Studies on cyromazine and diflubenzuron in the sheep blowfly Lucilia cuprina: Inhibition of vertebrate and bacterial dihydrofolate reductase by cyromazine

PESTICIDE

BIOCHEMISTRY

AND

27, ?(I-210

PHYSIOLOGY

(1987)

Studies on Cyromazine and Diflubenzuron in the Sheep Blowfly Lucilia cuprina: Inhibition of Vertebrate and Bacterial Dihydrofolate Reductase by Cyromazine K. C. BINNINGTON,*

A. RETNAKARAN,?

AND P SKELLY~

S. STONE,$

*CSIRO. Division of Entomology, G.P.O. Box 1700. Canberru. Austwlitr 2601: +Foresr Pest Manngeruent Institute. C’anndiun Forestry Service, 1219 Qwell St. EUSI. P.O. Box 490. Strrrlt Ste. Mnric. Ontctrio, Cutrrrdrr P6A 5M7; iDepartment of Biochemistry. John Cwtirz School qf Medical Research. A rtstrtrlian Natimtrl University. P.O. Box 334, Canberrcr. Austmliu 2600: and $Depariment of Biochemistry. Fnclrlt~ of Scirncr. Ausrr-ulinn Notionccl Unirsersity. P.O. Box 4, Cmberrtr. A/r.strct/itr 2600 Received

April

17. 1986; accepted

July

IS. 1986

Cyromazine causes epidermal cells of third instar Lwilicr cuprinn larvae to invade the cuticle and produce necrotic lesions whereas diflubenzuron inhibits chitin synthesis and thus results in the secretion of an imperfect cuticle. Ultrastructural as well as biochemical results indicate that cyromazine, unlike diflubenzuron, does not inhibit incorporation of N-acetyl-D-[l-‘4C]glucosamine into cuticle. Diflubenzuron and cyromazine-treated larvae weigh less and have cuticles which weigh less than those of controls. In the case of diflubenzuron this may be a consequence of decreased chitin production whereas cyromazine may have a more direct effect on protein synthesis. Electrophoresis shows that the inhibition did not change the pattern of cuticular proteins: both cyromazine and diflubenzuron apparently affect the production of all cuticular proteins to the same extent. Dihydrofolate reductase from chicken liver and bacteria is inhibited by cyromazine at a concentration of IO-’ M. It is suggested that. while inhibition of this enzyme is unlikely to be the primary mode of action of cyromazine possible effects of this insecticide on nucleic acid metabolism warrant further investigation. I 1987 Acadrmlc I’res\. Inc INTRODUCTION

The insecticide cyromazine (2-cyclopropylamino-4,6-diamino-s-triazine; CibaGeigy compound, CGA-72662 or Vetrazin) when fed to the sheep blowfly, Lmiliu c’w prim retards larval growth, causes malformations of the body, and inhibits molting, pupation, and emergence (1). Cuticles of affected larvae contain necrotic lesions which are contiguous with invasive processes of epidermal cells (2). Assays for chitin synthesis using in ciao preparations of insect integuments have provided equivocal results; cyromazine inhibited synthesis in cockroach leg cuticle (3) but not in cuticle of sheep blowfly larvae (4). An alternative hypothesis for the action of cyromazine is that it may affect folic acid metabolism in larval epidermal cells; aminopterin, a potent inhibitor of dihydrofolate

reductase causes malformations in cuticle of L. c~rprirzn which are similar in some respects to the lesions caused by cyromazine (2). In the present study cyromazine has been evaluated as an inhibitor of mammalian and bacterial dihydrofolate reductases and chitin synthesis has been assayed in Lucilia larvae fed on cyromazine, diflubenzuron, and chlorfluazuron (Ciba-Geigy compound CGA-I 1293). To determine if these insecticides result in quantitative or qualitative differences in protein composition cuticle proteins were analyzed by electrophoresis. Electron microscopy has been used to compare ultrastructural and biochemical effects. MATERIALS

Lucilia cuprim clrlturtr. Early third instar larvae ca. 1 hr after the molt were al-

202

BINNINGTON

lowed to feed on a modified artificial feeding medium (5) at 23°C for 12 hr; this period of feeding allowed the cuticle to grow or harden sufficiently to increase the survival rate of injected larvae (6). Chemicals. Cyromazine (95% technical grade) and chlorfluazuron (120 g/liter, emulsifiable concentrate) were gifts from Ciba-Geigy, Australia, and diflubenzuron from Duphar Ltd., Amsterdam. Aminopterin (95%) was purchased from Sigma Chemical Company, N-acetyi-D-[ IJ4C]glucosamine (55 Ci/mmol) from Amersham, Australia Pty Ltd., and NADPH from Boehringer. Dihydrofolate was prepared from folic acid (Calbiochem) by the method of Blakely (7). For incorporation in the diet, chemicals were dissolved in water or dimethyl sulfoxide before being added to molten feeding medium (2). Assay of labeled chitin. Larvae were injected with 0.05 mmol N-acetyl-D[I-r4C]glucosamine in 0.5 ~1 water using a front-fill microinjection system. They were then allowed to feed for 48 hr on either unadulterated feeding medium or medium with insecticides or inhibitors incorporated (2). After the feeding period body contents of larvae were removed, the cuticles hydrolyzed in 10% aqueous KOH and washed serially in water, 0.1 M HCl, and chloroform:methanol (3:l v/v). The residual chitinous membranes were hydrolyzed in a scintillation vial with 2 ml constant boiling HCl at 90°C for 24 hr. After evaporation of the acid, 2 ml water and 10 ml scintillation cocktail were added and the vials shaken. Radioactivities were then assayed in a Beckman LS-2800 scintillation counter [see Retnakaran and Hackman (6) for further details]. Larval and cuticle dry weight. The dry weights of larvae and isolated larval cuticles were estimated as described for third instar larvae by Skelly and Howeils (8). Cuticle phoresis.

protein

extraction

and electro-

Cuticles were removed from internal tissues and scraped clean of contaminating tissue as described by Skelly and

ET

AL.

Howells (8); protein was extracted by incubating isolated cuticles in a solution of 7 M urea (300 @cuticle) for 1 hr at room temperature. To ensure that this standard method of cuticle preparation did not cause greater damage to the cuticles of larvicidetreated larvae than to those of controls, a series of cuticles were isolated as follows. Each larva was immobilized by cooling on a microscope slide and its anterior end removed with a scalpel. The larva was then inverted and the internal tissues removed by gentle washing with water. The cuticle (contaminated with muscle and epidermal cells) was then processed as usual. The amount of protein extracted was determined by the method of Lowry et czl. (9). Electrophoresis of extracted proteins was performed on a lo-20% gradient nondenaturing polyacrylamide gel as previously reported (8). Dihydrafolate reductase assay. Dihydrofolate reductase was purified from chicken liver (10) and Escherichia co/i as previously described (11). The enzymes were assayed at 30°C using a Cary 118 spectrophotometer to follow the decrease in absorbance at 340 nm due to the disappearance of dihydrofolate and NADPH. Assays were performed at pH 7.4 in a buffer mixture that contained 2-(N-morpholino)ethanesulfonic acid (0.025 M), Tris (0.05 M), sodium acetate (0.025 M), and NaCl (0.1 M). The substrates, dihydrofolate and NADPH, were present at concentrations of 10 and 100 PM, respectively, and cyromazine was varied from 0 to 1.04 mM. The inhibition of the enzymes by cyromazine could be described by Eq. [ 11: I/V = I/V (I + I/Ki [app]),

[II

where v is the initial velocity, V is the apparent maximum velocity, I is the concentration of cyromazine, and Ki [app] is the apparent inhibition for the inhibitor. The apparent inhibition constant is related to the true inhibition constant [fl by Eq. [2]: K; = Ki [app] / (1 + A/K,),

where A is the concentration

[2]

of dihydrofo-

CYROMAZINE

FIG.

epidertnal

I. Thin section through cell (EC); procuticle

cuticle from (P). x 6370.

MODE

OF

(I control

late and K, is the Michaelis constant for dihydrofolate. Initial velocity data were fitted to Eq. [l] using weighted linear regression (12). The inhibition constants for cyromazine were calculated from the apparent constants using values for the Michaelis constants of 0.47 pAl for the enzyme from E. co/i (11) and 0.25 PM for the chickenliver enzyme (Stone, S. R., and Morrison, J. F., unpublished results). Electron microscopy. Larval cuticle was fixed and processed for electron microscopy as described previously (2). RESULTS

Electron microscopy. The ultrastructure of normal cuticle from a third instar larva

203

ACTION

48 hr fed

L. cuprina

larrja.

Epicuticle

(El:

fed for 24 hr is shown in Fig. 1 [see Filshie (13) and Binnington (2) for further details of normal L. cuprinn cuticle]. The effect of diflubenzuron was to inhibit completely normal cuticular growth. Only a thin layer of cuticle was laid down during third instar feeding and this layer lacked the lamellate appearance of normal cuticle (Fig. 2). Globular bodies were present in parts of the affected cuticle (Fig. 2, inset) but were less striking than those seen in first and second instar L. cuprina exposed to diflubenzuron during the molt (2). Cyromazine altered the ultrastructure in a completely different way than diflubenzuron. Cuticle with a lamellate appearance continued to be secreted during exposure to

BINNINGTON

the chemical but there was a deposition of necrotic tissue within the cuticle (Fig. 3). This necrotic tissue is probably from invading processes of epidermal cells; in earlier larval instars (2) invasive epidermal cells rich in electron-dense granules and microtubules have been observed asso-

ET

AL.

ciated with cuticular lesions. Abnormalities of the epidermal cells of third instar larvae included vacuoles within the microvillar layer and cytoplasmic vacuoles containing membranous bodies (Fig. 4). Chiti~ assay. Incorporation of r4C-labeled N-acetyl glucosamine into cuticle

CYROMAZINE

MODE

(Table 1) was reduced by diflubenzuron at concentrations of ca. 10 mg/kg of medium and was almost completely inhibited by 100 mgikg [concentrations of diflubenzuron

OF ACTION

305

were at approximately x Z and x 20 times the LDSO value for survival of f.. cc7prir7rr pupae exposed throughout larval development to food conaining diflubenzuron ( 14)].

206 Chlorfluazuron

BINNINGTON

(LD50 value not known for caused a marked reduction in incorporation at 4 mg/kg medium and thus may be a more effective insecticide against Lucilia than diflubenzuron. Cyromazine at 1 and 10 mg/kg medium did not cause a significant reduction in chitin synthesis [concentrations of cyromazine were at approximately x 2 and x 20 times the LD50 value for third instar larvae (IS)]. Larval and cuticle dry ,lseight. Both diflubenzuronand cyromazine-treated larvae weighed significantly less (P < 0.001, Student’s t test) than controls (Table 2). The cuticles of these larvae also weighed significantly less (P < 0.001) and yielded significantly less protein from isolated cuticles (P < 0.001) than the cuticles of controls (Table 2). When comparing diflubenzuron-treated with cyromazine-treated larvae, there was no significant difference between the larval dry weights of the two groups. However, the dry weights of the cuticles of the two larval groups differed significantly from each other (P < O.OOl), diflubenzurontreated larvae having the lighter cuticles. There was no difference between the amounts of protein extracted per cuticle. Protein electrophoresis. Although less total cuticular protein was extracted from diflubenzuron and cyromazine-treated larvae relative to controls, electrophoretic studies showed that the same types of major protein, in the same relative proportions, were extracted in all cases (Fig. 5). This result was verified when the proteins isolated from cuticles contaminated with muscle and epidermal tissue were examined by electrophoresis. Again the major cuticle proteins were discernible in all cases yet the amounts of these proteins were greatly reduced in the cuticle extracts of diflubenzuron and cyromazine-treated larvae (data not shown). Dihdtwfi~lc~te wduc,trrse irlhihilioll. Cyromazine inhibited dihydrofolate reductases from E. coli and chicken liver with inhibition constants of S.0 i I .O uM and 10.X 2 L. cuprina)

ET

AL.

TABLE

1

Incorporation of N-Acetyl-D[l-‘4C]R/u~~).~u~nine Chitin by Third Instar Lucilia cuprina Chemical (m&t) None (controls) Cyromazine 1.0 10.0 Diflubenzuron 10.0 100.0 Chlorfluazuron 0.4 4.0

Number surviving larvae

of

into Larvae”

Incorporation of N-acetyl-o[ I-‘4Cl glucosamine

SE

23

4.34

0.07

12 18

4.10 4.24

0.10 0.07

24 28

3.85 2.8

0.07 0.07

14 15

4.26 7.81

0.10 0.09

u N-Acetyl-o(l-14C) glucosamine was iqected mto larvae which were then placed on feeding medium containing chemicals at the concentrations shown. The chitin fraction of cuticle was then extracted and its radioactivity measured. The results are expressed as means of logarithms of the radioactivity counts.

I .7 FM, respectively. These inhibition constants are within the range previously found for other 4,6-diamino-s-triazines with enzymes from bacterial and vertebrate sources (16,17). Cyromazine differed from the triazine inhibitors of dihydrofolate reductase previously reported in that it showed a similar affinity for the bacterial and vertebrate enzymes. The majority of other triazines inhibit vertebrate enzymes more strongly (16.17). In this respect cyromazine is similar to pteridines such as methotrexate and aminopterin (IO). DISCUSSION

Previous findings (2) showed that in first and second instar larvae of L. crrprina exposed to cyromazine during growth, metamorphosis, and ecdysis, there were characteristic cuticular lesions: processes of epidermal cells appear to invade the cuticle and form necrotic areas. Similar, albeit less drastic. changes to the cuticle have now been documented for third instar larvae during the growth phase and in the absence of metamorphosis and ecdysis. This supports the findings of Price and Stubbs (18) who, using light microscopy, found that the

CYROMAZINE

EffcJcts of Lrrrlacid~d

Control (no treatment) Diflubenzuron ( IO0 mg/kg) Cyromazine ( IO mgikg)

Mean larval dry weight (mg i SD)

Mean cuticle dry weight (mg i SD)

Mean protein amount extracted per cuticle (mg i SD)

7.09

-c

0.78

1.31

2 0.22

0.31

5

4.85

k

0.90

0.37

k

0.06

0.08

2 0.01

4.58

-t

0.75

0.48

k

0.04

0.08

+ 0.02

of cuticular lesions in Mrrsca was not related to molting. Awad and Mulla (19). also using light microscopy, concluded that muscle insertions rather than cuticle were primarily affected in Muscn. However, in Lucilia the pattern of superficial cuticular lesions does not appear to be related to the pattern of muscle insertions (Binnington, unpublished data). As found previously (2) diflubenzuron does not produce localized necrotic lesions like those seen in cyromazine-treated larvae but has a generalized effect with the whole cuticle reduced in thickness and lacking a typical lamellate appearance. Diflubenzuron and another benzoyl phenylurea, chlorfluazuron, also differ from cyromazine in their ability to prevent incorporation of N-acetylglucosamine into the cuticle of feeding larvae; this supports the conclusions of Turnbull and Howells (4) for isolated integuments of L. cuprim. Inhibition of chitin synthesis by diflubenzuron is now well documented (20) although its precise mode of action is not yet understood. Both diflubenzuron and cyromazine caused a decrease in larval weight which exceeded the decrease in cuticle weight. The weight reduction caused by diflubenzuron here and in previous studies (21-24) could be a consequence of decreased chitin levels in the peritrophic membrane affecting food intake or digestive metabolism. Diflubenzuron-treated larvae also possess less 7 M urea-extractable cuticular protein than controls. An effect of benzoyl domestica

207

TABLE 2 Treatment on LUIUI D/y Weight, Cuticle Dry Weight, and Protein Amount E.utrac,ted from Isolated Cuticles with 7 M Urea

Larvacidal treatment

development

MODE OF ACTION

0.03

phenylureas on cuticular protein as well as chitin has been noted in previous studies [e.g., Post et nl. (25)] and other workers have reported that the endocuticle does not increase in thickness in diflubenzurontreated insects (26-28). In the present study on third instar L. clrprino larvae fed on diflubenzuron-treated mdium, there was little cuticle added to that already present in the unfed larva (cf. Figs. 1, 2). Thus it appears that not only is chitin synthesis inhibited by diflubenzuron but that protein synthesis or protein incorporation into the cuticle is similarly affected. It appears therefore that diflubenzuron either exerts an additional direct effect on cuticle protein

208

BINNINGTON

synthesis or that the effect on protein levels is a consequence of inhibition of chitin formation. However, not all results support this conclusion; e.g., Grosscurt (27) reported that in Leptinotcrrsrl decemlinentrr larvae, although diflubenzuron arrested chitin synthesis in the elytra, the cuticle still increased in thickness. He proposed that cuticle protein synthesis and deposition continues in the presence of diflubenzuron. Hunter and Vincent (29) reported no difference in the amount of protein extracted by 7 M urea from diflubenzurontreated and control adult locusts although, as expected, chitin production was inhibited in the treated locusts. Similarly, administration of diflubenzuron to last instar nymphs of Chrotogorllrs tr.trchypter.lrs or to larvae of Musccr domestica results in a reduction in cuticular chitin content but no decrease in protein (30,311. In these cases, it appears that protein deposition continues in diflubenzuron-treated insects and that the deposited proteins are stabilized even in the absence of chitin. This difference in results may reflect either a difference in how chitin and proteins are organized between the insects studied or a difference in cuticle preparation techniques. However. the possibility that it may merely reflect dosage effects should also be considered. It has been suggested that the globular material observed previously (26,321, and in the present study, in the endocuticle of diflubenzuron-affected cuticle may represent protein which has continued to be deposited. Perhaps proteins, secreted in the absence of chitin, associate with one another and cuticular lipids to form the observed globular structures. The dry weight reduction caused by cyromazine could be the result of an antifeedant effect since cyromazine is known to reduce water intake by adult L. cuprinct (15). Alternatively cyromazine may have a generalised toxic effect. However, the ability of cyromazine to produce cytopathic effects in the epidermis and cuticle but not in other tissues (Binnington, unpublished data)

ET

AL.

argues against such an hypothesis. Cuticle weight is decreased less by cyromazine than by diflubenzuron, a finding which conforms with ultrastructural effects of these chemicals. If protein secretion is inhibited by cyromazine, electrophoretic results indicate that the effect is quantitative rather than qualitative. One way in which protein secretion could be decreased is through an effect on nucleic acid metabolism. We have found that cyromazine inhibits bacterial and vertebrate dihydrofolate reductase. This finding differs from that of El-Oshar et rrl. (33). who concluded that cyromazine was not inhibitory against housefly or chicken liver dihydrofolate reductase. Certainly we do not find cyromazine to be as potent as the classical inhibitors of dihydrofolate reductase, methotrexate and aminopterin. However, the inhibition characteristics of cyromazine are closer to those of the classical inhibitors than other triazine inhibitors of dihydrofolate reductase (16,171. It has been known for many years that inhibitors of dihydrofolate reductase inhibit development of. and can be lethal to, larval dipterans (34-38). It is difficult to conceive, however, that an insecticide acting primarily through the inhibition of dihydrofolate reductase would not have drastic effects on egg production and hatchability. This is especially so since it is known that potent inhibitors of dihydrofolate reductase are able to sterilize female flies (39-40). When cyromazine is ingested by adult female L. cuprinu it reduces the number of eggs laid but does not affect hatchability of the eggs (15); apparently normal embryonic development takes place in an egg which contains sufficient cyromazine to cause subsequent death in larvae attempting to molt from the first to the second instar ( 1,151. Any hypothesis based on an interference with nucleic acid metabolism must invoke a differential susceptibility of epidermal cells compared with embryonic ones. In both cases there is a high rate of DNA replication but in the epidermal cells this is occurring by polyploid

CYROMAZINE

MODE

replication within polytene chromosomes (41’1 whereas in the embryo it is through mitotic division. There may also be an increased rate of production of RNA in the epidermal cells since there is a high level of cuticular chitin and protein synthesis during larval growth. This study has provided further evidence for different modes of action of cyromazine and diflubenzuron. Cuticle appears to be the primary target for both chemicals but only diflubenzuron inhibits chitin synthesis. The action of cyromazine remains unclear; the finding that it inhibits dihydrofolate reductase indicates that it may affect nucleic acid metabolism in some presently undefined way.

OF ACTION

11.

12.

13.

14.

15.

REFERENCES I. R. J. Hart. W. A. Cavey, K. J. Ryan, B. Moore, and M. B. Strong. Technical details of a new sheep blowfly insecticide, Wool Techno/. Sheep Breed. 21, 23 t 1979). 3. K. C. Binnington. Ultrastructural changes in the cuticle of the sheep blowfly. Llrcilicc. induced by certain insecticides and biological inhibitors. Ti.ts/re Crli 17, 131 (1985). 3. R. W. Miller. C. Corley. C. F. Cohen, W. E. Robbins, and E. P. Marks. CGA-19255 and CGA-72662: efficacy against flies and possible mode of action and metabolism. Sorrrirn,esr. Enrornol. 6. 273 (1981). 4. I. F. Turnbull and A. J. Howells, Effects of several larvicidal compounds on chitin biosynthesis by isolated larval integuments of the sheep blowfly L~cilicr c,lrpr-i/to. A/r.\!. /. Bid. Sf,i. 35, 491 (1982). 5. M. Brust and G. Fraenkel, The nutritional requirements of the larvae of a blowfly. Phorrnirr I’Cpinu (Meig.), PI~~siol. Zoo/. 2, 186 (1955). 6. A. Retnakaran and R. H. Hackman, Synthesis and deposition of chitin in larvae of the Australian sheep blowfly. Llrcilia c,rrpr-ins. Arch. INscat Bioc~hern. PIzy.sio/. 2, 35 I (19851. 7. R. L. Blakely. Crystalline dihydropteroylglutamic acid, Natrrre (Lottdon) 188, 23 I (19801. 8. P. J. Skelly and A. J. Howells, Larval cuticle proteins of L~cilirr clrprir~cr: electrophoretic separation, quantitation and developmental changes. lnsec~r Bioclwtn., in press. 9. 0. H. Lowry. N. J. Rosenbrough. A. L. Farr. and R. J. Randall, Protein measurement with the Folin phenol reagent, J. Bid. Cheeks. 193, 265 (1951). 10. S. R. Stone. J. A. Montgomery. and J. F. Mor-

rison, Inhibition of dihydrofolate reductase from bacterial and vertebrate sources by folate. aminopterin. methotrexate and their 5-deazoa analogues. Biochem. Pharrnacol. 33, 175 (1984). S. R. Stone and J. F. Morrison, Kinetic mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichirr cnli. Biochenzistr~ 16, 3757 (1982). A. Cornish-Bowden and L. Endrenyi, Fitting of enzyme kinetic data without prior knowledge of weights, Biochem. J. 193, 1005 (1981). B. K. Filshie, The fine structure and deposition of the larval cuticle of the sheep blowfly tL~i/icr cupr-ino), Tissrrv Ce// 2, 47!) (1970). G. W. Levot and E. Shipp, Reduction in offspring survival of Lwilia cuprina (Weidemann) following consumption of insect development inhibitors. J. Auf. Ent. Sm. 23, 85 (1984). T. Friedel and P. A. McDonell, Cyromazine: Its effect on reproduction and larval development in the sheep blowfly, Lucilicr crrprinu (Weid.), J. Econ. E~~romol. 78, 868 (1985). W. Dietrich. R. N. Smith, J. Y. Fukunaga, M. Olney. and C. Hansch. Dihydrofolate reductase inhibition by 2,4-diaminotriazines: A structure-activity study. Arch. Biocl7en7. Biopl1y.s. 194, 600 (1979). s W. Dietrich, R. N. Smith, S. Brendler and C. Hansch. The use of triazine inhibitors in mapping the active site region of Lrrc,toh~cillrr.v cusei dihydrofolate reductase. Arch. Biochen7. Biophys. 194, 612 (1979). N. R. Price and M. R. Stubbs. Some effects of CGA 72662 on larval development in the housefly, M~sc,u dome.sfictr (L.). 1171. J. Int,rrt. Rqwod. De\,. 7, I19 t 1984). T. 1. Awad and M. S. Mulla, Morphogenetic and histopathological effects induced by the insect growth regulator cyromazine in M~sccl dol?lestic.cr (Diptera: Muscidae), J. Med. E~fo~tw/. 21. 419 (1984). A. Retnakaran. J. Granett, and T. Ennis. Insect growth regulators. in “Comprehensive Insect Physiology, Biochemistry and Pharmacology” (G. A. Kerkut and K. I. Gilbert, Eds.). Vol. I?. p. 529. Pergamon Press, Oxford. L. Clarke, G. H. R. Temple, and J. F. V. Vincent. The effects of a chitin inhibitor-dimilin-on the production of peritrophic membrane in the locust. Locrlst~ /nigrutoric~. J. Insecr Ph~siol. 23, 241 (1977). T. Mitsui. C. Nobushawa. and J. Fukami. Mode of inhibition of chitin synthesis by diflubenzuron in the cabbage armyworm, Mnmr.sfrtr hrc~.s.\icrre L.. J. Pestic,. Sci. 9, I9 (1984). N. Soltani, Effects of ingested diflubenzuron on the longevity and the peritrophic membrane of

16. s

17.

18.

19.

20.

21.

22.

23.

209

210

24.

25.

26.

27.

28.

29.

30.

31.

BINNINGTON

adult mealworms (Terzehrio r,zo/itor. L. ). Pesti<,. Sri. 15, 211 (1984). B. Becker, Effects of 20.hydroxy-ecdysone. juvenile hormone. dimilin and captan on in vitro synthesis of peritrophic membranes in Ccr//iphoru eryvrhroccplrtrlrr. J. Insect Plr~sioi. 24, 699 (1978). L. C. Post, B. J. DeJong. and W. R. Vincent. I-(2.6-disubstituted benzoyl)-3-phenylurea insecticides: Inhibitors of chitin synthesis. Pestic. Biochetn. Plysiol. 4, 473 (1974). R. Mulder and M. J. Gijswijt. The laboratory evaluation of two promising new insecticides which interfere with cuticle deposition. J. Pe.\tic. Sci. 4, 737 (1973). A. C. Grosscurt. Diflubenzuron: some aspects of its ovicidal and larvicidal mode of action and an evaluation of its practical possibilities. J. Pestic,. Sc,i. 9, 373 ( 1978). N. Soltani, M. T. Besson. and .I. Delachambre. Effects of diflubenzuron on the pupal-adult development of Tctwhrio rrwlilor L. (Coleoptera. Tenebrionidae): growth and development, cuticle secretion. epidermal cell density and DNA synthesis. Pesfic,. Biochetn. Ph.vsiol. 21, 256 (1984). E. Hunter and J. F. V. Vincent. The effects of a novel insecticide on insect cuticle, Exprrietlticl 30, 1432 11974). S. C. Saxena and V. Kumar, Effects of difluron and penfluron on integumentary chitin. protein and lipid of CI~,-o~o.qor~rrs rruclypter-u.,\ (Orthoptera: Acrididael, indicttf J. Exp. Bid. 19. 669 (1981). I. Ishaaya and J. E. Casida. Dietary TH 60 40 alters composition and enzyme activity of housefly larval cuticle. Pastic. Bioc,hettf. Physiol. 4, 484 (1974).

ET AL.

31. R. F. Ker. Investigation of locust cuticle using the insecticide diflubenzuron, J. It~.~ert Physid. 23, 39 (1977). 33. M. A. El-Oshar. N. Motoyama, P. B. Hughes. W. C. Dauterman. Studies on cyromazine the housefly, M~sccl dorm~sfic~tr (Diptera: scidae). J. &on. Ento,n~~/. 78, 1203 (1985). 33.

and in Mu-

E. D. Goldsmith. M. H. Harnly, and E. B. Tobias. Folic acid analogs in lower animals. I. The insecta: Lhmophil~ rtdtrr~ogrrs~rr. Ant?. N. Y. Awd. .Sc,i. 52, 1342 (19.50).

35. N. Mitlin, M. S. Konecly. and P. G. Piquett. The effect of a folic acid antagonist on the house fly. .I. Econ. Et~iomol. 47, 932 (19541. 36.

G. C. LaBrecque, P. H. Adcock, Smith. Tests with compounds housefly metabolism. J. Ec,on. 802 (1960).

and C. N. affecting Etrrottwl. 53,

37. A. S. Perry and S. Miller. The essential role of folic acid and the effect of antimetabolite\ on growth and metamorphosis of housefly larvae Mo.sc~r~ dot?tP.stico L.. J. ltneci Ph,v.siol. 11. 1277 (1965). 38. S. Akov. Effect of folic acid antagonists on larval development and egg production in Ar~r1c.s ueqypfi. J. It~src,t Physid. 13, 913 (1967). 39. E. D. Goldsmith and 1. Frank, Sterility in the female fruit fly Dwwphiltr tnc/trtlo,~tr.slel.. produced by the feeding of a folic acid antagonist. Attlc1.. J. Plyviol. 171, 726 (1951). 40. M. M. Crystal. Insect fertility: Inhibition by folic acid derivatives. Sc.iarx~e 144, 308 ( 1964). 41, M. J. Pearson, The abdominal epidermis of Cal/iphorcl c,,lvrkroc,eplln/a (Diptera). I. Polyteny and growth in the larval cells. J. Cell Sci. 16, I I3 (1974).