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Cholinesterases of aphids—III. Sensitivity of acetylcholinesterases to several inhibitors as a possible phylogenetic character
Insect Biochem. Vol. 19, No. I, pp. 15-18, 1989 Printed in Great Britain.All rights reserved
0020-1790/89 $3.00+ 0.00 Copyright © 1989PergamonPressplc
CHOLINESTERASES OF APHIDS--III. SENSITIVITY OF ACETYLCHOLINESTERASES TO SEVERAL INHIBITORS AS A POSSIBLE PHYLOGENETIC CHARACTER K. V. NOVOZHILOV, 1 A. P. BRESTKIN, 2 A. E. KttOVANSKIKH,2 E. B. MAIZEL, 2 S. N. MORALEV,2 E. V. N I K A N O R O V A l and I. N. S A Z O N O V A l ~All-Union Research Instituteof Plant Protection, Shosse Podbelskogo 3, Pushkin-6, 188620 Leningrad and 21. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, U.S.S.R. Academy of Sciences, Thorez pr. 44, 194223 Leningrad, U.S.S.R. (Received 25 November 1987; revised and accepted 5 August 1988) Abstract--The influence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), three organophosphorous inhibitors (OPI) and physostigmine on the velocity of the acetylthiocholine enzymatic hydrolysis by the homogenates of 35 species of insects of the order Homoptera including 30 species of aphids (Aphidinea) was studied. Acetylcholinesterase (ACHE) of the representatives of the suborders psylla (Psyllinea), coccids (Coccinea), whitefly (Aleyrodinea) and leafhoppers (Cicadinea) is not inhibited by DTNB and has lower sensitivity to OPI and higher sensitivity to physostigmine as compared to the AChE of most species of aphids which have been investigated. It was found that only aphid AChE is sensitive to DTNB, this property being less pronounced for AChE of the aphid families Phylloxeridae and Lachnidae than for AChE of the aphid families Callaphididae, Pemphigidae, Chaitophoridae, Anoeciidae, Thelaxidae and Aphididae. AChE of all the aphids under investigation had very similar sensitivity to the OPIs studied and to physostigmine with the exception of AChE of the grape phylloxera Viteus vitifolii Fitch (Phylloxeridae) whose sensitivity to OPI was extremely low. It was concluded that the Lachnidae family is more ancient compared to other contemporary families of viviparous aphids. Key Word Index: aphid, Homoptera, phylogenesis, acetylcholinesterase, cholinesterase, SH-sensitivity, organophosphorous inhibitors, physostigmine
DTNB influence on the velocity of enzymatic hydrolysis of acetylthiocholine and butyrylthiocholine using homogenates of 30 aphid species and 5 representatives of other hornopterous suborders. We also determined the sensitivity of AChE in these species to three organophosphorous inhibitors and physostigmine.
INTRODUCTION Since 1972 a number of groups have shown that acetylcholinesterase (ACHE) of some aphid species is irreversibly inhibited by SH-reagents including 5,5'dithiobis(2-nitrobenzoic acid) (EUman reagent, DTNB) (Zahavi eta[., 1972; Smissaert, 1976; Sazonova et oi., 1978; Nikanorova, 1980; Manulis et al., 1981; Brestkin et ol., 1985). Cholinesterases of other animals, including AChEs of some insects, which are close relatives of aphids, such as the bug Triatoma infestans (Hemiptera) (Wood et al., 1979) and the rice leafhopper Nephotettix cincticeps Uhler (Homoptera) (Hama eta/., 1980) are not sensitive to DTNB. That is, these cholinesterases are not SH-sensitive enzymes. Also, some other unusual properties of AChE from aphids were revealed. Thus, AChE of the spring grain aphid Schizaphis gramina has a higher sensitivity to cationic OPI and derivatives of methylthiophosphonic acid, and a lower sensitivity to physostigmine compared to "typical" human erythrocyte AChE (Brestkin et al., 1985, 1986). Butyrylcholinesterase (BuChE) detected in homogenates of some aphid species proved to be insensitive to DTNB (Zahavi et al., 1972; Brestkin et al., 1985). We were interested in determining how these particular cholinesterases are distributed among insects of the order Homoptera, including aphids of various families. Particularly, we wanted to determine the relationship of these properties to the systematics and phylogeny of these insects. Therefore, we studied
MATERIALS AND METHODS
The majority of the insects were collected in field conditions in the Leningrad region. Whiteflies, Trialeyrodes vaporariorwn Westw., were collected on cucumbers in a hothouse. Grape phylloxera Viteus vitifolii Fitch. (from Kishinev), aphids Aphis craccivora Koch. and Pterochloroides persicae Chol. (from the Tashkent region) were transported to Leningrad in Dewar flasks with dry ice. Prior to investigation the insects were stored at - 15°C for < 1 month. After storage for 6 months, only 5-15% of the enzymatic activities were lost. To determine species specificity the insects were mounted and studied by light microscopy. Acetylthiocholine bromide (ATCh) and butyrylthiocholine bromide (BuTCh) were from Chemapol, 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) was from KochLight Lab. and diisopropylfluorophosphate(DFP), sodium dodecylsulfate (SDS) and physostigraine were from Serva. Organophosphorous inhibitors C2HsO(CH3)P(O)SC2H4S+(CH3)C2Hs. CH3SOf (Gd-42) and C2HsO(CH3)P(O)SC2H4SC2H5 (Gd-7) were synthesized at the A. N. Nesmeyanov Institute of Organoelement Compounds of the U.S.S.R. Academy of Sciences (Moscow), by the method of Volkova et aL (1961). 15
K.V. NOVOZHILOVet al.
16
Homogenates of whole insects were prepared in a glass homogenizer in 0.05 M sodium phosphate buffer, pH 7.5, at concentrations from I g/20 ml to ! g/100 ml depending on the enzyme's specific activity. Because of the high lipid content of the coccid, psylla and whitefly homogenates, they were centrifuged at 100,000g for 2 h at 4°C. Then, BuChE activity was measured in the supematant and AChE activity in the pellet after suspension in the starting volume of the buffer. The catalytic activity of the cholinesterases was determined by our modification of the Ellman method (Brestkin et al., 1985) in 0.07 M Na-phosphate buffer, pH 7.5, with the addition of 1.3 x 10-3 M substrate solution (ATCh for determination of AChE activity and BuTCh for BuChE activity). As stated before (Nikanorova, 1980; Brestkin et al., 1985), aphid BuChE does not hydrolyze ATCh significantly. Therefore, the use of these two substrates makes it possible to measure activity of the cholinesterases under study separately without recourse to specific inhibitors. In 10-15 rain the enzymatic reaction was stopped by addition of 3% SDS solution to a final concentration of 0.3%, followed by DTNB addition to a final concentration of 5 x 10-4 M. The reaction mixture was centrifuged for 10 min at 7000g. The optical density of the supernatant was measured at 412nm and the catalytic activity of the cholinesterases was calculated. When measuring enzymatic activity of coccid and psylla homogenates, an equal volume of ethanol was added to the reaction mixture before centrifugation to clarify the solution. The anticholinesterase potency of OPI and physostigmine was assessed by the magnitude of the bimolecular rate constant of interaction between cholinesterase and inhibitor (ka, M-~/min) calculated from the formula (Aldridge, 1950):
ka= l___lnV°
till v, where V0 is the catalytic activity of cholinesterase in the absence of the inhibitor and V, is the cholinesterase activity after interaction of the enzyme for t min with the inhibitor at a concentration [I] exceeding by many fold that of the active centres of cholinesterase. For V, determination the enzyme was incubated for 3-5 min with the inhibitor. Then the interaction between the enzyme and the inhibitor was stopped by the addition of substrate and the enzymatic reaction of substrate hydrolysis was carried out as outlined above. F0 was calculated from a similar experiment without an inhibitor. Various concentrations of inhibitors and several incubation intervals were used such that V, ranged from 20 to 80% of F0. The reaction corresponds to first-order kinetics as evidenced by the linear dependence In Fo/Ft vs t (Aldridge and Reiner, 1972). In the presence of DTNB the substrate hydrolysis by aphid AChE was nonlinear (Brestkin et al., 1985). Therefore we evaluated eholinesterase sensitivity to DTNB by comparison of two samples. In the first sample the homogenate was preincubated for 10rain at 25°C in 0.07 M Naphosphate buffer containing 7.7 x 10-4 M DTNB and incubated for 10rain with substrate. In the control sample DTNB was added after stopping the hydrolysis with SDS. Statistical evaluation of the results was performed by Miiller's method (1960). Each mean value was derived from 3 to 5 determinations. The SE was in the range of + 10%.
activity of AChEs are known with other groups of animals (Sazonova, 1966; Augustinsson, 1963). BuChE activity was detected only in 15 out of the 35 insect species under study and there is a 20-fold variation in its absolute value as well. In no case was BuTCh enzymatic hydrolysis inhibited by DTNB, nor could we find any relationship between BuChE activity and the taxonomy of the insects under study. Even within one Aphis genus this enzyme was found in four species and not detected in two others. It is likely that the BuChE of aphids as well as that of other animals does not take part in neuronal impulse conduction and cannot be classified as an enzyme of vital importance. Its physiological function is uncertain. From the data presented in Table 1 it follows that only AChE of aphids (Aphidinea) is sensitive to DTNB in contrast to that of the representatives of other suborders: psyllas (Psyllinea), coccids (Coccinea), whiteflies (Aleyrodinea) and leafhoppers (Cicadinea). The eight aphid families studied can be distinctly separated into two groups on the basis of AChE sensitivity to DTNB. AChE activity (measured in the presence of DTNB) of the aphid families Phylloxeridae and Lachnidae decreases only in the range of 3 to 8% following a 10 rain preincubation with D T N B whereas AChE activity of the representatives of the families of Pemphigidae, Chaitophoridae, Callaphididae, Thelaxidae, Anoeciidae and Aphididae is inhibited under the same conditions in the range of 33-84%. With the aim of further investigation of AChE properties of various insects, experiments were undertaken with three OPIs: Gd-42, Gd-7, DFP and physostigmine. Gd-42 is a cationic derivative of methylthiophosphonic acid, the specific and potent inhibitor of ACHE, especially of aphid AChE (Brestkin et al., 1986). Gd-7 is the non-cationic analog of Gd-42. DFP is a non-specific broad spectrum inhibitor, and physostigrnine, a methylcarbaminic acid ester, is a specific inhibitor of cholinesterases. The data obtained also revealed distinct dissimilarity in A C h E sensitivity of the aphids (Aphidinea) and that of the representatives of the psylla suborder (Psyllinea) and coccids (Coccinea). AChEs of psyllas and coccids are 1-2.5 orders of magnitude less sensitive to all three OPIs and are 1-2 orders of magnitude more sensitive to physostigrnine as compared to aphids. An exception is the AChE of grape phylloxera, a represenative of the family Phylloxeridae. For this insect the k a values following interaction with physostigmine approximate the k~ values for AChE of other aphids, whereas on interaction with OPI they are dramatically lower, being four orders of magnitude lower than that of the AChEs of other aphids under study. DISCUSSION
RESULTS
As follows from the data presented in Table 1, AChE activity was detected in all species of insects under study, but absolute activity values per g of live weight of representatives of various suborders of Homoptera as well as within the suborder of aphids, Aphidinea, differ considerably. Similar differences in
We made an attempt to correlate the observed differences in AChE sensitivity to various inhibitors of the insects under study with their systematics and phylogeny. The problem is complicated by the fact that there is no entirely unified viewpoint on aphid phylogeny. After the classical work of Mordvilko (1934), modern authors acknowledge the superfamily
Cholinesterases of aphids---Ill
17
Table 1. Velocity of enzymatic hydrolysis of ACTh and BuTCh in different insect homogenates and sensitivity of the AChEs to different inhibitors The degree of AChE inhibition in % as a V
Insect species Order Homoptera Suborder Psyllinea Psylla mall Schmidbg. Psylla ulmi Frst. Suborder Coccinea Pseudococcus maritimus Ehrh. Suborder Aleyrodinea Trialeyrodes vaporariorum Westw. Suborder Cicadinea Oncopsis flavicoUis L. Suborder Aphidinea Superfamilia Adelgoidea Familia Phylloxeridae Viteus vitifolii Fitch. Phylloxera coccinea Heyd. Superfamilia Aphidoidea Familia Laehnidae Cinara pinea Mordv. Schizolachnus pineti F. Pterochloroides persicae Chol. Familia Pemphigidae Eriosoma ulmi L. Familia Chaitophoridae Chaitophorus niger Mordv. Chaitophorus leucomelas Koch Familia Callaphididae Symydobius oblongus Heyd. Eucallipterus tilia L. Callipterinella betulae Kalt. Familia Anoeeiidae Anoecia corni F. Familia Thelaxidae Glyphina betulae Kalt. Familia Aphididae Pterocomraa salicis L. Rhopalosiphum padi L. Schizaphis gramina Rond.* Aphis gossipii Glov. Aphis grossularia Kalt. Aphis fabae Stop. Aphis farinosa Gruel. Aphis corniella H.R.L. Aphis craccivora Koch Dysaphis devecta Walk. Brevicoryne brassicae L. Brevicoryne crambe Bozh. Cavariella sp. Guere. Myzus persicae Sulz. Myzus padellus H.R. et Reg. Cryptomyzus ribis L. Acyrthosiphon pisum Harr.~"
(#mol/min/g)
ks (M-~ rain)
result of 10min ineincubation with Physostigmine 7.7 x i0 -4 M DTNB
ATCh
BuTCh
Gd-42
Gd-7
DFP
1.5 0.9
0.28 0.3
7.9 x 106 3.8 x 106
1.2 x 104 2.4 x 104
8.9 x 104 6.6 x 104
6.2 x 106 1.5 x 107
0 0
0.1
0
1.4 x l07
1.8 x 104
4.6 x 104
1.2 x 107
0
0.5
0
.
1.4
0
.
3.5 0.6
0 0
4.2 x l04 .
1.2 x 102 .
9.3 x 104
.
8 3
2.4 1.8 1.4
0.7 0 0
6,1 x l0 s 5.1 x 104 . . . 2.5 x l0 s --
3.9 x 106 . --
9.5 x 104 --
7 7 6
2.9
0
5.5 × 105
1.2 × 106
6.1 X 105
68
4.6 3.5
0 0
3.2 5.2 0.5
0 0 0
3.4 × l0 s . .
2.9
0
> 10 9
2.7
0
1.6 2.0 4.2 2.9 2.8 4.4 2.0 1.0 4.6 3.2 0.52 0.55 2.5 5.4 5.1 11.0 2.4
0.2 0.34 0.9 0.23 0.13 0.4 0 0 0.4 1.3 0.35 --3.7 1.1 1.5 1.3
.
.
. 6.7 x l0s 4.2 x l0s 1.6 × 109 > 10 9
. . . . > 109 4.3 x 108 3.4 x 10s 4.9 x 108 5.9 x 10a 9.3 x 108 . . > 109
.
.
0
.
> l0 t .
> 10 9
. .
.
.
0
.
.
.
.
. 8.1 × 105 . .
1.4 × 106 . .
2.2 × 105
. .
41 64 39
1.1 × 105
1.6 × 106
2.3 × 105
55
.
.
2.0 × 105 1,3 × 105 6.0 × 105 8.1 X 105 . . . . . . . . -7.4 x 105 7.5 x 105 8.7 x 105 7.8 x 105 2.0 x 106 . . . . 8.1 x 105
.
46
. 3.2 × 106 1.4 × 106 1.6 × 106 1.9 X 106 . . . . -3.6 x l06 2.5 x 106 1.4 x 106 1.6 x 106 2.5 x 106 . . 1.9 x 106
63 4.2 × 105 2.1 x 105 5.0 × 105 2.5 X 105
-5.3 x 3.2 x 4.5 x 5.6 x 2.1 x
105 105 105 105 105
2.5 x 105
73 73 75 75 59 64 74 75 43 84 33 35 30 59 41 42 74
*Brestkin et al. (1985). ?Nikanorova (1980).
o f o v i p a r o u s a p h i d s (Adelgoidea), including the f a m ily Phylloxeridae, to be the m o s t primitive a n d oldest in the a p h i d s u b o r d e r . T o all a p p e a r a n c e s a n o t h e r superfamily of true or viviparous aphids (Aphid o i d e a ) o r i g i n a t e s f r o m fossil o v i p a r o u s a p h i d s . N e v e r t h e l e s s , n o n e o f the k n o w n fossil o v i p a r o u s a p h i d families c o u l d be c o n s i d e r e d to be the direct a n c e s t o r o f a n y recent v i v i p a r o u s a p h i d families (Heie, 1967; S h a p o s h n i k o v , 1979). Several a u t h o r s h a v i n g a n a l y z e d a n d c o m p a r e d m o r p h o l o g i c a l structures o f recent a n d extinct a p h i d s d r e w different c o n c l u s i o n s c o n c e r n i n g the p h y l o g e n y o f the recent a p h i d s o f the s u p e r f a m i l y A p h i d o i d e a . T h u s , in the first w o r k o n a p h i d p h y l o g e n y (Baker, 1920), L a c h n i n i were d e s i g n a t e d as the m o s t p r i m i IB 19/I-B
tive, w h e r e a s the a u t h o r o f the next w o r k ( T a k a h a s h i , 1931) w h o h a d d e v e l o p e d the p h y l o g e n e t i c tree, c o n sidered the family C a l l a p h i d i d a e to be the m o s t primitive. M a c k a u e r (1965) h a v i n g u s e d a u n i q u e p a r a s i t o l o g i c a l classification m e t h o d d r e w the c o n c l u s i o n t h a t the m o s t p r i m i t i v e are a p h i d s o f the family T h e l a x i d a e a n d Heie (1967) places the family P e m p h i g i d a e b r a n c h i m m e d i a t e l y after the family P h y l l o x e r i d a e b r a n c h in his p h y l o g e n e t i c tree. M a m o n t o v a (1980) p r e s u m e s the family L a c h n i d a e to be the oldest. Recently, S h a p o s h n i k o v (1984), n o t i n g the subjective f a c t o r s w h i c h influenced the c o n c l u s i o n s o f several a u t h o r s , d e v e l o p e d a m o r e f o r m a l ized, s e m i q u a n t i t a t i v e m e t h o d o f a p h i d p h y l o g e n y analysis. F r o m the d a t a he r e p o r t e d t h a t P e m p h i g i d a e
18
K.V. NOVOZmLOVet al.
aphids have the most fossil signs and the least recent signs. From this it is inferred that the problem of phylogeny of the recent viviparous aphids is still open to argument. SH-sensitivity of AChEs of the aphids (Aphidinea), which was not detected with the insects of other suborders of Homoptera, suggests that this property originated and developed in the process of aphid evolution. As seen from the data presented in Table 1, SH-sensitivity of AChE of the older oviparous aphids of the family Phy!loxeridae is still very low whereas that of the majority of the more recent viviparous aphid families is much more prominent. The fact that SH-sensitivity of three aphid species of the family Lachnidae is low and is at the same level as that of the family Phyll.oxeridae, provides support for the authors (Baker, 1920; Mamontova, 1980) who consider the family Lachnidae to be the most primitive and oldest within the superfamily of viviparous aphids. Thus, SH-sensitivity can apparently be considered to be a specific biochemical character of aphid ACHE, capable of revealing phylogeny in the suborder Aphidinea. The changes in the structure of the AChE molecule, which occurred in the process of evolution and gave rise to SH-sensitivity, was accompanied by changes in other properties of the enzyme as well. In particular there are also changes in the velocity of irreversible inhibition by OPI and physostigmine. Sensitivity of AChE to DTNB in the aphid superfamily Aphidoidea coincides with higher sensitivity to the OPIs studied and lower sensitivity to physostigmine as compared with AChEs of psylla (Psyllinea) and coccid (Coccinea). Only the AChE of Lachnidae aphids, which we consider to be the oldest of the viviparous aphids, followed another pattern. Their OPI- and physostigmine-sensitivity is at the same level with the other more recent families whereas their SH-sensitivity is at a very low level characteristic of older oviparous aphids of the family Phylloxeridae. Other changes in molecular structure took place in phylloxera AChE leading to a considerable drop of its sensitivity to OPI (Table 1). Drastic differences of OPI-sensitivity of grape phylloxera, when compared to all AChEs of homopterous insects studied agree with the aphidologists' conclusions that this family branch is not an ancestor of the recent viviparous aphids. Thus, the study of AChE inhibitor specificity can provide important additional information for phylogenetic investigations. REFERENCES
Aldridge W. N. (1950) Some properties of specific cholinesterase with particular reference to the mechanisms of inhibition by diethyl-p-nitrophenyl thiophosphate (E605) and analogues. Biochem. J. 46, 451-460. Aldridge W. N. and Reiner E. (1972) Enzyme lnhibitors as Substrates. North-Holland, Amsterdam. Augustinsson K.-B. (1963) Classification and comparative enzymology of the cholinesterases and methods for their determination. In Handbuch der Experimentell Pharmakologie Vol. 15, pp. 89-128. Springer, Berlin. Baker A. C. (1920) Generic classificationof the hemipterous family Aphididae. U.S. Department of Agriculture Bulletin, No. 826.
Brestkin A. P., Maizel E. B., Moralev S. N., Novozhilov K. V. and Sazonova I. N. (1985) Cholinesterases of aphids--I. Isolation, partial purification and some properties of cholinesterases from spring grain aphid Schizaphis gramina (Rond.). Insect Biochem. 15, 309-314. Brestkin A. P., Khovanskikh A. E., Maizel E. B., Moralev S. N., Novozhilov K. V., Sazonova I. N., Abduvakhabov A. A., Godovikov N. N., Kabachnik M. I., Khaskin B. A., Mastryukova T. A. and Shipov A. E. (1986) Cholinesterases of aphids--II. Anticholinesterasepotency and toxicity of different organophosphorous inhibitors for spring grain aphid Schizaphis gramina (Rond.). Insect Biochem. 16, 701-707. Hama H., Iwata T., Mijata T. and Saito T. (1980) Some properties of acetylcholinesterase partially purified from susceptible and resistant green rice leafhopper, Nephotettix cincticeps (Uhler) (Hemiptera: Deltocephalidae). Appl. Ent. Zool. 15, 249-261. Heie O. E. (1967) Studies on Fossil Aphids (Homoptera: Aphidoidea), Vol. 26. Spolia Zool. Mus. Hauniensis. Copenhagen. Mackauer M. (1965) Parasitological data as an aid in aphid classification. Can. Ent. 97, 1016-1024. Mamontova V. A. (1980) The evolution, phylogenesis and system of Aphidinea, Lachnidae (Communication I). Vest. Zool. 1, 3-12. Manulis S., Ishaaya J. and Perry A. S. (1981) Acetylcholinesterase of Aphis citricola: properties and significance in determining toxicity of systemic carbamate compounds. Pestic. Biochem. Physiol. 13, 267-274. Mordvilko A. (1934) On the evolution of aphids. Arch. Naturgesch. 3, 1-60. Miiller K. N. (1960) Zur Anvendung der Variationsbreite bei der Beurteilungvon Versuchungsergebnisse.Z. landw. Vers.-u. UntersWes. 6, 195-198. Nikanorova E. V. (1980) New data on the properties of cholinesterase of pea aphid (Acyrthosiphon pisum). Byull. vses. nauchno-issled. Inst. Zaschity Rastenij 49, 31-32. Sazonova I. N. (1966) About cholinesterase activity in insect tissues homogenates. Ent. Obozr. 14, 793-797. Sazonova I. N., Brestkin A. P., Maizel E. B. and Moralev S. N. (1978) Acetylcholinesterasefrom spring grain aphid Schizaphis gramina (Rond.). Partial purification and some properties. Fourth International Congress of Pesticide Chemistry (IUPAC), Zurich. Shaposhnikov G. Kh. (1979) Some late Jurassic and early Cretaceous aphids. Paleontologich. Zh. 4, 66-78. Shaposhnikov G. Kh. (1984) Estimation of taxa and principle of their arrangement within the system. Zh. obshch. Biol. 45, 147-157. Smissaert H. R. (1976) Reactivity of a critical sulfhydryl group of the acetylcholinesterase from aphids (Myzus persicae). Pestic. Biochem. Physiol. 6, 215-222. Takahashi R. (1931) Aphididae of Formosa, Part 6. Department of Agriculture, Government Research Institute of Formosa, Report No. 53. Volkova R. I., Godovikov N. N., Kabachnik M. I., Magazanik L. G., Mastryukova T. A., Mikhelson M. Ya., Rozhkova E. K., Fruentov N. K. and Yakovlev V. A. (1961) Chemical structure and biological activity of organophosphorous inhibitors of cholinesterases. Vop. reed. Khim. 7, 250-259. Wood E., Zerba E., Piccolo M. and de Licastro S. (1979) Partial purification and characterization of Triatoma infestans head acetylcholinesterase. Insect Biochem. 9, 595-601. Zahavi M., Tahori A. S. and Klimer F. (1972) An acetylcholinesterase sensitive to sulfhydryl inhibitors. Biochim. biophys. Acta 276, 577-583.
0020-1790/89 $3.00+ 0.00 Copyright © 1989PergamonPressplc
CHOLINESTERASES OF APHIDS--III. SENSITIVITY OF ACETYLCHOLINESTERASES TO SEVERAL INHIBITORS AS A POSSIBLE PHYLOGENETIC CHARACTER K. V. NOVOZHILOV, 1 A. P. BRESTKIN, 2 A. E. KttOVANSKIKH,2 E. B. MAIZEL, 2 S. N. MORALEV,2 E. V. N I K A N O R O V A l and I. N. S A Z O N O V A l ~All-Union Research Instituteof Plant Protection, Shosse Podbelskogo 3, Pushkin-6, 188620 Leningrad and 21. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, U.S.S.R. Academy of Sciences, Thorez pr. 44, 194223 Leningrad, U.S.S.R. (Received 25 November 1987; revised and accepted 5 August 1988) Abstract--The influence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), three organophosphorous inhibitors (OPI) and physostigmine on the velocity of the acetylthiocholine enzymatic hydrolysis by the homogenates of 35 species of insects of the order Homoptera including 30 species of aphids (Aphidinea) was studied. Acetylcholinesterase (ACHE) of the representatives of the suborders psylla (Psyllinea), coccids (Coccinea), whitefly (Aleyrodinea) and leafhoppers (Cicadinea) is not inhibited by DTNB and has lower sensitivity to OPI and higher sensitivity to physostigmine as compared to the AChE of most species of aphids which have been investigated. It was found that only aphid AChE is sensitive to DTNB, this property being less pronounced for AChE of the aphid families Phylloxeridae and Lachnidae than for AChE of the aphid families Callaphididae, Pemphigidae, Chaitophoridae, Anoeciidae, Thelaxidae and Aphididae. AChE of all the aphids under investigation had very similar sensitivity to the OPIs studied and to physostigmine with the exception of AChE of the grape phylloxera Viteus vitifolii Fitch (Phylloxeridae) whose sensitivity to OPI was extremely low. It was concluded that the Lachnidae family is more ancient compared to other contemporary families of viviparous aphids. Key Word Index: aphid, Homoptera, phylogenesis, acetylcholinesterase, cholinesterase, SH-sensitivity, organophosphorous inhibitors, physostigmine
DTNB influence on the velocity of enzymatic hydrolysis of acetylthiocholine and butyrylthiocholine using homogenates of 30 aphid species and 5 representatives of other hornopterous suborders. We also determined the sensitivity of AChE in these species to three organophosphorous inhibitors and physostigmine.
INTRODUCTION Since 1972 a number of groups have shown that acetylcholinesterase (ACHE) of some aphid species is irreversibly inhibited by SH-reagents including 5,5'dithiobis(2-nitrobenzoic acid) (EUman reagent, DTNB) (Zahavi eta[., 1972; Smissaert, 1976; Sazonova et oi., 1978; Nikanorova, 1980; Manulis et al., 1981; Brestkin et ol., 1985). Cholinesterases of other animals, including AChEs of some insects, which are close relatives of aphids, such as the bug Triatoma infestans (Hemiptera) (Wood et al., 1979) and the rice leafhopper Nephotettix cincticeps Uhler (Homoptera) (Hama eta/., 1980) are not sensitive to DTNB. That is, these cholinesterases are not SH-sensitive enzymes. Also, some other unusual properties of AChE from aphids were revealed. Thus, AChE of the spring grain aphid Schizaphis gramina has a higher sensitivity to cationic OPI and derivatives of methylthiophosphonic acid, and a lower sensitivity to physostigmine compared to "typical" human erythrocyte AChE (Brestkin et al., 1985, 1986). Butyrylcholinesterase (BuChE) detected in homogenates of some aphid species proved to be insensitive to DTNB (Zahavi et al., 1972; Brestkin et al., 1985). We were interested in determining how these particular cholinesterases are distributed among insects of the order Homoptera, including aphids of various families. Particularly, we wanted to determine the relationship of these properties to the systematics and phylogeny of these insects. Therefore, we studied
MATERIALS AND METHODS
The majority of the insects were collected in field conditions in the Leningrad region. Whiteflies, Trialeyrodes vaporariorwn Westw., were collected on cucumbers in a hothouse. Grape phylloxera Viteus vitifolii Fitch. (from Kishinev), aphids Aphis craccivora Koch. and Pterochloroides persicae Chol. (from the Tashkent region) were transported to Leningrad in Dewar flasks with dry ice. Prior to investigation the insects were stored at - 15°C for < 1 month. After storage for 6 months, only 5-15% of the enzymatic activities were lost. To determine species specificity the insects were mounted and studied by light microscopy. Acetylthiocholine bromide (ATCh) and butyrylthiocholine bromide (BuTCh) were from Chemapol, 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) was from KochLight Lab. and diisopropylfluorophosphate(DFP), sodium dodecylsulfate (SDS) and physostigraine were from Serva. Organophosphorous inhibitors C2HsO(CH3)P(O)SC2H4S+(CH3)C2Hs. CH3SOf (Gd-42) and C2HsO(CH3)P(O)SC2H4SC2H5 (Gd-7) were synthesized at the A. N. Nesmeyanov Institute of Organoelement Compounds of the U.S.S.R. Academy of Sciences (Moscow), by the method of Volkova et aL (1961). 15
K.V. NOVOZHILOVet al.
16
Homogenates of whole insects were prepared in a glass homogenizer in 0.05 M sodium phosphate buffer, pH 7.5, at concentrations from I g/20 ml to ! g/100 ml depending on the enzyme's specific activity. Because of the high lipid content of the coccid, psylla and whitefly homogenates, they were centrifuged at 100,000g for 2 h at 4°C. Then, BuChE activity was measured in the supematant and AChE activity in the pellet after suspension in the starting volume of the buffer. The catalytic activity of the cholinesterases was determined by our modification of the Ellman method (Brestkin et al., 1985) in 0.07 M Na-phosphate buffer, pH 7.5, with the addition of 1.3 x 10-3 M substrate solution (ATCh for determination of AChE activity and BuTCh for BuChE activity). As stated before (Nikanorova, 1980; Brestkin et al., 1985), aphid BuChE does not hydrolyze ATCh significantly. Therefore, the use of these two substrates makes it possible to measure activity of the cholinesterases under study separately without recourse to specific inhibitors. In 10-15 rain the enzymatic reaction was stopped by addition of 3% SDS solution to a final concentration of 0.3%, followed by DTNB addition to a final concentration of 5 x 10-4 M. The reaction mixture was centrifuged for 10 min at 7000g. The optical density of the supernatant was measured at 412nm and the catalytic activity of the cholinesterases was calculated. When measuring enzymatic activity of coccid and psylla homogenates, an equal volume of ethanol was added to the reaction mixture before centrifugation to clarify the solution. The anticholinesterase potency of OPI and physostigmine was assessed by the magnitude of the bimolecular rate constant of interaction between cholinesterase and inhibitor (ka, M-~/min) calculated from the formula (Aldridge, 1950):
ka= l___lnV°
till v, where V0 is the catalytic activity of cholinesterase in the absence of the inhibitor and V, is the cholinesterase activity after interaction of the enzyme for t min with the inhibitor at a concentration [I] exceeding by many fold that of the active centres of cholinesterase. For V, determination the enzyme was incubated for 3-5 min with the inhibitor. Then the interaction between the enzyme and the inhibitor was stopped by the addition of substrate and the enzymatic reaction of substrate hydrolysis was carried out as outlined above. F0 was calculated from a similar experiment without an inhibitor. Various concentrations of inhibitors and several incubation intervals were used such that V, ranged from 20 to 80% of F0. The reaction corresponds to first-order kinetics as evidenced by the linear dependence In Fo/Ft vs t (Aldridge and Reiner, 1972). In the presence of DTNB the substrate hydrolysis by aphid AChE was nonlinear (Brestkin et al., 1985). Therefore we evaluated eholinesterase sensitivity to DTNB by comparison of two samples. In the first sample the homogenate was preincubated for 10rain at 25°C in 0.07 M Naphosphate buffer containing 7.7 x 10-4 M DTNB and incubated for 10rain with substrate. In the control sample DTNB was added after stopping the hydrolysis with SDS. Statistical evaluation of the results was performed by Miiller's method (1960). Each mean value was derived from 3 to 5 determinations. The SE was in the range of + 10%.
activity of AChEs are known with other groups of animals (Sazonova, 1966; Augustinsson, 1963). BuChE activity was detected only in 15 out of the 35 insect species under study and there is a 20-fold variation in its absolute value as well. In no case was BuTCh enzymatic hydrolysis inhibited by DTNB, nor could we find any relationship between BuChE activity and the taxonomy of the insects under study. Even within one Aphis genus this enzyme was found in four species and not detected in two others. It is likely that the BuChE of aphids as well as that of other animals does not take part in neuronal impulse conduction and cannot be classified as an enzyme of vital importance. Its physiological function is uncertain. From the data presented in Table 1 it follows that only AChE of aphids (Aphidinea) is sensitive to DTNB in contrast to that of the representatives of other suborders: psyllas (Psyllinea), coccids (Coccinea), whiteflies (Aleyrodinea) and leafhoppers (Cicadinea). The eight aphid families studied can be distinctly separated into two groups on the basis of AChE sensitivity to DTNB. AChE activity (measured in the presence of DTNB) of the aphid families Phylloxeridae and Lachnidae decreases only in the range of 3 to 8% following a 10 rain preincubation with D T N B whereas AChE activity of the representatives of the families of Pemphigidae, Chaitophoridae, Callaphididae, Thelaxidae, Anoeciidae and Aphididae is inhibited under the same conditions in the range of 33-84%. With the aim of further investigation of AChE properties of various insects, experiments were undertaken with three OPIs: Gd-42, Gd-7, DFP and physostigmine. Gd-42 is a cationic derivative of methylthiophosphonic acid, the specific and potent inhibitor of ACHE, especially of aphid AChE (Brestkin et al., 1986). Gd-7 is the non-cationic analog of Gd-42. DFP is a non-specific broad spectrum inhibitor, and physostigrnine, a methylcarbaminic acid ester, is a specific inhibitor of cholinesterases. The data obtained also revealed distinct dissimilarity in A C h E sensitivity of the aphids (Aphidinea) and that of the representatives of the psylla suborder (Psyllinea) and coccids (Coccinea). AChEs of psyllas and coccids are 1-2.5 orders of magnitude less sensitive to all three OPIs and are 1-2 orders of magnitude more sensitive to physostigrnine as compared to aphids. An exception is the AChE of grape phylloxera, a represenative of the family Phylloxeridae. For this insect the k a values following interaction with physostigmine approximate the k~ values for AChE of other aphids, whereas on interaction with OPI they are dramatically lower, being four orders of magnitude lower than that of the AChEs of other aphids under study. DISCUSSION
RESULTS
As follows from the data presented in Table 1, AChE activity was detected in all species of insects under study, but absolute activity values per g of live weight of representatives of various suborders of Homoptera as well as within the suborder of aphids, Aphidinea, differ considerably. Similar differences in
We made an attempt to correlate the observed differences in AChE sensitivity to various inhibitors of the insects under study with their systematics and phylogeny. The problem is complicated by the fact that there is no entirely unified viewpoint on aphid phylogeny. After the classical work of Mordvilko (1934), modern authors acknowledge the superfamily
Cholinesterases of aphids---Ill
17
Table 1. Velocity of enzymatic hydrolysis of ACTh and BuTCh in different insect homogenates and sensitivity of the AChEs to different inhibitors The degree of AChE inhibition in % as a V
Insect species Order Homoptera Suborder Psyllinea Psylla mall Schmidbg. Psylla ulmi Frst. Suborder Coccinea Pseudococcus maritimus Ehrh. Suborder Aleyrodinea Trialeyrodes vaporariorum Westw. Suborder Cicadinea Oncopsis flavicoUis L. Suborder Aphidinea Superfamilia Adelgoidea Familia Phylloxeridae Viteus vitifolii Fitch. Phylloxera coccinea Heyd. Superfamilia Aphidoidea Familia Laehnidae Cinara pinea Mordv. Schizolachnus pineti F. Pterochloroides persicae Chol. Familia Pemphigidae Eriosoma ulmi L. Familia Chaitophoridae Chaitophorus niger Mordv. Chaitophorus leucomelas Koch Familia Callaphididae Symydobius oblongus Heyd. Eucallipterus tilia L. Callipterinella betulae Kalt. Familia Anoeeiidae Anoecia corni F. Familia Thelaxidae Glyphina betulae Kalt. Familia Aphididae Pterocomraa salicis L. Rhopalosiphum padi L. Schizaphis gramina Rond.* Aphis gossipii Glov. Aphis grossularia Kalt. Aphis fabae Stop. Aphis farinosa Gruel. Aphis corniella H.R.L. Aphis craccivora Koch Dysaphis devecta Walk. Brevicoryne brassicae L. Brevicoryne crambe Bozh. Cavariella sp. Guere. Myzus persicae Sulz. Myzus padellus H.R. et Reg. Cryptomyzus ribis L. Acyrthosiphon pisum Harr.~"
(#mol/min/g)
ks (M-~ rain)
result of 10min ineincubation with Physostigmine 7.7 x i0 -4 M DTNB
ATCh
BuTCh
Gd-42
Gd-7
DFP
1.5 0.9
0.28 0.3
7.9 x 106 3.8 x 106
1.2 x 104 2.4 x 104
8.9 x 104 6.6 x 104
6.2 x 106 1.5 x 107
0 0
0.1
0
1.4 x l07
1.8 x 104
4.6 x 104
1.2 x 107
0
0.5
0
.
1.4
0
.
3.5 0.6
0 0
4.2 x l04 .
1.2 x 102 .
9.3 x 104
.
8 3
2.4 1.8 1.4
0.7 0 0
6,1 x l0 s 5.1 x 104 . . . 2.5 x l0 s --
3.9 x 106 . --
9.5 x 104 --
7 7 6
2.9
0
5.5 × 105
1.2 × 106
6.1 X 105
68
4.6 3.5
0 0
3.2 5.2 0.5
0 0 0
3.4 × l0 s . .
2.9
0
> 10 9
2.7
0
1.6 2.0 4.2 2.9 2.8 4.4 2.0 1.0 4.6 3.2 0.52 0.55 2.5 5.4 5.1 11.0 2.4
0.2 0.34 0.9 0.23 0.13 0.4 0 0 0.4 1.3 0.35 --3.7 1.1 1.5 1.3
.
.
. 6.7 x l0s 4.2 x l0s 1.6 × 109 > 10 9
. . . . > 109 4.3 x 108 3.4 x 10s 4.9 x 108 5.9 x 10a 9.3 x 108 . . > 109
.
.
0
.
> l0 t .
> 10 9
. .
.
.
0
.
.
.
.
. 8.1 × 105 . .
1.4 × 106 . .
2.2 × 105
. .
41 64 39
1.1 × 105
1.6 × 106
2.3 × 105
55
.
.
2.0 × 105 1,3 × 105 6.0 × 105 8.1 X 105 . . . . . . . . -7.4 x 105 7.5 x 105 8.7 x 105 7.8 x 105 2.0 x 106 . . . . 8.1 x 105
.
46
. 3.2 × 106 1.4 × 106 1.6 × 106 1.9 X 106 . . . . -3.6 x l06 2.5 x 106 1.4 x 106 1.6 x 106 2.5 x 106 . . 1.9 x 106
63 4.2 × 105 2.1 x 105 5.0 × 105 2.5 X 105
-5.3 x 3.2 x 4.5 x 5.6 x 2.1 x
105 105 105 105 105
2.5 x 105
73 73 75 75 59 64 74 75 43 84 33 35 30 59 41 42 74
*Brestkin et al. (1985). ?Nikanorova (1980).
o f o v i p a r o u s a p h i d s (Adelgoidea), including the f a m ily Phylloxeridae, to be the m o s t primitive a n d oldest in the a p h i d s u b o r d e r . T o all a p p e a r a n c e s a n o t h e r superfamily of true or viviparous aphids (Aphid o i d e a ) o r i g i n a t e s f r o m fossil o v i p a r o u s a p h i d s . N e v e r t h e l e s s , n o n e o f the k n o w n fossil o v i p a r o u s a p h i d families c o u l d be c o n s i d e r e d to be the direct a n c e s t o r o f a n y recent v i v i p a r o u s a p h i d families (Heie, 1967; S h a p o s h n i k o v , 1979). Several a u t h o r s h a v i n g a n a l y z e d a n d c o m p a r e d m o r p h o l o g i c a l structures o f recent a n d extinct a p h i d s d r e w different c o n c l u s i o n s c o n c e r n i n g the p h y l o g e n y o f the recent a p h i d s o f the s u p e r f a m i l y A p h i d o i d e a . T h u s , in the first w o r k o n a p h i d p h y l o g e n y (Baker, 1920), L a c h n i n i were d e s i g n a t e d as the m o s t p r i m i IB 19/I-B
tive, w h e r e a s the a u t h o r o f the next w o r k ( T a k a h a s h i , 1931) w h o h a d d e v e l o p e d the p h y l o g e n e t i c tree, c o n sidered the family C a l l a p h i d i d a e to be the m o s t primitive. M a c k a u e r (1965) h a v i n g u s e d a u n i q u e p a r a s i t o l o g i c a l classification m e t h o d d r e w the c o n c l u s i o n t h a t the m o s t p r i m i t i v e are a p h i d s o f the family T h e l a x i d a e a n d Heie (1967) places the family P e m p h i g i d a e b r a n c h i m m e d i a t e l y after the family P h y l l o x e r i d a e b r a n c h in his p h y l o g e n e t i c tree. M a m o n t o v a (1980) p r e s u m e s the family L a c h n i d a e to be the oldest. Recently, S h a p o s h n i k o v (1984), n o t i n g the subjective f a c t o r s w h i c h influenced the c o n c l u s i o n s o f several a u t h o r s , d e v e l o p e d a m o r e f o r m a l ized, s e m i q u a n t i t a t i v e m e t h o d o f a p h i d p h y l o g e n y analysis. F r o m the d a t a he r e p o r t e d t h a t P e m p h i g i d a e
18
K.V. NOVOZmLOVet al.
aphids have the most fossil signs and the least recent signs. From this it is inferred that the problem of phylogeny of the recent viviparous aphids is still open to argument. SH-sensitivity of AChEs of the aphids (Aphidinea), which was not detected with the insects of other suborders of Homoptera, suggests that this property originated and developed in the process of aphid evolution. As seen from the data presented in Table 1, SH-sensitivity of AChE of the older oviparous aphids of the family Phy!loxeridae is still very low whereas that of the majority of the more recent viviparous aphid families is much more prominent. The fact that SH-sensitivity of three aphid species of the family Lachnidae is low and is at the same level as that of the family Phyll.oxeridae, provides support for the authors (Baker, 1920; Mamontova, 1980) who consider the family Lachnidae to be the most primitive and oldest within the superfamily of viviparous aphids. Thus, SH-sensitivity can apparently be considered to be a specific biochemical character of aphid ACHE, capable of revealing phylogeny in the suborder Aphidinea. The changes in the structure of the AChE molecule, which occurred in the process of evolution and gave rise to SH-sensitivity, was accompanied by changes in other properties of the enzyme as well. In particular there are also changes in the velocity of irreversible inhibition by OPI and physostigmine. Sensitivity of AChE to DTNB in the aphid superfamily Aphidoidea coincides with higher sensitivity to the OPIs studied and lower sensitivity to physostigmine as compared with AChEs of psylla (Psyllinea) and coccid (Coccinea). Only the AChE of Lachnidae aphids, which we consider to be the oldest of the viviparous aphids, followed another pattern. Their OPI- and physostigmine-sensitivity is at the same level with the other more recent families whereas their SH-sensitivity is at a very low level characteristic of older oviparous aphids of the family Phylloxeridae. Other changes in molecular structure took place in phylloxera AChE leading to a considerable drop of its sensitivity to OPI (Table 1). Drastic differences of OPI-sensitivity of grape phylloxera, when compared to all AChEs of homopterous insects studied agree with the aphidologists' conclusions that this family branch is not an ancestor of the recent viviparous aphids. Thus, the study of AChE inhibitor specificity can provide important additional information for phylogenetic investigations. REFERENCES
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Brestkin A. P., Maizel E. B., Moralev S. N., Novozhilov K. V. and Sazonova I. N. (1985) Cholinesterases of aphids--I. Isolation, partial purification and some properties of cholinesterases from spring grain aphid Schizaphis gramina (Rond.). Insect Biochem. 15, 309-314. Brestkin A. P., Khovanskikh A. E., Maizel E. B., Moralev S. N., Novozhilov K. V., Sazonova I. N., Abduvakhabov A. A., Godovikov N. N., Kabachnik M. I., Khaskin B. A., Mastryukova T. A. and Shipov A. E. (1986) Cholinesterases of aphids--II. Anticholinesterasepotency and toxicity of different organophosphorous inhibitors for spring grain aphid Schizaphis gramina (Rond.). Insect Biochem. 16, 701-707. Hama H., Iwata T., Mijata T. and Saito T. (1980) Some properties of acetylcholinesterase partially purified from susceptible and resistant green rice leafhopper, Nephotettix cincticeps (Uhler) (Hemiptera: Deltocephalidae). Appl. Ent. Zool. 15, 249-261. Heie O. E. (1967) Studies on Fossil Aphids (Homoptera: Aphidoidea), Vol. 26. Spolia Zool. Mus. Hauniensis. Copenhagen. Mackauer M. (1965) Parasitological data as an aid in aphid classification. Can. Ent. 97, 1016-1024. Mamontova V. A. (1980) The evolution, phylogenesis and system of Aphidinea, Lachnidae (Communication I). Vest. Zool. 1, 3-12. Manulis S., Ishaaya J. and Perry A. S. (1981) Acetylcholinesterase of Aphis citricola: properties and significance in determining toxicity of systemic carbamate compounds. Pestic. Biochem. Physiol. 13, 267-274. Mordvilko A. (1934) On the evolution of aphids. Arch. Naturgesch. 3, 1-60. Miiller K. N. (1960) Zur Anvendung der Variationsbreite bei der Beurteilungvon Versuchungsergebnisse.Z. landw. Vers.-u. UntersWes. 6, 195-198. Nikanorova E. V. (1980) New data on the properties of cholinesterase of pea aphid (Acyrthosiphon pisum). Byull. vses. nauchno-issled. Inst. Zaschity Rastenij 49, 31-32. Sazonova I. N. (1966) About cholinesterase activity in insect tissues homogenates. Ent. Obozr. 14, 793-797. Sazonova I. N., Brestkin A. P., Maizel E. B. and Moralev S. N. (1978) Acetylcholinesterasefrom spring grain aphid Schizaphis gramina (Rond.). Partial purification and some properties. Fourth International Congress of Pesticide Chemistry (IUPAC), Zurich. Shaposhnikov G. Kh. (1979) Some late Jurassic and early Cretaceous aphids. Paleontologich. Zh. 4, 66-78. Shaposhnikov G. Kh. (1984) Estimation of taxa and principle of their arrangement within the system. Zh. obshch. Biol. 45, 147-157. Smissaert H. R. (1976) Reactivity of a critical sulfhydryl group of the acetylcholinesterase from aphids (Myzus persicae). Pestic. Biochem. Physiol. 6, 215-222. Takahashi R. (1931) Aphididae of Formosa, Part 6. Department of Agriculture, Government Research Institute of Formosa, Report No. 53. Volkova R. I., Godovikov N. N., Kabachnik M. I., Magazanik L. G., Mastryukova T. A., Mikhelson M. Ya., Rozhkova E. K., Fruentov N. K. and Yakovlev V. A. (1961) Chemical structure and biological activity of organophosphorous inhibitors of cholinesterases. Vop. reed. Khim. 7, 250-259. Wood E., Zerba E., Piccolo M. and de Licastro S. (1979) Partial purification and characterization of Triatoma infestans head acetylcholinesterase. Insect Biochem. 9, 595-601. Zahavi M., Tahori A. S. and Klimer F. (1972) An acetylcholinesterase sensitive to sulfhydryl inhibitors. Biochim. biophys. Acta 276, 577-583.