EXPERIMENTAL

PARASITOLOGY

42,

67-72

(1977)

Haemonchus conforfus: Effects of Compounds with Hormone Activity on the in Vitro Development of Infective Larvae R. J. BOISVENUE, T. L. EMMICK, Parasitology

Department, Lilly Research and Company, Greenfield, (Accepted

for

publication

AND

R. B.

GALLOWAY

Laboratories, Division Indiana 46140, U.S.A. 13 October

Juvenile

of Eli Lilly

1976)

BOISVENUE, R. J., EMMMICK, T. L., AND GALLOWAY, R. B. 1977. Haemonchus contortus: Effects of compounds with juvenile hormone activity on the in vitro development of infective larvae. Experimental Parasitology 42, 67-72. Inhibitory effects of farnesol, derivatives of farnesol, geraniol, 1-phenylnonane, and l-phenyl-2,6-nonodiene on the development of exsheathed, third-stage Haemonchuscontortus larvae were observed. Treatment of larvae with ,antibiotics prior to exsheathment provided control of bacterial contaminants for in vitro cultivation. Exsheathed larvae were placed in Earle’s balanced salt solution. The medium was saturated with 5% CO, and the larvae were incubated at 37 C. Candidate compounds were added at 10, 50, and 100 ppm. Activity was determined by comparing mortality and arrestment of larval development to fourth stage in treated media with nontreated controls at 48 and 72 hr of incubation. In vitro activity occurred in each of the four compound types with the greatest activity found in the famesol derivatives. Those compounds possessing 12- to 15-atom (carbon ,and oxygen) chains exhibited larval arrestment and larvicidal properties. Compounds that contained lo-atom chains were considerably less active. The four compound types did not affect the development of Ascaris suum migrating and Nematospiroicles dubius intestinal larvae in mice. All compounds that inhibited larval development possess juvenile hormone activity in insects. INDEX DESCRIPTORS: Haemonchus contortus; Parasitic nematode; Infective larva; In vitro nematode development; Evaluation technique; Insect juvenile hormone analogues; Farnesol; Terpene compounds; In viva activity.

Meerovitch 1970; Hitch0 and Thorson 1973; Rogers 1973), trematodes (Mors, DOS Santos, Montiero, Gilbert, and Pellegrino 1967; Gilbert, de Souza, Fortes, Santos, do Prad,o Seabra, Kitagawa, and Pellegrino 1970), and protozoa (Ilan and Ricklis 1969). Thorson, Digenis, Berntzen, and Kongalian (1968) and Frayha and Fairbairn (1965) reported on the presence of farnesol and farnesyl-like compounds in larval stages of cestodes. Evidence has been presented that nematodes require sterols or reIated derivatives for reproduction (Hieb and Rothstein 1968). Because of the interesting bio-

Terpene compounds such as farnesol and its ,derivatives have been shown to impede the metamorphosis of the normal pupation of insects and result in the formation of nonviable or steriIe insects (Bowers and Thompson 1963; Karlson 1963; Wigglesworth 1963; Schneiderman, Krishnakumaran, Kulkarni, and Friedman 1965). Insect juvenile hormones (JH) and juvenile hormone analogues have been shown to affect the development of nematodes (Meerovitch 1965; Meerovitch 1970; Hansen and Buecher 1970; Shanta and 67 Copyright All rights

@ 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0014-4894

68

BOISVENUE,

EMMICK

TABLE

AND

GALLOWAY

I

The Effects of Farnesol and Its Derivatives at 100 ppm in Vitro against Exsheathed Third-Stage Haemonchus contortus Larvae Incubated for 72 hr Chemical name

Compound number

Percentage of activity 48 hr

1 2 3 4 5 6 7 8 9 10 None

3,7,1l-Trimethyl-2,6,l0-dodecatrien-l-ol(farnesol) Methyl 3,7,11-trimethyl-2,6,10-dodecatrienoate Methyl 3,7,11-trimethyl6,10-dodecatrienyl ether 3,7,11-Trimethyl-2,6,10-dodecatrienyl acetate 3,7,11-Trimethyl-2,6,10-dodecatrienal 3,7,11-Trimethyl-2,6,10-dodecatrienoic acid Methyl lO,ll-epoxy-3,7,11-trimethyl-2,6-dodecadienoate lO,ll-Epoxy-3,7,11-trimethyl-2,6-dodecadien-l-01 Methyl lO,ll-epoxy-3,7,11-trimethyl-2,6-dodecadienyl ether 2-Propynyl-3,7,11-trimethyl-2,6,10-dodecatrienyl ether Untreated (control)

72 hr

LCa

LA”

LC

LA

56 16 19 32 23 10 23 10 16 13 0

92 62 60 42 56 42 98 30 61 29 0

85 60 74 77 70 47 33 74 69 84 2

98 92 97 93 76 80 98 85 94 79 0

a LC = larvicidal factor. b LA = larval arrestment fact,or.

chemical ‘control of the ,developmental process of insects in nature, studies were conducted to determine, ( 1) the effects of juvenile hormones and analogues on the development of Haemonchus contortus infective larvae cultured in vitro to the fourth stage and (2) the anthelmintic properties of these compounds against larval stages of Ascaris suum and Nemutospiroides d&us in mice. MATERIALS

AND

METHODS

Larval Preparation and Evaluation Technique Haemonchus contortus was obtained from male donor lambs with pure infections of a United States Department of Agriculture isolate BPLI. Feces containing eggs of the nematode were collected in fecal bags harnessed to the donor lambs for a 24-hr period, The fecal pellets were broken, mixed 1 to 1 with vermiculite, and moistened to provide a culture bed. The bed was oovered with ,clear plastic and kept at 72°F for 11 days. At this time, third-stage ( L3) ensheathed larvae were

collected from the cultures by a Baerman funnel. Larvae from the collections were concentrated by allowing to stand at room temperature for 12 hr. Using a modification of the method of Hansen, Silverman, and Buecher (1966), clean, acid-washed, antibiotic-treated suspensions of infective L3 ensheathed H. contortus larvae were gently spun down at 700 rpm and resuspended at a concentration of lO,QOO/ml in Earle’s balanced salt solution plus antibiotics (BSSA). A larval suspension of 10 ml was introduced into a 60-ml round bottle and gassedwith 100% COz until both liquid and gas phases were saturated. Then the bottle was slowly rotated at 25 rpm at 37 C for 16 hr. This procedure caused the Ls larvae to exsheath. The exsheathed larvae (XL3) were gently centrifuged, resuspended in physiological buffered saline of pH 7.2, and diluted with the same buffer so that 1 ml of the suspension contained 5000 larvae. The test compounds were added to the larval suspensions in phosphate buffered saline of pH 7.2 to give the appropriate final concentrations of compound at 10

Haemonchus

contortus:

INHIBITED

TABLE The

Effeck

of Juvenile Third-Stage

Hormone Compounds Haemonchus contortus Chemical

Compound number

69

DEVELOPMENT

II at 100 ppm ?.n V&o againsi, Exsheathed Larvae Incubated for 72 hr

name

Percentage

of activity

48 hr

11 12 13 14 15 16 17 18 19 20 21 22 23 None a LC 6 LA

Methyl 3,7-dimethyl-2,6-octadienoate Methyl 6,7-epoxy-3,7-dimethyl-2-octenoate 3,7-Dimethyl-2,&ocOadienyl 4-methoxyphenyl ether 3,7-Dimethyl-2,6-octadienyl 3-methoxyphenyl ether 9-(4-Methoxyphenyl)-2,6-dimethyl-2,6-nonadiene 9-(3,4-Dimethoxyphenyl)-2,6-dimethyl-2,6-nonadiene 9- (3-Methoxyphenyl)-2,6-dimethyl-2,6-nonadiene 2,6-Dimethyl-S(3,4-methylene dioxyphenyl)-2,6-nonadiene 9-(2-Methoxyphenyl)-2,6-dimethyl-2,6-nonadiene 9-(2-Bromo-4,j-methylenedioxyphenyl)-2,6-dimethyl2,6-nonadiene 2,6-Dimethyl-9(3,4-methylenedioxyphenyl)nonane 3,7-Dimet,hyl-2,6-octadienyl-3,4-methylenedioxyben~oate Geranyl2-tetrahydropyranyl ether Untreated (control) = larvicidal factor. = larval arrestment

72 hr

LC”

LAb

LC

LA

2 2 48 9 11 5 16 2 2 2

4 2 68 46 48 12 39 44 5 22

9 4 82 82 68 60 76 51 26 65

32 4 92 92 92 82 82 88 17 72

1 0 2 2

34 70 47 0

74 55 57 2

80 83 82 2

factor.

( 1 x 10W5), 50, and 100 ppm. The larval suspensions were then gassed with 5% CO2 and 95% air and incubated for 72 hr at 37 C while slowly rotating. Under these conditions a high percentage of the XL3 advanced to the fourth-stage ( L4) larvae. Less than 5% of the larvae died during the incubation period. After 45 and 72 hr, aliquots were withdrawn from the various suspensions and differential counts of the larvae were conducted to detect any effect on growth by the compounds. The effects of compounds were represented by percentages of XL3 larvae killed or arrested. The formula for Iarvicidal factor (LC) was the percentage of dead larvae in the treated suspension minus the percentage of dead larvae in the untreated (control) suspension equals the adjusted percentage of dead larvae. The arrestment factor (LA) was calculated as follows: The percentage of living XL3 larvae in treated suspension minus the percentage of living XL3 larvae in untreated (control) suspensions equals

the adjusted percentage ‘of arrestment. Four replicates of 100 larvae each were examined per compound at each dilution. Chemicals

Used

Twenty-three compounds were tested in the in vitro study. These compounds include a variety of farnesol, geraniol, lphenylnonane, and 1-phenyl-2,6-nonadiene derivatives having lo-15 atoms in both saturated and unsaturated chains. Isomeric mixtures were present in compounds possessing unsaturation. Examples exist in which parts ‘of the chain are tied back in phenyl or tetrahydropyranyl rings. Evaluation

in Vivo

Compounds active on H. contortus Iarvae in vitro were evaluated in mice against Ascaris swum larvae by a method of Boisvenue, Hendrix, and Scholz (1968) and against Nematospiroides &4.&s larvae according to Standen ( 1963).

70

BOISVENUE,

EMMICK

TABLl? The

Effects

of Selected Juvenile Exsheathed Third-Stage

a Refer

to Tables

III

Percentage Dilution

GALLOWAY

Hormone Compounds at 10, 50, and 100 ppm in Vitro Haemonchus contortus Larvae Incubated for 72 hr

Compound numbera

1 2 3 4 5 6 7 8 9 11 12 13 None

AND

: 100 ppm

against

of activity

Dilution : 50 ppm

Dilution

: 10 ppm

LC

LA

LC

LA

LC

LA

85 60 74 72 61 50 34 70 66 8 2 82 2

98 97 97 97 82 78 97 85 94 34 0 94 0

86 26 51 69 66 55 40 1 19 2 0 73 0

98 74 80 82 84 78 82 1 6 0 0 82 0

63 20 6 42 24 8 6 3 1 1 0 62 2

83 28 6 46 54 14 6 1 2 0 0 82 2

I and II for chemical

names.

RESULTS

The effects ‘of farnesol and its derivatives on the in vitro development of XL3 to L* HaRmonchus contortus larvae at a concentration of 100 ppm (1 X 10d4) are presented in Table I. Farnesol (compound 1) exhibited the greatest larvicidal and larval arrestment properties on XL3 larvae after 48 hr ‘of incubation. The epoxy derivative, compound 7, arrested 98% of the XL3 larvae, but had only a 23% larvicidal action compared to controls. At 72 hr, several derivatives of farnesol (compounds 2, 3, 4, and 9) markedly arrested development ( over 90% ) of exsheathed larvae but were not equal to farnesol in larvicidal activity. In one case, compound 10 possessed greater larvicidal than arrestment activity. Larvae inhibited by exposure to farnesol for 72 hr were washed with distilled water and resuspended in physiological buffered saline of pH 7.2. Growth of these larvae resumed. Data on the influence of some geraniol and l-phenyl-2-6-nonadiene derivatives on larval growth of H. contortus in vitro are

shown in Table II. The octadienoate esters (compounds 11 and 12) did not effectively inhibit development or cause mortality of growing larval populations. In general, 1-phenyl-2-6-nonadienes were noticeably active in arresting larval development with larvicidal action similar to farnesol derivatives. An ‘exception to this statement is compound 19 which was essentially inactive in both categories. The effects of isomer distribution on activity were not demonstrated. Compounds 21, 22, and 23 displayed equal inhibition of larval ‘development but were unequal in Iarvicidal action. In addition to farnesol and its derivatives, the geranoate esters and a I-phenyI2,6-nondiene derivative were selected to determine in vitro activity on XL3 larvae at 10, 50, and 100 ppm (Table III). The highest concentration was repeated to monitor reproducibility of the test system. Again, compounds I (farnesol) and 13 demonstrated the greatest activity on developing XL3 larvae at 100 ppm. When the concentration of farnesol was diluted to 50 ppm, the superior action was sustained,

Haemonchus

contortus:

However, the inhibitory action of compound 13 was reduced. At the dilution level of 10 ppm, both compounds (1 and 13) approached equal inhibitory and Iarvitidal activity, Individual differences in activity of compounds 5 and 6 were observed at the lo-ppm concentration only. Twenty compounds active on H. contortus larvae in vitro were evaluated against Ascaris suum migrating and Nematospiroides dub&s intestinal larvae in mice. Data on gross lung lesions due to migrating ascarid larvae and total numbers of immature and mature N. dubius worms indicated no inhibitory or larvicidal effects by th’e juvenile hormone mimics when incorporated in the diet at a concentration of 500 ppm for 8 days.

INHIBITED

vitro are either inactive or have poor activity in insects. The authors in vitro findings in biological terms suggested a biochemical control of the La to L4 developmental process in nature which may provide a clue for parasite control. However, there was no effect on the La to L4 stages of Nematospiroides dub& or on the larval development of the intestinal (i.e., second stage hatched from the egg) and migratory stages of Ascaris Suum in mice, The absence of larvicidal or arrestment activity may be due, in part, to chemical alteration ‘of the compounds by intestinal pH or enzymes. In vivo information on Haemonchus contortus is not available due to the need of increased amounts of juvenile hormone materials for extended prophylactic evaluation in sheep.

DISCUSSION

In vi&o inhibition of Haemonchus contortus larvae has been demonstrated with a variety of farnesyl and geranyl derivatives. It would appear that with these types of compounds a chain of greater than 12 atoms is necessary to maximize activity; for example, the octadienoate esters were much less active than other classes.In these systems activity is maintained in a system with 15 atoms in the chain (compound 22). Parts of the chain may be tied back in an aromatic or tetrahydropyranyl ring without appreciably ‘changing activity (compounds 13-23). The activity is not appreciably changed in chains having unsaturation in the 2 and/or 6 and/or 10 positions. The compounds having unsaturation in positions which give rise to cis, trans isomers are all mixtures with the trans isomer pre,dominating in all cases. The effect of isomer distribution on activity was not demonstrated. Substitution in the ortho position of a 1-phenyl-2,6-nonadiene markedly decreased activity ( compound 20). All compounds that inhibited larval development in vitro possessjuvenile hormone activity in insects; compounds inactive in

71

DEVELOPMENT

ACKNOWLEDGMENT The Henry

valuable technical is acknowledged.

assistance

of

Mr.

W.

REFERENCES BOISVENUE, R. J., HENDRLX, J. C., AND SCHOLZ, N. E. 1968. A Iarvicidal evaluation programme using Ascaris suum in mice. Parasitology 58, 465472. BOWERS, W. S., AND THOMPSON, M. J. 1963. Juvenile hormone activity: Effects of isoprenoid and straight chain alcohols on insects. Science 142, 1469-1470. FRAYHA, G. J., AND FAIRBALRN, D. 1965. Lipid metabolism in heIrninth parasites. 6. Synthesis of Cis, 6-trans farnesol by Hymenolepis diminuta ( cestoda). Comparative Biochemical Physiology 28, 1115-1119. GILBERT, B., DE SOUZA, J. P., FORTES, C. C., SANTOS, F. D., DO PRADO SEABRA, A., KITAGAWA, M., AND PELLEGRINO, J. 1970. Chemoprophylactic agents in schistosomiasis: Active and inactive terpenes. Journal of Parasitology 56, 397-398. HANSEN, E. L., SILVERMAN, P. H., AND BUECHER, E. J. 1966. Development of Haemonchus contortus in media designed for studies on Caenorhabditis briggsae. Journal of Parasitology 52, 137-140. HANSEN, E. L., AND BUECHER, E. J. 1970. Effect of insect hormones on nematodes in axenic culture. Experientia 27, 859-860. HIEB, W. F., AND ROTHSTEIN, M. 1968. Sterol re-

72

BOISVENUE,

EMMICK

quirement for reproduction of a free-living nematode. Science 160, 778-780. HITCH~, P. J., AND THORSON, R. E. 1973. In oivo effect of the Williams and Law mixture and ecdysterone on Nematospiroides &&us. Journal of Parasitology 59, ILAN, J., AND RICKLIS,

940-941.

S. 1969. Inhibition by juvenile hormones of growth of Crithidia fasciculata in culture. Nature (London) 224, 179180. KARLSON, P. 1963. Chemistry and biochemistry of insect hormones. Angewandte Chemie 2, 175182. MEEROVITCH, E. 1965. Studies on the in vitro development of TrichineZZa spiralis. II. Preliminary experiments ‘on the effects of farnesol, cholestrol and an insect extract. Canadian Journal of Zoology 43, 81-85. MEEROVITCH, E. 1970. Effects and possible implications of the action of insect juvenile hormone or its analogue on helminth cultures. Journal

of Parasitology

56,

234.

MORS, W. B., FASCIO DOS SANTO, F. M., MONTIERO, H. J., GILBERT, B., AND PELLEUUNO, J. 1967. Chemoprophylactic agent in schistosomiasis: 14,15-Epoxygeranylgeraniol. Science 157, 950-951.

AND

GALLOWAY

W. P. 1973. Juvenile and moulting hormones from nematodes. Parasitology 67, 105113. SCHNEIDERMAN, H. A., KRISHNAKUMARAN, A., KULKARNI, V. G., AND FRIEDMAN, L. 1965. Juvenile hormone activity of structurally unrelated compounds. Journal of Insect Physiology 11, 1641-1649. SHANTA, C. S., AND MEEROVITCH, E. 1970. Specific inhibition of morphogenesis in Trichinella spiralis by insect juvenile hormone mimics. Canadian Journal of Zoology 48, 617-620. STANDEN, 0. D. 1963. Chemotherapy of helminthic infections. In “Experimental Chemotherapy” (R. J. Schnitzer and F. Hawking, Eds.), Vol. I, pp. 701-892. Academic Press, New York and London. THORSON, R. E., DIGENIS, D. A., BERNTZEN, A., AND KONGALIAN, A. 1968. Biological activities of various lipid fractions from Echinococcus granulosus scolices on in vitro cultures of Hymenolepis diminuta. Journal of Parasitology 54, ROGERS,

970-973. WIGGLESWORTH,

V. B. 1963. The juvenile hormone effect of famesol and some related compounds: Quantitative experiments. Journal of Insect Physiology 9, 105-119.