iHl MINT \ J L W “WNW I1 I w J \ E M J 11mm;mummmnmnmnm THEE”? 3 1293 01074 5515 This is to certify that the thesis entitled . CHARACTERISTICS OF DDT- INDUCED PYRETHRO ID RESISTANCE IN THE GERMAN COCKROACH, BLATTELLA GERMANICA presented by Jeffrey Graham Scott has been accepted towards fulfillment of the requirements for M. S . degree in Entomology @me/CWM Major professor Date_Noyemher_LL._l981 0-7639 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE I DATE DUE DATE DUE OWMm‘ 1]” WWW“ CHARACTERISTICS OF DDT-INDUCED PYRETHROID RESISTANCE IN THE GERMAN COCKROACH, BLATTELLA GERMANICA By Jeffrey Graham Scott A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of M ASTER OF SCIENCE Department of Entomology 1981 ABSTRACT CHARACTERISTICS OF DDT-INDUCED PYRETHROID RESISTANCE IN THE GERMAN COCKROACH, BLATTELLA GERMANICA by Jeffrey Graham Scott The mechanism of cross-resistance between DDT and pyrethroids was studied using a DDT-resistant (VPIDLS) strain of Blattella germanica, a strain never exposed to pyrethroid selection. The resistance mechanism is likely a kdr type for the following reasons: (1) decreased sensitivity of the nervous system to DDT and permethrin, (2) cross-resistance to methoxychlor, and (3) lack of difference in the rate of permethrin metabolism between resistant and susceptible cockroaches in yit_r_o_. Kdr did not confer substantial cross-resistance to the recently developed synthetic pyrethroids, cypermethrin and decamethrin. The expression of poisoning by cypermethrin and decamethrin is different from pyrethrin and its synthetic analogue, allethrin, by the following criteria: (1) electrophysiological symptoms, (2) temperature effects on toxicity, and (3) the lack of substantial cross-resistance of kdr roaches to decamethrin and cypermethrin. Such differences suggest that at least a part of the mechanism(s) of action of these newly developed pyrethroids differs from the old type pyrethrin analogs. To Mom and Dad ii ACKNOWLEDGEM ENTS I would like to express my sincere appreciation to Dr. Fumio Matsumura for all his advice and direction during the course of this investigation. To Carol Thomson for her patience, support, faith and encouragement - thank you. I also wish to express my thanks to Dr. Brian Croft and Dr. Ralph Pax for all they taught me and for their assistance on numerous occasions. iii TABLE OF CONTENTS LIST OF TABLES .................... . . V LIST OF FIGURES ......................... Vi INTRODUCTION .................... . 1 MATERIALS AND METHODS .................... 3 Materials . . . ....................... 3 Animals and Chemicals ,,,,,,,,,, , ,,,,, 3 Methods ........................... 7 Susceptibility Studies ................... 7 Surface contact method . . . . , , , , ,,,,,,, 7 Abdominal topical application ......... , . . , 7 Cural topical application . . ,,,,,,,,,,,,, 8 Calculation ..... . . ........ . . 8 Metabolism Study ..................... 9 Electrophysiological Studies ............ , 10 Nerve sensitivity ,,,,,,,,,,,,,,,,,, 10 Responses to pesticides ,,,,,,,,,,,,, 11 RESULTS ............................ 12 Susceptibility Studies ..................... 12 Metabolism of Permethrin in Susceptible and Resistant Cockroaches ........ . ...... . . 17 Electrophysiological Studies .................. 26 NerveSensitivity...... ...... .....26 Responses to Pesticides .................. 26 DISCUSSION . . . . . . . .................... 34 REFERENCES . ........ . . . ............... 37 iv 10 11 12 LIST OF TABLES Sources and Purity of Compounds Studied ........... . Selection Process for VPIDLS ....... . ......... Susceptibility of a CSMA Strain of E. germanica to DDT and Six Pyrethroids byaSurface Contact Method . . . . . . . . . . . . Susceptibility of Four Strains of E. germanica to Methoxychlor in the Presence and Absence of the Synergist Piperonyl Butoxide (6 mg/l pint mason jar) byaSurface Contact Method . . . . . . . . . . Susceptibility of Five Strains of _E. germanica to Dieldrin (2 mg/l pint mason jar) by a Surface Contact Method . . . . . . . . . . . . . Comparative Toxicities of DDT and Six Pyrethroids to a CSMA Strain of German Cockroach by Topical Application to the Abdomen . . . Effect of Temperature on Toxicity of Allethrin and Cypermethrin by Cural Topical Application . . . . . . ........ . . . . . In vitro Metabolism of Permethrin by Microsomal Preparation from the Whole Body (minus head, legs and wings) Homogenate: the Data are Expressed as the Percentages of Originally-added Permethrin Remaining in the Incubate . . . . . . . ..... . ...... Comparison of Cytochrome Levels in a DDT-resistant and Susceptible Strain of Blattella germanica . . . . . . . . . . . . . . . . . Time to Onset of Nerve Poisoning Symptoms in the Abdominal Nerve Cord of Susceptible and Resistant German Cockroaches ...... Time to Onset of Nerve Poisoning Symptoms in the Abdominal Nerve Cord of German Cockroaches Before and After One Generation of SelectionbyDDT................ ..... . Effects of the Synthetic Pyrethroids Allethrin and Cypermethrin on the Spontaneous Activity of the Isolated American Cockroach Abdominal Nerve Cord . . . . . . ......... . . . . . 15 16 18 23 24 25 27 31 32 35 10 LIST OF FIGURES Chemical structures of DDT and six pyrethroid insecticides. . . . . Comparison of mortality resistance ratios in four substrains of B. germanica to DDT and six pyrethroid insecticides by a surface contact method, with P, 1, 2, and 3 referring to VPIDLS parental and successive DDT-selected generations, G1, G2, and G3, respectively. . Comparison of knockdown resistance ratios in four substrains of B. germanica to DDT and six pyrethroid insecticides by a surface contact method, with P, 1, 2, and 3 referring to VPIDLS parental and successive DDT-selected generations, G1, G2, and G3, respectively. . KD -time curves of allethrin by leg (---) and abdominal (——) topicalapplication. . . . . . ...... . . . . . . . . . . . KD -time curves of cypermethrin by leg (---) and abdominal ( —) top1calapplication....................... KD -time curves of allethrin at 31.0% (o), 24.5°c (o), and 11°C (A)50 KD -time curve for cypermethrin at 31.00C (O), 24.50C (a), and 1103(1). The pattern of a typical DDT-induced repetitive discharge as viewed by a slow scan (A) and a fast scan (b). Note the homogeniety in amplitude and frequency of individual action potentials. . . . . . . The pattern of a permethrin-induced repetitive discharge. These types of discharges are commonly observed at the beginning of the permethrin poisoning. (A) Entire burst of discharges viewed by a slow scan; (B) A front portion of the burst of discharge by a fast scan; (C) The tail portion of the discharge scanned by the same speed as (B). Note the difference in frequency of (B) and (C). . . . . . . . . . The pattern of a second type of permethrin-induced repetitive discharge which was often observed at the later part of nerve poisoning. (A) A slow scan indicating the gradual decrease in peak amplitudes; (B) A fast scan of the front; (C) A fast scan of the tail partoftheburst. .. vi 13 14 19 20 21 22 28 29 30 INTRODUCTION Due to the increased use of pyrethroids, the question of insect resistance to this new group of insecticides is of increasing importance. Some DDT- resistant insects are cross-resistant to pyrethrins (4,33,39). Several DDT- resistant species also are cross-resistant to the newly developed pyrethroids, to which they have never been exposed (11,26,34). This cross-resistance suggests the possibility of future problems in the use of pyrethroid insecticides (34). Pyrethroid cross-resistance is related to the kdr (knockdown resistance) factor in houseflies (9,11) which also confers DDT resistance through a decreased nerve sensitivity (9,31,41). The kdr factor appears also to be responsible for pyrethroid-resistance in mosquitoes (25,31), Spodoptera littoralis (13), and the predatory mite, Amblyseius fallacis (35). Understanding of cross-resistance problems requires basic knowledge on underlying physiological and biochemical mechanisms. The German cockroach, Blattella germanica, is uniquely suited for this type of study for three reasons. First, it has been shown that DDT-resistance in this species involves only reduced nerve insensitivity involving no metabolic or penetration factors (19). Second, it is well suited for electrOphysiological studies in an analogous situation with that of the well studied American cockroach. Third, and most important, a DDT-resistant strain (VPIDLS) has an altered Ca-ATPase in its nervous system which is more resistant to DDT inhibition i_n v_i_t£g (14). The natural pyrethrins were not effective pesticides for field use due to their low environmental stability and high degradability. Elliott and other 2 workers experimented with a great deal of structure modification to produce many new synthetic pyrethroids which have both greater stability and insecticidal properties. In this paper I will refer to "Type 1 pyrethroids" meaning pyrethrins and allethrin (esters of cyclopropanecarboxylic acids with alkenylmethyl cyclopentenolnes) and "Type 2 pyrethroids" referring to the newer synthetic pyrethroids, especially to cypermethrin and decamethrin (a- cyanophenoxybenzyl esters of dihalovinylchrysanthemic acid). Many previous studies on Type 1 pyrethroids revealed numerous similarities to DDT. In addition to cross-resistance, pyrethrins, like DDT, show a negative temperature correlation (2,6,16,18) (i.e., greater kill at lower temperature) and both cause repetitive discharges in axons (38). It is widely believed that all pyrethroids are similar in their mode of action and show cross—resistance due to kdr, even though central vs. peripheral action of pyrethroids is still widely debated (3,22). Recently, differences in insect responses to synthetic pyrethroids have been reported by Clements and May who found that a-cyano pyrethroids did not cause repetitive discharges or enhanced and prolonged muscle contractions as did other types of pyrethroids (8). Gammon (13) previously has shown a type of modified repetitive discharge caused by permethrin and further demonstrated a lack of repetitive discharges with cypermethrin, the a-cyano analogue of permethrin. Clark (7) recently has demonstrated a difference in the inhibitory powers of the Type 1 and Type 2 pyrethroids to two ATPase systems thought to be of importance in the maintenance of electrochemical gradients in neurons. The major goals of this research are to understand the nature of DDT induced cross-resistance to pyrethroids and to clarify the similarities and differences of the mechanism of action among various pyrethroids. MATERIALS AND M ETH ODS Materials Animals and Chemicals The sources and purity of all compounds used in this study are given in Table 1. The structures of the pesticides studied are shown in Figure 1. The three strains of Blattella germanica used in this study were: CSMA (Chemical Standard of Manufacturers Association) and Columbus (originally from the University of Western Ontario), both of which are susceptible strains, and VPIDLS, a DDT-resistant (kdr) strain selected from the original VPI (Virginia Polytechnic Institute) strain. All of these strains have been reared by our laboratory for many years (20,37). The CSMA strain was used for all tests except the temperature vs. toxicity tests where Columbus was used. The VPIDLS strain had shown considerable reversion from its previously high level of DDT resistance (14) and was, therefore, subjected to three further selections. Selections were accomplished by placing approximately 40 sixth instar nymphs in a one pint mason jar coated with the desired dose of DDT as described in the Methods section. After 24 hours the roaches were provided with a cube of Purina Dog Chow and a one inch piece of dental wick soaked with water. After about 50 percent mortality had been reached the survivors were placed in a rearing jar and allowed to mate. The offspring were removed and put in a new rearing jar for further testing. The doses and times of the selection process are shown in Table 2. VPIDLS never has been subjected to any pyrethroid insecticide selection. 4 Table 1. Sources and Purity of Compounds Studied Compound Source Purity DDT Aldrich Chemical Company 99% Pyrethrins Fairchild American Corporation S-Bioallethrin Permethrin Fenvalerate Cypermethrin Decamethrin Piperonyl Butoxide McLaughlin Gormley King Company 96 . 7% Penick 9196 (cis:trans ratio 35:65) Shell FMC Roussel UCLAF 99.696 Pfaltz and Bauer Inc. 5 CIHCI cr3 D DT / I l/O | o \ Pyrethrin \ l/O CI Cl 00 Permethrin (O :N . ° 6 O O Cypermethrin Figure 1. Ff Allethrin pita Fenvalerate 3. 325:0 Decamethrin Chemical structures of DDT and six pyrethroid insecticides 6 Table 2. Selection Process for VPIDLS Selection Resulting Generation Dose (mg DDT) Durationa Number 1 pt jar (Days) 1 VPIDLS G1 4 1 2 VPIDLS G2 10 14 3 VPIDLS G3 10 28 aFood and water were supplied after 24 hours. 7 Periplaneta americana used in some of the electrOphysiological studies were from a culture which has been maintained by this laboratory for many years. Adult male cockroaches were used exclusively for all of the experiments. Methods Susceptibility Studies Surface contact method The inner surfaces of one pint mason jars were covered with a uniform layer of pesticide delivered in 1.0 m1 of acetone. One control per test was run in a jar coated with 1.0 ml of acetone. German cockroaches (25 for the DDT tests, 10 for the dieldrin and pyrethrins tests and 20 for all other tests were placed in each jar and observed at room temperature (24-250C). Each jar was given a one inch piece of dental wick soaked with water after 24 hours and one cube of Purina Dog Chow after 48 hours. A cockroach was considered knocked down if it was on its back and unable to right itself. Death was defined as being nonresponsive to touch. Three replications were used for all experiments. For synergism studies, 6 u g of piperonyl butoxide (in 1.0 ul of acetone) was applied to the abdominal sternum one hour before exposure to pesticide. Abdominal topical application The appropriate amount of pesticide was delivered in 1.0 ul of acetone to the abdominal sternum of the male cockroach. Control cockroaches were treated with 1.0 ul of acetone. They were maintained in the same type of jar with food and water as above. After 4 min., 8 min., 15 min., 30 min., 1 hour, 2 hours, 5 hours, 12 hours, 24 hours, 48 hours and 96 hours, knockdown and mortality were recorded according to the definitions given above. All tests consisted of four doses replicated three times with ten cockroaches at each dose. Cural topical application The appropriate amount of pesticide was delivered in 2.0 ul to four legs with each leg receiving approximately 0.5 u l. The placement of the droplet was at the joint between the tibia and tarsus. Controls were treated with 2.0 ul of acetone by the same method. All tests consisted of five roaches per dose with at least four doses and done in duplicate for the cural vs. abdominal study (Figures 5 and 6) or triplicate for the temperature vs. toxicity tests (Figures 7 and 8, Table 6). For the cural vs. abdominal study, the abdominal dose was delivered in 2.0 ul. For the temperature vs. toxicity tests the following post-application conditions were employed: (1) low temperature-11°C, RH 50 to 6096, (2) room temperature-245°C, RH 50 to 60% and (3) high temperature-31.00C, RH 50 to 6096. Calculations When there was mortality in the control, the adjusted percent mortality was calculated using Abbott's formula. Data was then calculated by Finney's method (12). The parameters used in this study are: (1) resistance ratio (RR) (resistant strain susceptibility/susceptible strain susceptibility), (2) synergistic ratio (SR) (susceptibility without synergist/susceptibility with synergist), (3) KD50 (dose required to knock down 5096 of the individuals tested in 24 hours), (4) LD (dose required to kill 50% of the individuals tested in 24 hours or 96 hours 50 in the case of DDT), (5) KT50 (time required to knock down 5096 of the individuals tested at the given dose) and (6) LT50 (time required to kill 50% of the individuals tested at the given dose). Significance was determined by using a Student's T test for all experiments except the nerve sensitivity studies where the sign test was used. For either test * indicates significance (0.05), ** indicates highly significant (0.01), and "* indicates very highly significant (0.001). 9 Calculations of the knockdown activities of the pyrethroids were made by the method of Nishimura et al. (25). This model uses the assumption that first order kinetics can be used to describe initial pyrethroid penetration, therefore, when -1n(1-KD5"°0/KD;o ) vs. time is plotted, those compounds with the most potent knockdown activity will have the steepest slope. Metabolism Study After removal of the head, legs, and wings of 75 adult male cockroaches, their bodies were ground in ice cold insect saline (40) (2 mM phosphate buffer, pH 7.2) with sand for three minutes. The homogenate was first centrifuged at 1200 xg for 90 min. The supernatant fluid thus obtained was re-centrifuged at 100,000 xg for 60 min. The resultant pellet was resuspended in 2.5 m1 of saline for use. The incubation mixture contained 10 mM nicotinamide, 150 mM KCl, 8 mM Na HPO 2 4 2 2 4 treatment contained 1.80 mg of protein and 2.5 ug of permethrin in 2.0 ml of .7 H O, 2 mM KH PO and 0.3 mM NAPDH (pH 7.3). Each incubation mixture. All incubations were carried out at 30°C. Protein determinations were done by the method of Lowry et a1. (21). Quantitation of the permethrin remaining was accomplished by the use of an electron—capture detector (63Ni) coupled with a Varian 3700 gas chromatograph using a six foot class column of 596 QF-l on Chromosorb W, DMCS at a column temperature of 220°C, and was calculated as a percent of the control (timezO) peak area. Peak areas were calculated as height x width at one-half peak height. For analysis of cytochromes, the enzyme was prepared as described above and diluted with 0.2 M sodium phosphate buffer (pH 7.2). The CO—binding spectra were measured in the presence of NaZSZO4 at room temperature (24°C) with a Varian 635 scanning spectrophotometer. The amount of cytochrome P- 450 and cytochrome b was determined by the method of Omura and Sato (28), 5 10 using 91 mM'1 cm"1 at 450 nm (28) and 185 mM’1 cm’1 at 425 nm (29) as the molar extinction coefficients, respectively. Electrghysiological Studies Nerve sensitivity Tests were conducted on the intact abdominal nerve cord, exposed by opening the dorsal side of the male German cockroach. The dissections were performed essentially as described by Beeman and Matsumura (1). The saline used contained 210 mM NaCl, 3.1 mM KCl, 1.8 mM Na HPO °7HO,1.8mM 2 4 2 CaCl - 2 H O and 0.2 mM NaH P0 with a final pH of 7.4 (40). Air was 2 2 2 4 bubbled through the saline for at least one hour prior to use. Pesticide dissolved in 95% ethanol was added to the saline solution immediately before each treatment. The amount of ethanol added never exceeded 0.2%. After dissection, the specimens were treated by flooding the preparation with 7.0 m1 of the saline plus pesticide solution (or saline plus ethanol for the controls) for five minutes, prior to monitoring of nerve cord activity. The temperature range of the tests was 23.0 to 24.o°c for permethrin and 23.5 to 25.5°c for DDT. The temperature fluctuation is assumed to be of minimal importance as an equal number of susceptible and resistance cockroaches were run each day. Recordings of electrical activity were made by placing a pair of fine silver wire electrodes underneath the nerve cord midway between the fifth and sixth abdominal ganglia, and gently pipetting out the saline solution. The entire preparation was then enclosed in a chamber lined with moistened filter paper to prevent rapid desiccation and increase the life of the preparation. Observation of control insects indicated that the nerve preparation gives at least 35 minutes of reliable performance. No effort was made to continue the observations after this time period. A more in-depth discussion of the machinery used is given in Beeman and Matsumura (1). 11 Susceptibilities of the nerve cords were measured as a function of the time until onset of repetitive discharge. Repetitive discharge was defined as electrical activity of a uniform nature, firing at a frequency of ten milliseconds or less, being of greater amplitude than 10 u V (minimal detectable voltage) and lasting at least one second for DDT or two seconds for permethrin. Controls were run for the same length of time under identical experimental conditions. Responses to pesticides The nerve cord of Periplaneta americana was isolated from just anterior of the first abdominal ganglion to the end of the cereal nerves. The nerve was placed in a trough in a Sylgard 184 dissection dish and the spontaneous activity was monitored by the use of suction electrodes. The saline was the same as previously described, except that air was not bubbled through it before use. The nerve cord was rinsed with 1.0 ml of saline or saline containing pesticide every 10 minutes. Controls of this type of nerve preparation were found to last for many hours. RESULTS Susceptibility Studies It generally has been acknowledged that a kdr factor for DDT resistance confers a cross-resistance to pyrethroid insecticides in the housefly, ticks, Spodoptera littoralis, Amblyseius fallacis and some mosquito species (4,11,13,26,33,34,35,36,39). The data (Figures 2 and 3) confirm this tendency for the Type 1 pyrethroids, but disputes this tendency for the Type 2 pyrethroids as judged by the results of a surface contact test. In these tests resistance was manifested more clearly when assessed by knockdown than by death. The largest resistance ratio was 1000 for the VPIDLS G3 strain to pyrethrins (Figure 3). The susceptibility of the CSMA (susceptible) strain, which was used in the calculation of the resistance ratios in Figures 2 and 3, and the doses used are shown in Table 3. Two major mechanisms of DDT resistance are known to be kdr (target site insensitivity) and DDTase (metabolic dehydrochlorination) (30). Kdr in houseflies confers cross-resistance to methoxychlor (a DDT analogue) whereas DDTase does not (30,32). The resistance ratios for methoxychlor (Table 4) are similar to those for DDT (Figure 2). Penetration as a resistance mechanism confers cross-resistance to a large spectrum of pesticides, depending on their polarity. There was no resistance to dieldrin, a pesticide with similar polarity to the synthetic pyrethroids, in any of the strains tested (Table 5), indicating that penetration is not likely a mechanism of resistance in VPIDLS cockroaches. 12 1001 a- 50- <3 ID p- .-J v .2 b O M 3 ': 10- D - a: u: 0 5- K duels: aid-[H awwlua ,1 i *HJNa H 13 3M] N Hit-IL» P ¥_ i *firhv that] w P‘l Dose(ug/jar) 6000 Figure 2. N aelu: 3 1 3 P Pg: [1123 L. * DDT 'waxhckiS4unathnmrh1PEnmnmrhathwflennElamenmmhnkinaamedudn 150 200 200 Comparison of mortality resistance ratios in four substrains of B. 150 100 75 germanica to DDT and six pyrethroid insecticides by a surfac_e contact method, with P, 1, 2, and 3 referring to VPIDLS parental and successive DDT-selected generations, G1, G2, and G3, respectively. *Significantly greater than 1.0 (CSMA) at P 5 0.05. "Significantly greater than 1.0 (CSMA) at P g 0.01. *"Significantly greater than 1.0 (CSMA) at P 5 0.001. NS Not significantly greater than 1.0 (CSM A) at P _<_ 0.05. l4 ,.. § 1 ifiih» ”OI/ J; / 3 at 3 2% fi/ .3? Aioo- E 3. i g r .— 25 50-: .2 5 a 3 3 ti C 2 n: 5- 1 1 2E r 1 ** P E .2. i 1 3 if P‘ ‘ DUI‘ Pyrethrin S-bioallethrin Permetln'in Fenvalerate Cypermethrin Decmthrin Dose(ug/jar) 6000 200 200 150 150 100 75 Figure 3. Comparison of knockdown resistance ratios in four substrains of B. germanica to DDT and six pyrethroid insecticides by a surface contact method, with P, 1, 2, and 3 referring to VPIDLS parental and successive DDT-selected generations, G1, G2, and G3, respectively. *Significantly greater than 1.0 (CSMA) at P < 0.05. * 1"Significantly greater than 1.0 (CSMA) at P—< 0.01. §§*Significantly greater than 1.0 (CSMA) at P3 0.001. Not significantly greater than 1.0 (CSMA) at P _<_ 0.05. .J 15 Table 3. Susceptibility of a CSMA Strain of B. germanica to DDT and Six Pyrethroids by a Surface Contact Method Pesticide Dose (u g/ 1 pt mason jar) KT50a LT50a DDT 6000 9.65 _+_ 0.40 25.3 i 0.6 Pyrethrin 200 0.14 i 0.04 23.1 i 0.6 Allethrin 200 0.27 i 0.01 12.5 i 0.7 Permethrin 150 0.45 i 0.03 24.0 i 3.5 Fenvalerate 150 0.43 i 0.12 46.9 _+_ 0.2 Cypermethrin 100 0.24 i 0.02 32.9 i 1.4 Decamethrin 75 0.19 i 0.01 48.2 i 2.3 aValues are given as X- : SE. in hours. 16 Table 4. Susceptibility of Four Strains of B. germanica to Methoxychlor in the Presence and Absence of the Synergist Piperonyl Butoxide (6 mg/l pint mason jar) by a Surface Contact Method Resistance Ratio Strain Resistance Ratio with Piperonyl Butoxide SR CSMA 1.0 (46)8 1.0 (24)3 1.9 VPIDLS 1.8 1.7 1.0 VPIDLS G2 2.7 1.3 4.0 VPIDLS G2 > 8 > 14 — SRSynergistic ratio (Resistance ratio without synergist/Resistance ratio with ergist) a)ICHTSO in hours 17 The results of the comparative toxicity tests (Table 6) indicate that decamethrin was the most insecticidal of all the compounds tested, but that it had the lowest knockdown activity. Indeed the newer type pyrethroids had consistently greater toxicity and lower knockdown activity than the older, less synthetic types. The above findings on toxicity vs. structure are similar to those reported previously for houseflies (10), and American cockroaches (5) but vary somewhat from the order reported for Blattella germanica (5). Toxicity difference between cural vs. abdominal topical application indicates that both a Type 1 and a Type 2 pyrethroid cause greater knockdown much when applied to the legs versus the abdomen, although there was no significant difference in mortality between the two treatments (Figures 4 and 5). The results of effects of temperature on toxicity are shown in Figures 6 and 7. A highly significant negative temperature correlation to knockdown was obtained using allethrin, a Type 1 pyrethroid (Figure 6). By contrast there was no significant effect of temperature to knockdown by cypermethrin, a Type 2 pyrethroid (Figure 7). Allethrin also showed a negative temperature correlation to kill, while cypermethrin displayed a positive temperature correlation in terms of mortality (Table 7). Metabolism of Permethrin in Susceptible and Resistant Cockroaches There was no significant difference in the rate of in vitro metabolism of permethrin between the CSMA and VPIDLS strain (Table 8). In accordance with the above observation, there was no significant difference in the amount of either cytochrome P—450 or cytochrome b5 in the susceptible and resistant strains (Table 9). These results indicate that there is no interstrain difference in the activity of the mixed-function oxidase, the only enzyme system which has been shown to degrade both DDT and permethrin. 18 Table 5. Susceptibility of Five Strains of B. germanica to Dieldrin (2 mg/ 1 pint mason jar) by a Surface Contact Method Strain LTSOa Resistance Ratio CSMA 39.3 i 2.2 1.0 VPIDLS 37.6 i 0.6 1.0NS VPIDLS G1 41.1 i 2.0 1.0NS VPIDLS G2 34.9 i 5.5 0.9NS VPIDLS G3 36.9 i 3.1 0.9NS Egalues reported as X : S.E. in hours at 2 mg dieldrin per 1 pint mason jar. Not significantly different from 1.0 (CSMA) at P _<_ 0.05. 19 cofiw 2 one 1.638. TIIV 3580?? 98 Tiny mm: E 52:23 mo mezzo oESrchx .v 2sz «55.3.... 33¢ «3:55. 8.: 9: cm H 2: Nx M - M , \ Mr” N o . Se 0 \ . / m x m \ m \ e a , .. So a m. . . m 3 ’ m , . y a . eo- 20 5583a? Gammon Ox Alv BEEoBa EB Tiny m2 E3 523ansz Ho 3230 oESIo 2.25:2... ..o:< 03:55. 1 § (unto: zed urmamadfio 8n) OSCDI fir .m 9:63 § 21 3 06: Ba .3 Dog .5 ocean ea 5.52: .8 3:8 25-35. .e BEE «con—«ooh... 334‘ not—Es. c fco¢ Icon 1 cowa (qaeoz .rad utzqaauu Sn) 059' 22 75 0o: 28 .Q 0093 .on 0093 E 5.508.893 .8.“ 3.50 oEfirchM .p 9:33 23:52:... aoz< 23:55. eon co” on cu m N P II n P L b O .23 m S 0 m 0 M m 3 m. .63 w. d n 1 O . a m 1 Oco 23 Table 6. Comparative Toxicities of DDT and Six Pyrethroids to a CSMA Strain of German Cockroach by Topical Application to the Abdomen Knockdown Activityc . . a b 2 Pesticide KD50 LD50 Slope x 1000 r DDT -— 11,726 i 1142 —- —-—- S-Bioallethrin 390 i 113 696 i 116 29.4 .964 Permethrin 177 :65 275 _+_ 14 12.2 .996 Fenvalerate 134 _+_ 3 239 i 116 5.9 .998 Cypermethrin 75 _+_ 16 114 i 29 5.5 .935 Decamethrin 9.1 i 0.5 7.5 i 2.7 2.0 .971 a b cCalculated as explained 11; Methods. Values are reported as X : S.E. in ng per roach at 2 hours. Values are reported as X + S.E. in ng per roach at 48 hours. 24 Table 7. Effect of Temperature on Toxicity of Allethrin and Cypermethrin by Cural Topical Application LD 50 Compound 11°C 24°C 31°C Allethrin 249 i 138 636 i 163 982 i 182 Cypermethrin > 640 96.7 :31.9 38.8 :2.0 Values are reported as X 1 S.E. in ng per roach at 48 hours. 25 Table 8. In vitro Metabolism of Permethrin by Microsomal Preparation from 'tTie Whole Body (minus head, legs and wings) Homogenate: the Data are Expressed as the Percentages of Originally-added Permethrin Remaining in the Incubate Time Elapsed (minutes) 0 15 30 9o CSMA 100 i 2.0 69.3 i 0.6 74.3 i 3.0 60.8 _+_4.9 VPIDLS 100 : 2.1NS 70.1 : 0.3NS 68.0 : 2.2NS 59.6 i 13.4NS Numbers given represent X- : S.E. calculated as a percentage of the peak area relative to time = 0 as anaylzed by GLC. NSNot significantly different than CSMA at P 5 0.05. 26 Electrophysiological Studies Nerve sensitivity The VPIDLS strain took a significantly longer time to develop repetitive discharges than the CSMA strain for both permethrin and DDT (P <_ 0.10 for permethrin and P _< 0.01 for DDT, Table 10). It must be noted, however, that, at these doses, symptoms were induced more slowly with permethrin than DDT. Indeed, after one generation of selection the nervous system had become even less sensitive to both DDT and permethrin as can be seen by the results of the comparison test of VPIDLS and VPIDLS G1 in Table 11. The electrophsyiological effects of DDT and permethrin are different. DDT causes repetitive firings of single axons resulting in a burst of activity, uniform in amplitude, which ends abruptly (35) as shown in Figure 8a-b. Permethrin's poisoning symptoms were less predictable. The initial symptom noted in many, but not all, permethrin-treated nerve preparations was a repetitive disharge similar to DDT-type symptoms, except that it had less uniform amplitude and slightly decreasing frequency before it terminated (Fig. 9). The next symptom of permthrin poisoning to appear was a repetitive discharge of decreasing amplitude and slightly increasing frequency (Figure 10a- c). The latter symptom was frequently recurring, becoming progressively weaker with each discharge. These permethrin-induced discharges of decreasing amplitude (Figure 10a-c) were long lasting; i.e., they lasted an average of about seven seconds and some lasted as long as thirty seconds. This is in sharp contrast with DDT's repetitive activity which lasted an average of about two seconds with only rare bursts exceeding ten seconds. There was no difference in amplitude of sporadic action potentials observed between DDT and permethrin treatments. 27 Table 9. Comparison of Cytochrome Levels in a DDT-resistant and Susceptible Strain of Blattella germanica Strain Cytochrome P Cytochrome b 450 5 CSMA .782 .045 .253 + .025 VPIDLS .859 : .054NS .287 : .051NS |+ The above figures are if i S.E. in nmoles per mg protein. Cytochrome P 45 and Cytochrome b were measured at A = 450 nm and A: 426 nm, respectivePy, as described in Mithods. NSNot significantly different from CSMA at P _<_ 0.05. 28 A 1.0 mV 0. s B 1.0 mV 10 ms Figure 8. The pattern of a typical DDT-induced repetitive discharge as viewed by a slow scan (A) and a fast scan (b). Note the homogeniety in amplitude and frequency of individual action potentials. Figure 9. 29 B 1.0mV 10 ms C 1.0mV 'ww‘l- I‘ 3‘! m L The pattern of a permethrin-induced repetitive discharge. These types of discharges are commonly observed at the beginning of the permethrin poisoning. (A) Entire burst of discharges viewed by a slow scan; (B) A front portion of the burst of discharge by a fast scan; (C) The tail portion of the discharge scanned by the same speed as (B). Note the difference in frequency of (B) and (C). 30 B 1.0mV 10 ms C l.OmV 10 ms Figure 10. The pattern of a second type of permethrin-induced repetitive discharge which was often observed at the later part of nerve poisoning. (A) A slow scan indicating the gradual decrease in peak amplitudes; (B) A fast scan of the front; (C) A fast scan of the tail part of the burst. 31 Table 10. Time to Onset of Nerve Poisoning Symptomsin the Abdominal Nerve Cord of Susceptible and Resistant German Cockroaches Minutes to Repetitive Discharges CSMA VPIDLS -4 DDT (10 M) 10 28 14 15 10 17 13 25 15 22 15 > 35 H X:S.E. 12.4 11.0 >23.7 13.0 Permethrin (2 x 10-5M) 20.5 23.8 23.8 > 35 3 27.3 27.8 4 24.8 22.3 5 21.0 > 35 6 13.5 18.5 X:S.E 21.8 12.0 > 27.8 12.7a I,f,i$ignificantly greater than CSMA at P g 0.10. VPIDLS significantly greater than CSMA at P _<_ 0.01. 32 Table 11. Time to Onset of Nerve Poisoning Symptoms in the Abdominal Nerve Cord of German Cockroaches Before and After One Generation of Selection by DDT Minutes to Repetitive Discharges VPIDLS VPIDLS G1 -4 DDT (10 M) 14.6 32.3 > 35 14.7 27.8 > 35 16.3 > 35 13.7 16.7 20.2 > 35 X: S.E. > 21.3 i 3.5 > 28.1: 3.98l Permethrin (3 x 10-5M) 23.9 34.4 15.8 22.2 > 35 16.4 19.5 > 35 14.6 > 35 _ 19.5 21.4 b X:SE >214+30 >27.4:3.4 a b VPIDLS G1 significantly greater than CSMA at P VPIDLS G1 significantly greater than CSMA at P IAIA 33 Responses to pesticides The spontaneous activity of isolated American cockroach abdominal nerve cords in the presence of allethrin and cypermethrin is shown in Table 11. Allethrin caused repetitive discharges to occur in each nerve cord observed. These repetitive discharges could not be differentiated from those caused by DDT (Figure 8) in frequency of repetitive discharges, frequency of spikes during repetitive discharges or duration of repetitive discharges. Cypermethrin had no observable effect on the spontaneous activity of the isolated central nervous 10 3 system at concentrations of 10' M to 10- M, except in one nerve cord where repetitive discharges were observed (Table 12). DISCUSSION The three physiological mechanisms by which an insect may develop resistance to insecticides are: (1) decreased penetration, (2) target site insensitivity (i.e., kdr) and (3) metabolic differences. It has been established in this study that the mechanism of DDT-resistance in the VPIDLS strain of Blattella germanica is a kdr (target site insensitivity) type. Penetration was shown to be an unlikely mechanism of DDT-resistance because the VPIDLS strain showed no cross-resistance to dieldrin, a pesticide with similar polarity, but a different mode of action than DDT. It has been demonstrated that DDT-resistance in VPIDLS is correlated with a decreased nerve sensitivity toward DDT and permethrin which can be measured directly at the level of the central nervous system. It has been shown that VPIDLS is cross- resistant to methoxychlor, allethrin, pyrethrins, and permethrin, all compounds to which kdr houseflies are known to be cross-resistant (9,30,32,33). Lastly, there was no detectable difference in the mixed function oxidase system of susceptible and resistant (VPIDLS) roaches, either in the rate of metabolism of permethrin or in the levels of cytochrome P-450 or cytochrome b5, indicating that metabolism is not likely a mechanism of resistance in the VPIDLS strain of E. germanica. The mode of action of DDT has eluded investigators for decades. The mode of action of pyrethroid insecticides, despite intensive studies over recent years, also has evaded researchers. Insects which are resistant by means of a target site insensitivity (i.e., a change at the site of action) provide a useful tool 34 35 88:55 8 22-2 H momaanome 25:3; 2:2 TH zméaTE a 8258.. 262 w 5.255.553 8555 2-: sméfidé a 5.3.3an 2533me a 5.25: .820 @3880 098m omcoamom .8285 mo mama. onwam mouoo oEoSmom :25 223. 5322850 9:305 89:32 3.82 .«o 89:32 0.80 3.82 358003. 525.230 $2.823. won—20$ 05 .6 33:3 9882on 55 co 5.35533 23 5.522 "68.3553 ofifiém 9: mo 38% .2 538. 36 for the investigation of the modes of action of pesticides. Comparisons of the VPIDLS strain of the German cockroach to the susceptible counterparts could, therefore, yield helpful insights as to the mode of action of DDT and pyrethroid insecticides. This study has revealed that there are differences between cypermethrin and decamethrin pyrethroid insecticides (Type 2 pyrethroids) and those closely related to the pyrethrins (Type 1 pyrethroids). Type 1 pyrethroids have a negative temperature correlation for both knockdown and mortality factors, while Type 2 pyrethroids have no obvious temperature correlation for knockdown and a positive temperature correlation for mortality. Electrophysiologically, Type 1 compounds were shown to cause repetitive discharges while Type 2 compounds did not. Lastly, there was only very limited resistance of the VPIDLS strain of DDT-resistant (kdr) E. germanica to Type 2 pyrethroids while resistance to Type 1 pyrethroids was large. It should be noted that permethrin and f envalerate are intermediate pyrethroids, not clearly fitting into Type 1 or Type 2 classifications. 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