Insect Biochem., 1976, Vol. 6, pp. 541 to 547. Pergamon Press. Printed in Great Britain.

LARVAL AND ADULT HOUSEFLY CARBOXYLESTERASE: ISOZYMIC COMPOSITION AND TISSUE PATTERN

SAMI AI-~AD

Department of Entomology and Economic Zoology, Cook College, Rutgers--The State University, New Brunswick, New Jersey 08903, U.S.A.

(Received 17 May 1976)

Abstraet~Carboxylesterase (EC 3.1.1.1) isozymes of the adult and larval housefly Musca domestica, were separated electrophoretically on polyacrylamide gels. Of the 10 isozymes present in the 17,500 9 supernatant of the whole fly homogenate, 9 were detected in the gut and the thoracic muscles. Distribu- tion of isozymes was as follows: isozyme Et and Eto in the foregut as well as in the combined mid-and hindgut preparation, and Ez-s and Exo in the thoracic muscles. On the basis of the molecular sieving effect of the gels of different pore sizes, the approximate mol. wt of the isozymes El_ 9 were found to be in the range of 67,000 (+3000)-300,000. Isozyme Elo had the highest molecular weight of ca. 1 x 106. Ten isozymes were resolved from larval tissues, and these corresponded to Ea and E3 to of the adult, and another isozyme Ex with no correspondence to any of the adult isozymes. Isozyme E2 was absent. Distribution pattern of larval isozymes was as follows: Ex, E3_ 7 and E9-1o in the gut; Et, E6_s and Eto in the muscles; Ex, E3-s~ Elo and E~ in the fat body. Densitometric scans showed that the activity detected histochemically and by pH-titrinletric method (AI-IMAD, 1970a) was due primarily to isoz3qne Et. Although nothing further is known concerning this isozyme, its wide distribution in larval tissues and restriction to gut in the adult stage may be biologically significant. The molecular diversity and varied tissue pattern indicate several r61es for carboxylesterase in the housefly. The physiological r61es discussed are: (1) regulation of JH titre, (2) mobilization of fat in fat body, (3) energy-related catabolism of fatty acid esters in flight muscles, (4) cuticular wax synthesis and transport, and (5) degradation of undesirable, metabolically inert esters.

INTRODUCTION

CONSIDERABLE interest in insect carboxylesterase (EC 3.1.1.1) was generated following van ASPERFN and OPPENOOga~'S (1959) discovery that carboxylesterase activity in houseflies is negatively correlated with organophosphate insecticide resistance and VA~q ASPER~N'S (1960) indication that resistance is also accompanied by increased phosphatase activity. This led to the mutant-aliesterase theory of generalized organophosphate resistance (OPPENOORaTa, 1965). However, there are serious objections to this theory and O'BRtEN (1967) after carefully reviewing this sub- ject concluded that 'a minor increase in phosphatase activity, like other numerous changes in hydrolases (i.e. aliesterase and phosphatase) accompanies resist- ance rather than causes it'.

Several other physiological functions for this enzyme have since been advanced (review by SUDDER- UODIN, 1973). WHrrMORE et al. (1972) indicate that carboxylesterase is important in the regulation of juvenile hormone (JH) titre in pupal Hyalophora gloveri, thus insuring normal development. Carboxyl- esterase involvement in JH regulation has since been established for many insect species (AJAMI and RIDDIFORD, 1973; WEIRICH and WREN, 1973; WEIRICH et al., 1973; HIPPS and NELSON, 1974; WnITMORE et al., 1974; S~BURG et al., 1975). Recently, Yu and

TERRIERE (1975) have suggested a similar r61e for car- boxylesterase in the housefly. However, these authors made no attempt to determine if the carboxylesterase activity was the result of one or several isozymes. The present communication is the first report on the molecular diversity and the distribution of carboxyl- esterase isozymes in tissues of the larval and adult housefly. The data are discussed in the light of exist- ing theories on the physiological functions of carboxyl- esterase in insects.

MATERIALS AND METHODS

Enzyme preparation

One hundred 4 to 6-day-old standard susceptible house- fly Musca domestics adults of both sexes (50:50) (WHO strain, SRS) were dissected in 0.1 M phosphate buffer pH 7.0 to obtain the following tissues: foregut (including esophagus, crop and salivary glands), mid- and hindgut combined, thoracic muscles, and gonads. One hundred 1 to 2-day-old larvae were dissected to obtain whole gut, muscles and fatbody. One hundred of each tissue were homogenized in 4 ml of buffer using a glass homogenizer with Teflon ® pestle. The homogenate was centrifuged at 17,5000 for 20min. One hundred /d of the supernatant were used for electrophoresis. The method for obtaining enzyme preparation from whole fly has been reported (Ar~4AD, 1974).

541

542 SAMI AHMAD

Electrophoretic procedure Electrophoresis was conducted on vertical slabs of

polyacrylamide gel using a variable gel formation. The in- itial portion of 1.5 cm length was 3% w/v, followed by a main running gel of 7.5%. This is an adaptation of the system recommended for disc elcctrophoresis (ORr~r~IN, 1964). The details of the electrophoretic procedure have been reported (AHEAD, 1974).

Gel staining, densitometry and characterisation of car- boxylesterase

Esterases were rendered visible by staining for 20 rain with a 0.25% (w/v) solution of 1-naphthylacetate in 10% aqueous acetone. Afterwards the gel was incubated with 0.1% (w/v) diazo-dye, Fast Blue RR in 0.1 M phosphate buffer of pH 7.0. Within 40 min, deep brown esterase bands appeared. The gel was then washed in distilled water and stored in 7~/o acetic acid.

Freshly stained gels were scanned by a double beam microdensitometer MK III® made by Joyce Lobel & Co., Ltd., England.

Carboxylesterases (also known as aliesterase, B-esterase) were differentiated from arylesterases on the basis of inhibition by 1 × 105M tetraethylpyrophosphate. Aryl- esterases are not inhibited at this concentration. The car- boxylesterase isozymes were further distinguished from cholinesterases by 1 × 10 -5 M eserine which, at this con- centration, inhibits cholinesterases but not carboxyl- esterases. Peptidases were rendered visible by substituting DL-alanine-fl-naphthylamide for 1-naphthylacetate in the staining medium (AHMAD, 1974).

Isozyme designation Electrophoresis was continued until the bromothymol

blue marker dye front (MF) moved 10 cm from the cath- ode. The zymograms are based on three replicate separ- ations of each isozyme. Samples of the tissue preparations of both adults and larvae were subjected to electrophoresis simultaneously on a single gel slab. Under these condi- tions, bands with identical mobility were assumed to be homologous. The isozymes were numbered consecutively, with the lowest number given to the isozyme with the high- est mobility towards the anode. Thus, the leading carboxyl- esterase isozyme was labeled El, and the slowest isozyme was designated E~o. Isozymes of peptidase (P~-P4) were labeled similarly.

Approximation of tool. wt Approximation of molecular weights of carboxylesterase

isozymes was achieved on the basis of the molecular siev- ing effect of gels of different pore sizes. For this purpose, isozymes were resolved in polyacrylamide gels of 3.75 to 7.5% (w/v). For reference, mobility of each zone was com- pared to those of bovine serum albumin (mol. wt 67,000__+ 3000), human serum albumin (mol. wt 70,000) and globulin (mol. wt ca. 156,000), obtained from Armour Pharmaceutical Co., USA.

PS_,SULI~

Adult isozymes The results of electrophoretic separations are

shown in Fig. 1. Although in a previous paper ( ~ , 1974) the isozymic composition of whole fly

@ .,p

ADULT I S O Z Y M E S -

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, , • 17 I 16 I

E

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I

P2

A B C D E F

LARVAL ISOZYMES-

• eq E8 ms

• E7 e7 [ ] • e6 |6 [ ]

Ex

i n ' , 1 El _I G H I

Fig. 1. Zymogram of housefly adult and larval carboxyles- terases (solid bands E1-Elo) and peptidases (hatched bands PrP4). Arrow indicates sample slot. The dotted line (MF) represents the marker dye front. The enzyme sources are; whole fly (A), aduk muscles (B), adult foregut (C), adult mid- and hindgut (D), larval gut (G), larval fat body (H), and larval muscles (I); gut peptidase bands of whole fly

adult gut (E), and whole larva gut (F).

(SRS strain) was reported this was repeated in the present work to allow direct comparison between whole fly and fly-tissue patterns, and also to exclude the possibility of batch differences. Ten carboxylester- ase isozymes were resolved from whole fly prep- arations, as previously reported. Nine of these were detected in the adult tissues, with isozyme E 9 being absent. Therefore, this isozyme must be present in tissues other than gut, muscles, and gonads. Densito- metric scan (Fig. 2) suggested that the carboxylester- ase activity in the adult gut results mainly from the isozyme El, and to a lesser extent from Elo. Previous histochemical and pH-titrimetric determinations have shown the gut to be the principal site of carboxyles- terase activity; of the total activity per housefly, 53 to 55% is localized in the gut (AnMAO, 1970a). Taken together, these findings suggest that about one-half of the total carboxylesterase activity is attributable to isozyme El plus Elo, with the majority contributed by isozyme El.

In the preparations of thoracic muscle tissue, all isozymes except Ea were present. Densitometric scans showed that isozymes E 3 and E4 were more active than isozymes Ez, and Es_s (Fig. 3). Activity due to E~0 was greater than in the gut. Based on in vitro assays of total thoracic muscle activity (AnMAD, 1970a), these isozymes together contribute about 40%0 of the total carboxylesterase activity per fly.

The absence of carboxylesterase activity in gonads, and the failure to resolve any isozymes is surprising. In the adult female, the presence of JH is thought to be necessary for oogenesis (DoANE, 1973). Carboxyl- esterase apparently regulates JH titre; its activity is cyclic and varies with ovarian development (Yu and TERRmRE, 1975). These authors have shown that the carboxylesterase activity in the adult female housefly

543

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u ' l c

o t-,i i _

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8C

6(

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2C

71 El0 E l

Fig. 2. Densitometric scan and photograph of gel strip showing combined mid- and hindgut car- boxylesterase isozymes of adult housefly. Arrow indicates the isozyme area in the scan. Isozyme Elo

is at the base of the sample slot.

60 m

40 -

201 •

!

c o

<~ i I V .

0 n

II

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Eio E 8 E 7

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E 6 Es_ a

Fig. 3. Densitometric scan and photograph of gel strip showing thoracic muscle carboxylesterase isozymes of adult housefly, lsozyme E~o is at the base of the sample slot. Absorbancy relative to isozyme E1 as in Fig. 2. The photograph of the gel is black-and-white transfer from a color negative. Therefore, bands E2_s occurring closely are difficult to discern in this final illustration but the arrows

in the densitometric scan indicate the appropriate position of each of these bands.

Larval and adult housefly carboxylesterase 545

Table 1. Approximate molecular weights and distribution of isozymes of carboxylesterase in the adult housefly

Isozyme Approx. mol. wt Tissue source

El0 1000,000 Thoracic muscles, gut E 9 ca. 300,000 Unknown* E6_ 8 156,000-300,000 Thoracic muscles E2_5 70,000-156,000 Thoracic muscles E t 67,000 ___ 3000 Gut

* Isozyme present in whole fly extracts.

is lowest on the 4th day and, subsequently, rises to attain the highest titre on the 8th day. The average age of flies used in the course of present investigation was 5 days (range being 4 to 6) from emergence. Therefore, it is possible that carboxylesterase in the ovaries escaped detection due to assay at the time of low activity.

The peptidase staining of fly gut preparations showed the same three electrophoretic components (P~-P3) as previously reported for whole fly extracts (ArIMAD, 1974). The peptidase isozymes from gut preparations were quite distinct from the isozymes of carboxylesterase (Fig. 1). The peptidases P2 and P3 were scarcely distinguishable on the gel and could be isozymes of the same enzyme. This would mean 2, rather than 3, peptidases; possibly one is trypsin and the other enzyme intermediate between cathepsin and pepsin (GREENBERG and PARETSKY, 1955).

It is apparent that the tool. wt of the slowest iso- zyme El0 is about 1 × 106, as molecules of this size can pass through 3% gel but are stopped by 3.75% gel (technical information; disc electrophoresis appar- atus, Shandon Scientific Co., 1968). All the other iso- zymes passed through gels up to and including 7.5% concentration and, therefore, have a maximum mol- ecular weight of 3 × 105. The average pore size of 7.5% polyacrylamide gel is about 5 um (ORNSTEIN, 1964). Therefore, this gel exhibits greater frictional re- sistance to migration of heavier proteins such as globulin (ca. mol. wt 156,000, length 23.5um, dia- meter 4.4 um than to smaller proteins such as albu- mins (mol. wt 67,000 to 70,000, length 15nm, dia- meter 3.8 nm). In 7.5% gel, the mobility of the two albumins matched that of the leading isozyme (El), whereas the globulin moved half way through this gel. Therefore, the molecular weight of isozyme E~ was estimated to be about 67,000 __+ 3000 (Table 1). The isozyme E 9 moved freely through the 3.75% gel but was stopped by the 7.5% gel (1.5 cm from sample slot). Thus E 9 has a mol. wt between 3 × 105 and 1 x 106. Isozymes E2 to Es showed tool. wt in the range of 67,000 to 300,000, and were divided into those less than 156,000 and those greater than 156,000 based on their positions relative to the globulin band (Table 1). Molecular diversity of earboxylesterase iso- zymes has been recently reported for the cockroach Periplaneta americana, where the range for 7 isozymes is 58,000 to > 230,000 (Hn, PS and NELSON, 1974). A

possible explanation of such disparate molecular weights may lie in the aggregation of functional subunits (each with an active esteratic and, hence, acylation site). This has been postulated for mam- malian cholinesterase where molecules up to 3 x 106 are thought to be formed from aggregated subunits of 86,000 (OosxERBAAN and JANSZ, 1965).

Larval isozymes

Ten isozymes were present in larval tissues (Fig. 1). Isozyme E2 was absent from all larval prep- arations. In addition, isozymes E6 and E7 showed slightly lower mobility relative to corresponding adult isozymes, and one isozyme designated Ex did not cor- respond to any of the adult isozymes.

The larval gut showed isozymes E3_ 7 and E9 in addition to E1 and El0 the only two detected in the adult gut. Larval muscles also revealed a different type of pattern from that of the adult preparation (Fig, 1). Isozymes Ez_5 were absent and, in contrast to adult thoracic muscles, E1 was present. Isozyrnes E6-8 were common to both stages. Larval fat body contained 8 isozymes. In addition to E~_7 and E9_~o, an isozyme designated E~ was also resolved. As stated earlier, electrophoretic mobility showed this isozyme to be different from any of the adult isozymes.

In the larval gut, in addition to the two peptidases of the adult (comprising three bands as discussed above), another enzyme was resolved (Fig. 1). Although specific tests were not conducted, it may be the pepsin-type enzyme reported by G ~ E ~ G and PARETSKY (1955).

DISCUSSION

The biochemical aspects of the localization of iso- zymes within various tissues or cells are recognized (MARg_ERT, 1975). For instance, it is now known that different isozymes are needed to catalyse the same reaction under different metabolic conditions, in dif- ferent places in the same cell, or in different cells (MARKERT, 1975). On the other hand, UMBARGER (1969) has proposed that whenever an enzyme is under rigid end-product inhibition and repression, another isozyme(s) wilt be needed by the cell if there is some other essential role for that enzyme. Accord- ing to HOCHACm,:A (1973) this principle has found support from numerous studies. In view of the forego- ing account of a rather large number of isozymes showing a varied tissue pattern, it could be assumed that carboxylesterase has more than one physiological r61e in the housefly.

Carboxylesterase activity of crude extracts shows catalysis of a diversity of esters such as: fatty acid esters (up to C12 as acyl number), acylesters of naph- thol, butryl lactones, acylesters of glycerol and monoglyeerides (OosrERBAAN and JANSZ, 1965). Fur- thermore, different kinetic properties for isozymes are generally recognized (MARKERT, 1975). Therefore, it

LB. 6/5--h

546 SAMI AHMAD

is possible that not all the substrates mentioned above are hydrolysed by all isozymes, or that the substrates are hydrolysed to different degrees by different iso- zymes. Clarification of this point by future kinetic studies might shed light on the various physiological roles of carboxylesterase in the housefly.

Densitometric scans of the gel showed E1 to be the major isozyme of carboxylesterase in the house- fly. The high activity (based on intensity of histoche- mical reaction) of this isozyme could be a reflection of a greater amount of enzymic protein or in the spe- cific activity of the protein or both. This isozyme is widely distributed in larval tissues, and after meta- morphosis to adult, is restricted to the gut. Such a change in distribution of this isozyme must be of bio- logical significance. Yu and TERRIERE (1975) have studied carboxylesterase of the housefly in relation to hydrolysis of JH-analogues. They proposed that this enzyme may be involved in the regulation of endogenous JH, although they did not attempt to determine if the carboxylesterase activity was the result of one or several isozymes. Although direct ex- perimental evidence is not presented in this paper, it is possible that isozyme El, being the most active carboxylesterase, will be found to metabolize JH. In this connection it is interesting to note that the esti- mated tool. wt of this isozyme (6.7 + 0.3 x 10 4) appears close to that of the JH-specific esterase in the tobacco hornworm, Manduea sexta (6.7 × 104) (SAr~tYRG et al., 1975). Future studies directed towards the isozyme profile of carboxylesterase acti- vity during various stages of metamorphosis, and kin- etic studies, using JH as substrate, should identify the isozyme(s) involved in the regulation of JH titre in the housefly.

It is generally agreed that for many esters carboxyl- esterase activity overlaps that of lipase (OoSTERBhAN and JANSZ, 1965; LAKE, 1972; SELIGMAN, 1972). LAKE (1972) has suggested that fatty acid ester hydrolases, i.e., carboxylesterase and lipase should not be regarded as unrelated enzymes since they have one purpose--to cleave fatty acid esters into alcohols and acids. Furthermore, in a recent study of carboxylester- ase in the cockroach, Periplaneta americana, HIPPs and NELSON (1974) concluded that 'the distinction between esterases and lipases based on the idea that lipases are specific for long chain fatty acid glyceryl esters and that esterases are active on short chain esters of other alcohols does not hold with these enzymes'. Similarly, in the housefly, tributyrin (a classical lipase substrate) is hydrolysed by both car- boxylesterase and lipase, though predominantly by the latter (AHMAD, 1970b). In view of these facts, it is quite possible that some of the isozymes may be involved in fatty acid ester metabolism and, in par- ticular, in the mobilization of fats in the housefly. The presence of 8 isozymes in the fat body strongly sug- gests this possibility.

Insect esterases have been implicated in fat metabo- lism (GILBERT et al., 1965; STEVENSON, 1969), and in

insect reproduction (e.g. in the production of oothecae and in vitellogenesis in Blattella germanica) (HOOPER and WAN, 1969). These esterases may play a role in the mobilization of fats (OGITA, 1961; WAN and HOOFER 1967, 1969; HOOPER and WAN, 1969). Tan (in SUDDERUDDIN, 1973) has shown that the lipase system in fat body of Pycnoscelus striatus, which nor- mally breaks down triglycerides to diglycerides, has very low activity; whereas, carboxylesterases were very active and, presumably, also metabolised trigly- cerides. These studies, therefore, support the view dis- cussed above, that some of the carboxylesterase iso- zymes in the housefly may be involved in fatty acid ester metabolism.

It is of interest to note that adult thoracic muscle preparations revealed more isozymes than did larval preparations. This may be biologically significant and may be related to adult flight with its increased demand for energy (hence greater catabolism of fats). On the other hand, the histochemical observation of carboxylesterase activity in the epidermal region (AuMAD, 1970a) suggests yet another physiological role which is not clear at present. One possibility, based on the proximity of carboxylesterase activity to insect cuticle is the involvement of isozyme(s) in cuticular wax deposition. Esterases have been located in the cuticular pore canals and in the epicuticle of several insect species where it could be concerned in wax synthesis and transport (LocKE, 1961, 1974).

One other possible physiological role of carboxyles- terase in the housefly advanced earlier (A~AD, 1970a) deserves comment. In addition to lipase in mammals, there is another esterase, known as intra- mueosal monoglyceride lipase, that rapidly hydrolyses short-chain esters resulting in their selective removal (LANDS, 1965). Since the fatty acids released are not readily reactivated and reesterified, they are elimin- ated from the portal circulation and do not appear in the general circulation. Carboxylesterase in insects can hydrolyse glyceride esters and is present in the intramucosal region of the housefly gut. It was, there- fore, suggested that carboxylesterase may be involved in the removal of undesired esters (Ar~AD, 1970a). This hypothesis has interesting implications since gut is also the principal site for the oxidative degradation of foreign lipophilic substrates (WILKINSON and B~TTS~N, 1972). Furthermore, both microsomal oxidases and carboxylesterase are thought to metabo- lise JH (SLADE and ZmITT, 1972; WHITE, 1972; SLADE and WmgrNSON, 1973; TERRmRE and Yu, 1973; Yu and TERRIERE, 1974, 1975).

Acknowledgements---I am grateful to Dr. A. J. FORGASH, Dr. A. P. GUPTA, and Dr. R. B. ROBERTS for valuable advice and criticism of this paper.

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Larval and adult housefly carboxylesterase 547

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