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Effects of the chitin synthesis inhibitor diflubenzuron on development of Ascaris suum and Haemonchus contortus
Veterinary Parasitology, 32 (1989) 181-192 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
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E f f e c t s of the C h i t i n S y n t h e s i s I n h i b i t o r D i f l u b e n z u r o n on D e v e l o p m e n t o f A s c a r i s s u u m and Haemonchus contortus R.H. FETTERER, J.F. URBAN, JR. and R.W. MILLER
Helminthic Diseases Laboratory and Livestock Insects Laboratory, Livestock and Poultry Sciences Institute, Agricultural Research Service, U.S.D.A., BeltsviUe, MD 20705 (U.S.A.) (Accepted for publication 15 August 1988)
ABSTRACT Fetterer, R.H., Urban, J.F., Jr. and Miller, R.W., 1989. Effects of the chitin synthesis inhibitor diflubenzuron on development of Ascaris suum and Haemonchus contortus. Vet. Parasitol., 32: 181-192. The potential of the chitin synthesis inhibitor diflubenzuron (DFB) to alter the development of the parasitic nematodes (Ascaris suum and Haemonchus contortus was investigated. DFB given orally (10 mg kg -1 per day for 30 days) to sheep inoculated with H. contortus infective larvae did not prevent the establishment of adults or affect fecal egg output. However, there was a significant ( > 90% ) decrease in the number of infective larvae recovered from fecal cultures derived from lambs harboring H. contortus adults that were treated with DFB. DFB did not affect egg hatching. Oral administration (10 mg kg -1 per day for 20 days) of DFB to swine harboring adult A. suum adults had no effect on the adult worm burden or on egg morphology, but eggs removed from worms obtained from DFB-treated swine contained less chitin than eggs removed from untreated control swine. DFB also inhibited chitin synthesis in vitro in the isolated reproductive tract of A. suum adults. These results indicate that DFB at high doses can inhibit the subsequent development of H. contortus larvae in the feces. Since H. contortus larvae lack chitin, DFB may act on these larvae by a mechanism independent of a direct effect on chitin synthesis.
INTRODUCTION
Compounds of the benzoylphenylurea class such as diflubenzuron (DFB) are potent inhibitors of insect molting processes and are classified as insect growth regulators (IGR). Furthermore, ovicidal effects of DFB and other benzoylphenylureas have been reported in a number of insect orders including Coleoptera (Moore and Taft, 1975), Diptera (Wright and Harris, 1976) and Lepidoptera (Ascher and Nemny, 1974). Although the exact mechanism of action of benzoylphenylureas remains controversial, the general effect of these compounds is to prevent chitin synthesis and thus disrupt the normal devel0304-4017/89/$03.50
© 1989 Elsevier Science Publishers B.V.
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opment of insects (Deul et al., 1978; Hajjar and Casida, 1978). Chitin is an essential component of insect cuticle, but is not a constituent of nematode cuticle (Bird, 1971). However, chitin is an important component of the eggshell in many nematode species (Barrett, 1981). Although chitin has been thought to be of limited significance in nematode biology, previous studies on plant-parasitic and bacteria-feeding nematodes suggest that DFB and related compounds can inhibit egg laying and embryonation in these species. Relatively high concentrations (100 p.p.m.) of DFB, however, were required to demonstrate efficacy (Veech, 1978a,b; Townshend et al., 1983; Ibrahim, 1986). More recently, Waller and Lacey (1986) demonstrated that another chitin synthesis inhibitor of the benzoylphenylurea class, triflumuron, prevented the development of the infective larvae of Trichostrongylus colubriformis in the feces of sheep harboring a patent adult infection. It is unclear from the above studies whether there is a direct relationship between the inhibition of chitin synthesis and the inhibition of nematode development. Development and testing of compounds analogous to IGRs could lead to a desirable control strategy for livestock intestinal nematodes for at least two reasons: (1) the appearance of resistance to broad-spectrum anthelmintics has, in some regions, made conventional chemotherapy less effective and more expensive (Waller and Prichard, 1986); (2) compounds with IGR activity that also affect nematode development could simultaneously control both insect and nematode pests of livestock. With these practical benefits in mind, the effects of DFB on the in vivo and in vitro development of two major livestock parasitic nematodes, the large roundworm of swine, Ascaris suum, and the stomach worm of sheep, Haemonchus contortus, were examined. MATERIALS AND METHODS
Chemicals The compounds used in this study were obtained from the following sources: DFB (technical powder and 3.5% w/w feed pre-mix; Cyanamid Co., Princeton, N J) and parbendazole (Smith-Kline Animal Health, Westchester, PA). Unless otherwise noted, all other chemicals were of reagent grade and obtained from local sources. H. contortus injection of sheep
E[[ect o[ DFB on adult infections Polled Dorset weather lambs were raised helminth-free, except for minimal infections with Strongyloides papillosus, as determined by fecal examination (Whitlock, 1948) prior to the initiation of experiments. Ten helminth-free weather lambs (3 months of age, 17-23 kg in weight) were divided into groups
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of five animals each. Lambs were housed in individual raised wire pens, fed 2 pounds each per day of a standard maintenance ration and given water ad libitum. Both groups of sheep were inoculated with 10 000 H. contortus ( B P L strain) infective larvae. One group of lambs (treated group) was given a daily oral dose of DFB (10 mg kg -1 per day) in a gelatin capsule, starting 3 days before parasite inoculation and continuing through Day 27 post-inoculation (PI). The other group of lambs was not dosed, and served as the control group. Fecal egg counts were monitored from all lambs starting at 18 days PI. On Day 29 PI, fecal collections were made every 24 h on all lambs. Fecal egg counts (eggs g-1) were made on five random 2-g samples from each collection. The total egg output per 24 h was calculated by multiplying the average egg count (eggs g - l ) by the weight (g) of the fecal collection. On Day 31 PI, all lambs were necropsied and adult worms recovered from the abomasum. Worms from each lamb were counted and sexed. The egg output per female worm was obtained for each lamb by dividing the fecal egg output per 24 h by the number of female worms.
Effect of DFB on free-living stages of H. contortus In vivo assay. Helminth-free Polled Dorset or mixed-breed lambs (3 months of age ) harboring patent experimental infections of H. contortus ( B P L strain ) were given a daily dose of D F B at rates of either 1 or 10 mg kg-1 per day on Day 21 PI. The drug was given orally in a gelatin capsule. Fecal samples were collected daily during the first 7 days of D F B treatment and then on alternate days from 7 to 14 days. Standard 10-g fecal cultures were established simultaneously, from both treated and untreated infected lambs, and incubated for 7 days at 25 ° C. Third stage infective larvae were recovered from these cultures using a modified Baermann apparatus consisting of a glass funnel (25-cm diameter ) on top of which was placed a nylon screen (30-/~m mesh ). The number of larvae recovered from each culture was determined as the average number of larvae in triplicate 1-ml samples of the suspension X the suspension volume. In vitro assay. The effect of D F B on the development of H. contortus eggs to infective larvae in vitro was determined using a modified larval culture assay (Waller and Lacey, 1986). Briefly, a slurry of fresh sheep feces and water (20 g per 100 ml) was autoclaved, cooled and served as a nutrient suspension medium for assay. Eggs were isolated from feces obtained from lambs harboring patent adult H. contortus by flotation on a sucrose solution (150% w/v, specific gravity = 1.5). Eggs were added to the fecal slurry to give a concentration of 1000 eggs m1-1. The fecal slurry was divided into five replicate samples of 10 ml each and DFB and parbendazole (PBZ), a known inhibitor of nematode development, were dissolved separately in dimethylsulfoxide ( D M S O ) and
184 added to the slurry in a 100-#1 volume to give the desired final concentration (1, 2 and 10 ttg ml -~ for DFB; 0.1 pg m1-1 for P B Z ) ; D M S O alone (100 pl) was added to an equal number of samples and served as a control. Samples were agitated vigorously for 30 s and the entire sample poured into a 10-cm Petri dish lined with filter paper. Samples were covered and incubated in a humidified chamber for 7 days at 25 ° C. Larvae were recovered from cultures using a modified Baermann apparatus as described above. Egg hatch assay An egg hatch assay described by Donald et al. (1980) was used to determine the ovicidal activity of D F B on H. contortus eggs. Briefly, eggs were isolated from feces of infected lambs by flotation on sucrose. The eggs were suspended in Earle's balanced salt solution with 50 m M Tris, p H 7.0 ( E B S S ) and added to wells of a 24-well plastic tissue culture plate to give a concentration of 100200 eggs m1-1 E B S S in each well. Test compounds (DFB 10 #g ml-1; P B Z I pg m1-1 were dissolved in D M S O and added in 10-#l volumes to give the desired final concentration. D M S O only (10 pl) was added to control wells. At least six replicate wells per test compound or control were used. Plates were incubated at 25°C for 24 h and each well was examined microscopically (40 × magnification ). The hatching percentage was calculated as:
N u m b e r of first stage larvae observed/Total number of unhatched eggs and first stage larvae observed for each well)< 100 A. suum infection of swine In vivo experiments A group of 18 male and female crossbred (Yorkshire × Duroc ) pigs were born and maintained at our faculty, in pens in masonry buildings with concrete floors, under sanitary conditions that precluded helminth infection (confinement pigs). The pigs were randomly allocated to treatment groups and inoculated orally with 1000 infective A. suum eggs (Urban et al., 1981) for 5 successive days. Pigs carrying a patent adult infection were detected by examining the feces for eggs by standard flotation techniques from 6 to 8 weeks after inoculation. Ten of the 18 pigs that expressed a patent infection were housed individually, four of these remained untreated while six others were treated with D F B (10 mg kg-1 per day) by daily mixing of the drug with a small portion of dry feed. Pigs were not given additional ration until all of the drug-feed mixture was consumed. After 10 days of daily drug treatment, four pigs from the treated group and two from the untreated control group were killed, and adult A. suum were recovered from their intestine. U n e m b r y o n a t e d A. suum eggs were obtained from the uteri of adult female worms, freed from parasite tissues, examined microscopically and then embryonated (Urban et al., 1981 ).
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Eggs from five selected females derived from DFB-treated and five from untreated pigs were embryonated individually in 10-cm Petri dishes containing 20 ml of 0.1% formalin, at 25 °C for 28-32 days, with daily shaking of the dishes for aeration. Eggs from remaining females were embryonated as a pool derived from either DFB-treated or untreated pigs. The remaining pigs were killed after 21 days of drug treatment and processed similarly. Confinement pigs ( 10 weeks of age) were inoculated orally with aliquots of egg suspensions containing 10 000 second stage larvae derived either from DFB-treated or untreated pigs to test their infectivity; three pigs were used for each group. Third stage larvae were recovered from the lungs of pigs 7 days after inoculation and their numbers quantified (Urban and Romanowski, 1985). Chitin assay
The chitin content of eggs from A. s u u m was estimated by a modification of the method reported by Davidson (1966). Unembryonated A. s u u m eggs were obtained from the uteri of adult female worms from untreated control and DFB-treated pigs, and freed from parasite tissue (Urban et al., 1981). Eggs were extracted three times with chloroform:methanol (3:2, v/v), dried under vacuum and weighed. Dried material was resuspended in 4% perchloric acid (PCA) and centrifuged at 2000Xg for 10 min. The extraction with PCA was repeated three times. The acid-insoluble fraction was suspended in 30% KOH, heated at 100°C for 2 h and centrifuged at 5000Xg. The resulting pellet was suspended in 2 N HC1 and hydrolyzed overnight at 100 ° C. The hydrolyzed material was assayed for glucosamine by the method of Gatt and Berman (1966). The samples were then centrifuged, and the insoluble material was washed three times with water and assayed for chitin as described above. The results of the chitin assay were expressed as/tg glucosamine mg- 1 dry weight. Chitin s y n t h e s i s in vitro
Adult female A. s u u m obtained from experimentally infected pigs were maintained in artificial perienteric fluid (APF; Fleming and Fetterer, 1984) and were used for experiments within 1 day of collection. Chitin synthesis was determined by a procedure modified from a method used to measure chitin synthesis in insects (Mitsui et al., 1984). Female worms were dissected along the lateral line and the entire reproductive tract (RT) was removed. The RT was placed in tissue culture flasks containing 10 ml APF supplemented with 10% (v/v) fetal bovine serum. DFB was dissolved in DMSO and added in 100/~l volumes to give a final concentration of 10/~g m l - 1. Control flasks contained 100/11 DMSO (1% final concentration). All flasks contained 3H-glucosamine ( 100/tCi per flask; 10 Ci mmol- 1, New England Nuclear, Boston, MA ). Flasks were gassed with nitrogen and incubated in a shaking water bath (50 rev. rain -1) at 37°C for 18 h. Tissues were subsequently removed from flasks, washed with water, homogenized, and extracted three times with chloro-
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form:methanol (3:2 v/v). Extracted samples were dried under vacuum, weighed, resuspended in 30% (w/v) KOH, heated for 2 h at 100°C and centrifuged at 2000×g for 25 min. The resulting pellet was washed three times with water and then treated with tissue solubilizer (NCS, Amersham, Arington Heights, IL) for 1 h at 50 ° C followed by neutralization with acetic acid. Radioactivity of the samples was determined by liquid scintillation counting and all assays were performed in triplicate. Statistical procedures All values are expressed as means with one standard error. Statistical comparisons (P < 0.5) between treatment and control were made using non-parametric rank sum procedures (Hollander and Wolfe, 1973). RESULTS
H. contortus studies DFB effects on adult infections Daily oral treatment of lambs with DFB pre-mix did not prevent the establishment of patent adult H. contortus infections following an experimental inoculation (Table 1). No differences in worm number, male-female ratio, fecal egg counts (eggs g-1 ), total fecal output (g feces per 24 h) or egg output (eggs per female per 24 h) were observed. The small size of treatment groups, however, may have contributed to the lack of statistical differences between the two groups. TABLE1 The effect of daily oral treatment with DFB on the establishment of H. c o n t o r t u s infections in experimentally inoculated lambs
Adult worms Male-female ratio Eggs g-~ Eggs per female per 24 h Fecalweight (g fecesper 24 h)
Control untreated lambs
DFB treated lambs
1277 0.81 4726 4351 595
851 0.74 4854 9622 915
(293) (0.04) (698) (699) (120)
(275) (0.04) (1418) (2599) (43)
All values are means. Numbers in parentheses indicate one standard error; n = 5. DFB was given in a pre-mix formulation at a rate of 10 mg kg- ~per day starting 3 days before inoculation through 27 days following inoculation with 10 000 H, c o n t o r t u s infective larvae.
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DFB effects on free-living stages In vivo assay. Administration of DFB (10 mg kg -1 per day) to lambs with patent adult H. contortus infections prevented the development of eggs to infective larvae in fecal culture (Fig. 1 ). Significant inhibition of larval development was noted for 2-3 days after withdrawal of DFB. In vitro assay. The development of H. contortus eggs to infective larvae was partially inhibited by the addition of DFB to the fecal cultures (Fig. 2 ). The
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Fig. 1. The effect of in vivo treatment of lambs harboring patent H. contortus infections with DFB on the subsequent larval development in fecal cultures. DFB, either pure compound or in pre-mix formulation (PM), was administered daily during the periods indicated between arrows (7 days ). All values are means and the sample size is indicated in parentheses. Percent development is defined as: (number of larvae/number of eggs) × 100.
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Fig. 3. The chitin content of A. s u u m eggs. The A. s u u m eggs were from worms recovered from untreated infected pigs (control) and from worms recovered from DFB -treated infected pigs (DFB). Chitin content is expressed as #g glucosamine m g - 1 dry weight released upon acid hydrolysis of a partially purified chitin fraction. Values are means with a minimum sample size of 7. Vertical bars represent one standard error. The chitin content of H. c o n t o r t u s eggs obtained from infected untreated sheep is shown for comparison.
maximum inhibition (64%) was observed at a dose of 10 #g ml-1, while a dose 2 pg m1-1 caused 41% inhibition of development. PBZ, a known inhibitor of nematode development, caused a 98% inhibition of development at a concentration of 0.1 #g m1-1 (data not shown). D F B (10/~g m 1-1) did not significantly inhibit in vitro egg hatching (87.6%+1.4) compared to control (80.6% + 1.1 ). A. suum studies D F B effects on p a t e n t adult infections A. s u u m eggs passed in the feces of pigs treated for 10 or 21 days with D F B
were not morphologically different from eggs passed from untreated, infected pigs. There was also no apparent difference in the rate or degree of development of fertilized eggs to infective eggs in cultures derived from either individual females or a pool of female worms obtained from D F B - t r e a t e d or control pigs. Three pigs, which were inoculated with a pool of 10 000 infective eggs derived from worms obtained from a pool of untreated pigs, had an average of 2247 + 516 third stage larvae in their lungs 7 days after inoculation; three pigs, which were similarly inoculated with a pool of eggs derived from worms obtained from DFB-treated pigs, had an average of 2017 + 105 third stage larvae in their lungs. Chitin content A. s u u m eggs derived from female worms recovered from DFB-treated in-
fected pigs had a lower chitin content (18 pg m g - 1 dry weight) than eggs from female worms obtained from untreated pigs (43 #g mg -1 dry weight) (Fig. 3 ).
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Chitin synthesis in vitro
Chitin synthesis, as estimated by 3H-glucosamine incorporation into the alkaline-insoluble pellet from the isolated RT of female A. suum, was inhibited by 47% in the presence of 10 pg m1-1DFB (226 d.p.m, mg -1 dry weight +49; n-- 3 ) compared with DMSO-treated controls (424 d.p.m, m g - 1 dry weight +61; n = 3 ) . DISCUSSION
The current results demonstrate that DFB prevented the development in fecal cultures of the free-living stages of the sheep parasitic nematode H. contortus when administered to lambs in vivo at 10 mg day -1 for 30 days. This observation is consistent with previous findings of DFB effects on free-living and plant-parasitic nematodes (Veech, 1978a,b; Townshend et al., 1983). Since the administration of DFB in vivo did not alter the normal development of a patent infection in lambs and DFB does not prevent in vitro hatching of eggs, the action of DFB seems to be restricted to the free-living stages of H. contortus. Chitin in nematodes is found only in eggs and not in the larval stages (Bird, 1971; R.H. Fetterer, unpublished observations, 1988), therefore, the larvacidal effect of DFB is apparently not directly related to chitin synthesis. Chitin is found in measurable amounts in normal H. contortus eggs, with a glucosamine content of 7.3 pg mg-1 dry weight, but DFB did not affect egglaying by females in vivo. This suggests that either DFB does not affect chitin synthesis in H. contortus or that DFB is altered or fails to reach these parasites during in vivo treatment. It is possible, however, that a partial inhibition of egg laying did occur, but was not detected because of the small sample size used in these experiments. A detailed biochemical examination of the action of DFB on chitin synthesis in H. contortus is required to determine the exact mode of action of DFB. Complete inhibition of H. contortus larval development in fecal cultures was noted with eggs from lambs treated with DFB at a dose of 10 mg kg- 1day. The addition of DFB directly to fecal cultures, however, caused only a 64% inhibition of development at the highest dose used (10 pg m l - 1). Assuming a mean lamb weight of 20 kg, a mean fecal output of 915 g per day (Table 1 ) and little absorption of DFB, then an in vivo dose of 10 mg kg-1 DFB per day would translate to a DFB fecal concentration of > 200 p.p.m., which is more than 10fold greater than the highest dose used to demonstrate the inhibition of larval development in in vitro cultures. This suggests that the potency of DFB observed with in vivo treatment of lambs was due, at least in part, to high levels of DFB carried in the feces following a daily dose of 10 mg kg-1. It is also possible that DFB is either metabolized, concentrated or otherwise modified by passage through the sheep intestinal tract, making it functionally more effective against subsequent larval development. Since a relatively high in vivo
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treatment dose ( > 100-fold) is required to inhibit H. contortus larval development (10 mg kg -1 per day) compared to that used for the control of insect larvae in cattle feces (0.06 mg kg -1 per day; Miller and Miller, 1985), DFB appears to be of little practical value for the control of H. contortus larvae. In contrast to the present study, Waller and Lacey (1986) observed that a DFB analog, triflumuron ( T F M ) , was quite effective in vivo and in vitro in preventing the development of Trichostrongylus colubriformis. However, T F M was less effective in preventing the development of H. contortus and Ostertagia circumcincta. Differences in species susceptibility are considered to be characteristic of growth regulatory activity in insects and such a phenomenon may similarly occur in nematodes. Compounds such as DFB are potent inhibitors of chitin synthesis in arthropods. Since A. suum eggshell contains significant amounts of chitin, it seemed plausible that t r e a t m e n t ofA. suum-infected pigs should result in an alteration of egg production in normal infection. Supporting this hypothesis is the observation that t r e a t m e n t of Caenorhabditis elegans with DFB caused partial disintegration of the chitinous layer of the eggshell and marked alteration of normal egg morphology (Ibrahim, 1986). There is evidence in the present study that DFB interacted with A. suum in vivo, since the chitin content of eggs from worms obtained from DFB-treated pigs was lower t h a n from eggs from worms obtained from untreated pigs. Eggs exposed to DFB in vivo, however, were morphologically identical to eggs from worms derived from untreated pigs both before and after embryonation and they were equally capable of migrating to the lungs of pigs following experimental inoculation. It appears, therefore, that the observed reduction in egg chitin was not detrimental to the egg and larval infectivity. More severe natural or environmental stress, however, could have resulted in higher egg mortality than might be encountered under the laboratory conditions constructed in these experiments. Chitin synthesis in the isolated reproductive tissue ofA. suum was inhibited by DFB demonstrating that: (1) chitin synthesis in nematodes is a potential target for an inhibitor; (2) chitin synthesis may be biochemically similar in insects and nematodes. A previous study (Fuhrman and Piessens, 1985 ) demonstrated that in vitro chitin synthesis in the Filarid nematode Brugia malayi was partially inhibited by DFB and that the microfilaria (MF) produced by the females in vitro were malformed. This indicated a physiological effect of DFB on nematodes related to the inhibition of chitin synthesis. However, since no in vivo studies with DFB were performed by F u h r m a n and Piessens, it is not known if malformed MF would develop in a normal m a n n e r in vivo or if DFB would affect female production of MF within the host. It is now clear that compounds with IGR activity may be potential candidates for inhibitors of free-living stages of parasitic nematodes. The observation that DFB is inhibitory to larval development only at a high dose may, in part, reflect the fact
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that DFB and other IGRs have been developed by selection for activity on insects. Examination of other growth regulators may reveal compounds with enhanced efficacy against nematodes. The investigation of IGRs as possible nematode control agents remains an unexplored and potentially fruitful area for additional research. ACKNOWLEDGEMENTS
We are indebted to J. Corbin and A.W. Jones for their expert technical assistance. We also thank Dr. P.C. Allen and Dr. R.S. Rew for critical reading of this manuscript. Mention of a trade name, proprietary product or specific equipment does not constitute a guarantee of warranty by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may be suitable. REFERENCES Ascher, K.R.S. and Nemny, N.E., 1974. The ovicidal effect of PH60-4-1 (4-chlorophenyl)-3- (2,6difluorobenzolyl)-ureain Spodopterea littoralis Boisd. Phytoparasitica, 2: 131-133. Barrett, J., 1981. Biochemistry of Parasitic Helminths. University Park Press, Baltimore, MD, pp. 13-71. Bird, A.F., 1971. The Structure of Nematodes. Academic Press, New York, pp. 45-77. Davidson, E.A., 1966. Glucosamine-6-phosphate N-acetylase. Methods Enzymol., 9: 704-707. Deul, K.H., deJong, B.J. and Kortenbach, J.A.M., 1978. Inhibition of chitin synthesis by two l- (2,6, disubstituted benzoyl) -3-phenylurea insecticides. Pestic. Biochem. Physiol., 8: 98-105. Donald, A.D., Waller, P.J., Dobson, R.J. and Axelson, A., 1980. The effect of selection with levamisole on benzimidzole resistance in Ostertagia spp., in sheep. Int. J. Parasitol., 10: 381-389. Fleming, M.W. and Fetterer, R.H., 1984. Ascaris suum: continuous perfusion of the pseudocoelon and nutrient absorbtion. Exp. Parasitol., 57: 142-148. Fuhrman, J.A. and Piessens, W.F., 1985. Chitin synthesis and sheath morphogenesis in Brugia malayi microfilariae. Mol. Biochem. Parasitol., 17: 93-104. Gatt, R. and Berman, E.R., 1966. A rapid method for estimation of amino sugars on a micro scale. Anal. Biochem., 15: 167-171. Hajjar, N.P. and Casida, J.E., 1978. Insecticidal benzoylphenylureas: structure-activity relationships as chitin synthesis inhibitors. Science, 200: 1499-1500. Hollander, M. and Wolfe, D.A., 1973. Nonparametric Statistical Methods. Wiley, New York, pp. 26-48. Ibrahim, A.M., 1986. Studies on ovicidal activity of diflubenzuron on Caenorhabditis elegans. Dirasat., 13: 139-147. Miller, R.W. and Miller, J.A., 1985. Feed-through chemicals for insect control in animals. In: J.L. Hilfod (Editor), Agricultural Chemicals of the Future. BARC Symposium 8, pp. 355-365. Mitsui, T., Nobousawa, T. and Fukami, J., 1984. Mode of inhibition of chitin synthesis by diflubenzuron in the cabbage armyworm, Manestra brassicae L. J. Pestic. Sci., 9: 19-26. Moore, R.F., Jr. and Taft, H.M., 1975. Boll weevils: chemosterilization of both sexes with bisulfar plus Thompson-Hayward JH 6040. J. Econ. Entomol., 68: 96-98. Townshend, J.L., Pree, D.J. and Broadbend, A.B., 1983. Population development and reproduction of fungus and bacterium feeding nematodes in the presence of growth regulators. J. Nematol., 15: 105-110.
192 Urban, J.F., Jr. and Romanowski, R.D., 1985. Ascaris s u u m : protective immunity in pigs immunized with products from eggs and larvae. Exp. Parasitol., 60: 245-254. Urban, J.F., Jr., Douvres, F.W. and Tromba, F.G., 1981. A rapid method for hatching of Ascaris suum" eggs in vitro-Proc. Helminthol. Soc. Wasl., 48: 241-243. Veech, J.A., 1978a. The effect of diflubenzuron on egg formation of root knot nematodes. J. Nematol., 9: 184-185. Veech, J.A., 1978b. The effect of diflubenzuron on the reproduction of free-living nematodes. Nematologica, 24: 312-330. Waller, P.J. and Lacey, E., 1986. The effect of triflumuron (SIR 8514) on the free-living stages of sheep nematodes. Vet. Parasitol., 21" 119-126. Waller, P.J. and Prichard, R.H., 1986. Drug resistance in nematodes. In: W.C. Campbell and R.S. Rew (Editors), Chemotherapy of Parasitic Infections. Plenum, New York, pp. 330-362. Whitlock, H.V., 1948. Some modifications of the McMaster helminth egg-counting techniques and apparatus. J. Counc. Sci. Ind. Res. Aust., 21: 177-180. Wright, J.E. and Harris, R.L., 1976. Ovicidal activity of Thompson-Hayward TH 6040 in the stable fly and horn fly after surface contact by adults. J. Econ. Entomol., 69: 728-730.
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E f f e c t s of the C h i t i n S y n t h e s i s I n h i b i t o r D i f l u b e n z u r o n on D e v e l o p m e n t o f A s c a r i s s u u m and Haemonchus contortus R.H. FETTERER, J.F. URBAN, JR. and R.W. MILLER
Helminthic Diseases Laboratory and Livestock Insects Laboratory, Livestock and Poultry Sciences Institute, Agricultural Research Service, U.S.D.A., BeltsviUe, MD 20705 (U.S.A.) (Accepted for publication 15 August 1988)
ABSTRACT Fetterer, R.H., Urban, J.F., Jr. and Miller, R.W., 1989. Effects of the chitin synthesis inhibitor diflubenzuron on development of Ascaris suum and Haemonchus contortus. Vet. Parasitol., 32: 181-192. The potential of the chitin synthesis inhibitor diflubenzuron (DFB) to alter the development of the parasitic nematodes (Ascaris suum and Haemonchus contortus was investigated. DFB given orally (10 mg kg -1 per day for 30 days) to sheep inoculated with H. contortus infective larvae did not prevent the establishment of adults or affect fecal egg output. However, there was a significant ( > 90% ) decrease in the number of infective larvae recovered from fecal cultures derived from lambs harboring H. contortus adults that were treated with DFB. DFB did not affect egg hatching. Oral administration (10 mg kg -1 per day for 20 days) of DFB to swine harboring adult A. suum adults had no effect on the adult worm burden or on egg morphology, but eggs removed from worms obtained from DFB-treated swine contained less chitin than eggs removed from untreated control swine. DFB also inhibited chitin synthesis in vitro in the isolated reproductive tract of A. suum adults. These results indicate that DFB at high doses can inhibit the subsequent development of H. contortus larvae in the feces. Since H. contortus larvae lack chitin, DFB may act on these larvae by a mechanism independent of a direct effect on chitin synthesis.
INTRODUCTION
Compounds of the benzoylphenylurea class such as diflubenzuron (DFB) are potent inhibitors of insect molting processes and are classified as insect growth regulators (IGR). Furthermore, ovicidal effects of DFB and other benzoylphenylureas have been reported in a number of insect orders including Coleoptera (Moore and Taft, 1975), Diptera (Wright and Harris, 1976) and Lepidoptera (Ascher and Nemny, 1974). Although the exact mechanism of action of benzoylphenylureas remains controversial, the general effect of these compounds is to prevent chitin synthesis and thus disrupt the normal devel0304-4017/89/$03.50
© 1989 Elsevier Science Publishers B.V.
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opment of insects (Deul et al., 1978; Hajjar and Casida, 1978). Chitin is an essential component of insect cuticle, but is not a constituent of nematode cuticle (Bird, 1971). However, chitin is an important component of the eggshell in many nematode species (Barrett, 1981). Although chitin has been thought to be of limited significance in nematode biology, previous studies on plant-parasitic and bacteria-feeding nematodes suggest that DFB and related compounds can inhibit egg laying and embryonation in these species. Relatively high concentrations (100 p.p.m.) of DFB, however, were required to demonstrate efficacy (Veech, 1978a,b; Townshend et al., 1983; Ibrahim, 1986). More recently, Waller and Lacey (1986) demonstrated that another chitin synthesis inhibitor of the benzoylphenylurea class, triflumuron, prevented the development of the infective larvae of Trichostrongylus colubriformis in the feces of sheep harboring a patent adult infection. It is unclear from the above studies whether there is a direct relationship between the inhibition of chitin synthesis and the inhibition of nematode development. Development and testing of compounds analogous to IGRs could lead to a desirable control strategy for livestock intestinal nematodes for at least two reasons: (1) the appearance of resistance to broad-spectrum anthelmintics has, in some regions, made conventional chemotherapy less effective and more expensive (Waller and Prichard, 1986); (2) compounds with IGR activity that also affect nematode development could simultaneously control both insect and nematode pests of livestock. With these practical benefits in mind, the effects of DFB on the in vivo and in vitro development of two major livestock parasitic nematodes, the large roundworm of swine, Ascaris suum, and the stomach worm of sheep, Haemonchus contortus, were examined. MATERIALS AND METHODS
Chemicals The compounds used in this study were obtained from the following sources: DFB (technical powder and 3.5% w/w feed pre-mix; Cyanamid Co., Princeton, N J) and parbendazole (Smith-Kline Animal Health, Westchester, PA). Unless otherwise noted, all other chemicals were of reagent grade and obtained from local sources. H. contortus injection of sheep
E[[ect o[ DFB on adult infections Polled Dorset weather lambs were raised helminth-free, except for minimal infections with Strongyloides papillosus, as determined by fecal examination (Whitlock, 1948) prior to the initiation of experiments. Ten helminth-free weather lambs (3 months of age, 17-23 kg in weight) were divided into groups
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of five animals each. Lambs were housed in individual raised wire pens, fed 2 pounds each per day of a standard maintenance ration and given water ad libitum. Both groups of sheep were inoculated with 10 000 H. contortus ( B P L strain) infective larvae. One group of lambs (treated group) was given a daily oral dose of DFB (10 mg kg -1 per day) in a gelatin capsule, starting 3 days before parasite inoculation and continuing through Day 27 post-inoculation (PI). The other group of lambs was not dosed, and served as the control group. Fecal egg counts were monitored from all lambs starting at 18 days PI. On Day 29 PI, fecal collections were made every 24 h on all lambs. Fecal egg counts (eggs g-1) were made on five random 2-g samples from each collection. The total egg output per 24 h was calculated by multiplying the average egg count (eggs g - l ) by the weight (g) of the fecal collection. On Day 31 PI, all lambs were necropsied and adult worms recovered from the abomasum. Worms from each lamb were counted and sexed. The egg output per female worm was obtained for each lamb by dividing the fecal egg output per 24 h by the number of female worms.
Effect of DFB on free-living stages of H. contortus In vivo assay. Helminth-free Polled Dorset or mixed-breed lambs (3 months of age ) harboring patent experimental infections of H. contortus ( B P L strain ) were given a daily dose of D F B at rates of either 1 or 10 mg kg-1 per day on Day 21 PI. The drug was given orally in a gelatin capsule. Fecal samples were collected daily during the first 7 days of D F B treatment and then on alternate days from 7 to 14 days. Standard 10-g fecal cultures were established simultaneously, from both treated and untreated infected lambs, and incubated for 7 days at 25 ° C. Third stage infective larvae were recovered from these cultures using a modified Baermann apparatus consisting of a glass funnel (25-cm diameter ) on top of which was placed a nylon screen (30-/~m mesh ). The number of larvae recovered from each culture was determined as the average number of larvae in triplicate 1-ml samples of the suspension X the suspension volume. In vitro assay. The effect of D F B on the development of H. contortus eggs to infective larvae in vitro was determined using a modified larval culture assay (Waller and Lacey, 1986). Briefly, a slurry of fresh sheep feces and water (20 g per 100 ml) was autoclaved, cooled and served as a nutrient suspension medium for assay. Eggs were isolated from feces obtained from lambs harboring patent adult H. contortus by flotation on a sucrose solution (150% w/v, specific gravity = 1.5). Eggs were added to the fecal slurry to give a concentration of 1000 eggs m1-1. The fecal slurry was divided into five replicate samples of 10 ml each and DFB and parbendazole (PBZ), a known inhibitor of nematode development, were dissolved separately in dimethylsulfoxide ( D M S O ) and
184 added to the slurry in a 100-#1 volume to give the desired final concentration (1, 2 and 10 ttg ml -~ for DFB; 0.1 pg m1-1 for P B Z ) ; D M S O alone (100 pl) was added to an equal number of samples and served as a control. Samples were agitated vigorously for 30 s and the entire sample poured into a 10-cm Petri dish lined with filter paper. Samples were covered and incubated in a humidified chamber for 7 days at 25 ° C. Larvae were recovered from cultures using a modified Baermann apparatus as described above. Egg hatch assay An egg hatch assay described by Donald et al. (1980) was used to determine the ovicidal activity of D F B on H. contortus eggs. Briefly, eggs were isolated from feces of infected lambs by flotation on sucrose. The eggs were suspended in Earle's balanced salt solution with 50 m M Tris, p H 7.0 ( E B S S ) and added to wells of a 24-well plastic tissue culture plate to give a concentration of 100200 eggs m1-1 E B S S in each well. Test compounds (DFB 10 #g ml-1; P B Z I pg m1-1 were dissolved in D M S O and added in 10-#l volumes to give the desired final concentration. D M S O only (10 pl) was added to control wells. At least six replicate wells per test compound or control were used. Plates were incubated at 25°C for 24 h and each well was examined microscopically (40 × magnification ). The hatching percentage was calculated as:
N u m b e r of first stage larvae observed/Total number of unhatched eggs and first stage larvae observed for each well)< 100 A. suum infection of swine In vivo experiments A group of 18 male and female crossbred (Yorkshire × Duroc ) pigs were born and maintained at our faculty, in pens in masonry buildings with concrete floors, under sanitary conditions that precluded helminth infection (confinement pigs). The pigs were randomly allocated to treatment groups and inoculated orally with 1000 infective A. suum eggs (Urban et al., 1981) for 5 successive days. Pigs carrying a patent adult infection were detected by examining the feces for eggs by standard flotation techniques from 6 to 8 weeks after inoculation. Ten of the 18 pigs that expressed a patent infection were housed individually, four of these remained untreated while six others were treated with D F B (10 mg kg-1 per day) by daily mixing of the drug with a small portion of dry feed. Pigs were not given additional ration until all of the drug-feed mixture was consumed. After 10 days of daily drug treatment, four pigs from the treated group and two from the untreated control group were killed, and adult A. suum were recovered from their intestine. U n e m b r y o n a t e d A. suum eggs were obtained from the uteri of adult female worms, freed from parasite tissues, examined microscopically and then embryonated (Urban et al., 1981 ).
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Eggs from five selected females derived from DFB-treated and five from untreated pigs were embryonated individually in 10-cm Petri dishes containing 20 ml of 0.1% formalin, at 25 °C for 28-32 days, with daily shaking of the dishes for aeration. Eggs from remaining females were embryonated as a pool derived from either DFB-treated or untreated pigs. The remaining pigs were killed after 21 days of drug treatment and processed similarly. Confinement pigs ( 10 weeks of age) were inoculated orally with aliquots of egg suspensions containing 10 000 second stage larvae derived either from DFB-treated or untreated pigs to test their infectivity; three pigs were used for each group. Third stage larvae were recovered from the lungs of pigs 7 days after inoculation and their numbers quantified (Urban and Romanowski, 1985). Chitin assay
The chitin content of eggs from A. s u u m was estimated by a modification of the method reported by Davidson (1966). Unembryonated A. s u u m eggs were obtained from the uteri of adult female worms from untreated control and DFB-treated pigs, and freed from parasite tissue (Urban et al., 1981). Eggs were extracted three times with chloroform:methanol (3:2, v/v), dried under vacuum and weighed. Dried material was resuspended in 4% perchloric acid (PCA) and centrifuged at 2000Xg for 10 min. The extraction with PCA was repeated three times. The acid-insoluble fraction was suspended in 30% KOH, heated at 100°C for 2 h and centrifuged at 5000Xg. The resulting pellet was suspended in 2 N HC1 and hydrolyzed overnight at 100 ° C. The hydrolyzed material was assayed for glucosamine by the method of Gatt and Berman (1966). The samples were then centrifuged, and the insoluble material was washed three times with water and assayed for chitin as described above. The results of the chitin assay were expressed as/tg glucosamine mg- 1 dry weight. Chitin s y n t h e s i s in vitro
Adult female A. s u u m obtained from experimentally infected pigs were maintained in artificial perienteric fluid (APF; Fleming and Fetterer, 1984) and were used for experiments within 1 day of collection. Chitin synthesis was determined by a procedure modified from a method used to measure chitin synthesis in insects (Mitsui et al., 1984). Female worms were dissected along the lateral line and the entire reproductive tract (RT) was removed. The RT was placed in tissue culture flasks containing 10 ml APF supplemented with 10% (v/v) fetal bovine serum. DFB was dissolved in DMSO and added in 100/~l volumes to give a final concentration of 10/~g m l - 1. Control flasks contained 100/11 DMSO (1% final concentration). All flasks contained 3H-glucosamine ( 100/tCi per flask; 10 Ci mmol- 1, New England Nuclear, Boston, MA ). Flasks were gassed with nitrogen and incubated in a shaking water bath (50 rev. rain -1) at 37°C for 18 h. Tissues were subsequently removed from flasks, washed with water, homogenized, and extracted three times with chloro-
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form:methanol (3:2 v/v). Extracted samples were dried under vacuum, weighed, resuspended in 30% (w/v) KOH, heated for 2 h at 100°C and centrifuged at 2000×g for 25 min. The resulting pellet was washed three times with water and then treated with tissue solubilizer (NCS, Amersham, Arington Heights, IL) for 1 h at 50 ° C followed by neutralization with acetic acid. Radioactivity of the samples was determined by liquid scintillation counting and all assays were performed in triplicate. Statistical procedures All values are expressed as means with one standard error. Statistical comparisons (P < 0.5) between treatment and control were made using non-parametric rank sum procedures (Hollander and Wolfe, 1973). RESULTS
H. contortus studies DFB effects on adult infections Daily oral treatment of lambs with DFB pre-mix did not prevent the establishment of patent adult H. contortus infections following an experimental inoculation (Table 1). No differences in worm number, male-female ratio, fecal egg counts (eggs g-1 ), total fecal output (g feces per 24 h) or egg output (eggs per female per 24 h) were observed. The small size of treatment groups, however, may have contributed to the lack of statistical differences between the two groups. TABLE1 The effect of daily oral treatment with DFB on the establishment of H. c o n t o r t u s infections in experimentally inoculated lambs
Adult worms Male-female ratio Eggs g-~ Eggs per female per 24 h Fecalweight (g fecesper 24 h)
Control untreated lambs
DFB treated lambs
1277 0.81 4726 4351 595
851 0.74 4854 9622 915
(293) (0.04) (698) (699) (120)
(275) (0.04) (1418) (2599) (43)
All values are means. Numbers in parentheses indicate one standard error; n = 5. DFB was given in a pre-mix formulation at a rate of 10 mg kg- ~per day starting 3 days before inoculation through 27 days following inoculation with 10 000 H, c o n t o r t u s infective larvae.
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DFB effects on free-living stages In vivo assay. Administration of DFB (10 mg kg -1 per day) to lambs with patent adult H. contortus infections prevented the development of eggs to infective larvae in fecal culture (Fig. 1 ). Significant inhibition of larval development was noted for 2-3 days after withdrawal of DFB. In vitro assay. The development of H. contortus eggs to infective larvae was partially inhibited by the addition of DFB to the fecal cultures (Fig. 2 ). The
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Fig. 3. The chitin content of A. s u u m eggs. The A. s u u m eggs were from worms recovered from untreated infected pigs (control) and from worms recovered from DFB -treated infected pigs (DFB). Chitin content is expressed as #g glucosamine m g - 1 dry weight released upon acid hydrolysis of a partially purified chitin fraction. Values are means with a minimum sample size of 7. Vertical bars represent one standard error. The chitin content of H. c o n t o r t u s eggs obtained from infected untreated sheep is shown for comparison.
maximum inhibition (64%) was observed at a dose of 10 #g ml-1, while a dose 2 pg m1-1 caused 41% inhibition of development. PBZ, a known inhibitor of nematode development, caused a 98% inhibition of development at a concentration of 0.1 #g m1-1 (data not shown). D F B (10/~g m 1-1) did not significantly inhibit in vitro egg hatching (87.6%+1.4) compared to control (80.6% + 1.1 ). A. suum studies D F B effects on p a t e n t adult infections A. s u u m eggs passed in the feces of pigs treated for 10 or 21 days with D F B
were not morphologically different from eggs passed from untreated, infected pigs. There was also no apparent difference in the rate or degree of development of fertilized eggs to infective eggs in cultures derived from either individual females or a pool of female worms obtained from D F B - t r e a t e d or control pigs. Three pigs, which were inoculated with a pool of 10 000 infective eggs derived from worms obtained from a pool of untreated pigs, had an average of 2247 + 516 third stage larvae in their lungs 7 days after inoculation; three pigs, which were similarly inoculated with a pool of eggs derived from worms obtained from DFB-treated pigs, had an average of 2017 + 105 third stage larvae in their lungs. Chitin content A. s u u m eggs derived from female worms recovered from DFB-treated in-
fected pigs had a lower chitin content (18 pg m g - 1 dry weight) than eggs from female worms obtained from untreated pigs (43 #g mg -1 dry weight) (Fig. 3 ).
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Chitin synthesis in vitro
Chitin synthesis, as estimated by 3H-glucosamine incorporation into the alkaline-insoluble pellet from the isolated RT of female A. suum, was inhibited by 47% in the presence of 10 pg m1-1DFB (226 d.p.m, mg -1 dry weight +49; n-- 3 ) compared with DMSO-treated controls (424 d.p.m, m g - 1 dry weight +61; n = 3 ) . DISCUSSION
The current results demonstrate that DFB prevented the development in fecal cultures of the free-living stages of the sheep parasitic nematode H. contortus when administered to lambs in vivo at 10 mg day -1 for 30 days. This observation is consistent with previous findings of DFB effects on free-living and plant-parasitic nematodes (Veech, 1978a,b; Townshend et al., 1983). Since the administration of DFB in vivo did not alter the normal development of a patent infection in lambs and DFB does not prevent in vitro hatching of eggs, the action of DFB seems to be restricted to the free-living stages of H. contortus. Chitin in nematodes is found only in eggs and not in the larval stages (Bird, 1971; R.H. Fetterer, unpublished observations, 1988), therefore, the larvacidal effect of DFB is apparently not directly related to chitin synthesis. Chitin is found in measurable amounts in normal H. contortus eggs, with a glucosamine content of 7.3 pg mg-1 dry weight, but DFB did not affect egglaying by females in vivo. This suggests that either DFB does not affect chitin synthesis in H. contortus or that DFB is altered or fails to reach these parasites during in vivo treatment. It is possible, however, that a partial inhibition of egg laying did occur, but was not detected because of the small sample size used in these experiments. A detailed biochemical examination of the action of DFB on chitin synthesis in H. contortus is required to determine the exact mode of action of DFB. Complete inhibition of H. contortus larval development in fecal cultures was noted with eggs from lambs treated with DFB at a dose of 10 mg kg- 1day. The addition of DFB directly to fecal cultures, however, caused only a 64% inhibition of development at the highest dose used (10 pg m l - 1). Assuming a mean lamb weight of 20 kg, a mean fecal output of 915 g per day (Table 1 ) and little absorption of DFB, then an in vivo dose of 10 mg kg-1 DFB per day would translate to a DFB fecal concentration of > 200 p.p.m., which is more than 10fold greater than the highest dose used to demonstrate the inhibition of larval development in in vitro cultures. This suggests that the potency of DFB observed with in vivo treatment of lambs was due, at least in part, to high levels of DFB carried in the feces following a daily dose of 10 mg kg-1. It is also possible that DFB is either metabolized, concentrated or otherwise modified by passage through the sheep intestinal tract, making it functionally more effective against subsequent larval development. Since a relatively high in vivo
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treatment dose ( > 100-fold) is required to inhibit H. contortus larval development (10 mg kg -1 per day) compared to that used for the control of insect larvae in cattle feces (0.06 mg kg -1 per day; Miller and Miller, 1985), DFB appears to be of little practical value for the control of H. contortus larvae. In contrast to the present study, Waller and Lacey (1986) observed that a DFB analog, triflumuron ( T F M ) , was quite effective in vivo and in vitro in preventing the development of Trichostrongylus colubriformis. However, T F M was less effective in preventing the development of H. contortus and Ostertagia circumcincta. Differences in species susceptibility are considered to be characteristic of growth regulatory activity in insects and such a phenomenon may similarly occur in nematodes. Compounds such as DFB are potent inhibitors of chitin synthesis in arthropods. Since A. suum eggshell contains significant amounts of chitin, it seemed plausible that t r e a t m e n t ofA. suum-infected pigs should result in an alteration of egg production in normal infection. Supporting this hypothesis is the observation that t r e a t m e n t of Caenorhabditis elegans with DFB caused partial disintegration of the chitinous layer of the eggshell and marked alteration of normal egg morphology (Ibrahim, 1986). There is evidence in the present study that DFB interacted with A. suum in vivo, since the chitin content of eggs from worms obtained from DFB-treated pigs was lower t h a n from eggs from worms obtained from untreated pigs. Eggs exposed to DFB in vivo, however, were morphologically identical to eggs from worms derived from untreated pigs both before and after embryonation and they were equally capable of migrating to the lungs of pigs following experimental inoculation. It appears, therefore, that the observed reduction in egg chitin was not detrimental to the egg and larval infectivity. More severe natural or environmental stress, however, could have resulted in higher egg mortality than might be encountered under the laboratory conditions constructed in these experiments. Chitin synthesis in the isolated reproductive tissue ofA. suum was inhibited by DFB demonstrating that: (1) chitin synthesis in nematodes is a potential target for an inhibitor; (2) chitin synthesis may be biochemically similar in insects and nematodes. A previous study (Fuhrman and Piessens, 1985 ) demonstrated that in vitro chitin synthesis in the Filarid nematode Brugia malayi was partially inhibited by DFB and that the microfilaria (MF) produced by the females in vitro were malformed. This indicated a physiological effect of DFB on nematodes related to the inhibition of chitin synthesis. However, since no in vivo studies with DFB were performed by F u h r m a n and Piessens, it is not known if malformed MF would develop in a normal m a n n e r in vivo or if DFB would affect female production of MF within the host. It is now clear that compounds with IGR activity may be potential candidates for inhibitors of free-living stages of parasitic nematodes. The observation that DFB is inhibitory to larval development only at a high dose may, in part, reflect the fact
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that DFB and other IGRs have been developed by selection for activity on insects. Examination of other growth regulators may reveal compounds with enhanced efficacy against nematodes. The investigation of IGRs as possible nematode control agents remains an unexplored and potentially fruitful area for additional research. ACKNOWLEDGEMENTS
We are indebted to J. Corbin and A.W. Jones for their expert technical assistance. We also thank Dr. P.C. Allen and Dr. R.S. Rew for critical reading of this manuscript. Mention of a trade name, proprietary product or specific equipment does not constitute a guarantee of warranty by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may be suitable. REFERENCES Ascher, K.R.S. and Nemny, N.E., 1974. The ovicidal effect of PH60-4-1 (4-chlorophenyl)-3- (2,6difluorobenzolyl)-ureain Spodopterea littoralis Boisd. Phytoparasitica, 2: 131-133. Barrett, J., 1981. Biochemistry of Parasitic Helminths. University Park Press, Baltimore, MD, pp. 13-71. Bird, A.F., 1971. The Structure of Nematodes. Academic Press, New York, pp. 45-77. Davidson, E.A., 1966. Glucosamine-6-phosphate N-acetylase. Methods Enzymol., 9: 704-707. Deul, K.H., deJong, B.J. and Kortenbach, J.A.M., 1978. Inhibition of chitin synthesis by two l- (2,6, disubstituted benzoyl) -3-phenylurea insecticides. Pestic. Biochem. Physiol., 8: 98-105. Donald, A.D., Waller, P.J., Dobson, R.J. and Axelson, A., 1980. The effect of selection with levamisole on benzimidzole resistance in Ostertagia spp., in sheep. Int. J. Parasitol., 10: 381-389. Fleming, M.W. and Fetterer, R.H., 1984. Ascaris suum: continuous perfusion of the pseudocoelon and nutrient absorbtion. Exp. Parasitol., 57: 142-148. Fuhrman, J.A. and Piessens, W.F., 1985. Chitin synthesis and sheath morphogenesis in Brugia malayi microfilariae. Mol. Biochem. Parasitol., 17: 93-104. Gatt, R. and Berman, E.R., 1966. A rapid method for estimation of amino sugars on a micro scale. Anal. Biochem., 15: 167-171. Hajjar, N.P. and Casida, J.E., 1978. Insecticidal benzoylphenylureas: structure-activity relationships as chitin synthesis inhibitors. Science, 200: 1499-1500. Hollander, M. and Wolfe, D.A., 1973. Nonparametric Statistical Methods. Wiley, New York, pp. 26-48. Ibrahim, A.M., 1986. Studies on ovicidal activity of diflubenzuron on Caenorhabditis elegans. Dirasat., 13: 139-147. Miller, R.W. and Miller, J.A., 1985. Feed-through chemicals for insect control in animals. In: J.L. Hilfod (Editor), Agricultural Chemicals of the Future. BARC Symposium 8, pp. 355-365. Mitsui, T., Nobousawa, T. and Fukami, J., 1984. Mode of inhibition of chitin synthesis by diflubenzuron in the cabbage armyworm, Manestra brassicae L. J. Pestic. Sci., 9: 19-26. Moore, R.F., Jr. and Taft, H.M., 1975. Boll weevils: chemosterilization of both sexes with bisulfar plus Thompson-Hayward JH 6040. J. Econ. Entomol., 68: 96-98. Townshend, J.L., Pree, D.J. and Broadbend, A.B., 1983. Population development and reproduction of fungus and bacterium feeding nematodes in the presence of growth regulators. J. Nematol., 15: 105-110.
192 Urban, J.F., Jr. and Romanowski, R.D., 1985. Ascaris s u u m : protective immunity in pigs immunized with products from eggs and larvae. Exp. Parasitol., 60: 245-254. Urban, J.F., Jr., Douvres, F.W. and Tromba, F.G., 1981. A rapid method for hatching of Ascaris suum" eggs in vitro-Proc. Helminthol. Soc. Wasl., 48: 241-243. Veech, J.A., 1978a. The effect of diflubenzuron on egg formation of root knot nematodes. J. Nematol., 9: 184-185. Veech, J.A., 1978b. The effect of diflubenzuron on the reproduction of free-living nematodes. Nematologica, 24: 312-330. Waller, P.J. and Lacey, E., 1986. The effect of triflumuron (SIR 8514) on the free-living stages of sheep nematodes. Vet. Parasitol., 21" 119-126. Waller, P.J. and Prichard, R.H., 1986. Drug resistance in nematodes. In: W.C. Campbell and R.S. Rew (Editors), Chemotherapy of Parasitic Infections. Plenum, New York, pp. 330-362. Whitlock, H.V., 1948. Some modifications of the McMaster helminth egg-counting techniques and apparatus. J. Counc. Sci. Ind. Res. Aust., 21: 177-180. Wright, J.E. and Harris, R.L., 1976. Ovicidal activity of Thompson-Hayward TH 6040 in the stable fly and horn fly after surface contact by adults. J. Econ. Entomol., 69: 728-730.