Title Mechanism of the Selective Toxicity of Organophosphorus
Mechanism of the Selective Toxicity of Organophosphorus Compounds in the Armyworm, Leucania separata Walker. Port Title I. Topical Toxicity and Anticholincsterase Activity of Certain Organophosphorus Compounds
SINCHAISRI, Neungpanich; MIYATA, Tadashi; SAITO, Author(s) Tetsuo
Citation 防虫科学 (1977), 42(3): 125-132
Issue Date 1977-08-31
URL http://hdl.handle.net/2433/158971
Right
Type Departmental Bulletin Paper
Textversion publisher
Kyoto University Mechanism of the Selective Toxicity of Organophosphorus Compounds in the Armyworm, Leueania eeparat« Walker. Part I. Topical Toxicity and Anticholinesterase Activity of Certain Organophosphorus Compounds. Neungpanich SINCHAISRI, Tadashi MIYATA and Tetsuo SAITO (Laboratory of Applied Entomology and Nematology, Faculty of A~riculture, Nagoya University, Chikusa·Ku, Furo-Cho, Nagoya, 461) Received May 9,1977. Boiyu-Kagaku, 42,125,1977.
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4 f!l!:fHm ~ ~ ~Elt*,Jo.:> 7'7 ;l I-?, Leucania separate Walker .tIJEltII:;X",fi' ~ ~Elt:tJ ~ Il'Ombfliffl II:J; IJ WfjJ«~c c i,11: in vitro 1I::Io'1~ 7'7;l 1-?0.:>:1 ~ ~.x.;r.:r7--t! (ChE) mWf'Fffl~ HC-acetylcholine chloride ~mp"t"WfjJ«t=. LD,o filil;t. methyl parathion iJ{JR i, Ij, it <. "'?P'"'C' Ienltrothion, phenthoate, diazinon o.:>lIJii'"'C'ib -:>ti, 7'7;l I-? 0.:> ChE IHtfl ChE o.:>t,'Hl&~7f L tz; In vitro 11::1-3 It ~ ChE ~Il ;tHtm 1:1 phenthoate-oxon iJ{Ji"H ~ <, "'?P '"'C' methyl par aoxon, diazoxon, fenitroxon o.:>l!fl'i'"'C'ib -:> tz; 4 fiIi~rtU'i:Jo.:>~m:tJ c ChE m;tHtmo.:>IDMH~iHL t: c I:. -?, methyl parathion, diazinon, fenitrothion 0.:> 3 rn~m1fJ'"'C'o.:>mM l:tiI'!iiJ> -:>tz, -15, phenthoate o.:>.tJ~S methyl parathion 0.:> 5fffo.:>ChEmWf'Fm~7f-vt=iJ{~£!l:tJI;t. methyl parathion o.:>~ 1/3.8 i:ib-:>t=. UH,o.:>m1!l:1 phenthoate o.:>£!lU:J13"o.:>~~, ~ ~ PI;t.m14:J13'"'C'ft~l~o.:>~~iJ{ft!lo.:>3 fi11f1m~J c WI.. -:>"t"P ~ c ~'? 1iJfJlH't~IlIJ7f-v"t"P O.
Armyworm, Leucania separata Walker is one Despite the measurement of ChE activity by the of the important pests of corn, Zea mays L. and method described Hestrln!" showed the unsatisf rice, Oryza sativa L.1,2> Many ecological and ied result in the preliminary procedure due to physiological works have been reported so far the strong interference of melanization effect in but it seems that publications concerning with the armyworm enzyme, the radioisotopic assay, the toxicological study on this species are still exercised in this study, offered much advantages lack. The toxicity test with 31 insecticides to and Quite desirable for the micro-volume of the armyworm larvae indicated that most of solution in the measurement of ChE activity. organophosphorus (OP) insecticides were highly Materials and Methods toxic whereas carbamates were not and endrin and dieldrin, the only two organochlorine com Insect pounds gave relatively high effects>. The colony of the armyworm larvae was obtain It is generally acknowledged that OP insecti ed from the University Farm, Nagoya University, cides kill animals by inhibiting cholinesterase Togo, Aichi prefecture in 1974. The larvae were (ChE). Most of them are rather poor inhibitors reared at 25'C in the plastic box, 24x30x5cm, of ChE in vitro. They owe their potency in vivo fed with fresh corn leaves as their basic food to the fact that they are converted in the body (partly fed with the leaves of Italian Rye grass to give compounds which are presumed to be to keep the successive generations during winter the direct inhibltorsv". season). The mass rearing technics were essen Thus, as the first approach to clarify the tially the same as the previous describeds.v, The mechanism of selective toxicity of four OP com 5th-instar, 2-3-day larvae were provided for pounds, methyl parathion, phenthoate, diazinon experiments. and fenitrothion against the armyworm, topical Chemicals toxicity of them and anticholinesterase activity Over' 975'6 purity of four organophosphorus of their oxygen analogs in vitro were investigated. compounds were used. Methyl parathion and
125 fenitrothion were received from Sumitomo Chem nerve cord (B & VNC), from the 5th-instar larvae ical Co., Ltd., phenthoate was obtained from of the armyworm were prepared separately. Nissan Chemical Industry Ltd., diazinon was ob Hornogenates of Hand WB were centrifuged at tained from Nihon Kagaku Co., Ltd. 900g for 5 min. The supernatants were used as Cholinesterase inhibitors, P=O analogs of these enzyme sources throughout the study. B & VNC 4 OP compounds were synthesized by an oxida homogenate was directly applied as the enzyme tion of P=S compounds in bromine solution and source without centrifugation. purified by thin layer chromatography (tic). Buffer's solution.-Phosphate buffer's solution, Acetyl [1- HC]-choline chloride (l4C-AChCl), 1/15M, pH 7.4, was mainly used in the routine 13.7 mCi/mmole, was purchased from Amersham works. In the experiment of an effect of pH on
Co., England. The Amberlite CG-120, type 2, resin the ChE activity, buffer of Chadwick et al.8) was sodium salt was a product of Organo Co., Tokyo. used:NaCl, 26.30g; KH2PO" 3. SSg; NaOH, Chemicals for the preparation a scintillation 1.oog; water to make one litre. With all suspe solution, for instances, naphthalene (special grade), nsions, pH was adjusted to the desired value by 2,5-diphenyloxazole (PPO) , dimethyl POPOP and addtion of O.lN NaOH or O. IN HC!. The range l,4-dioxane were products of Katayama Chemical of pH values adjusted would cover enough in the ce., Ltd., Osaka. acidic and alkaline conditions from pH 6.1-9.2. Toxicity test Amberlite resin-dioxane mixture.-Preparation The evaluation of contact toxicity of 4 OP was essential the same as the method described compounds was accomplished by topical treating by Siakotos et al.w . Ten grams of dried resin the 5th-instar larvae of the armyworm with 0.7 was put into 100ml of dioxane. pi of acetone solution containing amount of OP Scintillation solution.-It was a modified Bray's compounds. In control, insects were treated with Cocktail!" containing 100g naphthalene,4g;PPO, the same volume of acetone. Insects were fed 200mg POPOP and 100ml absolute methanol, and with fresh corn leaves and kept in a constant then made to 1 litre with 1,4-dioxane. temperature chamber of 25·C. After 72hr, mor Assay procedure tality counts were taken. Badly affected or mori With an equal volume of 0.05ml, the mixture bund larvae were justified as dead insects accord of enzyme and HC-AChCl solution was put in a ing to the method of Ando and Sherman'!', At 20ml-tube, and the content of the tube was well least 4 replicates of 10 insects at 5-8 dosage mixed by shaking and incubated for 10 min at levels were provided for each insecticide in this 37·C. Non-enzymatic hydrolysis of the substrate test. The values of LD50 and LD 95 were calculated was measured with the solution containing of by the method of Finney12) and toxic index was 0.05ml of buffer instead of enzyme. ChE activity evaluated by the method of Sun!", was expressed as fl mole of the substrate hydro Radioisotopic assay of ekE activity lyzed/hr/g. Substrate solution.-Stock solution of l4C-AChCl, The proceeding steps of reaction were assayed 8 X1O-3M, containing approximate 3, 300, 000 as previously described by Siakotos et al.w . In cpm/ml was prepared by dissolving the contents order to stop the reaction, an approximately 5ml of AChCl .Irom a ampoule in distilled water. of the suspended resin-dioxane mixture was added. Stock solution of non-labelled AChCl, O. 55M, was Then, the enzyme-resin-dioxane solution was simultaneously prepared. The mixture of labelled brought to an exact volume of 10ml with dioxane. and non-labelled AChCl was provided to obtain The tube was tightly capped, and mixed by inver higher concentrations than 8 X 1O-3M of AChCl sion or shaking and allowed it to stand. The with highly enough radioactivity for the experi resin was allowed to settle by gravity for at ments. The mixture were always kept under least 2 hr. Five millilitres of the supernatant -20·C. solution was transferred to a scintilltion counting Source of enzyme--Three kinds of homogenate, vial, and 10ml of the modified Bray's scintilltion head (H), whole body (WB) and brain & ventral solution was added. Special care must be taken
126 to avoid transferring resin particles into the vial. HEAD BRAIN &VENTRAL' The samples, total volume of 15ml, were counted WHOLE BODY NERVE CORD. the radioactivity of the hydrolysis product in a U 10 100 U liquid scintillation spectrometer (Tricarb Model .<:: .<:: U U 3320 Liquid Scintillation Spectrometer) which < -e has an effeciency about 6O;'6at the optimized <3'" E HC setting. ::t...... 50 '-'~ For the inhibition study, inhibitors (P=O ~.E .- "- analogs of OP compounds) were dissolved in .~ "0 u .!::!'" acetone in widely different concentrations. With <3... w'" "0 the volume of O.Olml, an inhibitor solution was .<:: >. U .<:: delivered by means of a rnicrosyringe into the 20ml tube and allowed acetone to evaporate pS absolutely, 0.05ml of enzyme solution, thenafter, Substrate was added and pre-Incubated at 37°C for 30 min Fig. 1. Activlty-pS (-log mole of substrate) and then, 0.05ml of HC-substrate was added, curves for the enzymatic hydrolysis of incubated at 37°C for 20 min. The next procedure acetylcholine chloride in three sources for ChE activity measurement was carried out of armyworm enzyme. Reaction condi by the same manner as described above. tions;enzyme concentrations, head, 4%, whole body, 10% and brain & ventral Results nerve cord, 1;'6, pH adjusted at 7.4 with Toxicity test phosphate buffer. Incubation time, 10 LD~o and LDe5 values of 4 OP compounds were min at 37°C. shown in Table 1. Methyl parathion gave the highest toxicity to the 5th instar larvae of the presume that the maximum enzymatic activities armyworm. The relative toxicities of fenitrothion, of Hand WB are quite similar to each other at phenthoate and diazinon were lower than methyl 2.5 x 10-·M final concentration of the substrate, parathion, a standard, as expressed by toxic index but it needs higher concentration of 1. 0 x 10-3M value of 297, 383 and 1080 respectively. to produce the maximum activity for tissue Measurement of ChE activity homogenate of B & VNC. The data of this Substrate concentration.- The ChE activities experiment provided an additional evidence that of three homogenates were shown in Fig. 1. From ChE in the armyworm is true ChE. the bell shaped curves in Fig. 1, it is likely to Enzyme concentration.- From Fig. 2, enzymatic
Table 1. LD~o, LDes, regression equation of dosage-mortality and the relative toxicity of 4 organophosphorus insecticides topically treated on the 5th instar larvae of the armyworm.
Regression LD~o LDe5 Toxic Insecticides equation pg/g /tg/g index (LD~o)
Methyl parathion Y=5+3.9893 (x-0.9916) 0.97 2.51 100 Phenthoate Y=5+2.5934 (x-I. 5729) 3.72 15.85 383 Diazinon Y=5+3.1467 (x-2.0263) 10.48 34.75 1080 Fenitrothion Y=5+I. 7571 (x-I.4684) 2.88 25.20 297
Y=mortality in probit, x=log dosage (pg x 10)
127 activity was in the proportional rate to enzyme up to 20 min incubation. The hydrolysing rates concentration up to 10% in H,2096 in WB and 496 decrease with the time thereafter. The sharp in B & VNC enzymes. Higher enzyme concent decrease occurred from 60 min in Hand WB but rations yielded non-linear results. slow decrease was found in B & VNC homo Time course of the reaction-As illustrated in genates. Fig. 3, the time course of the substrate hydrolysis Optimum pH.- Based on the suitable condition by enzymes was demonstrated. Absolute linearity obtained from the previous experiments, the . was found in ChE activities of all hornogenates measurement of ChE activity as a function of pH was carried out and the obtained results were HEAD BRAIN & VENTRAL illustrated in Fig. 4. The effect of pH in the WHOLE BODY NERVE CORD reaction of enzyme and substrate caused different G enzymatic activity. The optimum pH of Hand ..c:: U Brain &ventral B & VNC homogenates were obtained in slight ~ nerve cord -, alkaline condition at the range of 8. 0-8. 6 while of WB homogenate was almost neutral at the 0.05 ~ Head range of 7.2-7.4 (Fig.4 and Table 2). The peaks of the curves in Fig. 4 would repre sent the maximum ChE activity of the army worm from different enzyme sources which were
o 4 10 20 Enzyme concentration (w/v) HEAD BRAIN & VENTRAL WHOLE BODY NERVE CORD Fig. 2. Linearity of enzyme activity as a func -e tion of enzyme concentration. Activities OJ .!::! 50 were measured after lO min incubation '0... -e at 37°C. Substrate concentrations: 2.5x >. ..c:: 10-4M final for head and whole body 40 G and 1. 0 X 10-3M for brain & ventral ..c:: U nerve cord homogenates, pH adjusted ~ so OJ to 7.4 with phosphate buffer. '0 E ..3- .20 HEAD BRAIN & VENTRAL o. WHOLE BODY NERVE CORD G 'r'~~ ..c:: 10 . .~ U Whole hod, ~ Brain& ventral nerve cord --.. "' OJ '0 1 Head E 1 ..3- 20 pH Whole body • 1 Fig. 4. Variation in ChE activity of 3 sources of armyworm enzyme as a function of pH. The reaction conditions were: 10:n so 120 substrate concentration for head and TIME (Minute) whole body homogenates, 2.5xlO-4M Fig. 3. Proportionality of substrate hydrolized final, brain & ventral nerve cord, 1. 0 X with time of incubation at 37°C. Enzyme lO-3M final, enzyme concentrations: concentrations: head,4 %,whole body, head, 4%, whole body ,1096, brain & 1096, brain & ventral nerve cord, 196. ventral nerve cord, 196. Incubationtirne, Substrate concentrations and pH were 10 min at 37°C. For full explanation the same conditions as Fig. 2. see Text.
128 Wi m ft ~ in 42 ~-III
Table 2. The different properties among the sources of enzyme in the armyworm larvae .s ... 0 0 ;u 8 N "'1' r-- 0::: ...: d N N Optimum ...... I .. I Source of enzyme I I 0 0 0 0 AChCl pH ~'i ...... XXX X "0 ~'-' 4M ... M 0'> 0'> Head 2.5xl0- 8.0-8.3 0 E u M r-: r-: ~ 4M ... 2.5xl0- 7.2-7.4 0 ~ Whole body ~ > .!: r-, l/'l >, ...... M N Drain & ventral ~ r: 8 8 e... c 0::: ...: d Lri r-: nerve cord 1.0xl0-3M 8.3-8.6 r: ;; ~ ...... I ; II I 0 0 0 0 '5 ~ ...... 5 > :ii x x X x Table 3. Cholinesterase activity in the ~ 0 co co t1J -'-' ... armyworm larvae ..c c ..; ...: N N U ';;j 0 ... N co 0 ~ (1) ~ ChE activity - ;u 8 ... c 0::: ...: d N ...: Source of enzyme ("mole AChCI .!:.... hydrolized/hr/g) ...... :.c I I I I 0 0 0 0 :.a e-, 0 ...... c "0 ... Head 53 0 z::E X X X X C ,Q .." ~ ~ l/'l Whole body 15 ~ ~ -'-' ...: ...: M N ...u '0 Drain & ventral nerve cord 265 ~ ..c e. .s 8 ~ (1) C5 0 ::: ;u co 0::: ...: d M M shown in Table 3. ChE activities were 53 /Imoles "0c ...... r: I .I I I 0 ...... 0 0 0 0 AChCI hydrolyzed/hr/g in H, 15 /1moles in WB, 0 ...... l/'l ~6 X X X X and the highest activity was found in B & VNC M ~ N 0 ...0 M at 265 "moles AChCI hydrolyzed/hr/g, r-: ...: ...: -III "0 Anticholinesterase activity c .s "'!' ::l ;; 8 ~ 8 0'> 0 ...: d ..; p=o analogs or oxon isomers of 4 OP com' ae. 0::: M 0 pounds were applied as ChE inhibitors in the u ...... I I I I I ChE inhibition test. The results were tabulated ~ 0 ...... 8 ...0 ....0 ....0 0 Z::E XX X X in Table 4. Methyl paraoxon wasgiven as a ""l' -'-' ~ co 0'> c '"...: ..; ..; r-: standard compound in the comparative inhibitory 0 "0 r: Il) c l/'l N 0 .g ... activities. The activity was expressed as 50;:'6 ~ :r: r: 8 ... 0'> ;; ...: d N inhibition (IN.a) and 80;:'6 inhibition (IN so) by the ... 0::: N C molar concentrations of the inhibitors. Il) ...... I . I II Uc 0 0 0 Diazoxon and fenitroxon gave lower inhibitory 0 .... 8 ...... u :ii X XX X activities than methyl paraoxon which, by all ... co M .." r: -'-' ..; r-: ...: ...: cases, were well corresponding with their P=S '0 ::E toxicities. But phenthoate-oxon showed an ex' ceptional result where it exhibited its anti ChE ..; Il) activity about 5 times higher than methyl para' :0 ~ til s ~ >, c oxon while its corresponding P=S toxiciy was N 0 C >< Il) c 0 3.8 times lower. 0 ., ... >< c III 0 0 ;u C 0 ...0 til 0 >< III ... 0 0 Il) .... til ..c >< Discussion o e. 0 -t: ... P- C N ::l ..c Il) til '2 0 c ..c Il) The basic properties of ChE in the armyworm Ul ~ c, is lLo enzymes from H, WB and B & VNC hornogenates - seemed to be the typical characteristic of true
129 ChE but it needs much lower concentration of ences of susceptibilities of insect ChE to toxi substrate (2.5XlO-·M) to produce a maximum cants and differences of activation and detoxifi enzymatic activity, particularly, in head and whole cation metabolism of toxicants. Metcalf and March body hornogenates than the other insects tissue 23l demonstrated the strong relationship between hornogenates (5x 1O-3M-1. Ox 1O-2M) I Table 5. The correlation of the toxicity of methyl parathion, phenthoate, diazinon and fenitrothion and the anticholinesterase activity of their oxygen analogs in the armyworm. Sources of Correlation coefficient (r) enzyme LD.o-IN.o LD.o-IN so LDu-IN•• LDu-INso r =0.1844 r =0.2509 r =0.2611 r =0.4109 Head r,=0.8036 r,=0.6367 rl=0.9855 rl=0.9158 r =0.3114 r =0.1933 r =0.3680 r =0.1991 Whole body rl=0.9019 rl=0.9846 r,=0.9997 r",=0.9578 Brain and r =0.4087 r =0.1903 r =0.5605 r =0.2828 ventral nerve cord r,=0.7508 rl=0.7595 rl=0.9678 rl=0.9711 r values were calculated from the logarithmic values of LD.oxlO,LD9.x10 IN.ox109, and INsox 109; r, is the values of correlation coefficient calculated without phenthoate 130 as the data are enable an explanation of unusual compounds, methyl parathion, fenitrothion and features of the toxic action of dimethoxon on diazinon. Unexpectedly, phenthoate was an Periplaneta americana L. comparing with diazoxon. exception where its antiChE activity was the From a small restriction of both compounds to highest, exhibiting about 5 times higher inhibi penetrate into the central nervous system(CNS), tory activity whereas it was 3.8 times less toxic dimethoxon was found 200 times less active than than that of methyl parathion, a standard diazoxon (both against cockroach ChE and in compound. This might indicate that the electrophyslologlcal test againstthe CNS of cock mechanism of selective toxicity of phenthoate roach). Yet, this weak neurotoxicity, however, against the armyworm is attributable to the is compensated for by relatively high stability different feature from those of other three OP to detoxication and allowing sufficient to accumu ompounds. late in insect to render it almost as toxic to cockroaches as a more active antiChE but more References rapidly being detoxified like diazoxon. 1) Wongslrl, T. and K. Kovitvadhi : In "The In order to clarify the mechanism of selective Major Insect Pests of the Rice Plant" IRRI, toxicity of these 4 OP compounds, other factors John Hopkins Press, Baltimore, Maryland, (metabolism, penetration and etc.) will be contri pp. 571 (196t). buted to the further outline of experiments. 2) Pholboon, P. and W. W. Cantalo:A host list of the insects of Thailand, Dept. of Agri., Summary Thailand, pp. 149 (1963). As the first step to clarify the selective toxicity 3) Sinchaisri, N.: The master thesis, Faculty of of 4 OP compounds in the armyworm, Leucania Agriculture, Nagoya University, Japan, pp. seporata Walker, the bioassay and in vitro anti 60 (1970). ChE activity were studied. The compounds were 4) Sinchaisri, N. and K. Sogawa: ]. Appl. Ent. treated topically to the 5th-instar, 2-3-day larvae. Zool., 21, 102 (1969). Comparing their relative toxicities by LD•• 5) O'Brien, R. D.: Insecticides, action and meta values, it was found that the toxic index of bolism, Academic Press, New .York and' Ienitrothion, phenthoate and diazinon to methyl London, pp, 332 (1967). parathion were 297,383 and 1080 respectively. 6) O'Brien, R. D.: Toxic phosphorus esters, chem Cholinesterase (ChE) activity was measured by istry, metabolism and biological effects. radioisotopic method using HC-acetylcholine Academic Press, New York, pp. 434 (1960). chloride (AChCI) as a substrate. ChE of the 7) O'Brien, R. D.: Adv. in Pest Cant. Res., 4, 75 armyworm shows the typical characteristic of (1961) . true ChE having an optimum substrate concent 8) Chadwick, L. E., J. B. Lovell and V. E. Egner: rations: 2.5xlO-~M with tissue hornogenates Bioi. Bull., 105, 139 (1954). of head and whole body and 1. 0 x 1O-3M with 9) Matsumura, F.: Toxicology of insecticides. brain and ventral nerve cord homogenate. Plenum Press, New York and London, pp. The results of in vitro antiChE activity with 513 (1975). oxon isomers of the compounds were orderly 10) Hestrin,S.: J, Biol:Chem., 180,249 (1949). accomplished; phenthoate-oxon>methyl paraoxon 11) Ando, K. and M.Sherman: Proc, Hawaiian >diazoxon>fenitroxon. Ent. Soc., 19, 349 (1967). The correlation between the toxicity of the 12) Finney, D. J.:Probit analysis, Cambridge Univ. OP compounds and the antiChE activities of Press, London and New York, pp. 318 (196t). their oxygen analogs was examined, Statistically, 13) Sun,Y.P.: ].Econ. Ent., 43, 45 (1950). basing on the correlation coefficient values (r), it 14) Siakotos, A. N., M. Filbert and R. Hester: seems that there is poor correlation between Biochem, Medicine, 3, 1 (1969). toxicity and antiChE activity data among 4 15) Kanehisa, K:Bull. of Lab. of Appl. Ent.and compounds but positive correlation among 3 Nema, Faculty of Agri..Nagoya Univ. Aichi, 131 Japan, 2, pp. 89 (1961). 21) Van Asperen, K.: Nature, 181,355 (1958). 16) Tahmisian, T. N.: J. Exp, Zool., 92, 199 22) Casida, J. E.: J. Agr. Food Chem., 4,772 (1943) . (1956) . 17) Glick,D.: J. General Physiol., 21,289 (1938). 23) Metcalf, R. L. and R. B. March: J. Econ. Ent., 18) Stegwee, D.: Physiol. Compo et Decol., 2, 241 42, 721 (1949). (1951) . 24) Fukuto, Y. R. and R. L. Metcalf: J. Agr, Food 19) Saito, T.: Bull. of Lab. of Appl. Ent. and Chem., 4, 930 (1956). Nema. Faculty of Agri, Nagoya Uniu.Aichi, 25) Devonshire, A. L., P. E. Burt and R. E. Japan, 1, pp. 53 (1961). Goodchild: Pest. Biochem. Physiol., 5, 101 20) Miyamoto, J.: Res. Rev., 25,251 (1969). (1975). JiJt!RrM)~Mtli!iLl::c:l:Q?:$I 7:t.l ~ J>. s- 0)Jl1i~, 'tT[ZSJZfa 7lilo.:> l- 7 ? :f'l&~~"? tt: 10!t~.Q[Z~li, Controlling the Pink Bollworm by Disrupting 'tT[ZSJZfa356i1lt b ~ ~~~, mn 985'6 ~~Pc M~ <:l Sex Pheromone Communication Between Adult tt~. viJ)U, ~fflilUiJ)~~'iUi, J,/J!R~tt'J1.to.:>~p Moths. L. K. GASTON, R. S. KAAE, H. H. SHOREY, >e1lm«t~tJttlit~~ t~~ '. fIiJ~H~J(ltu, fJJmo.:>ttA D. SELLERS: Science, 196, 904 (1977). ~1J1m>e1lm«t.:. 7 JHJ.l1U~~-A":.d{;l::m~~mt.il% ?~7~iA~~, illW~~Wm~m!R~~~~ ~Ii, v ; 0 'C :"-[z~15l1::ttW <:l ttt: bo.:>iJ;«-iJ)"? tz, 1 s-- A":..- 11111::, 'l&ill{~iJ;mtJ:-:> 't"~ I) j!&L "06.':1::.T "'Co.:>W:, 7J.l~1UiJ)>;'8J.rfij)~~, 7:r.o'C:,,-[Z~ 7.>. ? 7771 i A~~¥l:7:r. o'C:..-li, T~II::{~~m J,/Jm'l&iJ:{lf :..- l- 7 ? :fH1~~u't"i2Jlll:: 2 @m~~iJl'I&H,L:«t':iJ;, I!llfC 52:tjO 8 J3 25 S I'iJlj;U I!llfC 52:tjO 8 J3 31 S ~ff ~ffm MElli*.A. I!ti mH ~ liif :tE ftf ro!RH~ m42 J.§-III ~{jIfi ¥ 1000. Ji(tIli1Jtr:Ji([g;jtBIi I Ji(tIl*~."tI$Pi (ll'MDI11· Ji(tIl5899) 1ThlA~n:tj02000Pl Qlj.\;.~fi:tj03500Pl ?j.t!il~fi:tjOU .5. $10 ~u l?f! i1ij t.1i . 15:JHfCR5 1'iJlj;IJm lIE :f!l m IilrJ 606 Ji(tIlrtJtD!([g;jtslll Ji(tIl*~~~llIl Ji(tIlrtJ·fJi([gjU~iM-t~ T J1.' 132