expression of esterases during ontogenesis of the flour beetle tribolium castaneum (tenebrionidae;...

Biochemical Genetics, Vol. 15, Nos. 3/4, 1977

Expression of Esterases During Ontogenesis of the Flour Beetle Tribolium castaneum (Tenebrionidae; Coleoptera)

Ephraim Cohen, 1 Eliau Sverdlov, 1 and David Wool 1

Received 27 July 1976--Final 18 Aug. 1976

Two electrophoretieally fast-migrating, nonspecific esterases were detected in two strains of the flour beetle Tribolium castaneum and designated F(fast) and S (slow) according to their relative migration distances. Both isozymes are associated with the alimentary canal and display ontogenetic changes. Their activity is very low in the egg stage, increases in the larva, and deelines dramatically in the pharate pupa and pupa. The overall activity in the pupal stage is low, yet increases gradually throughout this period. In the adult, the activity of the esterases rises sharply. The larval and adult F and S isozymes were found to hydrolyze c~- and fl-naphthylacetate and cenaphthylpropionate with almost equal capacity, o~-Naphthyl laurate was cleaved by the F enzyme of both larvae and adults. The F and S were insensitive to inhibitors of arylesterases and cholinesterases and were markedly inhibited by the organophosphate diisopropylphosphorofluoridate ( DFP ) and could be classified as carboxylesterases. Differential sensitivities of larval and adult esterases to urea and heat treatment as well as to DFP may indicate the expression of different genes during metamorphosis.

KEY W O R D S : Tribolium castaneum; gel e lectrophoresis ; esterase isozymes; esterase inhibi tors .

INTRODUCTION

Isozymes are useful genetic markers in the study of gene expression as well as in studies on population genetics and evolution in plants and animals (Mark-

1 Depa r tmen t of Zoology, The George S. Wise Center for Life Sciences, Tel-Aviv Uni- versity, Tel-Aviv, Israel.

253

© 1977 Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011. No part of this publica- tion may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher.

254 Cohen, Sverdlov, and Wool

ert, 1975). Changes in isozyme pattern may be tissue or cell specific and indicate regulatory events at the genetic and epigenetic levels. These events are probably closely associated with morphological, physiological, or biochemical ontogenetic alterations.

Of all enzyme groups detected by gel electrophoresis, esterases are good markers for changes in gene expression because they are polymorphic in many organisms, stable under ordinary handling procedures, and easily detected. Insects are suitable material for studies of ontogenetic changes in gene expression because of their well-defined developmental stages. To visual- ize maximum bands by gel electrophoresis, nonspecific substrates are used. In most cases, the functional role of esterases is uncertain and their natural substrates are virtually unknown. Generally it is believed that insect esterases participate in food digestion, resistance toward pesticides, and fat metabolism. Recently, certain electrophoretically fast-migrating esterases were reported to be induced by juvenile hormone (Whitmore et al., 1972). It was suggested that they maintain and regulate the hormone level. This suggestion was supported by investigations of N~mec (1972) and Weirich et al. (1973) on the changes of hemolymph esterases with development.

Ontogenetic changes of esterase activity were reported in Drosophila species (Korochkin and Matveeva, 1974; Pasteur and Kastritsis, 1971), in the mosquito Anopheles albimanus (VedBrat and Whitt, 1975), in Ephestia kiihniella (Leibenguth, 1973), and in Hyalophora cecropia (Laufer, 1961). Changes in nonspecific esterase activity during the development of verte- brates were reported in rat liver (Kaeneko et al., 1972) and in the teleost Fundulus heteroclitus (Holms and Whitt, 1970).

The present communication deals with the appearance and activity of nonspecific esterases throughout the life cycle of the flour beetle Tribolium castaneurn. In particular, two electrophoretically rapidly migrating isozymes were investigated using various substrates and inhibitors. The stability of these esterases to heat and urea treatment was studied. The genetics and possible function of the enzymes are discussed.

MATERIALS AND METHODS

Animals

The Tribolium eastaneum "black" strain (CSbb) employed in this study was obtained from Stony Brook, New York, in 1970 and maintained in large stock cultures since then. The beetles were raised on wheat flour supple- mented with 5% brewer's yeast at 30 C and 70% RH. Additional substrains with specific esterase markers were selected from the CSbb stock (Sverdlov et al., 1976).

Esterase Activity During Ontogenesis of Tribolium castaneum 255

Enzymatic Assay

Insects were homogenized in cold 0.04 M phosphate buffer, pH = 7.0, in a glass-teflon pestle hand homogenizer. The homogenates were centrifuged for 15 rain at 10,000g and filtered through glass wool. Total proteins in each filtrate were determined spectrophotometrically (Gilford model 2400) by measuring the absorbance at 280 nm. Esterase activity was assayed by the method described by Gomori (1953) and Hipps and Nelson (1974) with some modifications. Incubation time was 30 min at 30 C. The azo-coupling reaction lasted 20 rain, and the absorbancy was measured colorimetrically at 560 nm. The e-naphthol standards were linear in the range of 0.014-0.070 mM.

Electrophoresis

Acrylamide Slab Gel Electrophoresis

Single beetles were homogenized in 0.2 ml of 0.1 M tris-borate-EDTA buffer, pH 7.0 (Shaw and Prasad, 1970), containing 10% w/v sucrose and bromo- phenol blue as tracking dye. Samples of about 25 pl were applied to pockets in a 6% polyacrylamide gel. A continuous 0.I ~ borate buffer system, pH 8.2, was used, and the gels were run at a constant current of 4 mA per centimeter of gel width for about 2 hr in the refrigerator. Gels were stained in 0.1 M phosphate buffer, pH 6.5, for about 2 hr at room temperature. Unless otherwise mentioned, ~-naphthylacetate (50 mg/100 ml) as substrate coupled with fast blue RR (50 mg/100 ml) was used.

After staining, the gels were fixed in water-methanol-acetic acid (5:5:1). The gels were then placed for preservation on filter paper and covered tightly with a piece of nylon bolting cloth. The gels were allowed to dry for about 3 days at room temperature. The nylon bolting cloth was removed and the dried gels remained attached to the filter paper.

Polyaerylamide Disc Eleetrophoresis

Samples of homogenates were loaded on 6% acrylamide gels (7.0-cm col- umns). Electrophoresis was carried out in 0.1 ~ borate buffer system (pH 8.2) at a constant current of 2.5 mA per gel for about 90 rain. The gels were stained for esterolytic activity with ~-naphthylacetate and fast blue RR salt as previously described. They were immersed overnight in the fixative and subsequently stored in 10% acetic acid. The gels were scanned in a Gilford model 2400 recording spectrophotometer equipped with a linear transport attachment at 500 nm. The relative activity of the esterase bands was determined by cutting and weighing the recorded peaks.

Snbstrate and Inhibition Studies

Substrate specificity was investigated by comparing the esterase activity on


the commonly used e-naphthylacetate with other substrates such as fl- naphthylacetate, ~-naphthylpropionate, and ~-naphthyl laurate. Staining was done in the same phosphate buffer with similar substrate and fast blue RR concentrations.

Inhibition studies were conducted by exposing the gels for 30 rain to inhibitor containing 0.1 M phsophate buffer (pH 6.5) prior to staining with the c~-naphthylacetate and the coupling dye. The inhibitors used were eserine, p-hydroxymercuribenzoate (PHMB), diisopropylphosphorofluoridate (DFA), EDTA, mercaptoethanol, and sodium fluoride.

The thermostability of esterases was examined by heating the gels at 55 C for various time periods prior to staining. The stability of the enzymes to hydrogen bond breaking substances was studied using high concentrations of urea.

Sources of Chemicals

Sigma (U.S.A.): a-Naphthylacetate, fi-naphthylacetate, e-naphthylpropionate, a-naphthyl laurate, eserine, p-hydroxymercuribenzoate (PHMB), mercaptoethanol.

Merck (West Germany): EDTA, sodium fluoride. Fluka (Switzerland): Diisopropylphosphorofluoridate (DFP).

RESULTS

Description of Tribolium Esterases

The soluble, nonspecific esterases in T. castaneum homogenates appeared as five to seven major bands on polyacrylamide gels. These isozymes were divided into four groups and numbered according to migration distances from the fastest (Est-1) to the slowest (Est-4). Ontogenetic changes in activity of the esterases are apparent (Fig. 1). Est-1, which stained intensively in the larva, disappeared in the pharate pupal stages and reappeared in the adult. No Est-1 could be detected in 4-hr-old pupae, but the activity increased gradually during pupal--adult transformation (Fig. 2). The latter is probably related to the formation of adult structures. Since Est-1 isozymes exhibited such dramatic changes during the insect developmental process, we decided to study them in more detail.

Two strains of Est-1 were selected from the laboratory stock of CSbb, one strain with a fast-migrating isozyme (F) and another with a slower esterase (Sverdlov et al., 1976). The strains were crossed to produce a hybrid (FS) showing both isozymes. F and S are probably coded for by two different loci, since some FS inbred lines show no segregation when crossed to each other

Esterase Activity During Ontogenesis of Tribollum castaneum 257

Fig. I. Slab gel electrophoresis of nonspecific esterases in larvae, pupae, and adults of Tribolium castaneum fast and slow strains. Est-1, -2, -3, -4, migration distances of the four esterase isozymes. (1-3) Adult, fast; (4-6) pupae, fast; (7-9) larvae, fast; (10-12) adults, slow.

Fig. 2. Slab gel electrophoresis of nonspecific esterases throughout the pupal stage of Tribolium eastaneum. Individual pupae of the fast strain we?e electro- phoresed at different stages of development. (1-3) 5 days; (4-6) 3 days ; (7-9) 1 day; (10-12) 0-7 hr.

or backcrossed to the parental strains. However, the functional role of the fast-migrating isozymes is at present unknown.

The F and S esterases are restricted to the gut. Isolated intestines exhibited all Est-1 activity and in addition showed a very slow-migrating band (Est-4). No Est-1 activity could be found in fat body and integument. It was suggested that the Est-1 isozymes function in digestion. However, larvae starved for 6 days displayed the same pattern and activity as the fed control larvae. Food ingestion is apparently not needed for the induction of esterase formation or activity.

Ontogenetic Changes

Figure 3 demonstrates the ontogenetic changes in total unspecific esterases.


12

I I I i i I I I I I I I I I I I 1 I I I I I I I I I I I I I I I I I I I I [

0 5 10 15 29 25 30 35 days 4 D 4 . . . . ) 4 m

egg larva -pp - pupa adult

Fig. 3. Total esterase activity during development in Tribolium castaneum. The esterase activity was determined colorimetrically. The bars indicate range of values within the repetitions.

._= 10

• 6

E

I

>" 4

,<

Newly laid eggs and embryos showed a relatively low esterolytic capacity. The level of activity increased dramatically after hatching. Throughout the larval period, the activity was very high and dropped considerably in the pharate pupal stage. The activity was low throughout the pupal period and increased in adults. Analysis of variance indicated no significant differences between larvae of different ages or between pupae 2 days old and older. The levels of activity in pharate pupae and 1-day-old pupae were similar. Similarity was found also between newly emerged adults and the older pupae. Adults of different ages differed significantly in the rate of esterase activity (P<0.001), which increased with the age (Fig. 3).

When the major five developmental stages of Tribolium, regardless of age, were compared by analysis of variance followed by a priori comparisons (Sokal and Rohlf, 1969) (Fig. 4), no significant differences were found among eggs, pharate pupae, and pupae, The levels of activity were much higher in larvae and adults, and a significant difference (P<0.001) was found between these two groups.

The relative activity of the F isozyme as percentage of total esterases in the five major stages in Tribolium development is shown in Fig. 5. A very low relative activity of F was found in embryos, pharate pupae, and pupae. In


10

._.q

2

o

=k

i 4

.>_ "6

2

17)

N e g g s

/26)

I

larvae

(Io}

T i

(61

p p pupae adults

Fig. 4. Total esterase activity in different developmental stages of Tribolium castaneum. The bars indicate sE values. Number of repetitions is given in parentheses, pp, Pharate pupae

I $ m

2si u_

"6

<

eggs eggs l a rvae pharate pupae adults (3-4 hrs] (4days) (last inster) pupae fl day)

Fig. 5. Relative activity of F esterase in different developmental stages of Tribolium castaneum. Polyacrylamide gels were stained for esterase activity and scanned at 500 nm, and the relative activity of the F isozyme was calculated,

newly laid eggs (3-4 hr), no measurable activity was found. The F band appeared later in embryonic development, and this was probably associated with the formation of the alimentary canal. Its relative activity reached a level of 15% just before hatching. In larvae and adults, the F band comprised up to 35~ of total esterase activity. In pharate pupae and pupae, it declined to the value of 4-day-old eggs.


Substrates and Inhibitors

T h e hyd ro ly t i c ac t iv i ty o f the F and S i sozymes on ~ -naph thy lace t a t e , fl-

naph thy l ace t a t e , and ~ - n a p h t h y l p r o p i o n a t e was s imi la r in l a rvae a n d adu l t s

(Tab le I). ~ - N a p h t h y l l au ra te was n o t c l eaved by l a rvae a n d adu l t es terases

o f the S strain. T h e a b o v e ind ica tes t ha t the F and S esterases differ in the i r

abi l i ty to ac t on large ester molecu les .

T h e di f ferent ia l sensi t ivi ty o f the esterases to v a r i o u s inh ib i to r s is sum-

m a r i z e d in Tab l e I. T h e ary les terase inh ib i to r , p - h y d r o x y m e r c u r i b e n z o a t e

( P H M B ) , d id n o t affect the F and S enzymes . S imi la r resul ts were o b t a i n e d

wi th 10 - 4 M eserine, k n o w n to inh ib i t chol ines te rases . Never the les s , a h ighe r

c o n c e n t r a t i o n (10 -3 M) inh ib i t ed to a g rea t ex ten t the F es terase b o t h in

l a rvae a n d in adul ts . T h e m o s t efficient i nh ib i t o r was the o r g a n o p h o s p h a t e

d i i s o p r o p y l p h o s p h o r o f l u o r i d a t e ( D F P ) at a level o f 10 - 4 M. I t is n o t e w o r t h y

t h a t a t 1 0 - 5 M the S b u t n o t the F was m a r k e d l y inhib i ted . Th is ind ica tes a

Table I. Effects of Inhibitors and Substrates on the Activity of Fast and Slow Esterases in Tribolium castaneum Larvae and Adults"

Fast Slow

Larvae Adults Larvae Adults

Inhibitors . . . . Eserine 10 -4 M Eserine I0 -a M + + + + + -- -- Diisopropylphosphorofluoridate

(DFP) 10-SM - - + + + + + + Diisopropylphosphorofluoridate

(DFP) 10-4 M + + + + + + + + + + + + p-Hydroxymercuribenzoate (PHMB)

1 0 - 4 M . . . .

EDTA 10 -z M . . . . fl-Mercaptoethanol 10 -a M . . . . Sodium fuoride 10 -2 M - - + + + + Urea 10 M, 5 rain . . . . Urea 10 M, 10 min . . . . Urea 10 M, 20 min + + -- + + -- Heating 55 C, 5 min + + + + + + - Heating 55C, 10min + + + + + + + + + +

Substrates c~-Naphthylacetate + + + + + + + + + + + + fl-Naphthylacetate + + + + + + + + + + + + c~-Naphthylpropionate + + + + + + + + + + + + c~-Naphthyl laurate + + + + + + - -

a The degree of inhibition is expressed as slight (+) , moderate (+ +), and strong (+ + +). When various substrates were used, the signs refer to the strength of the esterolytic activity.


possible structural difference between F and S. From the inhibition studies, we were able to conclude that the Est-1 isozymes are carboxylesterases.

Other candidate inhibitors such as EDTA and fl-mercaptoethanol had no effect, whereas sodium fluoride at a relatively high concentration (10 .2 r~) had a moderate effect on the S variant.

The F esterase was highly sensitive to heat treatment at 55 C for 5 min, whereas the S remained essentially unaffected. A prolonged thermal treatment destroyed the larval S isozyme activity but only slightly affected the adult S. Exposure of the enzymes to 10 M urea for 20 rain affected the activity of F and S only in larvae. We assume that the adult and larval esterases are dissimilar despite their identical migration distance on the gels.

DISCUSSION

Three electrophoretically distinct substrains were selected from the CSbb strain of Tribolium castaneurn. Two rapidly migrating esterases in the Est-1 group and their hybrid could be visualized (Sverdlov et al., 1976) (Fig. 1). A genetic analysis excluded a one-gene, two-alleles model, and supportive- evidence for the hypothesis that two genes, each with a "null" allele, was obtained (Sverdlov et al., 1976). Null alleles are known in esterase isozymes (Roberts and Baker, 1973).

When the F and S variants were ontogenetically analyzed (Fig. 5), a pattern of appearance and disappearance of Est-1 group was observed. The activity of the fast-migrating esterases was low in the egg stage, followed by a dramatic increase in the larvae. A decline in activity was observed in the pharate-pupal stage, which is a physiologically transient period where a non- feeding, motionless larva is about to pupate. In young pupae, the enzyme bands were invisible and reappeared gradually toward the end of the pupal period and the formation of adult structures. After adult emergence, the enzyme activity rose almost to the larval levels of stain intensity.

Korochkin and Matveeva (1974) reported similar alterations in the activity of nonspecific esterases in the fruit fly Drosophila virilis. They found a peak of activity in the middle of the last instar. In T. castaneum, however, equally high levels of enzyme activity were measured throughout the larval period (Fig. 3). An ontogenetically changing pattern of esterases was observed also in the mosquito Anopheles albimanus by VedBrat and Whitt (1975). They proposed several explanations for the phenomenon, such as cell types replacing one another during development, expression of specific genes, or activation and inactivation of preexisting enzymes. In fact, any posttranscriptional event might be associated with the observed phenomenon. At present, it is hard to distinguish and decide which of the previously mentioned mechanisms is in fact operating.

D


• Ontogenetic changes in enzyme activity are not restricted to esterases and were reported for isozymes such as acid phosphatases (Pasteur and Kastritsis, 1.971) as well as for several dehydrogenases (Rechsteiner, 1970; Fox, 1971) in Drosophila.

We have found that the Est-1 isozymes in Tribolium were associated with the alimentary canal. It may indicate that these esterases function in digestion. This possibility was supported by the observation that the F isoenzyme was capable of hydrolyzing long-chain acid residue esters such as e-naphthyl laurate.

Geering and Freyvogel (1974, 1975) reported an increase in total unspecific esterases in the midgut of the mosquito Aedes aegypti following a blood meal and suggested that these enzymes participated in lipid metabolism and peritrophic membrane formation. Nevertheless, Hipps and Nelson (1974) were unable to assign a physiological function to esterases isolated from midgut and cecum of the American cockroach, Periplaneta americana. Other functional roles for insect esterases were suggested. Electrophoretic analysis of esterases throughout the development of Drosophila pseudoobscura revealed a class of fast-moving enzymes in the pupal exuvia after adult emergence (Berger and Canter, 1973). It was proposed that those enzymes either participate in digestion of the pupal cuticle or modify the adult cuticle. Katzenellenbogen and Kafatos (1971) detected nonspecific esterases in silk- moth molting fluid, which might also point to their role in cuticle digestion.

A relationship between esterase activity and hormonal factors in insects was demonstrated by several investigators. Thomsen (1966) detected and localized esterase activity in hindgut epithelial cells of Calliphora females by histological methods. The cellular quantity of esterases was related to the action of medial neurosecretory cells in the brain. Similarly, Laufer (1961) showed a relationship between factors in Hyalophora cecropia brain and induction of esterases in the pupal intestine. The decline in activity of Est-1 in Tribolium pharate pupae and pupae, which do not feed, might be directly or indirectly associated with the lack of induction caused by food ingestion. However, this possibility seems unlikely because the activity in larvae starved for 6 days, with no food material in their alimentary canal, was similar to that in the fed control larvae. The changes in Est-1 activity with metamorphosis might be due to alterations in the hormonal milieu. Laufer (1961) and N~mec (1972) have suggested that hormones known to regulate growth and development were responsible for the pattern of esterase activity in the hemolymph of Hyalophora cecropia and Pyrrhoeoris apterus. A close relationship between hormones and esterases was proposed by Whitmore et al. (1972). According to their studies, juvenile hormone, which is an ester, induced the synthesis of rapidly migrating esterases capable of regulating the hormone titer. Supporting evidence for such a mechanism was provided by


Weirich et aL (1973). They have shown ontogenetic changes in juvenile hormone esterase activity in the blood of Manduca sexta which were associated with juvenile hormone metabolism.

Inhibition studies indicated that the F and S enzymes were insensitive to p-hydroxymercuribenzoate and eserine, which are inhibitors of arylesterases and cholinesterases, respectively. Both esterases were strongly suppressed by diisopropylphosphorofluoridate (DFP) and could be classified as hydrolases (E.C. 3.1.1), aliesterases, or carboxylesterases. It should be recalled that many soluble, nonspecific esterases studied in insects as well as juvenile hormone esterases were carboxylesterases (Whitmore et aL, 1972; Berger and Canter, 1973; Hipps and Nelson, 1974; Stephen and Cheldelin, 1970).

The F and S esterases effectively hydrolyzed esters with short acid moie- ties such as ~- and//-naphthylacetate and ~-naphthylpropionate. The F isozyme was also capable of cleaving c~-naphthyl laulate, indicating some lipase activity. Other differences between S and F were demonstrated by using esterase inhibitors. The S was highly sensitive to low concentrations of DFP but resistant to heat treatment for 5 min at 55 C. The larval S and F were affected by heat and urea. A prolonged exposure to 55 C destroyed the S activity in larvae but not in adults. We would like to propose that although larvae and adult isozymes migrated electrophoretically to the same distance, different genes were expressed in these developmental stages. Urea and heating ale known to break hydrogen bonds, and differential sensitivity to such treat- ments may indicate different three-dimensional arrangement of the molecule probably in or near the enzymatic active site. Williams and Kafatos (1971) proposed that three different gene sets are active in holometabolous insects, and are switched on and off with metamorphosis from larva to pupa and adult. Accordingly, adult esterases may be the product of adult genes and different from the larval ones. Nevertheless, we are not able to exclude the possibility of any posttranscriptional events which may eventually modify proteins and contribute to ontogenetic differences of isozymes activity.

ACKNOWLEDGMENT

The authors would like to thank Mrs. Ela Englander for her technical assistance.

REFERENCES

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Gomori, G. (1953). Human esterases. J. Lab. Clin. Med. 42:445. Hipps, P. P., and Nelson, D. R. (1974). Esterases from the midgut and gastric caecum of

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expression of esterases during ontogenesis of the flour beetle tribolium castaneum (tenebrionidae;...

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