Acetylcholinesterase of Aphis citricola: Properties and significance in determining toxicity of systemic organophosphorus and carbamate compounds

PESTICIDE

BIOCHEMISTRY

AND

Acetylcholinesterase in Determining

PHYSIOLOGY

15,

267-274 (1981)

of Aphis citricok Properties and Significance Toxicity of Systemic Organophosphorus and Carbamate Compounds1

SHULAMIT MANULIS,**~ ISAAC ISHAAYA,*-3 AND ALBERT S.PERRYt *Division of Entomology, The Volcani Center, ARO, Bet Dagan. Israel; and tlaboratory of Environmental Toxicology, Institute for Nature Conservation Research, Tel Aviv University. Ramat Aviv. Israel Received January 6, 1981; accepted March 30, 1981 In apterous adults of the spirea aphid, Aphis citricola van der Goot, the optimum conditions for determining acetylcholinesterase (AChE) activity consist of reaction mixture of 0.1 M phosphate buffer (pH 7.5). lo-$ M acetyhhiocholine (ASCh), and enzyme extract equivalent to 80 + 3 pg protein incubated for 15 min at 30°C. The K, value for ASCh (6.7 x 10e5 M) was much lower than that of butyrylthiocholine (Busch) (1.25 x lo-* M). The enzyme activity was atmost completely inhibited by 10e6 M paraoxon or 10e5 M eserine and was 84% inhibited by 10m5M BW284CSl (a specific AChE inhibitor). DTNB was found to inhibit the enzyme activity and was therefore added at the end of the reaction. AChE activity of A. citricola was inhibited in vitro and in vivo by dimethoxon > dimethoate, and aldicarb sulfoxide > aldicarb > aldicarb sutfone. The in vivo effect correlates well with the toxicity level of the various toxicants. A neurotoxicity index which combines both mortality and in vivo inhibition of the aphid AChE activity is suggested as a measure for determining the toxicity of organophosphorus and carbamate compounds toward aphids. INTRODUCTION

The toxicity of organophosphorus and carbamate insecticides is known to be due to inhibition of AChE.* In spite of ample information concerning the role and importance of AChE activity in various vertebrate and insect species (9), very little is known about the role of this enzyme in aphids and to the best of our knowledge nothing is known concerning A. citricola. During the course of this study we extracted AChE from this insect, and determined its optimum conditions along with its biochemical characteristics. This enabled us to develop a biochemical parameter in which AChE activity along with other

The spirea aphid, Aphis citricola van der Goot (A. spiraecola Patch), is a relatively new pest of citrus in Israel (1) and is an important pest of citrus and other crops in the United States (2-5). It is also an important vector of tristeza and other virus diseases (5, 6). Experiments carried out by Aharonson et al. (7) and Tashiro et al. (8) indicated that systemic organophosphorus and carbamate compounds, such as dimethoate, croneton, and aldicarb when applied to the soil were successful in controlling this pest without affecting the biological equilibrium in the citrus grove. ’ Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 324-E, 1980 series. p Submitted in partial satisfaction for the degree of M.Sc. in the Department of Zoology, Tel Aviv University, Ramat Aviv, Israel. 3 To whom requests for reprints should be sent. A Abbreviations used: AChE, acetylcholinesterase; ASCh, acetylthiocholine; BuSCh, butyrylthiocholine; BW284C51, 1,5-bis(4-alIyldimethylammoniumphenyl)pentane-3-one dibromide); ChE, cholinesterase; DTNB. 5.5dithiobis(2-nitrobenzoic acid); iso-OMPA,

tetraisopropyl pyrophosphoramide; aldicarb, 2methyl-2-(methylthio)propionaldehyde O-(methylcarbamoyl)oxime; aldicarb sulfoxide, 2-methyl-2-(methylsulfinyl)propionaldehyde O-(methylcarbamoyl)oxime; aldicarb sulfone, 2-methyl-2-(methylsulfonyl)-propionaldehyde 0-(methylcarbamoyl)oxime; dimethoate, O,O-dimethyl O-(N-methylcarbamoylmethyl)phosphorodithioate; dimethoxon, O,O-dimethyl S-(N-methylcarbamoylmethyl)phosphorothioate; paraoxon. O.O-diethyl 0-4-nitrophenyl phosphate. 267 0048-3575/81/030267-08!§02.00/0 Copyright All ri@ts

@ 1981 by Academic Press. Inc. of repmduction in any form reserved.

268

MANULIS,

ISHAAYA,

biological criteria were used to evaluate the effectiveness of some systemic organophosphorus and carbamate compounds toward A. citricola. MATERIALS

AND

METHODS

Chemicals. Aldicarb, aldicarb sulfoxide, and aldicarb sulfone were obtained from Union Carbide Corporation, Florida; dimethoate and dimethoxon from Nanogens International, California; paraoxon from Chemical Service Inc., Pennsylvania; DTNB, eserine sulfate, and BW284C51 from Sigma Chemical Company, St. Louis, Missouri; BuSCh and ASCh from BDH Chemicals Ltd., Poole, Dorset, England. All compounds were analytical or purified grade and the toxicants used contained over 9% active material. Rearing and bioassay. The aphids were reared on young plants of Viburnum suspensum L. (12). The infested plants were kept in a glass house at 22-24°C with a 12-hr photoperiod. Ten to fifteen apterous adults were transferred to a synthetic diet (13) for larviposition according to the method of Mittler and Dadd (14) and kept in a controlled environment of 24-25”C, 70-80% RH, and a 12-hr photoperiod. Groups of forty 4- to S-day-old aphids which developed on this diet were fed for 24 hr on an artificial diet containing the test compound. AChE activity of live aphids including moribund aphids was then determined. Mortality was recorded concurrently. LC,, was determined after 24 and 72 hr; moribund aphids were considered dead. Data are averages of three or four replicates with their SE values. The LC,, values were calculated according to Finney’s probit method (15) using an IBM 360 computer. Determination of enzyme activity. Forty aphids were homogenized in 1 ml 0.1 M phosphate buffer, pH 7.5, using a chilled glass-Teflon tissue grinder. The homogenate was filtered through glass wool to remove gross particles and used as the en-

AND

PERRY

zyme source. AChE activity was determined by the method of Ellman et al. (16), using conditions which were found to be optimal for A. citricola. The assay medium consisted of 0.7 ml 0.1 M phosphate buffer, pH 7.5, 0.1 ml 0.01 M ASCh, and 0.2 ml enzyme extract equivalent to eight aphids containing, according to the Lowry method (17), 80 it 3 pg protein. After 15-min incubation at 30°C the enzyme reaction was terminated by adding 0.1 ml low3 M eserine sulfate; 0.1, ml 5 x lo+ M DTNB was then added for determining free sulfhydryl groups released as a result of hydrolysis. For standardization, the activity was expressed as ASCh hydrolyzed/mm/aphid. Enzyme inhibition in vitro. The inhibition in vitro was carried out according to Ishaaya et al. (18). The test compound was dissolved in acetone at a concentration of 10,000 ppm. Freshly prepared solutions were diluted with water, as appropriate, to obtain the desired concentration of the compound in the preincubation medium containing the enzyme and the test solution. Enzyme reaction was initiated after 20 min preincubation at 30°C and determined as described above. The data are averages of three or four replicates, expressed as percentage of the control and plotted against PI (i.e., log molar concentration). Analysis of dimethoate and dimethoxon residues. Five hundred aphids reared on 5 ppm dimethoate for 24 hr were ground with sodium sulfate to a flowing dry powder and extracted for 1 hr with 15 ml of 20% acetone in hexane in a Soxhlet apparatus. The extract was evaporated to dryness and the residue was redissolved in 50 ~1 ethyl acetate. One to three microliters was injected into a Perkin-Elmer 3920 B gas chromatograph for analysis. The working parameters were as follows: temperature (on column injection), column, 24O”C, detector, 250°C; detector, FPD; carrier flow gas, N, at 15 ml/min; other gases: H, at 34 ml/min, air 55 ml/min; column make-up, 6 ft x ‘/4 in, glass column filled with 10% DC-200, on Gas Chrom Q 100/120 mesh.

ACETYLCHOLINESTERASE

269

OF Aphk ~itricola

Determination of neurotoxicity index. Groups of forty 4- to S-day-old aphids were fed for 24 hr on synthetic medium containing various concentrations of the test compound. Mortality and AChE activity in the surviving aphids were then determined as described previously. Neurotoxicity index was expressed as (percentage mortality x percentage AChE inhibition)/lOO. PH

RESULTS

TIME (min)

Optimum Conditions for AChE Activity In order to determine whether AChE activity ofA. citricola is located in the soluble or the particulate fraction, the aphid homogenate was centrifuged at various gravities (Table 1). The supernatant lost its activity gradually with an increase in gravity; the 90,OOOg supernatant exhibited only 16% of the total activity, while the 90,OOOg pellet exhibited 55.6%. The relatively low activity of the 90,OOOg precipitate resulted FIG. 1. Effeci oj pH, incuhution time. raution probably from difficulties in redissolving and enzyme level on AChE ucti~~ity. the pellet. Addition of Triton X- 100 for sol- temperature, ubilizing bound enzyme resulted in a strong Each arrow designates the stundud ~tsso~ conditiotz. inhibition of AChE activity. The whole hothe reaction time and 30°C as the reaction mogenate was found to be the most suitable Enzyme extract of eight enzyme source and was therefore used for temperature. aphids equivalent to 80 t 3 pg protein was the standard enzyme assay. used for each enzyme reaction. The optimum pH for the enzyme assay For determining AChE activity, DTNB was 7.5 and the activity was linear up to 20 reagent is usually added to the reaction min (Fig. 1). Fifteen minutes was chosen as medium prior to the initiation of the reaction (16). In our assays DTNB was found to TABLE 1 inhibit the enzyme activity similar to that AChE Acti\+v of Aphis citricolu in Homogenate, found for Myzus persicae (10, 11). This inSupernatnnts, and Pekt at Various Gravities hibition ranged from 35% at 5 x lo-” M to AChE activity relative to 80% at 5 x 10m3 M. DTNB reagent was whole homogenate therefore added at the end of reaction. (7%)

Whole homogenate Supernatant” 200x 9000g 90,ooog Pellet of 90,OOOg

100 78.8 32.0 16.4 55.6

c Centrifugation at 200g and 9000g was for 15 min, and that at 90,OOOg for 60 min. The activity of the whole homogenate is 0.40 2 0.01 nmol ASChimini aphid.

Enzyme Specificit) Substrates and inhibitors are used to distinguish between true and nonspecific ChE (19). Aphid homogenates appear to contain a mixture of cholinesterases. The enzyme preparation of A. citricola hydrolyzed ASCh much better than BuSCh, as indicated by a much lower K, value (Fig. 2). Enzyme - substrate saturation was obtained

270

MANULIS,

ISHAAYA,

with 1 mM ASCh, while no such saturation was found with BuSCh up to 8 mM. Enzyme activity of A. citricolu was completely inhibited by lo+ M paraoxon and was 95% inhibited by lo+ M eserine sulfate indicating that most of the activity is related to cholinesterases (Fig. 3). BW284CSl which is generally regarded as a specific inhibitor for AChE activity (20), inhibited at 10e5 M the enzyme activity of A. citricola by 84% with ASCh but not with BuSCh as substrate (Fig. 4). These results indicate that at least 84% of the activity is related to AChE. The aphid homogenate exhibits other ChE activity which hydrolyzes BuSCh and is not inhibited by BW284C51 (Fig. 4). This activity was not affected by the serum ChE inhibitor iso-OMPA at concentrations up to 1O-5 M in the enzyme assay. At 10m4M a slight inhibition of about 10% of BuChE activity was observed. The activity of this enzyme in the standard reaction is less than 16% of the total. AChE Inhibition and Toxicity of Dimetheate and Dimethoxon Dimethoxon inhibited in vitro AChE (Is,, = 1.7 x lo+ M) more than 150-fold the concentration of dimethoate (Iso = 2.7 x low4 M), while the in vivo assays indicated that the effect of dimethoxon (Iso = 8.0 X lo-’ M) was only 4-fold stronger than that of dimethoate (I,, = 3.2 x 10e6 M) (Fig. 5).

I -20

FIG.

ASCh

-lo

-202 -0.1 0 0.1

IO

20 ASCh 1.0 Bu!xh

PERRY

FIG. 3. Inhibition of AChE activity by paraoxon (A) and eserine (A). The enzyme preparation was preincubatedfor 20 min at 30°C with various concentrations of paraoxon and eserine prior to the initiation of the enzyme reaction.

The relatively stronger effect of dimethoate in vivo compared to that in vitro (Fig. 5), resulted probably from its rapid conversion to dimethoxon. Residue analysis of dimethoate and its oxymetabolite in 500 aphids fed for 24 hr on 5 ppm dimethoate revealed that 30-40% of the total residues was dimethoxon. Dimethoxon was about two- and fivefold more toxic than dimethoate after 24 and 72 hr of feeding, respectively (Table 2). AChE inhibition in vivo (Fig. 5) by dimethoate and dimethoxon correlates well with the toxicity results obtained after 24 hr in which the I,, value for dimethoate was about twofold higher than that of dimethoxon.

;

(0) (0)

2. Lineweaver-Burk plot for ChE activity and BuSCh as substrates.

AND

I

I

O7

6

5

I

4

PI

with

FIG. 4. Inhibition of ChE activity by BW284C51 using ASCh (I mM) and BuSCh (8 mM) as substrates. Enzyme inhibition and assay as in Fig. 3.

ACETYLCHOLINESTERASE

OF Aphis

‘~~~~~~

Y FIG. designate

forty the

q<:

I 6

o-

I 5

I 4

PI

5. Inhibition of AChE activity in vitro SE of the mean. The in vitro inhibition

4- to j-day-old determination

aphids of AChE

were fedfor

’

3

and

24 hr on an artificial

Index

6. Inhibition uldicrrrb .ru&mr

4

5

diet

(A) and dimethoxon 3. In the in vivo assay,

containing

the test

CA).Bars groicp~

ttf

prior

to

compound

activity.

The neurotoxicity index which combined mortality 24 hr after treatment, and AChE inhibition in the surviving aphids, takes into account all the individuals (the dead and the live aphids) of the test. It facilitates a rapid

F~ti.

I

6

in vivo by dimethoate and assay as in Fig.

Aldicarb sulfoxide inhibited AChE activity in vitro (Iso = 8 x lO+ M) to a greater extent than did aldicarb (Iso = lop6 M) and aldicarb sulfone (Iso = 4 x 10M6M) (Fig. 6). The relative potency of these compounds was less pronounced in the in vivo assays (Fig. 6). However, it is quite clear that at low in viva concentrations aldicarb sulfoxide is the most potent compound. The general pattern of activity correlates well with the toxicity of these compounds (Table 2).

and

I

o-7

PI

AChE Inhibition and Toxicity of Aldicarb and its Oxymetabolites

Neurotoxicity

271

cifricola

of AChE (a 1. The

activity in vitro

in vitro und

and accurate determination of the insecticidal effect which is due mainly to AChE inhibition (see Discussion). It is suggested as the most reliable parameter for evaluating toxicity of compounds acting as AChE inhibitors. The neurotoxicity indexes of dimethoate, aldicarb, and their oxymetabolites toward Aphis citricofa are given in Figs. 7 and 8. DISCUSSION

The use of various substrates and inhibitors for the enzyme assay demonstrates that at least 84% of the activity is related to AChE. This conclusion is supported by: (a) greater ability of the enzyme to hydrolyze ASCh in comparison with BuSCh; (b) almost complete inhibition of the enzyme activity by 10-j M eserine and by lo-” M 85% of paraoxon; and (c) approximately the enzyme activity is inhibited by lo-” M BW284C51. Dimethoate is a weak inhibitor

and in viva

ii3 viva

assays

by trldicurb c(re OX ifr Fig.

(0). 5.

uldicurb

su!fi)xidr

272

MANULIS,

ISHAAYA,

TABLE

LC’,,

of Dimethoate,

Aldicarb,

LC,” (ppm)

Insecticide Dimethoate Dimethoxon Aldicarb Aldicarb sulfoxide Aldicarb sulfone

and Their

AND

PERRY

2

Oxymetabolites

after

24 and 72 hr

of Feeding

24 hr

72 hr

95% Confidence limits of LC,”

95% Confidence limits of LC,”

Lower

Upper

6.16 2.73

4.82 2.48

8.57 3.00

7.44 1.53 23.93

6.12 1.36 21.71

8.84 1.72 26.60

LC,” (w-d

Lower

Upper

2.95 0.62

2.74 0.39

3.19 0.84

3.55 0.68 11.94

3.29 0.63 10.95

3.81 0.73 13.10

Note. Groups of forty 4- to 5-day-old aphids were fed for 24 and 72 hr on an artificial diet containing the test compound. Data are average of three or four replicates with their 95% confidence limits.

of in vitro A. citricola AChE, analogous to that found in mammalian species (21). On the other hand, dimethoate is a stronger inhibitor of AChE in vivo than in vitro. This seems to result from a rapid conversion of dimethoate to dimethoxon, as was evident from the large amount of dimethoxon found in aphids fed for 24 hr on ,a diet containing dimethoate. Similar results were found with other insects, such as Musca domestica (22, 23). The maximum in vitro inhibition of AChE by dimethoxon (-80%) is lower than the complete in vitro inhibition. Higher in vivo inhibition activity in the surviving aphids could not be obtained because of enhanced mortality at higher concentrations. The in vivo inhibition of AChE by dimethoate and dimethoxon cor-

0.313

0.625 LOG

FIG. 7. Neurotoxicity dimethoxon (A) after

1.25

2.5

CONCENTRATION

5

IO

(ppm)

index oj’dimethoate 24 hr of treatment.

relates well with the toxicity of these compounds. The in vitro Ijo of aldicarb sulfoxide, aldicarb, and aldicarb sulfone (Fig. 6) were lower than those reported for other insects, such as M. domestica and Anthonomus grundis (24). The maximum in vitro inhibition of over 90% of the enzyme activity obtained with all three carbamates indicates that almost no decarbamylation occurred during the 15min enzyme- substrate reaction. The relative potency of aldicarb and its oxymetabolites for AChE inhibition in vivo is evident at low concentrations in which aldicarb sulfoxide > aldicarb > aldicarb

(A) and

FIG. 8. Neurotoxicity index of aldicarb dicarb sulfoxide (0). and aldicarb suljime hr of treatment.

(O),

al-

(A) after 24

ACETYLCHOLINESTERASE

273

OF Aphis cirrirola

sulfone. This inhibition correlates well with the LC,, values of the respective compounds. The relatively low in vivo AChE inhibition of 50-60% might be due to decarbamylation that occurs in the intact aphid body during the feeding period. Extensive studies (25-27) were carried out to investigate the possible use of AChE inhibition as a parameter for the toxicity of organophosphorus compounds. The suggested “Neurotoxicity Index” in our study combined aphid mortality occurring 24 hr after treatment and AChE inhibition in the surviving aphids. In other words, it takes into account all the individuals of the test-the dead and the live (but intoxicated) aphids. Under our test conditions, aphid mortality extended over several days. Hence, the final LC,, values could not be used as an accurate measure of the insecticidal effect which is due solely to AChE inhibition. The “Neurotoxicity Index” which combined mortality and the degree of poisoning in the live aphids (as expressed by percentage of AChE inhibition), occurring a short time after treatment, facilitates a rapid and accurate determination of the toxicity or organophosphorus and carbamate insecticides toward aphids.

7. N. Aharonson. I. Neubauer, I. Ishaaya, and B. Raccah, Residues of croneton and its sulfoxide and sulfone metabolites in citrus (Clementine trees) following a soil treatment for the control of Aphis spirnecolu, J. Agr. Food Chem. 27. 265 (1979). 8. H. Tashiro, D. L. Chambers. J. G. Shaw, J. B. Beavers, and J. C. Maitlen, Systemic activity of UC-21149 against the citrus red mite. citrus thrips, California red scale, and spirea aphid on nonbearing orange trees. J. Econ. E~zromol. 62, 443 (1969). 9. A. Silver. “Frontiers of Biology: The Biology of Cholinesterase,” Vol. 36. North-Holland. Amsterdam, 1974. 10. M. Zahavi. A. S. Tahori, and F. Khmer, An acetylcholinesterase sensitive to sulfhydryl inhibitors. Biochem. Biophys. Acto 276, 577 (1972). 11. H. R. Smissaert, Reactivity of a critical sulfhydryl group of the acetylcholinesterase from aphids

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15.

(Mvzrrs

12.

13.

14.

16.

17.

18.

19.

20.

prrsictrr).

Pestic.

Biochem.

Ph~.siol.

6.

2 15 ( 1976). I. Neubauer, I. Ishaaya, N. Aharonson. and B. Raccah. Activity of soluble and membranebound trehalase in apterous and alate morphs of Aphi.\ citric&cl. Camp. Biochrm. Physiol. 668. 505 (1980). I. Neubauer. B. Raccah, I. Ishaaya, and N. Aharonson, A synthetic diet for the spirea aphid, Aphis cirricolo van der Goat. Pl~yropumsific~a 8, 19 (1980). T. E. Mittler and R. H. Dadd. Studies on the artificial feeding of the aphid My;rc.r Persia (I(’ (Sulzer). II. Relative survival, development and larviposition on different diets. ./. fr~.\(,c,r Physiol. 9, 741 (1963). D. J. Finney, “Statistical Methods in Biological Assay,” 2nd ed.. Griffin, London. 1971. G. L. Ellman. K. D. Courtney. V. Andres. Jr.. and R. M. Featherstone. A new and rapid colorimetric determination of acetylcholinesterase activity, Bidrem. Plrr~rmrtcwf. 7, 88 ( 19611. 0. H. Lowry. N. J. Rosebrough. A. L. Farr. and R. L. Randall, Protein measurement with the Folin phenol reagent. J. Bid. Chem. 193, 262 (1951). I. Ishaaya, N. Yablonski. K. R. S. Ascher. and J. E. Casida, Triphenyl and tetraphenyl derivatives of group V elements as inhibitors of growth and digestive enzymes of 7’rih~rliro~r c~ot>,fir.sumand TtYholiotti co~t~~ncum larvae. Pcvric. Biochcm. Physird. 13, 164 ( 1980). R. D. O’Brien. Acetylcholinesterase and its inhibition. in “Insecticide Biochemistry and Physiology” (C. F. Wilkinson. Ed.). pp. 271-296. Plenum. New York. 1976. L. Austin and W. K. Berry. Two selective inhibitors of cholinesterase. Birwhvu~. .I. 54. 695 (1953).

21. D. M. Sanderson and E. F. Ed\on. T~~~icol(tgi--

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cal properties of organophosphorus insecticide dimethoate, Brit. J. Ind. Med. 21, 52 (1964). 22. A. L. Devonshire, Metabolism of (methoxy-C”)dimethoate by houseflies, in “Rothamsted Report,” Part 1, p. 182, 1971 (as cited in Pestic. Biochem. Physiol. 5, 101, 1975). 23. T. Uchida, H. S. Rahmati, and R. D. O’Brien, The penetration and metabolism of H3dimethoate in insects, J. Econ. Entomol. 58, 831 (1965). 24. D. L. Bull, D. A. Linquist, and J. R. Coppedge, Metabolism of 2-methyl-2-(methylthio)propionaldehyde O-(methylcarbamoyl)oxime (Temik,

AND

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UC-21149) in insects, J. Agr. Food Chem. 15, 616 (1967). 25. D. C. Mengel and J. E. Casida, Biochemical factors in the acquired resistance of houseflies to organophosphate insecticides, J. Agr. Food Chem. 8, 431 (1960). 26. L. E. Chadwick, Actions on insects and other invertebrates in “Handbuch der Experimentallen Pharmacologic” (G. B. Koelle, Ed.), Vol. 15, pp. 741-798, Springer-Verlag, Berlin, 1963. 27. E. Y. Spencer, Organophosphorus insecticides, in “The Future for Insecticides: Needs and Prospects” (R. L. Metcalf and J. J. McKelvey, Jr., Eds.), pp. 295-307, Wiley, New York, 1976.