May 1951 E-818 United States Department of Agriculture Agricultural Research Administration Bureau of Entomology and Plant Quarantine DEVELOPMENT OP IHSECT BE SI STANCE TO INSECTICIDES— II A Critical Review of the Literature up to 1951 By Frank H. Babers and John J. Pratt, Jr. Division of Control Investigations In May 19l;9 the senior author (Babers 9) published a review covering the literature up to the fall of 19^8 on the subject of the development of resistance by insects to insecticides. At that time only a few cases of the failure of DDT to control house flies in the field had been reported from this country, and the potential signifi- cance inherent in these failures was apparently not universally recog- nized. Recently, however, these failures have received great publicity in scientific publications and in the press. In the earlier review it was noted that the following species had been reported as having developed resistance to insecticides: California red scale ( Aonidiella aurantii (Mask.)), black scale ( Saissetia oleae (Bern. )) , citricole. scale (Coccus pseudomagnoliarum (Kuw. )), codling moth ( Carpocapsa pomonella (L. ) ) , vinegar fly ( Drosophila melanogaster Meig. ), cotton aphid ( Aphis gossypii Glov. ), confused flour beetle ( Tribolium confusum Duv.), blue tick ( Boophilus decoloratus (Koch.)), citrus thrips ( Scirtothrips citri (Moult. )), screw-worm ( Callitroga americana (C. & P.)), an Italian subspecies of the northern house mosquito ( Culex pipiens autogenicus ), house fly (Musca domestica L. ), and San Jose scale ( Aspidiotus perniciosus Comst.). It is the purpose of the present review to bring the subject of the development of insect resistance to insecticides up to date and to include a critical discussion of some of the causes of the various discrepancies in results reported by the several observers. California Red Scale Several papers overlooked in the earlier review have been noted. Busbey et al. (35) found that second-stage nymphs of the strain of red scale ( AonidielTa aurantii (Mask.) resistant to hydrocyanic acid were more susceptible to trichloroacetonitrile than were the adult insects. The reverse is true when the insects are tested against hydrocyanic acid. The normal strain was not tested. Yust, Nelson, and Busbey ( 171 ) studied the effect of inbreeding on the susceptibility of the red scale to hydrocyanic acid. After several years no difference in susceptibility attributable to the inbreeding was noted. Cressman (1|0) found large differences in the mortalities of scales - 2 - from various groves when the insects were sprayed in the field with comparable dosages of oil, or collected in the field and sprayed in the laboratory. Scales collected from groves in which extreme dif- ferences were noted and reared in the laboratory for several genera- tions showed almost no differences in susceptibility when tested at intervals over a period of about 9 months. Cressman concluded that most of the observed variations in the susceptibility of field stocks of red scales to oil sprays were due to other than genetic differences. Whether or not any of the strains tested were those considered to be resistant to hydrocyanic acid was not stated. It is not clear how Cressman reached his conclusions concerning lack of genetic differences from the type and small amount of data presented. In the previous review (9) it was mentioned that the resistance of red scale to hydro- cyanic acid had been shown by several investigators to be inherited. According to Lindgren and Dickson (91) there was no difference between the susceptibility of laboratory-reared stocks of the two strains to oil sprays. Cressman did not refer to this earlier work. Hunger ( 119 ) compared the rate of development of red scales resistant to hydrocyanic acid with nonresistant insects and found no significant differences. He also ( 12C ) determined the effect of temperature on the biology of the two strains, and again no significant differences between the strains were noted. Drosophila Kalina (70) tried to develop a resistant strain of Drosophila melanogaster by rearing larvae produced from eggs laid by normal females in a medium containing $ parts per million of DDT. Larval development was somewhat slower than normal but up to the time of early pupation no real difference was noted. In the later stages of pupation, development was abnormal. Adults usually failed to emerge, and those that did exhibited signs of DDT poisoning and soon died. The specula- tion was made that DDT was stored in the larval fat and was released during histolysis in the pupae. Whether or not experiments were per- formed using lower concentrations of DDT was not stated. A strain of Drosophila resistant to DFDT (l,l,l-trichloro-2,2-bis (p-fluoro phenyl) -ethane) was developed by Reimschneider and Rohrmann (137 ). After 32 generations of selection, resistance had developed so that an exposure to deposits of DFDT that caused 70 percent mortality in the normal strain caused 30 percent mortality in the resistant strain. Resistance to carbon dioxide by Drosophila is apparently a normal condition, whereas susceptibility is abnormal. The sensitivity seems to depend on the presence in the cytoplasm of an agent called a genoid. L'Heritier and his associates (86 ,08,89,90) showed that the BtisceptiMliV to carbon dioxide could be transmitted by transplanting from susceptible to normal individuals ovaries, brain tissue, or imaginal discs of the eyes, legs or wings, or portions of the gut. Hemo lymph as well as the centrifuged supernatant of crushed susceptible flies at any stage was shown to be infectious. Under certain conditions the exposure of sus- - 3 - ceptible flies to heat will "cure" their germ cells, and it is thus possible to obtain a genoid-free stock from any sensitive stock. They also found that X-rays would inactivate the genoid (87). This and further work by L'Heritier raised the question as to the possible similarity of the genoid to a virus, and L'Heritier and his collaborators are now approach- ing the subject by using techniques applicable to virus research. Such an approach will, if successful, throw light on the relation between viruses and genes and will have far reaching applications. According to Goldstein (58, $9 ), if "normal" Drosophila males are made "sensitive" by the injection of sensitive hemolymph, they transmit the sensitivity to only a small percentage of their progeny, whereas if the males are normally sensitive a higher percentage of transmission occurs. It was found that the two types of males could be obtained from any stock. To explain this, it was suggested that the genoid of the synthetic sensitive stock had undergone a mutation limiting strongly its capacity to infect the male germ. Plus ( 133 ) determined the time required (incubation time) for Drosophila to become susceptible to carbon dioxide following injection with a suspension of genoids. If moderately high concentrations of genoids were used, a linear relation was observed between incubation time and the log of the number of in- fectious units injected. As an appendix to Plus's paper L'Heritier presents a mathematical treatment of these results. A review bv L'Heritier (_8£) covers the subject of carbon dioxide sensitivity up to about 19U7. There is considerable variation in the susceptibility to ether among Drosophila virilis , D. americana , and D. texana . Crow (Ul) found that when the dosage was varied and the time of exposure held constant virilis was the most resistant, followed by americana and texana . These observed differences were small. When the dosage was constant and the time of exposure varied, virilis was the most resistant, then texana , and finally americana . These differences were large. Fox (S3) prepared rabbit anti-sera from three mutant strains of lyophilized insects. By complement fixation tests, no differences in the anti-sera were noted. By precipitin ring tests, however, certain differences were noted. These differences were believed due not to the single major genie difference but to the multiple genie differences that must exist between such unrelated stocks. Ticks No reports of insecticide-resistant ticks in the United States have been noted. In sections of South Africa and to a lesser extent in Argentina, the problem is of considerable importance. Resistance to arsenic dips by the blue tick was first noted in 19I4.O in a small coastal area of South Africa. The resistance spread rapidly. It seemed to be inherited but other factors were definitely involved. Whitnall and Bradford ( 165 ) reported that if cattle infested with resistant ticks were transferred from the coastal area to an inland area, the ticks either disappeared because of their inability to survive in the new - h - environment or they succumbed to the normal arsenical dipping. Whitnall and Bradford in 19U7 (165), and again in 19h9 (166), and Thorbum (157) in 1°U7 reported that the resistant ticks were readily controlled with Gammexane dips. Whitnall and Bradford ( 166 ) ,also reported that the tick that cansed sheep paralysis, Ixode s pilosns Koch, seemed somewha f resis- tant to the Gammexane but succumbed to the usual arsenic dips. Later in 19h9 Whitnall and others (164, 16? ) and Haarer (60) reported that the arsenic-resistant tick had become resistant to gamma benzene hexachloride . In Argentina control of the blue tick with benzene hexachloride was still being obtained (51). Haarer (60) in discussing some of the economic aspects of the resistance of the blue tick stated^ "On one ranch alone, 600 head of stock died in one year because of the failure of arsenic to control the blue tick. The first year Gammexane was used, the figure dropped to 70 head. It was no wonder the cattlemen were jubilant with the material and it is also easy to understand their perturbation at the fact that resistance to benzene hexachloride has now developed." Red Spider Mite Smith, Fulton, and Lung ( 150 ) reported excellent control of many greenhouse pests, including the two-spotted spider mite ( Tetranychus bimaculatus Harvey), with parathion aerosols in 19^8. Complete control of the same mite with parathion was also reported by Blauvelt and Hoffman (22) about the same time. They state "Parathion shows promise of being ^e most important material yet discovered for greenhouse pest control." Parathion was effective in some greenhouses for only about a year, when Smith and Fulton ( H|7 , lU8 , i49) and Garman (56) found that resistance to the insecticide had developed. Garman (56) found that if resistant mites were transferred from greenhouses wEere they had infested roses to the laboratory where they were reared on bean plants, the resistance was lost in about U months. On the other hand, Smith and Fulton had reared resistant mites on beans for many months without exposure to insecticide and without any seeming loss of resistance. Garman found that the parathion resistance did not include resistance to tetraethyl pyrophosphate, p-chloropbenoxy methane, di(p-chlorophenyl) methyl carbinol, or 'alkyl sulTite" (trade product of unstated chemical composition). Except for tetraethyl pyrophosphate, ■which is distantly related chemically to parathion, the other materials are not structurally related to that compound. Smith's colony, however, showed considerable resistance to the following other insecticides : Hexaethyl tetraphosphate, tetraethyl pyrophosphate, tetraethyl dithio- pyrophosphate, l,l-bis(p-chlorophenyl)ethanol, 2-(p-tert-butylphenoxy- 1-methylethyl 2-chloroeThyl sulfite, and p-chloropEenyl p-chlorobenzene- sulfonate. Two of the above compounds were also tested by Garman, who reported his strain was not resistant to them. Fair control was still obtained by Smith using several of the above compounds although resistance was evident. Octane thyl pyrophosphoramide gave excellent control of resistant mites but whether or not there was any resistance to the com- pound was not determined. - 5 - In two greenhouse ranges in which Smith found "parathion-resistant" spider mites, resistance to hexaethyl tetraphosphate had been noted in 19U7 when it was first used as an insecticide. Repeated applications and heavy dosages had, however, maintained control. The development of resistance then could not be attributed to the use of either hexaethyl tetraphosphate or parathion. The history of these greenhouse ranges prior to the first use of hexaethyl tetraphosphate is not known, but undoubtedly the usual insecticidal practices for the control of green- house pests had been followed. E. W. Baker of this Bureau's Division of Insect Identification (personal communication) has been unable to find any morphological differences between the susceptible and resistant strains. Palm ( 126 ) reports that the red spider mite in New York State de- veloped resistance to ammonium potassium selenosulfide when this compound was used several years ago, and that cases of resistance to parathion are now occurring. Neiswander and Morris ( 121 ) after a period in which excellent control of two-spotted spider mites on roses grown in a nutrient solu- tion containing selenium was obtained, reported that a severe infesta- tion developed on the plants that had received a high dose of selenium. At that time, the infestation was not attributed to resistance. Re- cently, however, Neiswander, Rodriguez, and Neiswander ( 122 ) have decided that the insects had become more resistant to the insecticide. They further conclude that wide variations between populations of the mite exist. The rate of development as well as the resistance to acaricides was influenced by the host plant. A mite population reared on roses is generally more resistant than one reared on beans. A population subjected to an acaricide for three or more generations may develop a partial immunity to that material. Neiswander et al. ( 122 ) state, "It appears that such a heterogeneous organism as the two-spotted spider mite may require that acaricidal materials be changed at frequent intervals in order to insure effective control." Mosquitoes Salt-marsh areas along the east coast of Florida have been treated with oil solutions of DDT at regular intervals since 1?U5>. Before this period of treatment salt-marsh mosquitoes occurred in such numbers as to prevent full development of the area. After the treatment, freedom from mosquitoes was considered one of the shining examples of modern insect control measures. Recently the two most prolific insects in- volved, Aedes sollicitans (Wlkr.) and A. taeniorhynchus Wied. , have been reported as developing resistance to DDT (17, 27 . 37, 47, 48, 73, 106) Deonier and his associates (U7, U8) found that the DDT resistance was also apparent in the larvae. In addition to DDT the authors tested other insecticides — TDE, toxaphene, lindane, technical benzene hexa- chloride, and chlordane. They conclude, "Like DDT, TDE was much less effective against the adults from the treated area than it was against those from untreated areas,, an indication that the adults were about - 6 - equally resistant to TEE and DDT. At the lower concentration (0.5 percent) the other materials also showed less toxicity to the mos- quitoes from the treated area, but at the higher concentration (2 percent) this difference was not in evidence except possibly with toxaphene." The authors do not comment on the statistical significance of their data. In California, DDT has been reported as failing to control larvae of Culex spp. (72) and of Aedes nigromaculis (2U). Fay, Baker, and Grainger (50) attempted to develop resistance by exposing the common malaria mosquito (Anopheles quadrimaculatus Say) to low concentrations of insecticides in the laboratory. After one generation, some resistance had apparently been developed, but after several succeeding generations the resistance had not appreciably in- creased and the results did not seem to be conclusive. The experiments were then abandoned. The Malaria Eradication Program of the U. S. Public Health Service (26) has recently completed 5 years' operational work. Thousands of houses have been sprayed in the southeastern United States in an attempt to eradicate malaria by control of the mosquito vector. During this 5-year program, apparently no resistance to DDT has been evident. During the course of each season, however, there was a gradual decrease in the effectiveness of residual DDT. No indication as to the reason for this decrease was given but it is probable that loss of effectiveness was due to the normal aging of the deposited DDT. Mosna ( llli , 115) has reported that the DDT-resistant mosquito, Culex pipiens autogenicus ( 113 > 118 ), was effectively controlled with Octa-klor (chlordane) and hexachlorocyclohexane (gamma benzene hexachloride) . Only control dosages of these chemicals were used and no effort was made to determine whether any resistance to them was apparent. Losna noted that in DDT -treated houses, although there was an increase in the number of live insects, there was also an increase in the number of dead ones. The resistant insects were able to transmit the resistance through the eight generations tested without exposure to DDT. According to Verolini ( l6l ) the adults of C. pipiens from Latina were resistant to DDT, but the larvae showed high mortality from exposure to DDT. No comparison was made to determine whether or not any resistance was present. Starting a laboratory colony of C. fatigans Wied. from a single egg mass, Newman, Aziz, and Koshi ( T2l| ) found that successive generations showed greatly increased resistance to DDT and gammexane. The authors suggested that the increased resistance might be due to a rhythmic variation in the resistance of C. fatigans . Gahan and others (5U) compared the susceptibility of Anopheles psuedopunctipennis TheobT from areas in Mexico that had received four annual treatments with DDT to the susceptibility of mosquitoes from untreated areas. No difference was noted. Knipling (80) reported that there is no evidence that mosquitoes in the Pacific Northwest have developed resistance although resistant strains have been suspected in Oregon. - 7 House Flies Development of resistance in the field and in the laboratory House flies that have developed resistance to insecticides havt been collected in the field in Italy (110 ), Greece (61), Egypt (5>5>), Venezuela (160), Sweden ( 168 ) , Denmark (71) , England~~(6£), Sicily (h Peru (lUl) j Mexico (160), and the United States (12) . Strains with acquired resistance to various insecticides have been developed in several laboratories in the United States (1$, 23, 29, 9k, 103 , 13U), Sicily (j±2), and Great Britain (65). Busvine"" Q6"J~collected several strains of Musca vicina Macq., the house fly of Ceylon, and found no resistance although the area had been sprayed with DDT for 2 years. Surveys have been made in several sections of this country (12, 30, 33, 6U, 72, Ik, 81, 99, 126, l?4a, 139 , 160 ) and in Italy (TTJ2, llj.3T 7 ^d many strains of house flies resistant to insecticides found. Indeed, Upholt ( 160 ) states that his coworkers were unable to find a nonresistant strain in this country and contiguous areas of Mexico in 19U9. La Face (83), in a study of the biology of the fly and its impor- tance as a vector of various diseases, discussed resistance to insec- ticides. DDT-resistant flies — Musca domes tic a and Stomoxys calcitrans (L.) — in Sweden were discussed by Kjellander (75>, 76). No report of insecticide resistance in Russia has been noted. However, Rubtsov (ll;( after a visit to Missirolis' laboratory in Italy, summarized Missirolij results and speculated somewhat as to the probable causes for the appearance of resistant insects. Morrison and coworkers ( 112 ) reported that in a dairy barn in whic DDT has been applied as a space spray since 19k5, resistant flies have not developed. In contrast, a hog barn about 0.8 mile from the dairy barn has been repeatedly sprayed with DDT for residual treatment and here a decided resistance has developed. Pimentel and Dewey ( 130 ) found that the larvae of a field-collected resistant strain were also resistant. Barber and Schmitt (13) and Hansens and Goddin (63) found that in two colonies from the strain of flies used as a standard by the members of the Chemical Specialties Manufacturers Association (then the National Association of Insecticide and Disinfectant Manufacturers) there appear* to be resistance to methoxychlor. Such resistance has been reported by none of the many other laboratories using this strain. The list of insecticides used in the laboratory to develop resistar strains of flies includes DDT (9£), chlorriane (99), gamma benzene hexa- chloride (23, 131 ), parathion ( 103 ), pyrethrum, toxaphene, methoxychlor paraoxon, and dieldrin (33). Early in 191*6, long before resistant flies were reported from the field, Lindquist and Wilson (9k) began the de- velopment of the Orlando resistant strain. Flies from the regular laboratory colony were exposed to such a dose of DDT that about 90 perc - 8 - were killed. The survive s were used to carry on the colony. After three generations of such exposure, resistance was f ~und . Essentially their technique has been used in the development of all the other lab- oratory—resistant strains reported in the literature. In all but two cases noted* attempts to develop resistance in house flies by exposure to a given insecticide have been successful. The exceptions are the failure by D'Alessandro et al. (I4.2) 8 nfl the failure reported by Harrison (65) of attempt to develop a strain re- sistant to gamma benzene hexachloride from an Italian strain of resistant flies * In Harrison's case, only five generations were selected and this small number probably accounts for her failure. The development of strains resistant to benzene hexachloride, in the laboratory as well as in the field, is well known. Attempts by Bucher, Cameron, and Wilkes (3U) to develop a strain of house flies resistant to cold were unsuccessful. Loss of resistance Barber and Schmitt QJ4.) tested the Ellenville line of resistant flies after 10 and 11 generations in the laboratory without exposure to DDT. These flies were still resistant to DDT, but not to the same degree, as the third generation. After 2 hours' exposure to "l)|)| rag. of DDT per square foot, the 24-hour mortality reported for the third genera- tion was 10.9 percent and for the "10th and 11th" (which one was not specified) 59 • 2 percent. March and Metcalf ( 100 ) reared the Bellflower resistant strain for 35 generations without exposure to DDT and without loss of resistance. They state that "At present no indication has been found that resistant strains will revert to more susceptible strains following non-exposure to the insecticide." More recent tests, however, ( 102 ) "have shown a trend toward an increase in the number of more sus- ceptible flies in comparison with the number of highly resistant indi- viduals." In the writers' laboratory a colony started from pupae furnished by Metcalf and March in November 1°U° showed considerable resistance to insecticides in the first two generations. Within 10 generations without exposure to insecticide, hovrever, the resistance had fallen to the level of that of the normal laboratory strain (un- published results). At the Orlando, Fla., laboratory of this Bureau tests on the Bellflower strain showed that after 15 generations with- out exposure to insecticide a marked loss of resistance had occurred. The ratio, however, was still 10 times that of normal flies compared with 80 times when the strain was received (personal communication from It. V. King). The Orlando resistant strain loses resistance slowly but steadily if exposure to DDT ceases (72) . Keiding and Van Deurs (71) found that the resistance of their strain decreased considerably after 7 to 9 generations although it was still remarkably high. Knipling (77), speaking of work done at the Orlando laboratory, stated that 6 colonies of resistant flies collected in nature showed 20 to 5>0 times the resistance of normal flies to DDT. After 6 genera- - 9 - tions without exposure to DDT, some loss of resistance -was apparent but the extent of loss was extremely variable and most of the flies still showed some resistance. King (72) reports that nearly all wild resistant stocks tested showed some loss of resistance when exposure to DDT was stopped. The rate of loss of resistance was very inconsistent ♦ Bruce (30 ) on the other hand states "The several DDT-resistant strains of flies have shown no tendency to revert or lose their DDT tolerance through 3U generations of inbreeding." Bruce and Decker (33) state "All strains of DDT-resistant flies studied have retained their respective levels of tolerance when placed in a toxicant-free environment." They further state "It would appear by inference that if the chemical was entirely removed in the field, then the resistant strains might be effectively diluted by interbreeding with susceptible wild strains. This, of course, would be possible only if done before the entire population acquired a high degree of tolerance. Data obtained in a field survey may indicate this opportunity is rapidly passing if not gone." According to a later report, however, Bruce (6) now has experimental evidence that "dilution of one resistant strain with susceptible flies does not result in a strain with reduced resistant qualities." If this report is substan- tiated, the genetics involved are indeed unusual. Specificity of resistance Wilson and Gahan ( 170 ) tested the resistance of the Orlando resis- tant strain to several insecticides other than DDT, which was the insec- ticide used in the selection of the strain. The strain was more resistant to all the materials tested thanviras the regular colony. The materials were DDT, chlordane, pyrethrins plus $ percent of piperonyl butoxide (for composition see 162 ) , chlorinated camphene (toxaphene), rotenone, and thanite. Miller (109 ) compared the resistance to pyrethrins and pyrethrin mixtures of several strains of flies whose resistance had been developed by exposure to insecticides other than pyrethrins. No resis- tance to pyrethrins was found. By exposing DDT-resistant flies ( Orlando-Bel tsville resistant strain) to a mixture of DDT, chlordane, toxaphene, lindane, methoxychlor, and pyrethrins, Pratt and Babers (13U) developed a strain of flies that was resistant to these insecticides and to some extent to parathion as well. However, the resistance of this strain to the above insecticides was not statistically different from that of the Orlando-Belt sville resistant strain, whose resistance had been developed by exposure only to DDT. Bruce (29) reported that strains can be developed to be more or less specif icallyTesistant to one chemical or to closely related chemicals, but presented no data from which the degree of specificity could be de- termined. He found his resistant flies had a high tolerance for DDT and Rhothane D-3 (l,l-bis(p-chlorophenvl)-2,2-dicHoroethane) . A small amount of tolerance was evident for methoxychlor, and no "practical amount" for chlordane, heptachlor, toxaphene, aldrin, dieldrin, pyrenone, (piperonyl butoxide-pyrethrins mixture), or benzene hexachloride. He gave no data supporting these results. In his next paper Bruce (30) STATfc P^TBOARB - 10 - prosents data which indicate that the LD-50 of the most resistant strain is 776 times that of the normal strain for DDT, 1U«U times for methoxychlor, 3.2 times for lindane, 1.5 times for chlordane, 2.1 times for dieldrin, 1.6 times for piperonyl butoxide plus pyre thr ins, 2.5 times for toxaphene, 9.9 times for DDT plus methoxy- chlor, and 1.5 times for paraoxon (diethyl p-nitro phenyl phosphate). Here too then, some resistance for nonrelated insecticides seems apparent. In a third paper Bruce and Decker (33) state "The high resistance of the DDT strain I to DDT contributes resistance to other insecticides. DDT strains II and III with lower DDT re- sistance, show but little resistance to other insecticides." Earlier in the same paper, they state that DDT strain II was as resistant as DDT strain I. They state further "In general, the DDr -resistant strains exhibit a significant amount of tolerance for methoxychlor. On the other hand, the highly resistant methoxy- chlor strain shows little tolerance for DDT." Barber and Schnitt (13) also found no resistance to DDT in a strain of flies resistant to methoxychlor. Bruce and Decker continue "The lindane-resistant flies show some tolerance for chlordane, dieldrin, and toxaphene but no significant resistance to DDT. This may indicate some simi- larity in the physical or chemical structure in lindane, chlordane, dieldrin, and toxaphene molecules." The chemical structure and physical properties of the above compounds show no readily recog- nized similarities except for the presence of chlorine. The basis for this suggestion then is not clear to the writers. Sufficient research has not yet been done to determine whether or not these compounds show any similarity of physiological action. D'Alessandro §£ al. (U2) suggest that resistance to "other insecticides" may be due to inability to penetrate the chitinous covering rather than a resistance to the insecticide itself." They intimate that there is possibly a difference in reaction be- tween mists and residues. The reason for this suggestion is not clear, since the senior author and Scerrino (k3) had shown that resistant flies are resistant to both sprays and deposits. Numerous other authors have, of course, shown that resistance is independent of the method of application. The authors at that time did not have sufficient data to state whether or not their super-resistant strain was also resistant to chlordane. According to Brown and Rogers (28) the Ellenville DDT-resistant strain was markedly resistant to 1,1-dianisylneopentane, a compound in which three methyl groups are substituted for the three chlorine atoms in methoxychlor. Pimentel and Dewey ( 130 ) collected a field strain that was seven times as resistant as their normal strain on an LD-5C basis to DDT. There was possibly a slight resistance to methoxychlor, but none to dieldrin, gamma benzene hexachloride, aldrin, chlordane, or emulsifiable pyrenone ^piperonyl butoxide- pyrethrins mixture ) . - 11 - The strain of wild flies collected in Denmark by Keiding and Van Duers (71) was resistant to jDT and its analogs. No resistance to benzene hexachloride, chlordane, or toxaphene was evident. King and Gahan (7k) reported that the LT-70 (time of exposure in minutes re- quired for 70 percent mortality) for females of a wild strain (Lee) was 11 times that of females of the normal strain for DDT, 2.6 for methoxychlor, 1 for chlordane, and 0.6 for benzene hexachloride. March and Met calf (99) state "The resistance of the Bellflower strain to gamma benzene hexachloride, toxaphene, chlordan-type com- pounds, parathion, and pyrethrins is small or inappreciable." Later ( 102 ) they find that "There are now two types of resistant fly strains in the field in southern California. The Bellflower type is resistant only to DDT and related compounds, and the Pollard type is resistant to other chlorinated hydrocarbons such as lindane and dieldrin as well as to DDT and related compounds." The data presented, however, indicate that for the Bellflower strain for which specific resistance to DDT and its analogs is claimed, the LD-50 is 8 times that of the normal lab- oratory flies for benzene hexachloride, 2.8 times for toxaphene, 1.9 times for heptachlor, 2.2 times for aldrin, 1.6 times for dieldrin, 1.3 times for parathion, and 2.3 times for allethrin. Dr. Met calf , in a personal communication, explains that for "practical" purposes, they have not considered any insects as resistant until such resistance was 10 times that of the normal insects. These authors, therefore, evalu- ate resistance in terms of effectiveness of control rather than as a physiological difference between strains. Knipling (7§) reviewed some of the work that has been done on flies resistant to insecticides. He stated that tests have been made at Orlando to determine whether DDr-resistant flies were resistant to other insecticides, and the tests showed the flies had considerable resistance to methoxychlor, some slight resistance to chlordane, but little if any to benzene hexachloride. Apparently he referred only to wild resistant strains, since Wilson and Gahan ( 170 ), as indicated above, considered the "Orlando resistant strain" resistant to all other insec- ticides tested. Pimentel et al. ( 130 ) state ,T! ^hen DDT-resistant flies were subjected to lindane, dieldrin, or parathion pressure and not DDT, the DDT re- sistance did not decrease, and in some cases even increased. If this occurs in the field, then the control of flies with residual materials is not a bright prospect." That such events do occur in the field is indicated by the work reviewed by Knipling (79), who, again referring to tests made at Orlando on a strain of flies collected from a local dairy, stated that the approximate magnitude of resistance compared to normal flies was as follows (assuming a level of 1 for the normal strain): "DDT 8, methoxychlor U, chlordane U, toxaphene 8, lindane 8, dieldrin greater than 16, and aldrin greater than 16. The flies exhibited no resistance to parathion, pyrethrins plus piperonyl butoxide, and alleth- rin plus piperonyl butoxide." The dairy from which these flies were - 12 - collected had been treated with DDT from 19U5 through 1?U7. In 19U8 one application of methoxychlor was made. In the spring of 19U9 two treatments of pyrethrins plus piperonyl butoxide were made at 1-week intervals. Dieldrin emulsion was then used during the remainder of the 19h9 season. In 1°£0 fixed aerosol equipment was installed for the daily application of lindane aerosols. A pyre thrum-piper onyl butoxide aerosol was then substituted, but results were unsatisfactory as most of the flies left the building before knock-down occurred. Physiological and morphological differences between resistant and normal strains Wiesmann ( 168 ) reported that the "Arnas" strain of resistant house flies had thicker pulvilli and articular membranes of the joints than did his normal laboratory strain, thus indicating that resistance was due to failure of a lethal quantity of DDT to reach a site in which physiological action would occur. Bettini (18) and March and Metcalf (99) agreed that when DDT solutions were injected directly into the hemocoele of the flies, the same relative degree of resistance was noted, thus demonstrating that resistance was not due to lack of pene- tration alone. Hookum was quoted (7) as being unable to detect any structural differences between normal and resistant flies. D'Alessandro and his coworkers (U2) measured the diameter of the pulvilli of the pretarsi of normal and resistant flies and found that on the average, the diameter of the resistant insect's pulvilli was less than that of the susceptible flies. It was clear, however, that the great difference in suscepti- bility was not proportional to the size of the pulvilli. March and Lewallen (98) compared normal and resistant (Bellflower) strains and found no gross morphological differences. In general the tarsi of the Bellflower strain tended to be shorter than those of the normal flies. The tarsal widths were variable. No significant differences in the thickness of the cuticula from the first tarsal segment were found. Neither was the slight difference in life cycle noted significant. The average life cycle of the Bellflower strain was 18.1 days and that of the laboratory strain 17.9 days. Over a period of 7 months, no dif- ference in the weight of female flies was observed. Both strains were similarly immobilized by heat and cold, an indication that there was no significant difference in the general vigor of the two strains . The authors concluded then that neither thickness of the cuticula nor general vigor are concerned in the increased resistance of the Bellflower strain. "The differences in the dimensions of the tarsal segments of the two strains are not uniform enough or large enough to indicate they contribute to the resistance." Neri ( 123 ) observed that when DDT was applied to different portions of the body of the fly, the resulting mortality varied greatly. However, when resistant flies were used, except for applications to the antennae, no differences were noted. Applications of DDT to the antennae of re- sistant insects were in no cases lethal. Wilson (169) also found - 13 - variation in the mortalities obtained from applying pyrethrum solutions to varying parts of the bodies of normal flies but did not use resis- tant insects. Bruce (29) reported that the total development time from egg to adult of his - resistant strains appears to be 1 to 2 days longer than that of the normal laboratory strain. He concludes that under field conditions the resistant flies would then tend to disappear when in competition with flies of a shorter life cycle. Bruce did not elab- orate on the reasoning by which this conclusion was reached. Pimentel et al. ( 131 ) also report that the life cycle of several strains of resTstant flies was longer than that of non-resistant strains. The difference was most noticeable during the larval period and the length of this period seemed to be definitely related to the degree of resistance. "DDT-resistant flies from larvae which pupated the first I48 hours were less resistant to DDT than flies from larvae which pupated the last 1;8 hours in a given larval population for the strains studied. No significant differences between a highly DDT-resistant strain and a non-resistant strain were found in the number of eggs laid, length of egg stages, percent hatching, length of pupal stage, pupal weights, sex ratio, preoviposition period or length of adult stage." Babers and Pratt (10) found that the cholinesterase activity of the heads of resistant "(O r l an do-Beltsville) adult flies was consistently lower than that of the normal flies for the first 5 days after emergence. The enzymatic activity of both strains with respect to acetyl choline was inhibited by excess substrate. Differences due to strain were not determined since only 1 resistant and 1 normal strain were studied. Sacktor (])|J|) determined the cytochrome oxidase activity of a strain of resistant flies derived from the Ellenville strain of Barber and Schmitt (IJ4) and the normal laboratory strain of the Army Chemical Center. The enzyme activity of the brei from whole flies increased rapidly immediately after emergence. A peak was usually reached about the second day and a gradual decrease followed until about the fifth day. The changes after the first day were much more apparent in the resistant strain. The resistant strain consistently had a greater cytochrome oxidase activity than did the normal insects. In the normal strain, the male and female insects on a per fly basis had similar enzyme activities. However, on a weight basis, the male normal flies showed more oxidase activity than did the females. In the resistant strain, the females had about 20 percent more activity than did the males. Sternberg, Kearns, and Bruce ( 15U ) applied ethanol solutions of DDT externally to the pronotum of both normal and resistant flies (Bruce and Decker (33) multi-strain I) and after a specified time the remaining external DDT was removed by washing. The flies were then crushed, ex- tracted with ether, and the ether analyzed for DDT and the suspected meta- bolic products DDE (2,2-bis-(p-chlorophenyl)-l ,1 dichl oroethylene) and DDA (2,?-bis(2-chlorophenyl) acetic acid). The rate cf absorption of DDT - lU - was highest the first hour after application, possibly due to the effects of the alcohol used as solvent. The normal, or susceptible, strain absorbed DDT at a fairly constant rate for k hours. The rate then decreased sharply, coinciding with the appearance of paralysis. Unlike the susceptible flies, resistant flies continued to absorb DDT at a steadily diminishing rate so that by 28 hours after treatment, no DDT could be recovered from outside the body when the original dosage was 0.5 microgram per fly. In the susceptible flies, analysis showgd that there was a very slow metabolism of DDT but that neither DDE nor DDA were metabolic products. However, in the resistant strain, the absorbed DDT was rapidly metabolized, essentially to DDE but some DDA was also found. The evidence for tho identification of the metabolic products was based mainly on spectrophotometric data and cannot be considered as conclusive. After as reported below, found that both susceptible and resistant flies"~rapidly metabolized DDT to a product nontoxic to mosquito larvae. Sternberg and Kearns ( 153 ) found that both susceptible and resistant flies absorbed DDE at comparable rates and neither strain was able to further metabolize this compound. Both strains were also unable to further metabolize DDA. Degradation of DDT to DDE in vitro by the cuticle -hypo derm of resistant flies was demonstrated, but the conversion was below 20 percent. No other tissues tested were able to degrade the DDT. It was also found that resistant larvae could metabo- lize externally applied DDT to DDE and store it in considerable quantity. Susceptible larvae on the other hand metabolized a considerable portion of the DDT but the metabolite, as in the case of the adult, was unknown. It was not, however, DDE or DDA. Yilhen 10 micrograms of DDT were applied to each of 10 susceptible and 10 resistant larvae, the data presented indicate that the susceptible larvae absorbed the DDT at a very much greater rate than did the resistant larvae. The authors did not comment on this difference. When larvse were placed in medium containing DDT for varying periods and then analyzed for DDT and DDE, it was found that both DDE and DDT were present on the external portions of the resistant larvae. Only DDE was found internally in these larvae. On the external portion of susceptible larvae, only DDT was found, but internally both DDT and DDE were found. If the DDT-fed larvae were allowed to pupate and the pupae analyzed, similar results were obtained. The resistant adults emerged normally and contained DDE both externally and internally. The few susceptible adults that emerged contained DDE internally only. If DDT was applied topically to resistant pupae, they also were able to absorb and degrade it to DDE. Susceptible pupae were also able to de- grade DDT to DDE to some extent. In many of the above experiments, the quantities of DDT and DDE determined were considerably below those for which the analytical methods available are usually considered accurate. The authors, however, appar- ently encountered no difficulty in this respect. Perry and Hoskins ( 128 ) found that DDT absorbed by both normal and resistant flies was metabolized to what was first thought to be DDA (5) but later presumed to be DDE ( 128 ). This was contradictory to the results of Sternberg et al . ( 15U ) who found that only a resistant strain could metabolize DDT to DIE but agreed in general with the results reported by March and Metcalf ( 103 ). From the normal strain in one experiment Perry and Hoskins recovered 30 percent of the amount of DDT applied externally as internal DDT and 66 percent as DDE. With the resistant strain the applied DDT recovered as DDT was 7»5 percent and DDE 33 »1 percent. These authors conclude "It is obvious that increased ability to convert absorbed DDT into DDE is characteristic of the resistant strain.!' The authors analyzed both survivors and dead insects and found "In a given experiment, the survivors on the average always had converted more DDT - 16 - than those that had died, and hence ability to make this conversion is a major factor in variation in resistance within individuals of a giver- strain. " From dead resistant insects treated with DDT plus piperonyl cyclonene, only 3»h percent of the applied DDT was recovered as DDE con- trasted with the 17.5 percent without the synergist. They conclude then that in flies treated with DDT-synergist mixture the conversion to _Jj£ was largely prevented. They state also that the total recovered DDT from the resistant strains was at least a third less than that applied. Larch and Letcalf ( 102 ) tested the compounds commonly used as synergists for pyrethrum to determine the effect on DDT-resistant flies. Little or no effective synergism for DDT was observed. They ( 103 ) also studied the metabolism of DDT and found that the super-Bellflower strain of resistant flies was able to metabolize larger quantities of DDT at a greater rate than were normal flies. DCE was the principle degradation product and there was no evidence of the formation of DDA. The end products as yet have not been completely defined. Lindquist and his associates (93), using radioactive DDT and bio- assay methods, have found that DDT applied externally to flies is slowly absorbed. Once absorbed, however, it is rapidly metabolized by both normal and resistant (Orlando) insects to a material nontoxic to mosquito larvae . In the writers' laboratory (unpublished results) little difference in mortality was obtained when dosages varying from 5>0 micrograms of DDT per fly to 300 micrograms were applied externally to female resistant (0rlando-3eltsville) flies. The volume of solution applied was l.lli microliters per fly. From the first dosage the mortality at 2k hours was 18 percent and from the highest dose 16 percent. Only a small per- centage of the DDT absorbed was accounted for by analysis of extracts of the acetone -washed flies. From such extracts small amounts of DDT and a compound presumed to be DDE were recovered. In no case was T)Dk found. Results obtained from the analysis of normal flies have been inconclusive to date. In some cases small amounts of DDT and DDE have been recovered and in other cases neither material was found. The Schechter-Haller method of analysis which was used is not well adapted to the small quan- tities of DDT involved when normal flies are used, and some difficulty with interfering materials was encountered. Transmission of resistance Bruce (29) states "Further study with the resistant flies has shown by several reciprocal crosses of susceptible and resistant flies that resistance is neither dominant nor recessive, but rather than being a gene characteristic is probably a cytoplasmic characteristic." No data was given to support this statement. Bruce (29) and Bruce and Decker (33) interpret data obtained by crossing resistant insects with normal insects to mean that both male and female flies carry the resistant character. "Tolerance might simply be described as a multiple-gene character which causes indifferent physiological and perhaps morphological changes." Later in the same paper they state "There is reason to believe - 17 - that resistance to toxicants by house flies may be attributed to indif- ferent genetical changes, ephemeral acquired tolerance, morphological alterations or even changes in habit." The genetical data presented is limited to the LD-^O of the male and female progeny of the F]_, F2> and F15 generations resulting from the reciprocal crossing of a resistant and normal strain of flies. Bruce, in a discussion of Knipling's paper (78), commented that information on crossing strains shows not so much a Lendelian inheritance as a dilution or cytoplasmic inheritance. Bruce (29), Decker (6), and Bruce and Decker (33) observed that "fly resis- tance to DDT is not all chemical in nature" and that in certain insec- ticide-treated barns, house flies no longer rested on the treated surfaces but preferred to rest on the floor or on the animals. No data were presented. D'Alessandro and his coworkers (1|2) started a strain of susceptible flies from a single pair of flies captured in a locality which had not been treated with DDT. This strain ( partinicensis ) showed a relatively homogeneous sensitivity to DDT and all individuals were killed after a 35-minute exposure to BDT-treated cages. In a strain ( tiberina ) secured from Llissiroli's laboratory, the knock-down time varied between 20 min- utes and 2 hours and thus contained both susceptible and resistant indi- viduals. After repeated selection to eliminate the susceptible individu- als, a strain ( sup ertiber ina ) was obtained capable of living many days in DDT-coated cages. On the other hand, the attempt to select a resis- tant strain from partinicensis was unsuccessful. Apparently the strain had been deprived of resistant characters. The authors were unable to increase resistance in individual flies of the strain partinicens is by repeatedly exposing them to nonlethal doses of DDT. The toxicity of DDT was not accumulative after such series of exposure, an indication that the flies were able to detoxify or metabolize the material. The authors define as susceptible flies those unable to survive 35 minutes in a DDT-treated cage, as medium-resistant those able to survive 1 to 2 hours, and as super-resistant those able to survive 2i| hours in the cage. In a study of the transmission of the resistance, sufficient data was not obtained to establish a strict definition of a phenomena within the laws of heredity. However, the strain of susceptible flies remained homo- geneously susceptible. The strain with individuals of varying resistance so remained, and the super-resistant strain remained homogenously re- sistant. By pairing a super-resistant female and a sensitive male, 177 individuals were obtained, %,$ percent sensitive, 90 percent medium- resistant, and I4..5 percent super-resistant. The mixed generation thus produced was allowed to interbreed promiscuously and the F3 generation consisted of 21;. 2 percent susceptible, 73.1 percent medium-resistant, and 2.7 percent super-resistant. All the super-resistant flies were females. The arrival then of super-resistant female flies in a sensi- tive population would lead to a diffusion of the resistant character. The authors concluded that the phenomenon of DDT resistance does not represent a definite quantitative characteristic but one that is ex- tremely varied in degree. Also that by a series of selective isola- tions beginning with moderately resistant individuals, it is possible to - 18 - obtain generations that are highly resistant to the poison. The data presented in the manuscript are very meager and few of the techniques used are described. According to the authors, supporting data will be published elsewhere. Until such information is available the work cannot be properly evaluated. In the writers' laboratory, after about 92 generations of inten- sive selection by exposure of each generation of house flies to DDT sprays or residues, the population is still by no means homogeneous (unpublished results). Pupae to start this line of flies, now designated Orlando-Bel tsville resistant, were from the 55th generation of the Orlando resistant colony. For about the last 20 generations selection has been made by holding adult flies in cages coated with continuous deposits of DDT (about 2 grams per square foot) for several days after their emergence. The colony is maintained from the survivors. Although the percentage of survivors has increased, many flies are still killed by the treatment. Control of resistant flies That the extent of development of resistance to insecticides by the house fly was not quickly realized by all entomologists is indicated by Hatfield's (66) statement in ±9h9'. "Personally, I think DDT produced in 19U8 or 19h9 applied like the 19U6 and 19U7 DDT was applied, will give results comparable to the 19h6 or 19U7 results." He implied that if DDT was applied at the rate of 200 mg. of DDT per square foot it would give excellent control of resistant flies. The many laboratory reports of testing resistant strains against this and even higher dosages shows the error of his supposition. Following the failure of DDT to control flies in certain areas, recommendations were often made to change insecticides. Ayars (8) stated "Fortunately flies resistant to DDT are relatively susceptible to lin- dane, chlordane, toxaphene, and dieldrin. ... Entomologists expect lindane to be effective against house flies for about 2 years. 3y about 1952 or 1953 perhaps a new insecticide will be needed." Losna (115, 116 , 117 ), Patrissi, Barbieri, and Bessler ( 127 ), Bettini and Barachini (20), and others (1, 2, 19, j4, 57, 141) a^ree^ that the DDT- resistant flies could be effectively controlled with chlordane, lindane, or toxaphene. Decker (6) proposed alternate use of lindane, chlordan, and dieldrin. Instead of the 2 years' effectiveness predicted by Ayars for lindane, March and Metcalf (IOC, 101 , 102 ) found that in the field several DDT-resistant strains had in a few months become resistant to lindane and dieldrin as well as to DDT. They state (101) "These new developments, though as yet only on a limited scale, indicate that eventually standard fly control procedures may have to be re-evaluated and revised, and that emphasis may have to be placed on sanitary measures, repellent materials, and space sprays, rather than on residual appli- cation of insecticides." Fjelddalen (52) discussed DDf resistance and the use of methoxychlor as a control chemical for the reristant flies. - 19 - Gahan and Weir (55) used gamma benzene hexachloride for the control of L'.usca domestica viclna Lacq. in Egypt. It was not stated whether or not other insecticides had ever been used in the area. For the first 10 months, fresh applications were made once a month with very effective results. After 1 year, the intervals between treatments were shortened to 1 week and even then effective control was not obtained. Laboratory tests confirmed the field tests and showed that resistance had developed. Mo other insecticides were tested. The authors conclude that "the re- sults obtained . . . indicate that any benefit derived from a change to this insecticide (in order to control DDT-resistant flies) might be only temporary." Bruce (30) stated that the substitution of methoxychlor for use against DDT-resistant flies gave unsatisfactory results due to the rapid acquisition of tolerance for the compound. Bruce (6) suggested that "extensive use of one insecticide will produce a tolerant strain more quickly," and later he (31) stated "Probably the development of insecti- cide tolerance can be avoided by not relying upon a single highly effec- tive material for the control of any particular pest. Use a chemical to supplement other kinds of control methods and change chemicals as soon as there is any evidence of resistance developing to an insecti- cide." To date little evidence has appeared which indicates that chang- ing residual type insecticides under natural conditions will in any way prevent the appearance of resistant strains. Catalano and Llariani (39) report that the addition of fats or oils to DDT solvents remarkably increases the toxicity of the material and that "flies that are most resistant to residuums of the usual solutions of DDT behave like sensitive flies on being exposed to residuums obtained from solvents containing olio. Davidovici et al. (U5) also report that the addition of lanoline to DDT-kerosene solutions enhances the toxicity of the residue to resistant flies and mosquitoes. The increased toxicity was lost after about 3 weeks when crystals of DDT began to appear in the residues. D'Alessandro et al. (k2) report that 5 gamma of DDT in a fat solution is lethal to super-resistant flies that, according to Bettini (18), are able to survive 5 gamma in olive oil when injected directly into the thorax. Perry and Hoskins ( 128 ) found that piperonyl cyclonene (for chem- ical composition see 162 ) increased the toxicity of DDT for two strains of resistant flies. Thus 5 micrograms of DDT alone per fly gave U2.1 percent mortality and 5 micrograms of DDT plus 25 micrograms of piper- onyl cyclonene gave 87*9 percent. If the synergist was increased to 50 micrograms, the mortality dropped to 76.8 percent. With the normal strain, no synergism was observed. This work has not yet been confirmed. Because DDT failed to control i'lies due to the development of resistance and partly because of recommendations that DDT be avoided in dairy barns or on dairy animals, Hansens (62) investigated several other insecticides. Lindane or methoxychlor was recommended as replace- ment for DDT in these circumstances. - 20 - According to Upholt ( l6o ) , "the often expressed opinion that the situation could be solved by rotating insecticides is ... without foundation." He believes that resistant strains whose degree of re- sistance is low will lose resistance if kept out of contact with in- secticides. Practically, "it is a vain hope to expect under field conditions to maintain any population of flies out of contact with DDT, or with any other insecticide, which is used to such an extent as to become a factor in resistance." A strain of flies from an area in which resistance was pronounced, quickly lost their resistance in the laboratory when not exposed to DDT. In the field, however, after more than a year after stoppage of the DDT program, the resistance was not diminished. The explanation of course was that the stoppage of the use of DDT for fly control did not stop the use of DDT in the area for other agricultural pests and did not remove the DDT from any areas in which large quantities had been applied. In Upholt' s opinion, the development of resistant insects had been greatly accelerated by the substitution of DDT for adequate sanitation as a fly control measure. Other Species In Illinois, filter flies ( Psychoda alte^nata Sty) were no longer controlled by treatment of the larval habitat with DDT, according to Bruce (30) and Bruce and Decker (33) , after 2 years of excellent con- trol. The first chlordane treatment was highly successful, the second only moderately so, and the third and fourth were near failures. Schaerffenberg ( 11+5 ) studied the absorption of DDT and the resistance to the compound in certain May beetles. Contrary to results in the United States, Raucourt (136) reported that .lead arsenate was still very effective in France for the control of the codling moth. According to him, the failure of lead arsenate to control the insect in the United States had not, in published reports, been attributed to resistance. This statement is in error, since several publications have appeared, as noted in the earlier review (9), on the subject of such resistance. More recently Palm ( 126 ) states that the entire Hudson Valley orchard area has developed a codling moth problem that lead arsenate will not handle. He quotes S. W. Harman as finding in 1°U5> that only the very high prices received for the fruit at that time enabled growers in western New York to make a profit because of the failure of lead arsenate to control the insect. Up to 19k9 it was the opinion of several entomologists familiar with the subject (3) that no case of codling moth resistance to DDT or of other DDT- resistant fruit insects had been proved. No more recent references have been noted. Pielou ( 129 ) states that "Investigations are in progress to de- termine the possibility of breeding strains of beneficial insects resistant to DDT and other materials." These attests have not pro- gressed far enough to predict their outcome. - 21 - That resistance to lead arsenate by the peach twig borer ( Anarsia lineatella Zell.) had developed in a small area in California was sus- pected by Bailey (11). Laboratory experiments by Summers ( 15>5 ) tended to confirm these suspicions. No tests have been conducted with other in- ecticides, nor is it clear whether economic control of the insect with lead arsenate is still indicated. Shepard (II4.6 , p. 327) quotes 0. Peters as finding that an exposure to a small quantity of hydrocyanic acid causes a stupefying effect on the granary weevil ( Sitophilus granarius (L.)) that results in cessa- tion of respiration. The cyanide can thus only enter the insect by diffusion and hence the high resistance of the species. Kono (82) found that prefumigation of the rice weevil ( Sitophilus oryza (L.)) with low doses of carbon disulfide for 2k hours lowers sus- ceptibility. There was no significant difference in oxygen consumption. Berim and Edelman (16) studied the resistance of several insects to benzene hexachloride and DDT and found that it varied greatly with time of day and the season. Ricci ( 138 ) studied the action of DDT on the oriental cockroach (ftlatta orientalis L. ) and found that strains obtained from areas pre- viously "treated with DDT showed increased resistance to the insecticide. In a colony of body lice ( Pedi cuius human us corporis Deg.), after the exposure of 8 generations to DDr, no increased resistance to the insecticide was noted (72). According to D'Alessandro et al. (h£) , Mariani found that after two years of treatment with DDI the dictyospermum scale (Chrysomphalus dictyospermi (Morg ♦ ) ) had become resistant to a considerable degree ~ After the first treatment with DDT only 2 to 5 percent survived, but after 2 years of treatment 25' to 30 percent survived equivalent dosages. Johnson and Hill (69) report that in Hawaii bed bugs (Cimex lectularius L.) have become resistant to DDT. Stanley W. Bromley (personal communication) , from the results of field tests, has concluded that the bark beetle (Scolytus multistriatus (Marsh.)), the carrier of Dutch elm disease, has developed a considerable degree of resistance to DDT and to a lesser extent to methoxychlor. The beetles were from an area that had been treated with DDT for several seasons. According to Jayewickreme (68) field-collected larvae of Anopheles subpictus Grassi showed resistance" in laboratory tests to films of mineral oil (Shell Malariol). The resistance was attributed to in- creased vigor caused by favorable environmental conditions. A strain of the cockroach ( Blattella genaanica (L.)) resistant to DDT has been developed by J. L. Grayson (personal communication). - 22 - Following 7 generations of selective exposure to DDT, la. 3 times as much DDT was required for an LD-£C as was necessary in the unselected insects. Follov/ing 7 generations of selective exposure to benzene hexachloride . only a slight increase in tolerance was noted. In the DDT-resistant insects, no difference in reproductive performance was noted but the f-ven^e number of nymphs per egg case was lower in the DDT-resistant strain than in the normal strain. A species of bug, species not given but apparently the large milk- weed bug, Cn copeltus fasciatus (Dall.), has been reported ~oy Turner (l£9) as developing resistance to nicotine in the laboratory. Discussion Icelander ( 108 ) is generally credited with the first publication on insects showing resistance to insecticides. According to Bruce (32) » Piper in 1913 found the San Jose scale to be resistant to lime-sulfur. The reviewers have searched the usual reference sources but have been unable to locate a reference to this work by Piper. However, it appears that John 3. Smith ( 15>2 ) observed resistance some years previously. Smith in 1337 stated "Insects which succumb readily to kerosene in the Atlantic States defy it absolutely in Colorado, while we are just as likely to find the food plant much more sensitive to it. ./ashes that easily destroy the San Jose scale in California are ridiculously ineffec- tive in the Atlantic States. This very scale is changing its life his+oryand habits in the East materially in several directions. I will venture the prediction that in half a dozen years it will not be considered a first class pest in Mew Jersey, though I would not like to extend this prophecy to localities with which I am less familiar." I.onro ( 111 ) has discussed the subject of resistant insects and speculates to some extent on various aspects of the problem. Variations within insect species was suggested in 186U by Walsh (163). Certain wood-boring and plant-feeding beetles could to some ex- tent be divided into groups by food preference. They occurred in the same locality and had definite differences in biology, but often showed no or at the most very minor structural differences. Thorpe ( l£8 ) and later Smith (151) reviewed the occurrence of biological races in insects. In some insect species, the European fruit lecanium ( Lecanium corni Bouche), for example, the external appearance varies considerably depending on the host plant. On the elm this scale has a large pruinose form and on the arbutus it is small and shiny. Ebeling (U°), although unsuccessful in the field, »vas able to transfer the insect from its normal host to other plants in the laboratory. Succeeding generations conformed to the appearance of those insects regularly found on the new host plant. No report concerning the development of resistance to insecticides by the hessian fly ( rhytophaga destructor ^Say)) has been observed. Some of the work with this insect, however, seems to warrant comment in this review, because of the possible bearing on the resistance problem. Data presented by Painter ( 12!? ) in 193C tended to show that the hessian fly population in any one locality is a mixture of two or more genetically distinct strains that seem to differ only in their ability to infest dif- ferent varieties of wheat. Cartwright and Noble (38) further explored - 23 - this observation and found that the Dawson variety of wheat was resis- tant to the hessian fly in California and Kansas but was susceptible to the fly in Indiana. Thus the California and Indiana strains of flies seem to be physiologically different races. No reference has been noted indicating the effect of testing the California strain on Dawson wheat in Indiana or that of the Indiana strain on Dawson wheat in California. There have been a number of reports of insects, particularly house flies, having specific resistance to one insecticide or its analogs. Other reports indicate that high resistance to one insecticide confers some resistance to most other insecticides, regardless of structure. The two apparently divergent views would be reconciled to a considerable degree if a standard definition of the term resistance and uniform methods of testing were adopted. Quayle ( 135 ) proposed that an insect species be termed resistant if there was a decreased effectiveness of an insecticide compared with that of former years. This definition seems to the writers to infer at least a direct relationship of resis- tance to economic control. Tests under field conditions have many variables that determine to a considerable degree the effectiveness of an insecticide. Under laboratory conditions several of the more important variables are eliminated, temperature, humidity and nutrition of the insects being relatively constant. The writers have considered that two strains of insects vary in resistance if, when tested under similar conditions, there is a statistically significant difference in their response to a given quantity of insecticide. Physiologically, a slight variation in response may be of considerable importance but at the same time have no appreciable bearing on the effectiveness of the material as an economic poison, since complete control may still be obtained with readily usable quantities. In some cases the dosage of insecticide used has been such that all insects of the strains tested were killed and the conclusion reached that there was no difference in resistance between the two strains. It is obvious that such data cannot be considered a valid test for resis- tance. Small differences in rearing methods might often account for some differences in results between laboratories. Swingle ( 15>6 ), Larkos and Campbell ( I0I4. ), and McGovran and Gersdorff ( 107 ) have pointed out the effect of variations in susceptibility resulting in changes of diet. It is also necessary of course that temperatures to which the insects are exposed during the tests be relatively constant and also comparable to those used in other laboratories. Lindquist and his associates (92, 95>) have shown the great effect of temperature on the knock-down and mor- tality obtained from given doses of DDT. Many factors other than resistance often cause the failure of an insecticide to effect control. The term resistance then should not indicate in any way failure to obtain economic control. When house fly - 2ii - resistance to DD 111 was first observed, the failure to recognize as danger signals the small increases in resistance to other insecticides led to many such statements as "DDT-resistant flies are readily con- trolled by", etc. Such statements^ while perfectly correct at the time, in only a few instances carried warnings that such control would probably be only temporary. Indeed, if the data of V/ilson and Gahan ( 170 ) showing that the Orlando resistant strain was al60 resistant to several other unrelated insecticides had not been largely ignored, re- searchers concerned with the field control of flies might have recog- nized that substitution of other highly toxic residual insecticides would result only in short term effectiveness. At present, however, it seems that almost any positive statement concerning resistance will probably have to be rescinded or modified. The thought has been expressed in several publications that resistance follows repeated exposure to insecticides. Resistance of the red scale to hydrocyanic acid appeared in several isolated areas in California in 1916. Effective control of these resistant insects was obtained with oil sprays and the use of hydrocyanic acid was gradually supplanted by them. One would have expected that the resistance to hydrocyanic acid would disappear after the continued use of oil. However, the scales in these areas today are still resistant to hydrocyanic acid and the areas are appreciably larger than they were in l°l6. Oil sprays are still used on them, and no resistance to the oil has apparently developed. Also after 2£ to 30 years use of oil sprays against the San Jose scale, there is no evidence that lime-sulfur resistant scales have developed resistance to oil sprays. However, as Upholt (l6o) points out, the scale insect as well as the codling moth are highly specialized species restricted to a limited number of host plants. Especially with the scale insect, the species is not particularly adaptable. On the other hand, the house fly hes adapted itself to all conditions to which man has adapted himself. Thus the house fly might be expected to develop resistance, w}-preas *ith the scale inserts the development of resistance to hydrocyanic acid might be considered an accident of nature. Such a specialized insect would not be expected to rapidly adapt itself to changes in environment. "The house fly has a very high biotic potential, and when a population seems essentially to be wiped out, a very few survivors are able to restore the popula- tion rapidly to its normal size." "Adaptability is associated with a high frequency of gene mutation: and when these adaptations do become inherited through mutations, we have a situation which may be similar to the observed resistance in flies." Citrus thrips became highly resistant to tartar emetic in a comparatively short time but have not become resistant to other insecticides used before or since the intro- duction of tartar emetic. Another anomaly is the arsenic-resistant tick. Apparently only one species is so far involved. Yet the infested cattle have been heavily infested with several other species of ticks that, although treated in the same manner, are still readily susceptible to arsenic. - 25 - ■Why 3 too, should the resistance largely disappear when the tick-infested cattle were moved inland? Similar anomalies occur in the case of the house fly. Strains that maintained resistance on the West coast for many generations lost the resistance rather rapidly when pupae were sent East to start a colony. Other cases have occurred in which appar- ently contradictory results were obtained when pupae or eggs from re- sistant strains of house flies were sent from one laboratory to another. Errors in sampling and even misidentification of strains have been sus- pected. To the writers, it seems more logical to assume that the results as obtained are correct and that more attention should be given to the indications and interpretations of such data. The great difference in response to insecticides by the various populations of flies is emphasized by Upholt (l6o). He reports that in two areas in which dieldrin applications were made late in 19h9 or early 19$0 } a high degree of resistance was soon developed and essen- tially eliminated the practicability of residual chemical control of flies in those areas with known insecticides. However, there are areas other than the above two in which dieldrin has been used for a longer period and with more frequent applications but no resistance is appar- ent. All populations therefore do not develop resistance to dieldrin at the same rate. but it seems probable that within a year from the time of its first use resistance can be expected. At times positive statements have been widely disseminated that, from the results published in the literature, seem to have little or no data to support them. In a recent editorial (Ij.) it is stated "How the latest reports hint that after 20 or so generations of immunity (to insecticides) by flies, DDT and other chlorinated insecticides again become effective." There has been no evidence published that in the slightest degree seems to indicate such a trend. It may be that the author intended to say "20 or so generations without exposure to in- secticides" but that is not what was published. Plackett and Hewlett ( 132 ) and Hewlett and Plackett (6?) statis- tically treated data obtained following the application of mixtures of two insecticides to insects, and discussed the effects on resistant insects of such mixtures if the two toxicants acted independ- ently. According to their reasoning, if two insecticides having in- dependent physiological action were applied jointly to insect popula- tions, the development of strains of insects highly resistant to either insecticide would be prevented because the insects most resistant to each toxicant would be killed: only those insects with intermediate resistance would survive and any survivors could be expected to give rise to descendants also of intermediate resistance. No error in the statistical treatment is at once apparenti but it is difficult to under- stand how a dose of insecticide which kills the tougher individuals of a population would fail to kill the 7/eaker ones first. For many years insecticides have for convenience been classified according to their generally accepted mode of entry into the insect as - 26 - stomach poisons, contact poisons, or fumigants. The implication has sometimes been made erroneously that the mode of entry really consti- tutes mode of action. There seems to be widespread misconception that changing the mode of entry of the insecticide into resistant flies would drastically change or even overcome resistance. Since it has been shown that resistance is maintained when the insecticide is in- jected, physical factors such as thickness of integument or lack of penetration are not the cause of resistance in house flies and once an insecticide reaches its site of action, no evidence has ever been presented to indicate that mode of action varies with mode of entry. The role that the mixing of various strains of insects in the field plays in the development of resistance has yet to be determined. However, because Bishopp and Laake (21) reported that marked house flies were recovered as far as 13 to Ik miles from the point of re- lease, considerable effect must be expected. In other species of insects even greater dispersions are found. No adequate genetical study of resistance in the house fly has yet been reported in the literature. Eruce (29), Bruce in Knipling (78), and Bruce and Decker (33) presented a hypothesis for the method of transmission of inheritance. To date, however, their published data consist only in the determination of dosage-mortalities for the Fi, F2t an d Fl5> generations resulting from the reciprocal crosses of a strain of normal with a resistant strain of flies. Whether such crosses were made with individuals or whether they were mass crossings is not clear. Neither is it clear how the meager data presented justify the conclus- ions made. According to D'Alessandro and his associates (U2) DDT sensitivity and DDT resistance are hereditarily transmitted to the progeny and apparent modifications are not met with in the course of many genera- tions. Here again, the data so far presented do not seem sufficient to justify the drawing of definite conclusions. The difficulties involved in the interpretation of uata obtained from mass crossings are considerable. Several questions that rust be considered in mass crossing are: What percentage of the population is resistant? What is the percentage of susceptible individuals? These questions apply equally to the normal strain, for it is well known that all insects of a given population do not respond in the same way to the same dose of insecticide. Are the results obtained from mass crossings with subsequent determination of the LD-5>0 of the Fi population com- parable to results from individual crossings with determination of the resistance of each individual progeny? How can the resistance of an individual insect be determined? It has been generally accepted that antigens, the substances that disturb protein synthesis in the organism in such a way that antibodies are produced, are macromolecules and are generally proteins or protein - 27 - complexes. However, Loiseleur (96 , 97) has reported that antibodies are formed in the serum against such simple substances as ethyl alcohol, ethylamine, sodium acetate, xylose, and arginine. The presence of the antibodies was demonstrated, except in the case of sodium acetate and ethylamine, by an increased viscosity of the serum after addition of the injected substance. When sodium acetate and ethylamine were used, flocculation was observed. Viscosity tests are generally not accepted as conclusive proof of the formation of antibodies. In the event that Loiseleur is correct in the interpreation of his results, his findings may elucidate to a considerable degree the phenomena of the development of resistance to various materials by vertebrates and possibly inver- tebrates as well. Such an explanation might also explain certain cases of cross tolerance, since the injection of a uniform antigen solution leads to the formation of multiple antibodies directed against the dif- ferent determinant groups of the antigen molecule. In the recent literature there are numerous references to "DDT and other chlorinated hydrocarbon insecticides." Often it is obvious that the author considers that "chlorinated hydrocarbon insecticides" are closely related chemically and should be expected to behave in a similar manner physiologically. Possibly this reasoning is based on the assump- tion of the correctness of the hypothesis of Martin and Wain ( lCj? ) con- cerning the mode of action of DDT. According to this hypothesis, the chlorophenyl rings confer lipoid solubility on the molecule whereas the remainder of the molecule liberates hydrochloric acid at vital centers and is responsible for toxicity. Although such a possibility must be considered, little experimental data have appeared in its support and much of the data suggest that the theory is not correct. Dieldrin and aldrin, for example, are excellent chlorinated hydrocarbon insecticides but are little affected by strong alkali in vitro. Such stability in vitro does not preclude breakdown in vivo but the probability is strong. Admitting for a moment the possibility that the toxicity of DDT was due to the liberation of hydrochloric acid, it would seem probable that DDT-resistant flies would be generally better buffered than the susceptible insects. The authors and M. Beroza of the division of Insecticide Investigations (unpublished results) therefore ran titra- tion curves on the brei obtained by grinding whole flies in distilled water under deobase. No difference was noted between the curves ob- tained from the brei of normal ( Belts ville normal) and resistant flies ( Orlando -Belts ville) . Further doubt on the validity of the theory arises when one considers the isomers of DDT. It is well known that a shift of the chlorine atom on one of the benzene rings of the L'DT molecule to any other position markedly lowers the insecticidal value of the compound but these isomers readily de- hydrochlorinate . Several hundred analogs of DDT have been prepared and in none of them has the insecticidal value of DDT been approached. Methoxychlor and the fluorine analog of DDT are also often included in lists of such "chlorinated hydrocarbons." To assume that all chlori- nated hydrocarbons should act pysiologically in a similar manner is - 28 - about as logical as assuming that all plant material should act on in- sects in a manner similar to pyrethrum. The situation is further confused by the several reports that methoxyclor-resistant flies are not resistant to DDT, but that DDT-resistant flies usually show high cross tolerance for the methoxy compound. Haarer (60), speaking of the development of resistance to benzene hexachloride by the already arsenic-resistant blue tick, states: "It seemed that news such as this quietly trickling through scientific circles, and appearing in small sections of the overseas press, is enough to make headlines as big as those concerning the new atomic bomb, if only the significance of the matter were properly understood." In the United States flies, mosquitoes, cockroaches, and bark beetles have been definitely reported as developing resistance to the organic insecticides currently used in large quantities for the control of in- sects by contact with residual deposits. The development of resistance to benzene hexachloride by the blue tick in South Africa, of Drosophila to DFDT, and of bed bugs to DDT, proves that other species can and have developed resistance, and indicates further that the development of similar resistance by more species can be expected to occur in nature at any time. It should also be pointed out that all work so far reported on the development of resistance in insects has been done on populations; never, apparently, has resistance been developed in an individual insect. In conclusion it may be pointed out that the phenomenon of resist- ance is not much better understood now than it was at the time of the first report of its occurrence. The method of development or of trans- mission is not clear, and no adequate suggestion has been made as to what should be done to overcome the resistance. An extensive and coordi- nated long-range program seems to be indicated, but currently most of the research efforts seem to be directed toward temporary expedients rather than toward the solution of the problem. If one thing in the picture is clear, it may be summed up in Decker's suggestion (i|6) — "Remember what has happened to DDT and proceed cautiously in the use of other insecti- cides." It is obvious from the various reports reviewed that there is var- iation between the response of the various strains, both normal and re- sistant, to the several insecticides. It is not possible, apparently to make any statement that will cover all cases but the following gen- eralities begin to appear : 1. A very high level of resistance to one insecticide confers a certain amount of cross tolerance for other insecticides of widely dis- similar structure. 2. When the level of resistance is low, the resistance is almost - 29 - specific and is limited to other insecticides of closely related structure. 3» Strains of house flies resistant to all insecticides for which the attempt was made have been developed in the laboratory. This in- cludes materials of plant origin as well as synthetic organic materials. U» The resistance level of wild strains of house flies is by no means as high as that of laboratory-developed strains. 5» The degree of resistance in house flies varies considerably from generation to generation. 6, Most strains of resistant house flies, either laboratory or field, tend to lose their resistance if exposure to the chemical ceases, but some strains maintain resistance for many generations. 7. Adequate evidence for the formulation of a theory concerning mode of transmission of resistance from parent to offspring has not been presented. 8, Once resistance to any insecticide is developed by house flies, the development of resistance to other insecticides proceeds at an accelerated rate. 9. Resistance by other species of economic importance will probably be developed, 10. The several resistant strains of house flies do not behave in an analogous manner, and progeny from the same parent strain often have different responses to insecticides when tested in other laboratories. It is almost impossible, therefore, to compare directly results between laboratories. 11. The resistance of house flies is not due to the failure of the insecticide to penetrate the cuticle since resistance is evident when the insecticide is injected directly into the body cavity. Resistance is also apparent regardless of whether the insecticide is applied as a mist, dust, or residue. -30 - Literature Cited (1) Anonymous. 19U9. Methoxychlor, a manual of technical data. E. I. du Pont de Nemours & Co., July 1. (2) (3) 00 (5) (6) (7) 19U9. Controlling flies with 3HC. Agr. Chem. ^(l): 30-32, 73, 75. 19l|.9. Are insects rallying against DDT. Amer.. Fruit Growers 69(2): 17, 39. 19^0. How's your supply of chemicals? Do you have enough? Pest Control 18(9): 7. 1950. Pepper extract lets DDT kill super-flies. Farm Jour., March: 17. 19^0. Switch chemicals in 195>0 Dr. Decker says at Purdue con- ference. Pest Control 18(3): 12, li|, 16, 18, 20. 19^0. Says tolerant fly structures same as others. Pest Control 18(H): 22. (8) Avars, James S. 19^0. Flies thrive on DDT? Kill them with lindane. Successful Farming J4-8 ( 7 ) : 18, I0J4. (9) Babers, Frank H. 19^9. Development of insect resistance to insecticides. U.S. Bur. Ent. and Plant c^uar. 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