applied topically to the insects. Larvae were obtained from infested onion bulbs which were brought to the laboratory from an onion field in Michigan. Larval treatment: Second and third instar larvae were taxen from infested onion bulbs and treated with the follow- ing doses of dieldrin: 1000, 2000, and 4000 micrograms per gram of larvae. also, third instar larvae were treated with 15 and 100 micrograms of ethion per gram of larvae. The individual weights of 20 second instar larvae and 40 third inStar larvae are recorded in table 25 in the appendix. Two to 4 replicates of 20 larvae were tested at each dose level. Also, a control treatment was conducted with each dose level. After treatnent the larvae were neld in pint- size ice crean cartons and transferred to a constant . o i . . _ . . temperature cabinet at 77 g. The larvae were frOVlded With 57 slices of onions for food. Mortality counts were taken 24 hours after treatment. The number of larvae pupated, larvae survived and larVae Killed are recorded in tables 14, 15 and 18. Tupal treatment: lupae tere obtained by allowing sone of the larVae which uere brought from the field to pugate. The concentrations of insecticides used were 1000, 2000, and 4000 nicrograns of dieldrin and 100 micrograms of ethion per gran of pupae. The individual weights of 20 pupae are shown in table 25 in the appendix. Insecticide solutions were dissolved in acetone and applied tOpically to the pupae. Four replicates of 20 pupae were treated at each dose level. Also, a control treatment was conducted along With each replicate. After treatment the pupae were held in pint-size ice cream cartons and placed in a tempera- ture cabinet at 770 F. The pupae were allowed to develop, and the emerged flies were counted. The number of adults that emerged and pupae vhich failed to develop are shown in tables 10 and 19. adult treatment: adults were obtained by allowing sone EUpae which were obtained for pupal treatment to develop. Doses of 1000 and 2000 micrograms of dieldrin per gram of fly were applied to 2 to 5 day old adults. Fifteen and 100 micrograns of ethion per gram of fly were applied. The insecticides were dissolved in acetone and applied to the mesonota of individual insects. after treatment the flies were held in pint-size ice cream cartons and transferred to a tenperature cabinet at 770 E. Mortality data obtained from treating the onion naggot adults with different doses of both dieldrin and ethion is recorded in tables 17 and 20. Table l4 -- Number of survived and dead larvae after treathent of the second instar larvae of the onion fly with different doses of dieldrin. Each lot consisted of 20 larvae. hicrograms of dieldrin per gram of larvae Larvae survived Larvae dead 1000 20 0 1000 so 0 1000 20 0 Control 30 O 2000 18 3 2000 13 2 2000 20 O 2000 17 5 Control 19 l 4000 19 l 4000 17 3 4000 20 O 4000 13 2 20 0 Control 00 Table 15 -- Number of larvae pupated, larvae survived, and larvae dead after treating the third instar larvae with different concentrations of dieldrin. Eacn lot consisted of 30 larvae. Micrograns of dieldrin Larvae Larvae larvae per gram of larvae pupated survived dead 1000 7 lo 0 1000 8 2 0 1000 4 10 0 1000 0 l7 0 Control 5 15 0 2000 5 15 0 2000 0 20 O 2000 3 14 3 Control 3 18 O 4000 3 17 l 4000 4 1° 0 4000 l 19 0 Control 2 18 O 61 Table 10 -- Number of adults emerged and undeveloped pupae alter treatnent with different doses of dieldrin. Each lot consisted of 80 pupae. micrograns of dieldrin per gran of pupa adults emerged Undeveloped pupae 2000 5 15 Control 9 11 2000 6 14 Control 7 15 2000 10 10 Control 0 14 8000 7 15 Control 5 15 4000 5 15 Control 5 15 4000 10 10 Control 10 4 4000 14 6 Control 6 14 4000 14 6 9 11 Control Table 17 -- huhber of adult onion naggots Killed when eXposed to different concentrations of dieldrin. Each lot consisted of 20 flies. Micrograms of dieldrin per gram of fly Dead Alive 1000 12 8 1000 14 6 1000 11 9 C Iii/I'D]. l 19 2000 15 5 2000 15 7 2000 14 6 ‘) (4 Control 0 Table 10 -- Number of pupae, larvae dead, and larvae survived after treating the third instar larvae with different concentration of ethion. Each lot consisted of 30 larvae. Micrograns of ethion per gram of larvae lupae Larvae survived Larvae dead 15 5 l4 1 15 b 10 1 Control 6 12 2 100 2 0 13 100 1 O 19 100 2 0 18 100 O O 20 Control 15 O 04 Table 19 -- Nuhber of adults that energed and pupae which failed to develOp after treatment with ethion. Each lot consisted of 20 pupae. hierograms of ethion iupae failed per gram of pupa adults emerged to develop 100 9 11 Control 9 11 100 8 2 Control 9 11 100 10 10 Control 8 12 100 6 14 Control 8 12 Table 20 —- number of adult onion naggots hilied when exposed to different concentrations of ethion. Each lot consisted of 20 flies. Micrograns of ethion per gram of fly Dead Alive 15 15 7 15 16 4 15 14 6 Control 0 20 100 20 0 100 20 0 100 20 0 Control 2 l8 an inSpection of tables 14 and 15 indicates that the second and third instar larvae of the onion maggot were extremely resistant to the action of dieldrin. A dose of dieldrin as large as 4000 micrograms per gram of larvae was ineffective against the second and third instar larvae. also, table 10 snows that a dose of a000 micrograms of dieldrin per gran of pupae was ineffective, because more treated pupae developed than in the control group. This would not be the case if dieldrin was effective against the pupa. 0n the other hand, table 17 reveals that dieldrin is effective at 1000 and 8000 nicrograns per gran of onion maggot adults. This indicates that the adult stage of the onion maggot is less resistant to dieldrin than the other stages tested. Tables 10, 19 and 20 demonstrate that a dose of 100 micrograms of ethion per gran of third instar larva, pupa or adult was very effective against the third instar larvae and adults, but was ineffective against the pupae. nlso, tables 18 and 20 show that a dose of 15 micrograms of ethion per gran of third instar larva or adult was ineffective against third instar larvae but effective against the adults. Therefore, it was concluded that the order of resistance of the life stages of onion maggot tested with ethion was pupa) third instar larva) and adult. Experiment VIII This eXperiment was carried out in 1959 to deteraine Whether or not the site of application has any effect on the toxicity of 00th ethion and dieldrin to the onion maggot adults. Doses of 12 micrograms of ethion or 800 nicrograms of dieldrin per gram of fly were applied. Four replicates of 10 fehales and 10 males at each dose level and locus of application were used. The insecticides were dissolved in acetone and applied to the nesonota or to the middle of the abdominal dorsuh of individual flies. The insecticide solutions were topically applied by the hodified microburet described previously. After treatnent the flies were neld in pint-size ice creah cartons and transferred to a constant temperature cabinet at 770 F. Food and water were supplied to the treated insects. Jortality counts were made 2 hours after treatment. The nunber of dead insects obtained from applying ethion or dieldrin to different locations of the onion maggot adults are recorded in tables 21 and 22. Table 21 -- lumber of adult onion maggots Killed by apply- ing dieldrin to the thoracic nesonotum or dorsun of abdomen of the insect. Each lot consisted of 30 flies. ,q . . Site of application micrograms of dieldrin per gram or fly Mesonotun of Dorsun of abdomen thorax OO 7 10 (I) C) 0 L0 (.0 800 8 8 800 6 3 mean 7.5 7.5 Analysis of Variance Source of variation D.E. 8.3. Mean 5. E. Site of application I O O O Replicate 8 25 8.3 Error 3 9 3 Conclusion: The difference in toxicity of dieldrin topically applied to the thorax or abdomen of the onion fly is not significant. a a Table 22 -- humber of adult onion naggots Killed by apply- ing ethion to the thoracic mesonotum or dorsum of abdomen of the insect. Each lot consisted of 20 flies. W. . . Site of application micrograms of ethion per gran of fly mesonotum of Dorsun of abdomen thorax 12 4 9 12 l 10 12 2 6 l2 5 6 Mean 5 7.75 Analysis of Variance Source of variation D.E. 5.8. Mean 8. F. F.95 Site of application 1 45.15 a5.13 8.27 10.13 Replicate 5 6,58 (£1 H 03 to Q 01 as 0 Error Conclusion: The difference in toxicity of ethion applied topically to the thorax or the dorsum of abdomen of the onion fly is not significant DISCUSSIOR In measuring the relative toxicity of a number of insecticides to a particular pest, it is important to test such insecticides under field conditions where they will ultinately be used. This was recognized by Busvine (1957) when he wrote "Logically, of course the best criterion is a full scale field trial; but such trials are expensive, Slow and owing to the difficulty of providing adequate replications to naxe up for great Variability, do not always produce unanbiguous conclusions. Therefore they should be reserved till laboratory tests have narrowed the choice down to two or three substances." Laboratory tests to eValuate several insecticides against the onion maggot were carried out in 1958 in nichigan. Table 1 indicates that the organic phoSphate insecticides were much nore effective against the onion maggot than the chlorinated hydrocarbon insecticides used. Also table 1 strongly indicates that tae pest was resistant to the chlorinated hydrocarbon insecticides. lncreased resistance in an insect population is first s'spected because of the failure of a standard treat- ment to give control equal to that obtained previously. This, houever, is not a positive proof that the insect is 70 71 actually resistant to a certain insecticide. In order to ascertain if a particular insect is resistant to a certain insecticide, laboratory tests are usually carried out to give a direct comparison between a Known normal strain and the one under suSpicion. although there was a strong indication that the onion fly in certain areas in Michigan was resistant to the chlorinated hydrocarbon insecticides it'uas not experimentally confirmed. To confirm if the onion maggot was resistant to dieldrin, samples of the suSpected resistant and non-resistant flies from wo lobalities were tested in the laboratory, and the LDSO for the two papulations was established. Table l Snows that the LDCO of dieldrin for the onion nagéot adults obtained from nichigan was 542 micrograms per gran of fly. The LDSO of dieldrin for the Cornell strain of flies was 1.38 micro- grass per gran of fly. This confirmed that the samples of Michigan onion maggots tested we e positively resistant to dieldrin. From the scanty reports on the effect of pre-treatnent temperature on the toxicity of insecticides, no conclusions can be reachel. however, Cotton (19a2), Manson (1&53, 1954) and Craufurd-Benson (1958) reported that insects were less susceptible to the action of chemicals wnen pre-conditioned at lower temperatures. On the other hand, iuayle(1934) and Lindgren (1941) revealed that insects were less resistant to insecticides when pre-conditiohed at lower temperatures. Tables 2 and o of this work shows that the pre-treatnent temperature had no significant effect on the toxicity of either ethion or dieldrin to the onion na~sot adults. 22?) It seems that temperature is the most important factor affecting the insect during insecticidal tests. Also, the temperature affects the killina efficiency of insecti- cides. A brief review of literature indicated three general trends of the effect of post-treatment temperatures on the toxicity of insecticides to insects. first, is an increase in toxicity at lower temperatures, an second an increase in toxicity at the higher temperatures. a153, there were Cases in which the post-treatment temperatures have no effect on toxicity. Experiment 111 of the present research Showed that the post-treatment temperature has a profound effect on toxicity of both dieldrin and ethion to onion flaggot adults. Dieldrin and ethion here much more toxic to the insect at the higher temperature. Careful inspection of tables 4 and 5 revealed that at 600 F. dieldrin and ethion were ineffective agains: the onion fly. The toxicity of the materials increased as the tenperature increased. Guthrie (1950) reported on the effect of post-treatment temperature. he indicated that dieldrin was more toxic at higher temperatures when applied topically to the German cockroach. This is the only report found on the effect of tenperature on the toxicity of dieldrin to insects. No reports are available in the literature on ethion. According to the few reports on the effect of relative humidity on the action of insecticides, it seemed that increasi g humidity increased or decreased toxicity depend- ing on the poison and the test insect. nlSO, there were cases in which relative humidity had no significant effect on toxicity of insecticides. Tables a and 7 in experiment 1V shows that relative humidity after treatment had no significant effect on the toxicity of either dieldrin or etaion to the onion maggot adults. lotter and say (195a) reviewed the literature on the effect of relative hunidity before and during treatment on the toxicity of insecticides. They concluded that the relative humidity before and during treatnent does not affect the subsequent toxicity of a_ poison. It has previously been shown that the susceptibility of insects to insecticides increased as they became older (Simanton 1957, Craufurd-Benson labs, mcLeod 1944, David 1945, Collins 1955, and herr 1954). All of the literature available and known to the writer on the effect of age of insects on the toxicity of insecticides indicated that older insects here more susceptible than their younger counter- parts. hlthough there was no significant difference in 74 the effect of age on susceptibility of the onion fly to either dieldrin or ethion, greater kills were obtained with the older flies. Also, there were more deaths due to natural mortality. This is shown in tables 8 and 9. It appears that the slightly lower hills obtained with the younger flies was due to vigor resistance. The majority of reports in the review of literature on the effect of sex on the toxicity of insecticides to insects indicated that the female insects were more resis- tant than the male. This is in agreement with the present research. Tables 12 and 15 show that the LDSO of dieldrin for female and male onion flies was 594 and 529.2 micrograms per gram of fly reSpectively. Also, the LDSO of ethion for female and male onion flies was 11.5 and 5.9 micrograms of ethion per gram of fly respectively. It is important to note that the difference in weight between the female and male insects was not significant. This eliminated any suggestion that the difference in resistance may be due to significant difference in weight between male anu female insects. The significant difference in resistance of the onion fly to both dieldrin and ethion appears to be a case of physiological resistance, and it would seem worthwhile to investigate. After the difference in resistance between males and females of the onion fly was found to be signifi- cant it was decided that a determination of the sex ratio \7 (I I would be important. nn investigation to deternine the sex ratio of the onion fly revealed that the sex ratio is 55 per cent males and a5 per cent females. This is not significant from a 1:1 ratio. There are no reports available in the literature on this subject. hhen it is stated that an insecticide is effective against a certain insect Species, it usually means that the insecticide has been proved toxic to a certain stage in the life cycle of the insect. Little is Known of the extent of variation in susceptibility of the different stages in the life cycle. There are no reports dealing with this subject Specifically on the onion aaggot. The literature review on the effect of the stages of develOpnent on resistance of insects to insecticides revealed that large variation in susceptibility may occur throughout the life cycle. The present research found this to be true with the onion maggot. The adult stage of the onion maggot was less resistant to the action of dieldrin than the second instar larva, third instar larva and pupa. also, the order of resistance in the life stages of the onion maggot to ethion were pupa) third instar larva)»and adult. This snould be tanen into consideration in devising control measures. Aside from biological factors, there are physical factors wnich may affect the results of insecticide tests. \‘l 0. One of these factors is the site of application. The majority of reports in the preceding review of literature on the effect of location of dose on toxicity of insecti- cides to insects indicated that the closer the site of application to the central nervous system, the higher the mortality. Although the present investigations showed no significant difference in the effect of site of application on the toxicity of either dieldrin or ethion, inspection of table 22 reveals that more kills mere obtained when ethion was applied to the nesonotun than when applied to the dorsum of the abdomen. It is to be mentioned that the possibility of a slight Spreading of the insecticide solutions to the neighboring areas nay have contributed to the insignifiCant difference in the effect of site of application on the toxicity of both dieldrin and ethion to onion naggot adults. H SULLQtiY 111.3 C ‘I‘xibLUSION: The reSpective toxicities of 15 insecticides to the onion maggot adults Hylenya antiqua (Meig.), were determined by topical application. A modified syringe-microburet was used to apply the insecticide solutions to the insects. also, a laboratory strain of onion maggot adults was compared with samples of the suSpected dieldrin-resistant onion fly in nichigan. Experiments were conducted to determine the effect of different pre-treatnent and post-treatment temperatures, Various after-treatment relative humidities, age, sex, Stage of development, and site of application on the individual toxicity of dieldrin and ethion to the onion maggot. The resalts indicated: 1. Of the insecticides tested, methyl parathion, dimethoate, and Diazinon (0,0-diethyl O- (Z-isopro;yl-4-methyl—d-pyrimidinyl) phos- phorothioate) were the most toxic to the onion maggot adults. heptachlor, dieldrin, endrin, and DDT were the least toxic. 2. The samples of flies obtained from hichigan were found to be nucn nore resistant to dieldrin 77 7.1; than a normal strain of flies reared in the laboratory. Different pre-treatnent temperatures did not effect the toxicity of either dieldrin or ethion to the onion maggot adults. Post-treatment temperature had a profound effect on the toxicity of dieldrin and ethion. The higher the temperature the higher the toxicity. Various after-treatment relative humidities did not effect the toxicity of dieldrin and ethion to the onion maggot adults. There was no significant difference in the effect of age on the toxicity of either dieldrin or ethion to the onion maggot adults. more Kills were obtained with the older flies than young ones. Also, there was more natural mortality with the older flies. The lesser kills obtained with the younger insects was attributed to vigor resistance. Che female flies were significantly more resistant to the action of both dieldrin and ethion than the males. There was no signifi- cant difference in weight between female and male flies. It was suggested that the difference in resistance between females and males may be a case of physiological resistance and worth investigating. Q The onion maggot adults were less resistant to the action of both dieldrin and ethion than the second instar larvae, third instar larvae, =nd pupae. Because at the doses used, dieldrin was not effective against the second instar larvae, third instar larvae, and pupae no comparison could be made between these stages. The order of resistance in the stages of development of the onion maggot to etnion was pupa) third instar larva) and adult. On a weight oasis it was concluded that adults were the least resistant stage tested. 9. although there Was no significant difference in the effect of the site of application on the toxicity of either dieldrin or ethion to the onion fly, more kills were obtained uhen etnion was applied to the nesonotum of individual insects than when applied to the dorsum of the abdomen. (‘1‘. ln toxicological investigations on the onion maggot, temperature after treatment, age, sex, and site of application should be standardized. Al'PELfiDIX (n H The Mathehatical Irocedure in Determining the 1050 for Thinet by the Probit Analysis netnod Ier cent 10g3dose 2 irovisional ExPected Probit 2 Dose dead 1 A Probit Probit Y Y 20 98 1.501 1.695 7.954 6.600 6.914 47.705 15 80 1.176 1.585 5.842 0.180 5.770 55.295 12 82 1.079 1.164 5.915 5.915 5.915 54.987 8 51 0.905 0.815 5.025 5.400 5.005 25.050 2 9 0.501 0.091 5.659 5.450 5.700 15.690 Control 15 2 . 2 - NW th HJX NJ. ILAT hde 2.002 2.605 5.589 15.842 95.702 18.011 5.142 5.695 4.545 18.129 104.607 21.520 5.859 4.142 4.469 22.708 154.515 24.500 to P C23 (1‘ H 4.751 4.272 5.856 25.679 118.512 0.651 0.196 0.059 2.409 8.912 0.725 14.565 14.910 16.118 80.767 462.048 85.957 1after correction for natural nortality by Abbott's formula. 82 ' E35 - $34319 - 1.008 NJ 140365 Y NVY - 221222 . 5.622 IIX - 54 << n ' .641 hung - A(nw1) 16.118 - 1.055(14.910) - .641 hHKY - Y(kaX) 85.957 - 5.622(14.910) . 2.115 .296 (.3 fix The equation for the probit regression line is: The above values of Y on the Iolloving grain to draw Y = i + b(x - ) I ()1 O C {‘3 {a + (33 O [‘0 IL.) OJ IF’ 0 I" Q (7 I I" (— C 3 (0 II 02 O \‘1 \1 : 5.532 + 3.395(1.079 - 1.058) - 4“. t r, |fi"" = 000(1)); + Q’tho(ov 5.296(.501 - 1.058) . 3.195 for 5 Values of i are plotted the log.dose-probit line. C) (xx .5 .1nu the logar1thm 1350 as the value of K WfllCh wives Y 5' n = o + bx - Y b - 5 1 0.296 x 1.03 - 5.632 - ”fl = .849 0.4.5 Jariance of (a) Atli l U ”4 {‘0 l ‘ CC ". 1H - «to Irr- , 1118 St Q ndard error of n is WJT01129JL3 = .1065 W1~ '\- _ .o . o rhe 95 per cent coniidence limits are: t ts- x antilogarithn of .849 = 7.06 nntilogarithn of 1.057 - 11.40 Antilogarithn of .641 - 4.58 Lhencethe LD50 for Thinet is 7.06 nicrograms per gram of fly and the 95 per cent confidence limits are 11.40 and 4.o8. m.a m.a 93% pfloonmuomowéoq I I I I mafia assoamfwomg IIII mmOfiohOH 10. A m. . . 1!QOJ{ fable 25 —- The individual weights in milligrans of onion flies obtained from hichigan, onion flies from Cornell University, male and female Lichigan onion flies, second instar larvae, third instar larvae, and pupae. Second Third El1es from 21192 from Male Female lnstar Instar nicnigan Cornell U. flies Flies Larvae Larvae Pupae 11 11 19 12 5 17 14 11 9 12 11 5 1a 14 15 12 11 11 5 19 15 19 9 11 11 5 1e 12 11 a 11 9 4 14 2 19 9 22 19 5 19 12 12 19 11 1s 4 19 12 12 19 19 15 5 17 15 19 19 11 12 6 29 11 9 9 9 12 5 l8 2 11 19 19 14 6 17 12 12 9 19 9 6 17 11 11 15 19 9 5 1e 11 12 11 19 19 5 16 11 19 9 11 19 4 17 15 12 9 12 12 5 19 2 11 19 11 12 4 l8 2 15 19 11 19 5 1a 2 12 19 19 11 5 16 15 1s 11 1o 19 4 15 11 19 19 15 11 19 16 9 14 19 19 19 16 15 11 17 10 lo 18 19 9 16 12 11 17 12 12 14 19 12 17 9 14 17 9 19 18 11 12 12 19 9 17 11 9 l6 2 11 17 9 9 ' 14 9 9 14 19 11 15 11 9 15 hean 11.45 9T95 19.59 19.92 479' '16735 'IBTIU LITERAQUKE CITEJ Ball, L. J. and 3. 3. Beck. 1951 The role of circulatory and nervous systems in the toxic action of parathion. Jour. Econ. Ent. 44: 556-564. Barber, G. H. and J. 3. ochmitt. 1945 house flies resistant to DDT residual Sprays. New Jersey Agr. Expt. Sta. Bul. Lo. 742. R hp k bJ. Benschoter, C. A. 1960 susceptibility of life stages of the Lexican fruit fly to funigation with ethylene chloro- bromide. Jour. Econ. Ent. 53: 325-525. Busvine, J. R. 1957 A Critical Review of the Iechniques.for 'festing Insecticides. Commonwealth Inst. Ent. London. 203 pp. Campbell, F. L. 1926 Relative susceptibility to arsenic in successive instars of the silkworm. Jour. Gen. lhysiol. 9: 727-753. Cochran , D . G . 1955 Differential susceptibility of the sexes and developmental stages of the Aherican cockroach to several insecticides. Jour. rrr— dcon. Ent. 45: 131- oo. COlliflS , ‘J o E o Llfld 1:. Lo iii-fig. 1955 n nodified technigue for laboratory evaluation of contact insecticides. Jour. Econ. Ent. 46: 51-530 Cotton, R. T. 19o2 The relation of respiratory metabolisn of insects to their susceptibility to fumigahts. Jour. Econ. Ent. 25: 1088—1103. Craufurd-Bensoh, h. J. 1955 an improved method for testing liquid contact insecticides in the laboratory. Bul. Int. (es. 29: 41—56. 1" be Cressman, n. 1995 David, J. 1926 no Deane, J. F. 1957 1. Dl'e‘v'v' v tilid H9 w (J1 w. DuCnanois, 1947 Einney, D. 1958 Eisner, R. N 1932 Gains, J. C. 1949 Glynn-Jones, 1952 Cough, 1"1. C. 19&O E . . n. broadbent, and E. aunger. factors influencing the effectiveness 1 parathion against California red scale. our. Econ. Ent. 46: 1071-1074. L., and P. dracey. Factors influencing the interaction of insecticidal mists on flying insects. 111. Biological factors. Bul. Ent. R‘s. 57: 177—190. Ft. and d. x. Chapman. Onion naggot resistance to chlorinated hydrocarbon insecticides. Bul. Ent. Soc. Amer. 5: 40. G. E. Guyer. _ Test methods for evaluating aaainst the onion Maggot. Q Bro Eflt. SCC. Amer. A111: insecticides Proc. E. Central 45‘440 R. Toxicity of gamma-benzene hexachloride to the preinaginal stages of the house fly. Jour. Econ. Ent. 40: 749-751. lrobit Analysis. Cambridge Univ. Eress. 313 pp. The importance of the locus of application on the effectiveness of DDT for the house fly, Musca domestica L. Canad. J. Zool. 30: 254’2660 and ii. 11. Dean. Effect of temperature and humidity on the toxicity of certain insecticides. Jour. Econ. Ent. 2: 429-493. G. D. and R. a. Edwards. Studies of toxicity of 5:5 dinitro-ortho- cresol and its sodium salt to thelnneybee. Bul. Ent. Res. 45: 67—78. Toxicity of sulfur dioxide to the bed bug, Cimex lectularius L. Ann. appl. Biol. 2 101-1090 Gunderson, H. and n. L. Strand. 1959 Toxicity of hydrogen cyanide, chloropicrin and ethylene oxide to eggs, nymphs and adults of the bed bug. Jour. Econ. Ent. 38: 105-110. Guthrie, E. E. 1950 Effect of temperature on toxicity of certain organic insecticides. Jour. Econ. Ent. 45: 559-560. Guyer, G. E. and A. Wells. 1959 Evaluation of insecticides for control of the chlorinated hydrocarbon resistant onion maggot. Mich. Agr. EXpt. Sta. guart. Bul. 41: 614-685. "T" 1 o nailiger, E. 1949 Comparative toxicity of various insecticides to the honeybee. Jour. Econ. Ent. 42: 525-533. harris. T. U. 1852 A Treatise on Some of the Insects of New England which are lnjurious to Vegitation. Boston, finite and Lotter. 515 pp. nodgman, C. D. 1949 handbooh of Chemistry and lhysics. p. 1951. Chenical Rubber Co., Cleveland, Ohio. hoffman, R. a. and A. J. Lindquist. 1949 Effect of temperature on Knockdown and mortality of house flies exposed to residues of several chlorinated hydrocarbon insecti- cides. Jour. Econ. Ent. 42: 891—895. Koshins, h. n., J. n. ”itt and U. 4. Erwin. 195B Bioassay of l,2,5,4,5,5-hexach1orocyclohexane (Lindane), Some factors influencing the contact of chemical and test insect and methods of standardizing the process. Analyt. Chem. 24: 555-560. Hoskins, J. m. and H. T. Gordon. 1955 Arthropod resistance to chemicals. In Annual Review of fintomology. Vol. 1. Annual Reviews, Inc. Stanford, California. pp. 89-123. Howitt, A. J. 1958 Chenical control of gylemya antiqua (neig.) in the Tacific Northwest. Jour. Econ. Ent. 51: 835-387. herr, d. a. 1954 Variation with age in the susceptibility to DoT and the respiratory rate of male and female Drosophila melanogaster meig. Eul. Ent. des. 45: 585-528. Lindgren, D. L. 1955 The reSpiration of insects in relation to the HGating and the fumigation of grain. Minnesota Agri. Expt. Sta. Tech. Bul. Ho. 109. 52 pp. ‘ Lindgren, D. L. and d. C. Dickson. 1941 Eumigation of purple scale with hydrocyanic acid. Jour. Econ. Ent. 54: 59-04. Lindquist, A. n., A. h. Madden, H. G. Hilson and H. a. Jones. 1944 The effectiveness of DDT as a residual Spray Lindduist, A. W., n. G. Scnroeder, H. O. and A. H. Madden. 1945 Effect of temperature on Knockdown and Kill of house flies exposed to DDT. Jour. Econ. Ent. 38: 251-254. Maan, Hillem Jan. 1947 Biology and phonology of Chortophila antiqua, the onion fly, and ncrolepia assectella, the leex moth, as a basis for control. Ph.D. Tiesis. University of Amsterdam. 120 numb. leaves. (Abstracted in Biological abstracts. 21: no. 20814.) MacCuaig, R. D. 1956 Determination of the resistance of locusts to DNC in relation to tneir weight, age and sex. Ann. Appl. Biol. 44: 654-642. McLeod, n. S. 1944 Further refinement of a technique for test- ing contact insecticides. Canad. J. Res. 1D) 22: 87-104. 1943' Effect of nexacnlorocyclohexane on onion seedlings. Jour. Econ. Ent. 59: 815. MGHUSdn, n. 1948 Comparative toxicity of insecticides admini- stered in various ways to several Species of insects. Jour. Econ. Ent. 41: 503-515. Merrill, L. G. and a. hutson. 1955 maggots attacking Michigan onions. Jour. SOON. Eflt. 46: 078-6800 Metcalf, n. L. 1958 Methods of topical application and injection. In Methods of Testing Chemicals on Insects. Vol. 1. Burgess iublishing Co. Minneapolis, ninn. pp. 95-114. morrison, H. E. and a. H. Crowell. 1953 Soil insecticides in Oregon. Jour. Econ. Ent. 45: 1005-1010. muIlI‘O, 11. J9 1947 DDT as an insecticide against the onion maggot. h. Dax. agri. Expt. Sta. Bimon. Bul. 9: 81-88. munson, S. C. 1955 Some effects of storage at different tempera- tures on the resiSLance of the .nerican cocxroacn to DDT. Jour. Econ. Ent. 45: 754- 750. Munson, S. 0., C. M. ladilla, and m. L. Zeissmann. 1954 Insects lipids and insecticidal action. Jour. Econ. Ent. 47: 575-557. Murray, 0. a. 1957 a statistical analysis of fly mortality data. Soap and Sanit. Chem. 15: 89-105. O'hane, d. C., G. L. Walker, R. G. Guy and O. J. Smith 1955 Studies of contact insecticides. New Hamp- shire Agr. Expt. Sta. Tech. Bul. he. 54. 25 pp. Potter, C. and M. J. Hay. 1958 Precision Spraying. In methods of Testing Chemicals on Insects. Vol. 1, Jurgess Publishing Co. Minneapolis, Minn. pp. 92- 114. 91 lradhan, S. 1949 Studies on the toxicity of insecticide films. 11. Effect of temperature on toxicity of DDT films. Bul. Ent. Res. 40: 259-255. 1949 Studies on tne toxicity of insecticide films. 111. Effect of relative humidity on the toxicity of films. Bul. Ent. Res. 40: 451- 444. guayle, H. J. and L. h. Ronrbaugn 1954 Temperature and humidity in relation to hCE fumigation for the red scale. Jour. Econ. Ent. 27: 1085-1095. Roth, 2. 3., n. W. Lindquist, and L. C. Terriers 1955 Effect of temperature and the activity of house flies on their absorption of DDT. Jour. Econ. Ent. 49: 127-150. Snircx, F. H. 1957 Experiments for the control of the onion maggot. Jour. Econ. Ent. 50: 577-580. Simanton, U. A. and A. C. miller 1957 house fly age as a factor in susceptibility to pyrethrum Sprays. Jour. Econ. Ent. 50: 917-921. Snedecor, G. U. 1956 Statistical Methods. Iowa State College Tress, Ames, Iowa. 554 pp, fozlosxi, A. E. 1954 Control of the onion maggot on seed sets in the Connecticut Valley. Jour. Econ. Ent. 47: 494-497. TreVan, J. n. 1922 an apparatus for measuring small quantities of fluids. Lancet 202: 786. Vinson, E. B. and C. W. Kearns . 1952 Temperature and the action of DDT on the American roach. Jour. Econ. Ent. 45: 484-495. weaver, N. 1949 The toxicity of certain organic insecticides to honeybees. Jour. Econ. Ent. 42: 975-975. fiilson, C. S. 1949 Workman, R. B. 1958 'Jright, 1). W. 1958 Piperonyl butoxide, piperonyl cyclonene and pyrethrun agplied to selected parts of individual flies. Jour. Econ. Ent. 42: 425—428. The biologj of the onion naggot, hflenya antiqua (Miegen), under field and greenhouse conditions. Unpublished ih.D. Thesis. Corvalis, Oregon State College. 88 hunb. leaves. The control of the onion fly. Jour. Ministry of Agric. 44: 1051-1087.