Esterases during postembryonic development of the larva of Chilo zonellus

7. Insect Physiol., 1970, Vol. 16, pp. 2385 to 2399. Pergamon Press. Printed in Great Britain

ESTERASES DURING POSTEMBRYONIC DEVELOPMENT OF THE LARVA OF CHILO ZONELLUS H. N. SINGH” Division of Entomology,

and K. N. MEHROTRA

Indian Agricultural Research Institute, Delhi, India (Received 27 February

1970)

Abstract-Presence of esterase activity in the whole body homogenate of the larva of the maize stem borer, Chile zonellus, was demonstrated using indophenyl acetate and ortho-nitrophenyl acetate as substrates, the former being hydrolysed at a higher rate than the latter. There was no apparent increase in the esterase activity during different larval instars on a fresh weight basis, but the esterase content present in a fifth instar larva was found to be about 22.5 to 255 times higher than in a first instar. The effect of different concentrations of substrates, e.g. indophenyl acetate and O-nitrophenyl acetate, and inhibitors, e.g. DFP and diazoxon, on esterase activity indicated the presence of at least two esterases, one susceptible and the other resistant to OP-inhibition. This was further confirmed by studying the effect of various substrate concentrations on each esterase after separating them by selective inhibition. Michaelis-Menten constants and bimolecular rate constants are calculated in each case. DFPresistant esterase was found to increase during the larval growth. Possibilities for the presence of cholinesterase, ah-esterases and aromatic esterases have been discussed. INTRODUCTION

STUDIESon e&erases particularly cholinesterases are fundamental for the physiology of nerve function in insects. Although extensive studies have been undertaken on the properties of esterases in both adults as well as during embryonic development of insects (COLHOUN, 1963; SMITH and SALKELD,1966), very little is known of the various esterases during post-embryonic development (SMALLMANand MANSINGH, 1969). The present work was therefore undertaken to study the esterases hydrolysing indophenyl acetate (IPA) and ortho-nitrophenyl acetate (0-NPA) during larval development of Chile xonellus (Swinhoe). MATERIALS

AND METHODS

Insects used in the present study were the larval stages of Chilo xmllus. Larvae used were either reared in the laboratory or collected from the field. The method of rearing of insects in laboratory was that of PANT et al. (1961). Pupae and pharate adults collected from the field or obtained from the laboratory were placed in oviposition jars which were provided with a piece of cotton soaked in sugar solution to provide food for emerging adults and also to maintain humidity inside the jars. The eggs laid were collected at X&hourly intervals and the papers * Present address: Department of Entomology, Faculty of Agriculture, Banaras Hindu University, Varanasi-5, India. 2385

H. N. SINGHANDK. N. MEHROTRA

2386

containing these were cut out and placed in Petri dishes to hatch. The hatched larvae were reared on the maize stem cuttings under laboratory conditions and the food was changed every fourth day. Various stages of larvae collected from the culture as well as those collected from the field were used in experiments. Preparatimc

of homogenate

The esterase activity was measured in the homogenates of the whole larvae. The larvae were homogenized with the help of an all glass Potter-Elvehjem type homogenizer in phosphate buffer. The concentration of tissues in the homogenate was kept at 10 mg/ml and the homogenate was centrifuged for 4 min at ca. 1000 g. The supematant was collected and diluted with the buffer to the desired concentrations used in the experiments. The diluted homogenate was divided into small aliquots and kept frozen at - 5°C until used. Enzymatic

assay

Esterase activity in the homogenate was measured by using two different substrates viz. IPA and 0-NPA. The method of KRAMER and GAMSON (1958) as modified by ARCHERand ZWEIG (1959) was used to estimate esterase activity using IPA as substrate. The hydrolysis of IPA was followed by measuring an increase in absorbance at 625 my, and the amount of IPA hydrolysed was estimated using molar extinction coefficient of indophenate ions as 1.16 x 104. The method of MAIN et al. (1961) was used to estimate the esterase activity using 0-NPA as substrate. The rate of hydrolysis of 0-NPA in this method was followed by measuring the increase in absorbance at 415 mp. The rate of reaction was calculated taking molar extinction coefficient of 0-nitrophenol as 4.95 x 103. The studies on the inhibition of esterase activity present in the homogenate by organophosphorus compounds were undertaken with a view to see (a) if more than one esterases are present in the homogenate and (b) to find out the bimolecular rate constant of the esterase(s) with the inhibitors. The method of SMISSAERT(1965) was followed to resolve the various esterases present in the homogenate. The bimolecular rate constant of the various esterases was determined according to the method developed by MAIN and IVERSON(1966). Two inhibitors used in these studies were (a) DFP (di-isopropyl phosphorophosfluoridate) and (b) D iazoxon (diethyl 1-2-isopropyl-4-methyl-6-pyrimidyl phate). The solution of DFP was prepared in ethylene glycol and that of diazoxon in 3.5% sodium chloride. First, the stock solution of 3.2 x 10~~ M of each inhibitor was prepared and, from this, the successive concentrations were made by further dilution. The preparations were stored at - 5°C when not in use. Fresh inhibitor solutions were prepared every twelfth day. RESULTS Esterase activity

in larvae

The esterase activity in different instars as measured by the hydrolysis of IPA and 0-NPA are given in Table 1. These results were determined in the whole body

ESTERASESIN POSTBI'ABRYONIC DEVRLOPMENT OF

CHILO

2387

LARVA

homogenate of larvae and the figures represent the average values of 5 determinations. The data indicate the presence of sufficient amount of esterase activity during all the instars of larval development. In general, the e&erase activity was found to be lower with 0-NPA than IPA and the ratio of IPA versus 0-NPA activity was found to range from 1.5 to 2.2 in different instars of developing larvae. TABLE I-THE

ESTBRASEACTIVITYIN THEVARIOUS LARVAL INSTARSUSING IPA AND 0-NPA AS SUBSTRATE Indophenyl

Instars

1 2 3 4 5

Per mg body wt. 0.225 0.201 0.224 0.290 0.255

f 0.0009 + 0.0025 zk0.0003 f 0.0002 & 0.0016

acetate Per larva 0.11 2.01 6.72 17.40 28.05

0-nitrophenyl Per mg body wt. 0.121 0.135 0.161 0.130 0.124

* 0*00088 +0.00113 & 0.00218 * 0~00104 + 0.00050

acetate Per larva 0.06 1.35 4.83 7.80 13.64

Ratio of activity IPA/O-NPA 1.86 1.49 1.39 2.23 2.06

Unit: pmoles of the substrate hydrolysed/mg of body tissue or pmoles of the substrate hydrolysed/larva per hr. Note (a) The incubation mixture for IPA hydrolysis contained homogenate equivalent to I.0 mg body tissue, 0.033 M KH,PO, and I.24 x 10V4 M IPA in 3*3 ml of total incubation mixture. (b) The incubation mixture for 0-NPA hydrolysis contained I.5 mg of body tissue, O-066 M phosphate buffer and 10e4 M 0-NPA in 3.3 ml of total incubation mixture.

It is also apparent from the data given in Table 1 that there was no significant difference in the esterase activity of various instars on wet weight basis; the hydrolytic activities were found to be 0.20 to 0.30 pmoles IPA and 0.12 to 0.16 pmoles 0-NPA hydrolysed/mg of tissue per hr. If, however, the total esterase activity is calculated on per larva basis, rather than tissue weight basis, a significant difference in the esterase content was obtained between the different instars. Thus, a full grown fifth instar larva exhibits as much as 225 to 250 times higher activity with 0-NPA and IPA than shown by the first instar. The increase in total esterase activity during development could be explained on the basis of an increase in various nervous and non-nervous tissues in successive instars. These results on the esterase activity are similar to those of MANSINGH and SMALLMAN (1967a, b) who demonstrated the ChE hydrolysing ACh in a number of lepidopterous larvae. In all, the total esterase activity with ACh as substrate was found to increase from one instar to another. The presence of an esterased hydrolysing 0-NPA has also been demonstrated in Tmebrio larvae (LORD and POTTER, 1951). However, our results are differentfrom those of HASAN and ZAYED (1965) who failedto demonstrate the esteraseactivityin the blood, head, and total homogenate of Prodenia litura F.

2388

H. N.

Effect

ofsubstrate

SINGII ANDK. N. MEHROTFCA

concentrations on esterase activity

Difference in the hydrolysis rate of IPA and 0-NPA by larval homogenate suggests two possibilities (a) either the esterase hydrolysing IPA and 0-NPA possesses different affinity towards the two substrates, i.e. one is preferably hydrolysed more than the other due to its structural closeness to natural substrate, or (b) there may be two or more separate esterases hydrolysing these two substrates independently or simultaneously. In order to resolve different esterases present in larvae hydrolysing IPA and 0-NPA and also to find out the affinity of enzymes to these substrates, the method of HOFSTEE (1952) was utilized, by observing the rate of reaction at various substrate concentrations. When the rate of reaction (v) was plotted against the velocity upon substrate concentration (v/s), a curved line was obtained with IPA as substrate, for each instar (Fig. 1A). For a single enzyme catalysed reaction, the above plotting should ideally give a straight line with the slope -K,. A curved line is taken to represent at least two or more different enzymes hydrolysing the same substrate (HOFSTEE, 1952). The results given in Fig. 1A indicate that at least two esterases having

4

8

32

16 v/s

FIG. 1. Effect of different concentrations

x

104

of indophenyl acetate and 0-nitrophenyl acetate on e&erase activity in the homogenate of fifth instar larvae of ChaZo eon&us Incubation mixture for indophenyl acetate contained (Swinhoe). Note: O-033 M KHBPOl at pH 8.0, homogenate equivalent to 1.0 mg of body tissue and different concentrations of indophenyl acetate in 3.3 ml of total volume. The incubation mixture for 0-nitrophenyl acetate contained 0.066 M KH,PO, and NarHPO* mixed in the ratio to give pH 7.6, homogenate equivalent to 2-O mg body tissue and different concentrations of 0-nitrophenyl acetate in the total volume of 3.3 ml. V = pmoles of the substrate hydrolysed/mg of body tissue/hr. S = con0, esterase activity with indophenyl centration of the substrate in pmoles. Oacetate; O----- 0, esterase activity with 0-nitrophenyl acetate.

ESTERASESIN POSTEMBRYONIC DEVELOPMENT OF

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LARVA

2389

significantly different affinity for the IPA were present in the larval homogenate. The Km values of the two esterases were calculated and are presented in Table 2. From the data presented, it is evident that esterase giving slope a in Fig. 1A has nearly three to eight times lower affinity for the substrate than the esterase giving slope 6. In contrast to IPA, the results obtained with different concentrations of Q-NPA (Fig. 1B) gave a straight line for all stages of larval development. These results may, therefore, suggest that there is only one enzyme hydrolysing 0-NPA in various stages of larval development and if there are two enzymes, they must have very similar affinity for 0-NPA. A compa.rison of the Michaelis-Menten constant values for the two enzymes hydrolysing IPA and one hydrolysing 0-NPA (Table 2) TABLE 2-MICHAELIS-MENTEN CONSTANTS (M) OF ESTERASESFROM THE LARVAE HYDROLYSING IPAAND 0-NPA Indophenyl acetate Instars 1 2 3 4 5

Slope a 4.3x10-4 7.1x10-4 3.6~10-~ 3.3x10-4 2.1x 10-a

Slope b 8.7x10-5 9.1x10-5 9.3x10-5 8.3x10-5 8.1x 1O-5

O-nitrophenyl acetate 3.4x10-4 3*0x10-4 2.5x10-4 2+3x10-4 2.9x10-*

shows that there was no significant difference in the Km value of the same enzyme from one instar to another. Moreover, the Km value of the 0-NPA hydrolysing enzyme was found to be similar to that of a enzyme hydrolysing IPA. ESfect of DFP and diaxoxon on the esterases The effect of substrate concentrations showed that there may be more than one esterase present in the homogenate. A confirmation of this was sought by studying the susceptibility of these enzymes to inhibition by organophosphates, because it has been conclusively shown that various esterases have different I,, (inhibitor concentration required to inhibit 50 per cent of the total enzymic activity) for various organophosphorus compounds. Moreover, SMISSAERT (1965) has shown that by proper use of inhibitors, not only the proportions of various esterases showing difference in susceptibility to organophosphates can be established but their kinetic properties could also be studied. The effect of various concentrations of DFP on the esterases hydrolysing IPA and 0-NPA in the fifth instar larvae is presented in Fig. 2. The esterase activity was considerably reduced and about 50 per cent activity was inhibited by as low as 10-T M DFP with IPA and 0-NPA as substrates within four minutes of incubation period. DFP inhibited the esterase hydrolysing IPA with its subsequent increase in concentration up to 10e6 M and about 70-80 per cent of the total esterase activity

H. N. SINGH AND K. N. MEHROTRA

2390

was inhibited (Fig. 2A). However, above 5 x lO-5 M, there was no such proportional increase in the degree of inhibition with increase in inhibitor concentration. This remaining esterase activity seems to represent a separate esterase less sensitive to inhibitor and produces almost a straight line (Fig. 2A, b). The esterase hydrolysing 0-NPA seems to be more sensitive to DFP inhibition and about 35 per cent of the total activity was inhibited with as low as 1O-s DFP but the increase in

I 169

FIG. 2. Effect of different concentrations of DFP on esterase activity in the homogenate of fifth instar larvae of Chilo zonellus (Swinhoe) using indophenyl acetate and 0-nitrophenyl acetate as substrates. Note: Reaction mixture similar to that mentioned for Fig. 1. Incubation period of DFP with homogenate, 4 min. Concentration (final) of indophenyl acetate, 1.24 x lo-* M and that of O-nitro0, results 0 phenyl acetate, 1O-3 M in 3.3 ml of total incubation mixture. 0, results using 0-nitrophenyl using indophenyl acetate as substrate; 0 acetate as substrate.

inhibitor concentration above 10ms M had very little or no effect on the esterase activity. Thus, a straight line formed over a wide range of DFP concentrations indicates the organophosphate resistant esterase (Fig. 2B). The nature of both the curves remained exactly alike in all the instars of larval development. The effect of diazoxon on the inhibition of esterases hydrolysing IPA (Fig. 3A) was exactly similar and comparable to that of DFP. Two esterases, one being inhibited between 10-s to 10e6 M following first order reaction kinetics (Fig. 3A, a) and the other, less affected up to 5 x 10e5 M diazoxon (Fig. 3A, b) are present. The effect is somewhat different in pattern with 0-NPA hydrolysing esterase complex: unlike DFP, the diazoxon continued to inhibit the 0-NPA hydrolysis with further increase in concentrations and a concentration equal to lo4 M hardly left any esterase activity (Fig. 3B).

ESTBRASES

IN

POSTBMBRYONIC

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2391

The diazoxon seems to inhibit even some proportion of so called resistant esterase. This is substantiated by the fact that the proportion of the OP-resistant esterase hydrolysing 0-NPA as indicated by the per cent remaining esterase activity after complete inhibition of OP-sensitive esterase by DFP in the fifth instar larvae is obtained only between lo-” to 5.5 x 10e6 M concentrations (nearly 45-50x) of diazoxon, and the further increase in diazoxon concentration inhibits none but resistant esterase.

D I AZ

OXON

CONC.CM)

FIG. 3. Effect of different concentrations of diazoxon on esterase activity in the homogenate of fifth instar larvae of Chilo mn.e&~s (Swinhoe) using indophenyl acetate and 0-nitrophenyl acetate as substrates. Note: Reaction mixture similar to that mentioned for Fig. 1. Incubation period of diazoxon with homogenate, 4 min. Concentration (final) of indophenyl acetate, 1.24 x low4 M and that of O-_-O, O-nitrophenyl acetate, 10ea M in 3.3 ml of total incubation mixture. results using indophenyl acetate as substrate; O0, results using O-nitrophenyl acetate as substrate.

The ratio of, OP-sensitive and resistant e&erases hydrolysing IPA as well as 0-NPA in different instar larvae has been calculated and the per cent esterase activity after complete inhibition of OP-sensitive esterase, has been given in Table 3. The ratio of percentage of OP-resistant esterase seems to increase l-5fold from first to fifth instar and this increase is evidenced by both the substrates. The data clearly indicate that the ratio of OP-resistant to OP-sensitive esterase is about 1.5 times higher for 0-NPA hydrolysis as compared to that of IPA in all the instars. The data for diazoxon and 0-NPA could not be taken to represent actual

2392

H.N.

SINGHAND

K.N.

MEHROTRA

values since at 5 x 1O-5 M diazoxon, a portion of the OP-resistant inhibited. TABLE

esterase was also

3-PERCENTRRMAININGACTIVITYOFESTERASEHYDROLYSING IPAand 0-NPA, INHIBITIONOF OP-SENSITIVEESTERASEIN EACH LARVAL INSTAR BY 5 X1@-5 M DFP ORDIAZOXON

AFTER

Instars Inhibitor

Substrate

1

2

3

4

5

DFP

IPA 0-NPA

18.7 27.6

23.5 34.7

24.0 38.8

27.6 43.7

29.0 44.9

Diazoxon

IPA 0-NPA

22-o 8.9

28.0 8.9

29.6 10.1

29.6 5.6

33.2 6.0

The bimolecular rate constants of both the inhibitors with both the esterases has been calculated and has been presented in Table 4. From the data it can be seen that with organophosphate-sensitive-IPA-hydrolysing-esterase the diazoxon is ten times more reactive than DFP and this reactivity of diazoxon increases to TABLE 4-Ki (M-lmin-l)~~u~ OF DFP AND DIAZOXONWITH THETWOESTERASES PRESENT IN THE HOMOGENATE OF VARIOUS LARVAL INSTARSUSING IPAAND 0-NPAAS SUBSTRATES Larval instars Inhibitor Substrate Esterases DFP (Fig. 2)

IPA 0-NPA

Diazoxon (Fig. 3)

IPA 0-NPA

1

2

3

4

5

Slope Q b B

2.2 x 106 1.8 x lo5 1.7 x 10’

2.0 x 106 1-l x 105 4.4 x 10’

2.1 x 106 3.5 x 105 3.1 x 10’

1.7 x lo6 8.4 x lo* 1.4 x 107

2.4 x lo6 9.4 x lo* 7.9 x 106

a

3.4 x 10’ 3.0 x 107 3.0 x 105

2.4 x 10’ 1.4 x 10’ 2.8 x lo5

1.1 x 10’ 4.0 x 106 3.1 x 105

2.8 x 10’ 8.3 x lo6 3-0x lo5

4.3 x lo7 1.6 x lo6 2.4 x lo5

b B

about one hundred times the DFP for OP-less sensitive or resistant esterase. This is in accordance with the idea that diazoxon inhibits a portion of the so called OP-resistant esterase. The lower bimolecular rate constant of diazoxon with esterase hydrolysing 0-NPA may indicate that the latter refers to other than OPsensitive esterase. The hundred times greater value of esterase hydrolysing 0-NPA with DFP as compared with diazoxon can not be explained at present. The lack of comparable data may be due to the fact that bimolecular rate constant is a function of affinity constant as well as phosphorylation constant (MAIN, 1964) which are completely independent of each other for any particular enzymatic reaction.

ESTERASES IN POSTEMBRYONIC DEVELOPMENT OF CHILO LARVA Michaelis-Menten constants of the esterases hydrolysing IPA and O-NPA by selective inhibition

2393

separated

The results presented so far indicated that the hydrolysis of IPA in the larvae is catalysed by two separate enzymes differing in their Km values or in other words in their affinities. Inhibitor studies also showed that there may be two separate enzymes hydrolysing IPA differing in their susceptibility to OP-inhibition. The present set of experiments were, therefore, undertaken to determine the Km of both the resistant as well as susceptible esterases and compare them with the originally two suspected esterases present in the normal homogenate. The procedure adopted for this was according to SMISSAERT (1965). The sensitive esterase was selectively inhibited by either 5 x 10e5 M DFP or diazoxon for 4 min of incubation and the effect of different substrate concentrations on the rate of reaction was studied. The activity of OP-sensitive e&erase was interpolated by subtracting from the activity of total homogenate, the activity of resistant esterase on each concentration of substrate according to the method of HOFSTEE (1952). Side by side, the total esterase activity was also determined from the same untreated homogenate preparation and at the same substrate concentration level. The plottings of z, vs. v/s were done as in the previous experiment (Fig. 1) for the normal homogenate as well as for the treated homogenate preparations. The slopes of both the sensitive and resistant esterases were fitted with the least square method. The results with IPA as substrate and DFP as inhibitor are shown in Fig. 4. The figure shows that in normal homogenate there are two IPA hydrolysing esterases present similar to the results of the previous experiment (Fig. 1A). When the sensitive esterase is absent from the homogenate, the activity of esterase remains very little and yields a curve (Fig. 4, a’) similar to slope a of Fig. 1A. On the other hand, the slope representing the OP-sensitive esterase (Fig. 4, b’) shows higher activity and is similar to slope b of Fig. 1A. The Km values for the curves corresponding to normal homogenate as well as for both esterases separately have been calculated and presented in Table 5. These values are in agreement with the results obtained in earlier experiments with different IPA concentrations and normal homogenate. These results not only confirm the K,, values previously obtained, but also indicate that esterase having 3 x 10B4 M Km value for IPA is resistant to organophosphate and that with Km 3 x 10” M for IPA is susceptible to OP-inhibition. The results with both the inhibitors are exactly similar for all the 5 instars of larval development clearly demonstrating the presence of two esterases with different Km for IPA. The six different concentrations of 0-NPA were used to see the effect of both the esterases separately using 5 x 10-S M DFP (Fig. 5). The results obtained with untreated homogenate are similar to those obtained earlier (Fig. 1B) i.e. the plot ZJvs. v/s gave a straight line. The 0-NPA hydrolysing esterase not inhibited by 5 x lop5 M DFP also gave a straight line indicating the Km as 3 x 10-h M (Table 6). The activity of the DFP susceptible 0-NPA hydrolysing esterase, when interpolated, also gave a straight line of a Km value equal to 4 x 1O-4 M. Because

2394

H. N. SINGH AND K. N. MEHROTRA

of such a small difference in the two Km values of OP-susceptible and OP-resistant 0-NPA hydrolysing enzymes it was unlikely that the combined activity of two esterases as observed in the total homogenate would have been any different.

16

8

16

24

v/s

32

40

48

56

x IO4

FIG. 4. Effect of different concentrations of IPA on the esterase activity in the homogenate of fifth instar larvae of ChiZo zonellus (Swinhoe) with and without the inhibition by DFP. Note: Reaction mixture contained in 3.3 ml of total volume, O-033M KHZP04 buffer of pH 8.0, homogenate equivalent to I.0 mg of body tissue and the different concentrations of substrate. In treated homogenate 5 x 10m5M (final) DFP was incubated with enzyme for 4 min. V, pmoles of indophenyl acetate hydrolysed/mg of tissue/hr; S, indophenyl acetate concentration in pmoles; O0, total homogenate activity; l 0, activity of homogenate after complete inhibition of OP-sensitive esterase; @0, the activity of OP-sensitive e&erase.

TABLE S-MICHAELIS--MENTEN CONSTANTSFORTHE TWO ESTBRASES SEPARATED BY SELECTIVE INHIBITION USING IPA AS SUBSTRATE JCm(M) values Untreated homogenate

Treated by DFP

Untreated homogenate

Instars

Slope a

Slope b

Slope a’

Slope b’

Slope a

1 2 3 4 5

2.39x IO4 3.55x IO+ 3.20x lO-4 4.88x lO-4 4.01x IO-4

4.78x lO-5 5.31x IO” 3.38x lO-6 5.59x lO-6 444x IO”

2.54x IO4 4.06x IO+ 4.53x lO-4 1.73x 104 3.11x lO-4

3.82x 10-5 2.57x 10--5 2-94x lO-6 4*64x lOA 2.04x 10--5

3.00x IO-4 6.06x 10-k 1.90x IO4 3.30x lO-4 4.20x IO4

Slope b 4.00x IO-5 4.50x IO-5 4.60x lo-+ 2.25x IO” 5-13x 1O-5

Treated by diazoxon Slope a’ 5.45x IO-4 3.00x IO-4 3.25x lO-4 2.25x lO-4 I.50 x IO4

Slope b 5.00x 10-s 3.85x IO-5 4.54~ lOA 3.35x lO-5 6.15x IO4

ESTERASS

IN POSTEh4BRYONIC

DRVRLOPMENT

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2395

LARVA

I 48-

3

12

6 v/s

x

I04

FIG. 5. Effect of different concentrations of 0-nitrophenyl acetate on esterase activity in the homogenate of fifth instar larvae of Chile zonellus (Swinhoe) with and without the inhibition by DFP. Note: Reaction mixture contained 0.066 M phosphate buffer of KH,PO, and Na,HPO, of pH 7.6 homogenate equivalent to 2-O mg body tissue and the different concentrations of 0-nitrophenyl acetate in 3-3 ml of total volume. In treated homogenate, 5 x 10u5 M DFP (final) was incubated with enzyme for 4 min. V, pmoles of 0-nitrophenyl acetate hydrolysed/mg body tissue/hr; S, 0-nitrophenyl acetate concentrations in pmoles; eactivity; a, activity of homogenate after o----- O, total homogenate complete inhibition of OP-sensitive esterase; O-0, the activity of OPsensitive esterase.

TABLE 6--K,(M)vAI.uEs OFDIFFERENTESTRRASES HYDROLYSING 0-NPAFROMTHE:VARIOUS LARVAL INSTARS AFTER THEIR SEPARATION BY SELECTIVE INHIBITION BY 5 x~O-~M DFP

Larval instars Treatments

1

2

3

4

5

Untreated homogenate OP-resistant esterase OP-sensitive e&erase

3.37 x 1O-4 3.13 x IO-4 5.00 x 10-4

3.00 x 10-4 3.48 x 1O-4 1.95 x 10-a

2.52 x 10-d 2.03 x lo-* 3.37 x 1O-4

2.82 x 1O-4 2.62 x lo-* 2.66 x 1O-4

2.58 x 1O-4 2.40 x lo-* 5.10 x lo-4

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H. N. SINGHANDK. N. MEHROTRA

In conclusion, it can be said that the homogenate of the larvae contains at least two esterases hydrolysing IPA and 0-NPA with different affinities, and showing different characteristics when inhibited by organophosphorus compounds. The results also indicate that the organophosphates may differ among themselves not only in the over all rate of reaction of inhibition as indicated by bimolecular rate constant but, also in their aflinity and phosphorylation constants. DISCUSSION

The present demonstration of various esterases, both resistant and susceptible to organophosphate poisons in the various larval stages suggests that the OPsusceptible esterase may have physiological function in the working of the nervous system, and that it is analogous to that of specific ChE described from insects. This view finds support in the work of DAUTERMANet al. (1962) who demonstrated that the purified AChE from houseflies hydrolyses IPA and similar work of AFSHARPOURand O’BRIEN (1963) s h owed that AliE (ali-esterases) can also utilize IPA as substrate. If this is true, it is conceivable that at least part of the IPA hydrolysing enzyme activity seen in the larvae must be due to specific ChE. It is possible that the OP-susceptible IPA hydrolytic activity was due to the specific ChE and the OP-resistant enzyme was most probably aliphatic esterase or a mixture of aliphatic and aromatic esterases. HOPF (1954) c 1aimed that 0-NPA was hydrolysed by acetyl esterase in locust. VAN ASPEREN (1959) al so arrived at a similar conclusion that 0-NPA was hydroIysed by a esterine-sensitive ChE, 70 per cent of which was shown to be localized in the head and the remaining distributed in the trunk of the fly. In the trunk more than 83 per. cent of the total 0-NPA hydrolysis’was due to AliE. These workers also demonstrated that OP-resistant esterase, perhaps the ArE (aromatic esterase), also hydrolyses a small portion of 0-NPA. Similar results have been obtained for this ester by MAIN et al. (1961). Assuming that the physiology of larval stages is similar to that of adults and eggs, a similar substrate specificity may be expected and the presence of a number of esterases hydrolysing these esters may not be ruled out. Thus, the results obtained in the present studies suggest that all the three types of esterases, viz. ChE, AliE, and ArE are present in the homogenate. The IPA was probably hydrolysed predominantly by AChE and AliE, whereas 0-NPA was predominantly acted upon by ArE and AliE. The various esterases taking part in the hydrolysis of various substrates were distinguished by studying the effect of different substrate concentrations on the esterase activity according to the method followed by HOFSTEE (1952) which is based upon the fact that in a z, vs. U/Splot, a straight line is obtained, cutting the X axis on Vm/I& and Y axis on V,,,, with its slope equal to the value of -K,. A curved line thus, will be exhibited only by more than one esterase activities together, with different a&nities or X, values. However, a straight line may also be the resultant of more than one enzyme having similar K, for the substrate. The results obtained in these studies according to the method of HOFSTEE (1952) not only indicated the presence of various esterases but also provided some

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useful parameters, viz. Km for comparison. If the Km values obtained in the present study are compared with those of literature, the affinity of esterase hydrolysing 0-NPA is about 15 times higher than that reported for phenyl acetate by METCALF et al. (1956). The OP-sensitive esterase exhibits the affinity with IPA even higher than that with ACh which is supposed to be the natural substrate of AChE. The activities of esterases hydrolysing IPA and 0-NPA do not vary significantly when the activity is estimated on per mg fresh weight basis, but this difference becomes higher between the instars on per larva basis. This may be due to the fact that a certain amount of esterase would be essential for physiological functioning in each functional unit, i.e. cell or organ. In the first instar, the total esterase activity is lower and the activity on weight basis is higher than in second and third instars. This can be interpreted on the basis that at least two types of esterases would be acting upon these esters. The ratio of nervous and non-nervous tissues will be important in this connexion. If it is assumed that in first instar where the larvae are small, the proportion of nervous tissues are higher than nonnervous tissues in 1 mg of these larvae, the ratio of the esterase present in nervous tissues would be higher than one associated with non-nervous tissues. But as the development proceeds the bulk increase takes place in the non-nervous tissues. If this be so, one would observe a progressive increase in aliphatic and aromatic esterase types from first to fifth instars. Indeed, our results show that the OPresistant aliphatic and aromatic types of esterases increase during the postembryonic development. The results reported also show that a comparatively higher titre of esterases are present in larvae than that obtained by MANSINGH and SMALLMAN (1967a, b). From the practical point of view, the amount of esterase specially ChE activity is important in consideration of the toxicity levels. HASAN and ZAYED (1965) failed to show any effect of an extremely high dosage of Dipterex on the larvae of Prodenia Zitura and ascribed this due to the absence of the ChE in this insect. The results showing an increase in the OP-resistant aliphatic and aromatic esterases from the first to fifth instar would suggest that resistance of larvae to OP-insecticides will perhaps increase in later larval stages. The various esterases present in the larvae were further distinguished by selective inhibition. Such studies on the esterases in human erythrocytes (MOUNTER and WHITTAKER, 1953) and in a number of insect species (METCALF et al., 1956) ascribed the esterases inhibited below 10T5 M of OP-inhibitors as ChE, between 10” to 10m4 M as AliE and non-inhibited esterases as ArE. The method followed here was that of SMISSAERT (1965). The studies with DFP and diazoxon on the esterases hydrolysing IPA showed the presence of two esterases, one OP-sensitive and the other resistant. It was also shown that OP-resistant esterase hydrolysed the 0-NPA preferably at higher rate with Km 3 x 10” M. It was also observed that, unlike IPA, the 0-NPA gave a straight line which was inhibited by diazoxon but not by DFP. It is evident, therefore, that the homogenate contains (1) an esterase highly sensitive to DFP and diazoxon, perhaps being ChE, (2) an esterase

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which is less sensitive to organophosphorus compounds and inhibited only after 5 x 1O-5 M of DFP or diazoxon. Although this esterase has equal affinity for both the inhibitors, DFP seems to be more effective due to its high phosphorylation constant than diazoxon. The inhibition studies indicate it to be AliE. (3) There appears a very little amount of OP-resistant esterase which is not inhibited even by 1O-4 M of DFP. This can be presumed to be ArE. Acknowledgements-Thanks are due to Dr. S. PRADHAN,Head of the Division of Entomology for providing the facilities and for taking a keen interest in the problem. Appreciation is also expressed to Dr. W. R. YOUNG for the gift of many chemicals used in the present study. One of us (H. N. SINGH) is especially indebted to the Rockefeller Foundation for grant of fellowship. REFERENCES AFSHARPOUR F. and O’BRIEN R. D. (1963) Column chromatography of insect esterases. r. Insect Physiol. 9, 521-529. ARCHERT. E. and ZWEIG G. (1959) Direct colourimetric analysis of cholinesterase inhibiting insecticides with indophenyl acetate. J. agric. Fd Chem. 7, 178-181. COLHOUNE. H. (1963) The physiological significance of acetyl choline in insects and observations upon other pharmacologically active substances. Adv. Insect Physiol. 1, l-46. DAUTERMANW. C., TALENS A., and VAN ASPEREN K. (1962) Partial purification and properties of fly head cholinesterase. r. Insect Physiol. 8, 1-14. HASANA. and ZAYEDS. M. A. D. (1965) Enzymatic studies in Prodenia Zitura F.-I. Cholinesterase activity in relation to insect control. Naturwissenschaften 52, 18-19. HOFSTEEB. H. J. (1952) On the evaluation of the constants Vm and K, in enzyme reactions. Science, Wash. 116, 329-331. HOPF H. S. (1954) Studies in the mode of action of insecticides-II. Inhibition of the acetylcholinesterase of the locust nerve cord by some organic phosphorus esters. Ann. appl. Biol. 41, 248-260. KRAMER D. N. and GAMSONR. M. (1958) Calorimetric determination of acetylcholinesterase activity. Analyt. Chem. 30, 251-254. LORD K. A. and POTTER C. (1951) Studies on the mechanism of insecticidal action of organophosphorus compounds with particular reference to their anti-esterase activity. Ann. appl. Biol. 38, 495-507. MAIN A. R. (1964) AtBnity and phosphorylation constants for the inhibition of esterases by organophosphates. Science, Wash. 144,992-993. MAIN A. R. and IVERSONF. (1966) Measurement of the aflinity and phosphorylation constants governing irreversible inhibition of cholinesterase by diisopropyl phosphorofluoridate. Biochem. 3. 100,525-531. MAIN A. R., MILES K. E., and BRAID P. E. (1961) The determination of human serum cholinesterase activity with ortho-nitrophenyl butyrate. Biochem. J. 78, 769-776. MANSINGHA. and SMALLMANB. N. (1967a) The cholinergic system in insect diapause. J. Insect Physiol. 13, 447-467. MANSINGHA. and SMALLMANB. N. (1967b) N europhysiological events during larval diapause and metamorphosis of the European corn-borer, Ostvinia nubilalis Hubner. J. Insect Physiol. 13, 861-867. METCALFR. L., MAXONM. G., FUKUTOT. R., and MARCHR. B. (1956) Aromatic esterase in insects. Ann. ent. Sot. Am. 49, 274-279. MOUNTERL. A. and WHITTAKERV. P. (1953) The hydrolysis of esters of phenol by cholinesterase and other esterases. Biochem. 3. 54, 551-559.

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PANT N. C., PATHAKM. D., and PANT J. C. (1961) Resistance to Chile xonellus (Swim) in different host plants. IndiunJ. Ent. 23, 128-136. SMALLMANB. N. and MANSINGHA. (1969) The cholinergic system in insect development. A. Rev. Ent. 14, 387-408. SMISSAERT H. R. (1965) Esterases in spider mites hydrolysing ca-naphthyl acetate. Nature, Lord. 205, 158-160. SMITH E. H. and SALKELDE. H. (1966) The use and action of ovicides. A. Rev. Ent. 49, 777-783. VAN ASPERENK. (1959) Distribution and substrate specificity of esterases in the housefly, Musca domestica L. J. Insect Physiol. 3, 306-322.