Some properties of the haemolymph juvenile hormone esterases in Galleria mellonella larvae and Tenebrio molitor pupae

Insect Biochem., Vol. 10, pp. 273 to 277. © Pergamon Press Ltd. 1980. Printed in Great Britain.

0020-1700/80/0601--0273 $02.1)0/0

SOME PROPERTIES OF THE HAEMOLYMPH JUVENILE HORMONE ESTERASES IN GALLERIA MELLONELLA LARVAE AND TENEBRIO MOLITOR PUPAE* DAVID C. MCCALEB, GUNDA REDDYt and A. KRISHNAKUMARAN Department of Biology, Marquette University, Milwaukee, WI 53233, U.S.A. (Received 20 August 1979)

Abstract--The apparent molecular weight, the Kin,and the sensitivityto different organophosphates of the haemolymph juvenile hormone esterase (JHE) in the larvae of Galleria mellonella was determined. The apparent molecular weight, determined by gel filtration methods, was estimated to be 60,000. The apparent Kmof the enzymewas 1.79#M. The enzymewas insensitiveto diisopropylphosphofluoridate (DFP) but was sensitive to paraoxon and 0-ethyl-S-phenylphosphoramidothiolate(EPPAT). The sensitivity of JHE in pupae of Tenebrio molitor to organophosphates was also determined. It was found to be insensitiveto DFP and EPPAT. Key Word Index: Juvenile hormone esterase, Galleria mellonella, Tenebrio molitor

INTRODUCTION JUVENILE hormone esterase (JHE) in lepidopteran insects is primarily responsible for the initial degradation and inactivation of juvenile hormone (JH) in the haemolymph (HAMMOCK and QUISTAD, 1976). Several studies have shown the presence of JHE in last instar larvae of diverse lepidopterans (WEIRICH et al., 1973; SANBURGet al., 1975a, b; HAMMOCKet al., 1975; HWANG-Hsu et al., 1979; SPARKSet al., 1979). JHE activity in all these insects shows a specific developmental pattern; it increases just before the release of 20-hydroxyecdysone in preparation to metamorphosis. JHE activity has also been reported to increase before moulting in orthopterans and coleopteran insects (RETNAKARANand JOLY, 1976; KRAMER and DE KORT, 1976a; WEIRICHand WREN, 1976; REDDY and KUMARAN, 1980). Furthermore, studies in Galleria mellonella larvae showed that the production of JHE during the last instar is closely coupled with the commitment for metamorphosis (McCALEB and KUMARAN, 1980), suggesting a developmental role for this enzyme. Even though JHE activity has been reported from a variety of insects, the enzyme has not been well characterized except in Manducta sexta. Further, SPARKS and HAMMOCK (1979) studied the effect of various inhibitors on JHE activity in Trichoplusia ni, the cabbage looper. The present report deals with some of the properties of JHE activity in larvae of the wax moth, Galleria mellonella. In these investigations the effects of different organophosphate inhibitors of esterases, and other agents that affect esterase activity were determined. In addition, the molecular weight and K m of the crude enzyme were estimated. * Supported by N.I.H. Grant No. G.M. 22429 and funds from the Johnson Wax Foundation, Racine. t Present address: Department of Pathology, V.A. Hospital, Madison, Wisconsin 53706. 273

Although JHE activity in lepidopterans has been studied in great detail by a number of workers, very little information is available regarding this enzyme in other groups of insects. WEIRICH and WREN (1976) reported changes in JHE activity in Tenebrio molitor during pupal-adult development, but no information is available on the properties of JHE in this insect. We report here the effect of different organophosphate and other esterase inhibitors of JHE activity in pupal haemolymph from T. molitor. These data on the properties of JHEs from G. mellonella and T. molitor are discussed in the context of the classical views on the classification of carboxyl esterases (EC.3.1.1) into A (EC.3.1.1.2) and B (EC.3.1.1.1) type esterases (ALDRIDGE, 1953a, b; AUGUSTINSSON, 1964). A preliminary account of some of these data has been presented previously (McCALEB and KUMARAN, 1978).

MATERIALS AND METHODS Insects

Wax moths, Galleria mellonella, were reared in the laboratory and the appropriate stages were collected (BECK, 1960; KRISHNAKUMARAN,1972). The day of ecdysis is clesignatedas day 0 and the followingday as day 1. Under the conditions used in this study the last larval instar (7th) lasted 7.5 ___0.5 days; that is a range of seven to eight days. For all data in this study only haemolymph from day 4 animals was used. Mature mealworms, Tenebrio molitor, were purchased from bait dealers and maintained in the laboratory as reported earlier (REDDYand KRISHNAKUMARAN,1972). In this study haemolymph from day 1 pupae was used. Chemicals

Juvenile hormone (CI8JH), a 99.5~o pure isomeric mixture, was a gift from Hoffman LaRoche, Nutley, N.J. [10-aH]-CI8JH (13.5 Ci/nmole) was purchased from New England Nuclear Coroporation. EPPAT (O-ethyl-S-phenyl phosphoramidothiolate) was kindly supplied by Dr. B. D. Hammock. All other inhibitors were obtained from Sigma

274

DAVID C. MCCALEB,GUNDA R.EDDYAND A. KRISHNAKUMARAN

Chemical Corporation. Sephacryl S-200 is a product of Pharmacia.

Biochemical assays JHE activity was assayed as described by HAMMOCKand SPARKS (1977) using a phase partition of the radiolabelled substrate and product. Incubations of the haemolymph samples from G. mellonella were run for 10 min at 30°C in 0.05 M phosphate buffer, at pH 7.0 (HWANG-Hsu et al., 1979). Thc enzyme preparation was diluted so that only 10-15% of the 1.7 nmole of C18JH was hydrolyzed and the data are presented as nanomoles of JH acid formed per 10 min per microlitre of haemolymph. In studies with haemolymph from T. molitor a methoxy assay (VINcE and GILBERX,1977) was used with a slight modification. Because of the quenching effect of chloroform in scintillation counting, 10/~1aliquots were used to determine the amount of methanol released instead of 50 #1. Some of the inhibition studies on JHE activity of T. molitor haemolymph were repeated using the more reliable procedure of HAMMOCKand SPARKS(1977). For inhibition studies the inhibitor was incubated with the diluted enzyme solution for 15 min at 23-24°C immediately preceding the enzyme assay. To test for calcium activation and ethylenediaminetetra-acetic acid (EDTA) inactivation, Tricine buffer (0.05 M, pH 7.0), was used since calcium precipitates with the phosphate buffer. Similar enzyme activities were seen in this buffer with untreated samples as with phosphate buffer. The enzyme solution was incubated with 1 mM calcium chloride for 10 min at 23-24°C immediately preceding the JHE assay (EcoBICHON, 1970). For EDTA chelation of endogenous calcium the haemolymph solution was dialyzed at 4°C, against six to eight changes of EDTA (0.1 mM) in Tricine buffer overnight, before assaying the enzymatic activity. For molecular weight determination, haemolymph collected from 150-200 day four last instar larvae was used. The pooled haemolymph provided approximately 2 ml of supernatant after centrifugation at 10,000 g for 10 min at 4°C. The haemolymph was diluted in cold phosphate buffer and a 50% saturated ammonium sulphate precipitate was obtained by adding dropwise an equal volume of cold saturated ammonium sulphate solution in buffer. The precipitate was collected by centrifugation at 10,000g for 10 min at 4°C and redissolved in buffer. The sample was then loaded onto a 90 × 1.5 cm i.d. gel filtration column of Sephacryl S-200 equilibrated with the phosphate buffer at 4°C. The flow rate

of the buffer was adjusted to 25 ml/hr, and 2.3 ml fractions were collected. Protein in the fractions was determined by continuously monitoring at 280 nm and by the protein determination of LOWRY et al. (1951). JHE activity in the fractions was identified by means of the JHE assay of HAMMOCKand SPARKS(1977) in the presence of 5 x 10 4 M DFP. RESULTS

Effect o f inhibitors on J H E activity in G. mellonella larvae and T. molitor pupae Since esterases are classified on the basis of their sensitivity to different o r g a n o p h o s p h a t e and other esterase inhibitors, in the first series o f experiments the effects of various esterase inhibitors on the J H E activity in the h a e m o l y m p h o f G. mellonella larvae and T. molitor were investigated. These data (Table 1) show that J H E from G. mellonella and T. molitor is insensitive to eserine, a carbamate. In both species J H E activity was only slightly inhibited ( < 5%) by the prototype organophosphate, diisopropylphosphofluoridate (DFP) at a concentration of 5 × 10 4 M, suggesting that the JH-specific esterase is not o f the B type. However, J H E activity in G. mellonella was inhibited totally by another o r g a n o p h o s p h a t e , paraoxon, at a c o n c e n t r a t i o n o f 1 × 10 4 M. Only 1~o o f the activity observed in untreated h a e m o l y m p h was found in preparations incubated with paraoxon. Even at a concentration o f 10 6 M, P a r a o x o n inhibited over 60~o J H E activity in G. mellonella. In T. molitor, P a r a o x o n at a concentration of 1 x 10 ~ M had no effect on J H E activity. Even at a concentration o f 1 × 10 -4 M only a small reduction in activity was observed. In its sensitivity to a third o r g a n o p h o s p a h t e , EPPAT, J H E activity from the two species could be more easily distinguished. While J H E from the wax m o t h larvae is totally inhibited by E P P A T at a concentration of 1 x 10 s M, J H E in T. molitorpupae appears to be insensitive at this concentration. Furthermore, J H E activity in T. molitor, like that in Leptinotarsa decimlineata (KRAMER and DE KORT,

Table 1. The sensitivity of Galleria mellonella and Tenebrio molitor haemolymph JHE activity to various chemicals. Agent Untreated Eserine DFP Paraoxon Paraoxon Paraoxon EPPAT Calcium EDTA Triton X100 Triton XI00

Concentration

1 5 1 1 1 1 1 1

x × × x x x x x

-10 4 M 10 -4 M 10 -4 M 10 -6 M 10 =~ M 10 -8 M 10 -3 M 10 -4 M 0.1~o 0.5%

JHE activity* Galleria mellonella Tenebrio molitor 0.70 0.69 0.67 0.01 0.21

_+ .03 (100) + .02 (99) + .02 (96) + .02 (1) + .01 (30) -0.05 _+ .004 (7) 0.66 ± .03t (94) 0.67 ± .03t (96) --

1.76 1.76 1.78 1.60

± ± + +

.08 (100) .08 (100) .03 (101) .03 (90)t

1.80 ± .04 (102) 1.76 ± .03 (100)t 1.38 ± 04 (79) 1.04 ± ~.04 (59)

* JHE activity in G. mellonella was measured according to the procedure described by HAMMOCKand SPARKS(1977) and in T. molitor the modified procedure of VINCE and GILBERT(1977) was used. JH acid nmole/10 min/#l haemolymph + SEM based on four to six estimations. The number in parentheses represents percent activity remaining after treatment with the chemical. t In these studies JHE was determined using the method of HAMMOCKand SPARKS (1977) and the data were normalized.

275

Properties of haemolymph JH esterases - 0.7

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0.1

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35

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Fig. 1. The elution profile of haemolymph JH esterase in Galleria mellonella larvae on a 90 x 1.5 cm Sephacryl-200column eluted with phosphate buffer pH 7.0. The void volumewas 35 ml and the total volume of the fractions was 92 ml.

1976b), appears to be sensitive to Triton X-100, although to a lesser extent. Effect o f calcium on J H E activity in Galleria mellonella Since JHE in G. mellonella is not inhibited by DFP, according to the classification scheme of ALDRIDGE (1953a, b) and AUGUSTINSSON(1964) it is not a B type esterase but could be an A type esterase. With a view to determining whether JHE from the wax moth larvae had any other characteristics of A type esterase we examined the effect of the divalent cation calcium on the enzyme activity. Endogenous calcium was chelated by dialysis against a buffer containing EDTA according to the published methods (EcoBICHON, 1970; ERDOSet al., 1959). JHE activity of haemolymph samples in which calcium was chelated by dialysis against EDTA buffer was determined in the presence and absence of added calcium. The data shown in Table 1 indicate that unlike the classical A type esterases the wax moth JHE activity is not sensitive to calcium.

known standards, cytochrome c, chymotrypsinogen, ovalbumin and bovine serum albumin, and the ratio of their eluant volumes to void volumes indicates that the protein species associated with the peak of JHE activity has an apparent molecular weight of 60,000. JHE was eluted from the Sephacryl column after bovine serum albumin and before ovalbumin. The apparent K m of JHE was.deterrflined by construction of a Lineweaver- Burk reciprocal plot of data obtained with diluted larval haemolymph. The data were fitted using least squares regression analysis and are presented in Fig. 2. The results show that the apparent Km is 1.79 pM. Thermal sensitivity In Sephacryl-200 fractionation JHE activity was eluted as a broad peak (Fig. 1). To determine whether

Kin= 1.79 xlO'S M x T .c

Molecular weight and K m o f J H E To determine whether the enzyme is similar to that of other insects, the apparent K mand molecular weight of the JHE in larval haemolymph was investigated. To determine the molecular weight of the enzyme, haemolymph was first fractionated with 50~o saturated ammonium sulphate as described in Materials and Methods. Precipitate containing high JHE activity was loaded onto a 90 x 1.5 cm i.d. Sephacryl-200 gel column. The elution profile of the JHE is shown in Fig. 1. Cytochrome c, chymotrypsinogen A, ovalbumin, bovine serum albumin and human ~ globulin were used as standards of known molecular weight. The elution profiles of each of the standards and of JHE were determined separately. A plot of the log molecular weight of the

E x

IE >

/ I -I0

~ -5

0

I

I

I

5

IO

15

I / r S ] (M)-'x I0 -s

Fig. 2. The apparent Km of haemolymph in Galleria mellonella as determined by a Lineweaver-Burk reciprocal plot; the data was fitted using the least squares regression analysis.

DAVID C, MCCALEB, GUNDA REDDY AND A. KRISHNA KUMARAN

276 IO0 i 9O 80 70 60 50 40 30 •

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Fig. 3. Thermal sensitivity ofJH esterase peak shown in Fig. 1. Log O/oactivity remaining after heating the enzyme at 68°C for different periods of time was plotted against time in min. the broad peak contains more than one molecular species of the enzyme the time course of inactivation of the enzyme at 68°C was studied. The data presented in Fig. 3 show that the enzyme activity as assayed in the presence of 5 × 10 -4 M D F P decreased linearly with time through 4'~o of remaining activity. This suggested that the enzyme activity may be caused by a single molecular species, or if more than one molecular species of JH esterase were present they had identical thermal sensitivity properties, or the other molecular species represents less than 4'),i, of measurable activity. DISCUSSION The data presented in this report show that the haemolymph J H E in G. mollonella larva is similar to that of other lepidopterans studied. The apparent molecular weight of J H E is estimated at 60,000, very similar to that of M . sexta, Trichoplusia ni and other lepidopterans in which the J H E molecular weight is estimated to be between 52,000-67,000 (SANBURG et al., 1975a, b; SPARKS and HAMMOCK, 1979; KRAMER and CH1LOS, 1977). The apparent K m of the wax moth J H E is 1.79 ~M which is very close to the 1.43 #M of M. sexta (VINCE and GILBERT, 1977) and 0.5-1.0 #M in Leptinotarsa decimlineata (KRAMER and DE KORT, 1976a, b). In its sensitivity to various esterase inhibitors the wax moth J H E is similar to that of the other lepidopterans. As with the enzyme in M . sexta (SANBURG et al., 1975) and T. ni (SPARKS et al., 1979), the wax moth larval J H E is insensitive to eserine, an acetylcholinesterase inhibitor and to DFP, an inhibitor of the B type of carboxyl esterases

(EC.3.1.1.1). However, these enzymes are sensitive to Paraoxon and EPPAT, suggesting that the wax moth JH esterase does not belong to the typical B class esterases according to the classification of ALOR1DGE (1953a, b) and AUGUSTINSSON (1964). Furthermore, the enzyme does not belong to the typical A type or aryl esterases (EC.3.1.1.2) either, because the enzyme activity is not dependent on calcium ions and also because the enzyme does not appear to metabolize D F P (data not shown). Thus, the J H E in lepidopteran haemolymph appears to belong to the C type of esterases (EC.3.1.1.6; BERGMANN et a/., 1957: BERGMANN and RIMON, 1960). The limited studies on the effect of various inhibitors on J H E activity in the pupal haemolymph suggest that the enzyme in the latter species may be different from that of the lepidopterans. It is not inhibited by eserine, D F P , Paraoxon or EPPAT, Since the enzyme is insensitive to both the prototype organophosphates and also to EPPAT, another organophosphate, this enzyme is tentatively classified as an A type esterase. Thus it appears that the J H E enzyme in different orders of insects may differ in some of its properties, although all of them seem to be insensitive to DFP, thereby suggesting that they may have evolved from classical A type esterases. Acknowledgements The authors thank Dr. Bruce HAMMOCK of the University of California, Riverside, sending us copies of manuscripts that are in press, and Walter W. FREDRICKS for helpful discussions on methodology.

D. for Dr. the

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