Camp. Biochem. Physiol. Vol. 99C, No. I/2, pp. 15-20, 1991 Printed in Great Britain

0306-4492/91 $3.00 + 0.00 0 1991 PergamonPress plc

EFFECT OF BENZYL-1,3-BENZODIOXOLE DERIVATIVES ON JUVENILE HORMONE BINDING BY COMPONENTS OF THE HEMOLYMPH FROM THE MEDITERRANEAN

FRUIT FLY, CERA TITIS CAPITA TA WIEDEMANN

FRANKLIN CHANG, ERIC B. JANG,~ CHIOU-LING Hsu and LEONARD JURD$

Department of Entomology, University of Hawaii at Manoa, Honolulu, HI 96822, U.S.A.

(Received 25 June 1990)

Abstract-l. Competitive displacement assays showed the presence of a single saturable high-affinity binder for JH III in hemolymph from adult female C. cupitata.

2. The dissociation constant Kd and total binding capacity B,,,,, for the binder were 0.93 x lo-’ M and 7.46 nmol/mg protein respectively.

3. The order of affinity of JH homologs and an analog for the hemolymph binder was JH III > JH II > JH I > (7S)methoprene.

4. Three benzyl-1,3-benzodioxole derivatives (BBDs), coded 52581, 52710, and 53263, were ineffective in blocking JH III binding to hemolymph components.

5. The relationship between BBDs, vitellogenesis, and hemolymph binders and a proposed mode of action of BBDs are discussed.

INTRODUCTION

Multiple biological functions have been attributed to methylenedioxy derivatives of benzyl-1,3-benzodi- oxole (BBDs) and related benzylphenols. These include inhibition of cytochrome P-450 monooxygen- ase activity (Matolcsy et al., 1986), tubulin polymeriz- ation and mitosis (Batra et al., 1984; Roach et al., 1987; Hsu et al., 1990a), in uivo tumor activity (Jurd et al., 1987), and juvenile hormone (JH) activity in insects (De Loof et al., 1982; Chang et al., 1988; Song et al., 1990). BBDs were shown to be biologically active against several insect species, often resulting in interference with reproduction (Jurd et nl., 1972, 1979; Rawlins et al., 1979; Chang et al., 1980; Flint et al., 1980; Langley et al., 1982; Chang et al., 1984, 1988; Matolscy et al., 1986; Hsu et al., 1989, 1990b). BBDs blocked the action of JH in the Galleria cuticle wax bioassay (De Loof et al., 1982) which suggested an anti-JH effect. The induction of precocious metamorphosis in Zfyphantriu cunea Drury larvae (Darvas et al., 1988) and failure of vitellogenesis to occur in Drosophila melanogaster L. (Song et al., 1990) after treatment of both insects with BBDs further suggested an anti-JH function for BBDs. Vitellogenesis in D. melanogaster and the Mediter- ranean fruit fly, Ceratitis capitata Wiedemann was not blocked after simultaneous application of BBDs with a JH analog, (7S)methoprene (Chang et al., 1988; Song et al., 1990). These results suggested that

*Correspondence to: Dr Franklin Chang, Department of Entomology, 3050 Maile Way, Gilmore 407, University of Hawaii at Manoa, Honolulu, HI 96822, U.S.A.

tUSDA-ARS Tropical Fruit and Vegetable Research Lab- oratory, Hilo, HI 96720, U.S.A.

SUSDA-ARS Western Regional Research Center, Berkeley, CA 94710, U.S.A.

blocking of JH binding sites by BBDs may not be involved. However, the finding that JH homologs and analogs apparently share different nuclear receptor proteins in the tobacco hornworm, Manduca sexta Johannsen (Riddiford et al., 1987), implied that interference by BBDs on JH binding could not be entirely ruled out.

In adult insects, JH is transported by the hemo- lymph to the ovaries where it initiates vitellogenesis (uptake of yolk proteins). Transport of JH to target organs is thought to be accomplished by proteins in the hemolymph which bind specifically or non-specifi- cally with JH (Hammock, 1977; Goodman and Chang, 1985). Of importance are those proteins which specifically bind JH in the hemolymph, the JH binding proteins (JHBPs), whose role may involve transport of the naturally-occurring hormones to target organs and their protection from hemolymph esterases (Goodman and Gilbert, 1974; Kramer et al., 1974). Interference with JH binding to carrier proteins could result in a decrease in the availability of JH to target organs that could affect physiological functions, e.g. vitellogenesis, dependent on critical titers of the naturally-occurring hormones. In this study, we characterized JH binding in hemolymph from adult female C. cupitata and examined the effect of BBDs on this binding.

MATERIALS AND METHODS

Insects

C. cupiratu puparia were obtained from the mass-rearing facilities of the USDA-ARS Tropical Fruit and Vegetable Laboratory in Honolulu. The sex of flies was determined upon emergence. Flies were allowed to feed ad libitum on a sugar-yeast hydrolysate (3: 1, w/w) mixture (Tanaka et al., 1969) and water.

15

16 FRANKLIN CHANG et al.

Chemicals Binding assay

Radiolabelled racemic [IO-‘H]JH III (spec. act. 12Ci/mmol) was obtained from NEN Research Products (Boston, MA). Radioinert racemic JH I, II, and III (Sigma Chemical Co., St Louis, MO) were further purified by silica gel TLC (chloroform : acetone, 9: 1, v/v) before use. (7S)methoprene ( > 99%) was a gift from Dr G. B. Stahl (Zoecon Research Inst., Palo Alto, CA). Radioinert racemic BBDs coded 52710 (5-methoxy-6-(l-[Cmethoxy- phenyl]ethyl)- 1,3-benzodioxole), 52581 (5-ethoxy-6(4- methoxyphenyl)methyl-1,3-benzodioxole), and 53263 (5- methoxy-6-(1-[4-ethoxyphenyl]ethyl-1,3-benzodioxole) were synthesized by one of us (L.J.). Structures of BBDs were confirmed by elemental (C,H), proton NMR, and mass spectral analyses (Jurd et al., 1979). A purity of > 99% for the three BBDs used in this study was determined by silica gel TLC (ethyl acetate: hexane, 3: 7, v/v) and subsequent densitometric analysis. Dimethylsulfoxide (DMSO, > 99.9%) (Sigma Chemical Co.) was used as the diluent for [3H]JH III and to increase the solubility of the JH homologs, (7S)methoprene, and BBDs in the assay mixtures. Hydrox- ylapatite (HAP) (DNA-Grade, Bio-Rad Laboratories, Richmond, CA) in TMK buffer (1OmM Tris-HCl, 5mM MgCl, 150mM KCI, pH 7.4) was used to separate bound and free hormone. Phenylmethylsulfonylfluoride (PMSF) (Sigma Chemical Co.), was used to inhibit general esterase activity. Samples were counted in ScintiVerse II scintillation fluid (Allied Fisher Scientific, Fairlawn, NJ). Other chemicals used were reagent grade or higher.

Preparation of hemolymph

The binding assay used was a modification of the HAP method of Roberts and Wyatt (1983). A typical assay consisted of [‘H]JH III (105,OOODPM) in 10~1 DMSO, 10 ~1 buffered hemolymph containing SO-85 pg protein, 10~1 of a given concentration of radioinert competing ligand, and 0.2 ml TMK buffer, pH 7.4. Assay mixtures were contained within 7 x 1 cm borosilicate tubes previously treated with a 5% polyethylene glycol (mol. wt 20,000) solution to prevent adhesion of JH to the inside walls of the test tube. Incubation was carried out at 23°C with moderate agitation in a water-bath. After incubation (30 or 60 min), 0.5 ml cold 5% HAP in TMK buffer @H 7.4) was added to each tube to precipitate proteins, the tubes were vortexed three times over a 15 min period, and allowed to sit for an additional 15 min. The tubes were then centrifuged at 6000g for 10 min at 4°C. One ml of cold TMK buffer (pH 7.4) was added to the pellet after the supematant was discarded, and the tube vortexed and centrifuged as above. This step was again repeated. After discarding the supernatant, 1 ml of a cold buffer solution (pH 7.0) containing 0.5 M KH,PO,, 8 mM MgCl,, and 15 mM I-monothioglycerol was added to each tube, the tubes again vortexed, and the contents of each tube mixed with 15 ml ScintiVerse II scintillation fluid and counted in a Beckman LS 8100 liquid scintillation spectrometer. Counting efficiency (%) was determined using a standard quench curve and all counts were converted to DPMs. Reproducibility among replications using the HAP method of separating bound from free hormone was excellent, never exceeding +2% of the individual counts.

Hemolymph was withdrawn from 6-day-old vitellogenic adult female C. capitata by puncturing between the metathorax and first abdominal segment with a capillary pipet having a drawn-out tip. The hemolymph (ca. 200 ~1) was pooled into a polyethylene microcentrifuge tube (on ice) containing a few crystals of phenylthiourea to inhibit tyrosi- nase activity. A 1: 10 dilution of hemolymph with TMK buffer t&H 7.4) containing PMSF was made and the mixture centrifuged at 10,OOOg for 15 min at 4°C to remove hemo- cytes. The supernatant was then filtered through a low protein binding 0.2 pm membrane filter (Gelman Sciences, Ann Arbor, MI). A total protein determination was made on an aliquot of hemolymph using a bicinchoninic acid (BCA) protein reagent kit (Pierce, Rockford, IL) with bovine gamma globulin serving as standard. A volume of hemolymph containing SO-85 pg protein was used for the binding assays.

Data analysis

Data were analyzed by Scatchard analysis (Scatchard, 1949). Specific binding was defined as the portion of the total binding that was prevented by a IOOO-fold excess of radioinert hormones or BBDs. Correction for non-specific binding was made according to Chamness and McGuire (1975).

The rate of specific binding of [3H]JH III in hemolymph from vitellogenic female C. capitata at 4°C was not appreciably different from 23°C (Fig. 1 A,B). Therefore, all later assays were carried out at 23°C. Under our assay conditions, equili- bration of [3H]JH III with hemolymph binders occurs

8 0.6 E

4 0.5

0.7

4 s 0.4

z m 0.3 0

E Y 0.2

a 0.1

30 60 90 120 150 180

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

TIME (rainy 30 60 90 120 150 180

RESULTS

Fig. 1. A: Specific binding of [‘H]JH III to hemolymph components from 6-day-old adult female C. capitata. Radiolabelled JH III in DMSO containing 100,000 DPM was added to TMK buffered hemolymph containing 80 pg protein and the mixture incubated at 23°C for 180 min. Bound and free hormone were separated by HAP at 30 min intervals. B: Same conditions as (A) except incubated at 4°C.

Each point represents mean of duplicate samples run under the same conditions.

Effect of BBDs on JH binding 17

a 100 z s 90

z 80

= 70

5 60

$ 50 s 30 40

g 20

8? 10

0 0 5

Owith PMSF

I without PMSF

10 25 30

Fig. 2. Percent of maximum binding of [3H].lH III to hemolymph components from 6-day-old adult female C. capirara. Conditions same as in Fig. 1B except incubation for a total of 30min in the presence and absence of 6 x 10m4 M PMSF. Each point represents mean of duplicate

samples run under the same conditions.

quite rapidly. Maximum binding of [‘H]JH III was achieved within 5 min after initial mixing of assay reagents and was not affected by the absence or presence of 6 x 10d4 M PMSF (Fig. 2). General conditions for incubation were therefore set at 30 min at 23°C.

To monitor any degradation of [3H]JH III, ether extraction of assay mixtures incubated for up to 5 hr at 23°C were conducted. The ether extract components were resolved by silica gel TLC and a radiochemical purity of 99.3% and 97.8% was obtained for [‘H]JH III before and after incubation respectively (Fig. 3). This result showed that virtually none of the [‘H]JH III was appreciably degraded in our assay and that the ligand bound to putative hemolymph binders was JH III. We assumed that the radioinert JH homologs likewise remained unaltered.

Specific binding increased linearly up to at least 12Opg total hemolymph protein (Fig. 4). For each subsequent assay, 80-85 pg hemolymph protein were added.

The binding affinities of hemolymph were determined by measuring specific binding over a

1 0m5 - Ether extraction of 3H- JH III

JH III and metabalites

10T4- mixture (5 hr ot 23 C) after incubation of gssoy

8 Radiochemical

A 1 o-3- purity = 97.80%

1.50 r

8 E 1.25 - 4 CJ 1.00 . z

8 s 0.75 -

0.00 0.06 0.12 0.16 0.24

TOTAL PROTEIN (mg)

Fig. 4. Specific binding of [“H]JH III as a function of increasing amounts of total protein from hemolymph of C. capitata. Conditions same as in Fig. 1B except incubation for 30 min in absence of PMSF. Each point represents mean

of duplicate samples run under the same conditions.

range of [3H]JH III concentrations (O-175 nM). The binding sites of hemolymph were saturable (Fig. 5). After correction for non-specific binding, a straight line was obtained when bound/free hormone was plotted against specifically-bound hormone (Fig. 6). This showed the presence of a single saturable site on the putative receptor. The dissociation constant, Kd, for the binding reaction was calculated to be 0.927 x lo-’ M, which is within the range for a high-affinity binder. The total binding capacity, B,,,,,, was 7.46 nmol/mg protein. The correlation coefficient over 7 assay points was 0.81.

The ability to displace [‘H]JH III by JH homologs and an analog were in the order JH III > JH II > JH I > (7S)methoprene over a concentration range of 10-4-10-* M (Fig. 7). Figure 7 also shows that over the same concentration range, J2581, 52710, and 53263 were unable to displace or block [3H]JH III binding whether BBDs were added first or last to the assay mixture.

As expected, binding for [3H]JH III was destroyed when hemolymph samples were boiled for 1 min before incubation (data not shown).

1o-5

/

0

I’ JH Ill

J

10 -4

3H-JH III t

lo-3 Radiochemical purity = 99.33% I I

1 Id c , 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0

Rf Fig. 3. Radiochromatogram of (a) ether-extracted [3H]JH III and metabolites (left) after 5 hr incubation at 23°C of radiolabelled ligand with hemolymph from 6-day-old adult female C. capitata and (b) radiolabelled JH III standard (right). Ether extracts of the incubation mixture and [3H]JH III standard were separated on silica gel G plates. After development, radioactivity in 2-mm sections of the plate were counted. Radiochemical purity was defined as the percentage of total radioactivity present in the stated

chemical form.

CBPC 99,1,*-a

18 Fumtcmi CHANG et al.

(q 140

I O-O Non-specifically bound A-A 41 120 _ l -•Specifically bound

X A- A Total bound 0 -0

z 100 - n

9 80-

2 m 60-

Q

0 20 40 60 80 100 120 140 160 180

TOTAL JH III (nmole)

Fig. 5. Specific binding of [)H]JH III as a function of increasing amounts of radiolabelled JH III (O-175 nM) in hemolymph of 6-day-old adult female C. capitata. Conditions same as in Fig. 4 except the incubations carried out in the presence and absence of a lOOO-fold excess of radioinert JH III. Each point

represents mean of triplicate samples run under the same conditions.

DISCUSSION

Topical or oral administration of BBDs to insects resulted in failure of vitellogenesis to occur (Chang et al., 1988; Song et al., 1990). These findings led us to believe that BBD treatment was, in some way, interfering with JH function since JH is known to regulate vitellogenesis in insects.

In vertebrates, carrier proteins for steroid hormones, besides serving a solubilization function, appear to protect and transport these hormones in the blood. Similar proteins in insect hemolymph also serve the same function (Goodman and Chang, 1985). In this study, the effect of three BBDs on competitive displacement of [3H]JH III from putative hemolymph binders from adult female C. capitata, was tested. Competitive displacement assays showed the presence of a single-site saturable high-affinity

0.45

0.40 K,=0.927 Y 1 O-7M

0.35

8 0.30

E \ 0.25

9 0.20

2 0.15

E&,=7.463 nmole/mg

R=0.615

Y=0.346-0.00001x

0 4 6 12 16 20 24 26 32

SPECIFIC BOUND (fmole/ml) x 10 3

Fig.’ 6. Representative Scatchard plot for hemolymph binders from 6-day-old adult female C. capitata using HAP for separation of bound and free hormone. Conditions for incubation same as in Fig. 4. Lines were fitted with linear regression. The dissociation constant (&) and total binding capacity (E,,,,,), both calculated from the regression line, and correlation coefficient (r), are shown on the graph. Each point represents triplicate samples run under the same

conditions.

binder in the hemolymph of C. capitata. The dissociation constant Kd = 0.9 x lo-’ M for the hemolymph JH binder in C. capitata is within the range (10-6-10-9 M) of hemolymph binders found in other insect species (Goodman and Chang, 1985) and is within the magnitude of the dissociation constants found in two dipteran species, Drosophila hydei Sturtevant (1 x 10m7 M) (Klages and Emmerich, 1979) and Sarcophaga bullata Parker (1.5 x 10m7 M) (Van Mellaert et al., 1985). Maximal binding of [3H]JH III in C. capitata hemolymph occurs very rapidly, within seconds of mixing of the reagents. Competitive displacement assays using radioinert JH homologs and an analog showed the ability to displace [3H]JH III from the hemolymph binder in the order JH III > JH II > JH I > (7S)methoprene. Similar results were obtained by Van Mellaert et al. (1985) with Sarcophaga. However, J2581,52710, and

90 -

60

70

60 - 50 - 40 - 30

20 - 10

a--•J2561 A-AJ2710 m-¤J3263 q -OJH I V-VJH I, V-VJH III

0’ 9 6 7 6 5 4 3

-LOG OF COMPETITOR CONC. (M)

Fig. 7. Competitive displacement curves for competition by radioinert JH homologs, an analog, and BBDs, for binding of [‘H]JH III by hemolymph from 6-day-old adult female C. capitata. Competed binding expressed as percent of maxi- mum binding. Incubation mixtures contained radiolabelled JH III (105,000 DPM), TMK buffered hemolymph (81 pg). and increasing amounts of radioinert ligands. Incubation conditions same as in Fig. 4. Each point represents mean of

triplicate samples run under the same conditions.

Effect of BBDs on JH binding 19

Chang F., Hsu C. L., Jurd L. and Williamson D. L. (1984) Effect of precocene and benzyl-1,3benzodioxole derivatives on sex attractancy in the Mediterranean fruit fly (Diptera: Tephritidae). Ann. en?. Sot. Am. 77, 147-151.

Chang F., Hsu C. L. and Jurd L. (1988) Effects of topical application of benzyl-1,3benzodioxole derivatives on reproduction in the Mediterranean fruit fly. Insect Sci. Appl. 9, 381-388.

Darvas B., Varjas L., Farag A. I. and Van Mellaert H. (1988) Effects of benzyl- 1,3-benzodioxoles on develop- ment of sensilla trichodea of Hyphuntriu cunea (Lepidoptera: Arctiicdae) larvae. In Endocrinological Frontiers in Physiological Insect Ecology, Vol. 1, pp. 581-585. Wroclaw Technical University Press, Wroclaw, Poland.

De Loof A., Van Mellaert H. and Jurd L. (1982) Two new compounds with anti-juvenile hormone activity as shown by the Galleria bioassay. Gen. camp. Endocr. 46, 371.

Flint H. M., Jurd L. and Merkle J. (1980) Pink bollworm: sterilizing effects of benzylphenols and benzyl-1,3-benzo- dioxoles. J. econ. Ent. 73, 710-714.

Goodman W. G. and Gilbert L. I. (1974) Hemolymph protein binding of juvenile hormone in Manduca sexta. Am. Zool. 14, 1289.

Goodman W. G. and Chang E. S. (1985) Juvenile hormone cellular and hemolymph binding proteins. In Comprehen- sive Insect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), pp. 491-510. Pergamon Press, Oxford.

53263, were unable to displace [3H]JH III from its hemolymph binder. This result suggests that in C. capitata, BBDs do not block hemolymph binding sites with an affinity for JH III. If BBDs could block JH binding sites, then one would expect JH homologs and analogs to be likewise blocked, if they share identical binding sites. However, the finding that JH homologs and analogs apparently share different nuclear receptor proteins in Manduca sexta Johannsen (Riddiford et al., 1987) require that this assumption be tested.

In C. capitata, BBDs apparently do not block JH III binding in hemolymph and probably in other tissues as well. That the effect of BBDs on vitellogenesis could be negated by concomitant ad- ministration of (7S)methoprene to adult C. capitata (Chang et al., 1988) and D. melanogaster (Song et al., 1990) suggested that endogenous JH may not be available to target organs but could be supplied by an exogenous source of JH or its mimic. That JH biosynthesis or release, rather than JH binding sites, may be blocked by BBDs is supported by the finding of Brooks et al. (1985) who showed the absence of JH in the culture medium following incubation of CA glands from Periplaneta americana (L.) with benzylphenol derivatives, compounds closely related to BBDs. Further, our radiochemical assays with CA preparations from C. capitata showed that JH release, rather than its biosynthesis, was affected (Jang, in preparation).

In conclusion, the mode of action of BBDs in C. capitata and probably in other insects as well, apparently does not involve blocking of JH binding sites. BBDs can inhibit microtubule assembly from vertebrate tubulin in vitro (Batra et al., 1984; Roach et al., 1987) and has been shown to affect axoneme structure in sperm flagellae from the oriental fruit fly, Dacus dorsalis Hendel (Hsu et al., 1990b). It is not unreasonable to speculate the physiological functions dependent on cell movement or secretory activity, e.g. “patency” in follicular cells before vitellogenesis or JH release by the CA, may be inhibited because of lack of cytoskeletal integrity. Further study will be needed to establish such a role.

Acknowledgements-This research was supported by the USDA under CSRS Special Grant No. 85-CRSR-2-2652 managed by the Pacific Basin Advisory Group (PBAG). Journal Series No. 3466 of the Hawaii Institute of Tropical Agriculture and Human Resources.

REFERENCES

Batra J. K., Jurd L. and Hamel E. (1984) Structure-function studies with derivatives of 6-benzyl-1,3-benzodioxole, a new class of synthetic compounds which inhibit tubulin polymerization and mitosis. Molec. Pharmac. 27,94102.

Brookes G. T., Pratt G. E., Mace D. W. and Cocks J. A. (1985) Inhibition of juvenile hormone biosynthesis in corpora allata of the cockroach, Periplaneta americana (L.) in vitro. Pesticide Sci. 16, 132-142.

Chamness G. C. and McGuire W. L. (1975) Scatchard plots: common errors in correction and interpretation. Steroids 26, 5388542.

Chang S. C., Borkevec A. B. and Demilo A. B. (1980) Effects of substituted benzylphenols on reproduction of house flies. J. Econ. Ent. 73, 7455747.

Hammock B. D.. Soarks T. and Mumbv S. (1977) Selective inhibition of JH* esterases from cockroach hemolymph. Pestic. Biochem. Physiol. 7, 517-530.

Hsu C. L., Chang F., Mower H. F., Groves L. and Jurd L. (1989) Effect of orally-administered 5-ethoxy-6-[4- methoxyphenyllmethyl-1,3-benzodioxole on reproduction of the Mediterranean fruit fly (Diptera: Tephritidae). J. econ. Ent. 82, 10461053.

Hsu C. L., Chang F., Mower H. F. and Jurd L. (1990a) Effect of top-soil treated with 52581 (5-ethoxy-6-(4- methoxyphenyl)methyl-1,3-benzodioxole) on reproduc- tion in the Oriental fruit fly, Dacus dorsalis Hendel (Diptera: Tephritidae). J. econ. Em. 83, 1261-1266.

Hsu C. L.. Chane F.. Hunter W. and Hou Roger F. (1990b) Cytological elects of 52581 (5-ethoxy-z-ethoxy-6-[4: methoxyphenyllmethyl-1,3benzodioxole) on spermatoge- nesis in the Oriental fruit fly, Dacus dorsalis Hendel. Camp. Biochem. Physiol. 96C, 27-31.

Jurd L., Stevens K. and Manners G. D. (1972) Quinonoid constituents of Dalbergia retusa heartwood. Phytochem- istry, 11, 3287-3292.

Jurd L., Fye R. L. and Morgan J. (1979) New types of insect chemosterilants. Benzylphenols and benzyl-1,3-benzodi- oxole derivatives as additives to housefly diet. J. agric. Fd Chem. 27, 1007-1016.

Jurd L., Narayanan V. L. and Paul1 K. D. (1987) In uivo antitumor activity of 6-benzyl-1,3-benzodioxole deriva- tives against the ~388, L1210, B16, and M5076 murine models. Med. Chem. 30, 1752-1756.

Klages G. and Emmerich H. (1979) Juvenile hormone binding proteins in the haemolymph of third instar larvae of Drosophila hydei. Insect Biochem. 9, 23-30.

Kramer K. J., Sanburg L. L., Kezdy F. and Law J. H. (1974) The juvenile hormone binding protein in the hemolymph of Munduca sexta Johannson (Lepidoptera: Sphingidae). J. biol. Chem. 251, 49794985.

Langley P. A., Trewern M. A. and Jurd L. (1982) Sterilizing effects of benzyl-1,3benzodioxoles on the Tsetse fly Glossina morsitans morsitans. Bull. ent. Res. 72, 47348 1.

Matolcsy G., Feyereisen R., Van Mellaert H., Pal A., Varjas L., Belai I. and Kulcsar P. (1986) Molecular modifications of benzylphenol and benzyl-1,3-benzodioxole types of insect chemosterilants. Pesticide Sci. 17, 13-24.

20 FRANKLIN CHANG et al.

Rawlins S. C., Jurd L. and Snow J. W. (1979) Anti-fertility effects of benzylphenols and benzyl-1,3-benzodioxoles on screwworm flies. J. econ. Ent. 72, 674-677.

Riddiford L. M., Osir E., Fittinghoff C. and Green J. M. (1987) Juvenile hormone analog binding in Manduca epidermis. Insect Biochem. 17, 1039-1043.

Roach M. C., Trcka P. P., Jurd L. and Luduena R. F. (1987) The effects of 6-benzyl- 1,3-benzodioxole derivatives on the alkylation of tubulin. .I. Pharmac. exp. Ther. 32, 432436.

Scatchard G. (1949) The attraction of proteins for small molecules. Ann. N. Y. Acad. Sci. 5, 6-72.

Song Q., Ma M., Ding T. and Ballarino J. (1990) Effects of benzodioxole 52581 on vitellogenesis and ovarian devel- opment of Drosophila melanogaster. Pesticide Biochem. Physiol. 37, 12-23.

Tanaka N., Steiner L. F., Ohinata K. and Okamoto R. (1969) Low-cost larval rearing medium for mass production of Oriental and Mediterranean fruit flies. J. econ. Ent. 62, 967-968.

Roberts P. E. and Wyatt G. R. (1983) Juvenile Van Mellaert H., Theunis S. and De Loof A. (1985) Juvenile hormone binding by components of fat body cytosol hormone binding proteins in Sarcophaga bullata hemo- from vitellogenic locusts. Molec. Cell. Endocr. 31, lymph and vitellogenic ovaries. Insect Biochem. 15, 5369. 655661.