Growth Inhibition of and Panagrellus redivivus by Selected Mammalian and Hormones

V. H. DROPKIN, W. R. LOWER, AND J. ACEDO 1

Abstract: Caenorhabditis elegans and Panagrellus redivivus were cultured in axenic medium in microwells. The addition of selected steroids and terpenoids to the medium caused quan- titative inhibition of numbers of offspring produced per well. Three out of 14 vertebrate sex hormones and analogs, and seven out of 10 insect juvenile hormones and analogs inhibited growth at 25 or 50 micrograms per ml. In addition, two insecticide synergists which mimic juvenile hormones, propyl 2-propynyl phenyl phosphonate and piperonyl butoxide, inhibited growth at 7 ~g/ml. Total lipids from Panagrellus and from Nematospiroides dubius were inhibitory. Separation by silicic acid column chromatography yielded active and inactive portions. We concluded that the inhibition observed was non-specific. Key Words: Microwells, Steroids, Sesqui-terpenoids.

Recent data on arthropod endocrines have tion of growth of two species of free-living shown insect hormones and analogues to be in axenic culture by a number of valuable in insect control. The discovery that mammalian sex hormones, insect hormones, certain plants contain compounds with molec- and related compounds. In addition, we ular structure and activity resembling insect examined the effects of lipid extracts from hormones suggests that search for parallel two species of nematodes and from other systems against parasites might be sources. fruitful ( 13 ). Very little is known about growth and MATERIALS AND METHODS differentiation in nematodes. Nerve cells of Second-stage larvae of Panagrellus redivi- certain nematodes exhibit a secretory cycle vus (Linn) T. Goodey or Caenorhabditis correlated with molting (3). Hypodermal elegans (Maupas) Dougherty were cultured and gonadal growth abnormalities were in- in groups of two or three, respectively, at duced by exposure of sugarbeet nematode 23 C. The medium consisted of 3% soy- larvae to an exogenous steroid androgen and peptone, 3% yeast extract, with 10% crude to insect juvenile hormone mimics (8). Al- yeast supplement (9). Stock cultures were though there is no critical proof of direct maintained in 2 ml of medium in 25 × 100- effects of mammalian host hormones on mm screw-cap test tubes. -parasitic nematodes, there is an ill- Microtiter plates of flexible vinyl plastic defined relation between the host's reproduc- were sterilized for 12 hr in sealed Mason tive cycle and the growth of nematode para- jars by chlorine gas generated from NaOC1 sites (4). and HC1. Each well received 0.0625 ml of The purpose of the studies reported here medium. The microtiter plates were sub- was to measure the effect of selected mam- sequently enclosed in sterile plastic dishes malian sex hormones, insect hormones, and sealed with wide rubber bands to reduce lipid extracts from nematodes on the growth evaporation. of free-living nematodes. We report inhibi- Second-stage larvae of P. redivivus and of C. elegans were collected for assay by filtra- Received for publication 26 February 1971. tion of mixed populations through sand (11). a Professor, post-doctoral fellow, and technician, Department Filters were prepared from acid washed sand of Plant Pathology, University of Missouri, Columbia, Mo. 65201. sieved through a 60-mesh, and caught on an 349 350 Journal o[ Nematology, Vol. 3, No. 4, October 1971

80-mesh sieve. A 1.8-cm diam glass cen- Vertebrate sex hormones and analogs were trifuge tube (12-ml capacity), drawn into a donated by J. Fried, Syntex Corp., and insect cone with a 4-mm opening at the bottom, hormones and analogs by G. B. Staal, Zoe- was loosely packed with about 0.5 cm of con Corp., both at Palo Alto, California. fiberglass topped with a 2-cm column of dry, sieved sand. The assembly was fitted into RESULTS a conical screw-capped centrifuge tube and LIPID EXTRACT: Unmated female P. red- sterilized by autoclaving. Nematode suspen- ivivus contained relatively few oocytes in sions from axenic cultures were transferred the uterus, whereas mated females produced onto the sand and washed into the centrifuge eggs which were fertilized as they enter the tube with 10 ml of sterile Fenwick's solution uterus. Six days after isolation of a pair of (5). When the small larvae which emerged larvae, the uteri of mated females contained settled to the bottom of the tube, they were 50 --- 5 (SE) eggs and embryos compared transferred by pipette to a well of a micro- with 7 -+ 1 oocytes in uteri of unmated titer plate. Individual larvae were aseptically females. To test whether lipid extracts might dispensed into the microwells by micro- stimulate an increase in oocyte production pipette under 12× magnification in a laminar of unmated females, chloroform-methanol flow hood. and ether extracts of P. redivivus were added In each experiment, the effects of three to culture media containing unmated females. test substances or three concentrations of a No effect was detected. single substance were compared with one or Lipid extracts of both P. redivivus and two controls on unamended medium. Each Nematospiroides dubius, an intestinal para- treatment and control medium was dispensed site of mice, inhibited reproduction of P. to 21 replicate microwells. In the P. red- redivivus. Reproduction failed completely in ivivus series, larval counts were recorded cultures exposed to 5.76 mg/ml of lipids directly; in the C. elegans series, a scoring from P. redivivus. An ether extract of N. system permitted more rapid collection of dubius completely blocked reproduction of data. Wells with no larvae were scored -1; P. redivivus at 0.7 mg/ml and reduced larval with 1-10 larvae, +1; with 11-20 larvae, production to approximately half that of the +2; and with more than 20 larvae, +3. controls at a concentration of 0.07 mg/ml. Lipids to be tested were dissolved in The extracts were not lethal to the assay appropriate solvents (ether, acetone, or organisms. Table 1 shows the total scores ethanol) and added to the medium by one in a dilution series of lipid extracts assayed of several methods: (i) addition to the on C. elegans. Results are comparable to lyophilized soy-peptone-yeast portion; (ii) those with P. redivivus. addition to lyophilized crude yeast supple- The medium used for cultivation of P. ment; and (iii) addition to liquid soy-pep- redivivus mass cultures was a mixture of tone-yeast portion. The medium was recon- oatmeal, yeast and bacteria. Total lipid ex- stituted or completed after evaporation of tracted from this mixture incubated in the the solvent at 40 C (ether and acetone). absence of nematodes, and lipid from yeast Ethanol solvent was removed by desiccation alone were inhibitory to both species of in a lyophilizer. Method (iii) was used for nematodes. We might conclude that lipids most experiments since it proved to be as in general are toxic to these nematodes. But effective as the other methods and much total lipids extracted from mouse liver were simpler. inactive at 0.25 to 0.063 mg/ml. Further, GROWTH INHIBITION aV STEROIDS AND TERPENOIDS ° Dropkin, et al. 351

TABLE I. Effect of lipid extracts from nematodes some chromatography fractions of Panagrel- and yeast on reproduction of Caenorhabditis lus lipids separated on silicic acid columns elegans were highly active, but other fractions caused Extract Total Replicate no depression of reproduction in bioassays Source of conc. reproduction micro- extract (#g/ml) scorea wells with both species. MAMMALIAN SEX HORMONES: Table 2 Panagrellus redivivus 530 -3 28 summarizes results of bioassays with C. 265 39 elegans of 14 mammalian sex hormones and 132 76 analogues. Compound-4 (17 a-acetoxy-6- 0 72 chloro-pregna- 1,4,6-triene-3,20-dione) was Nematospiroides dubius 350 -11 21 the most inhibitory steroid tested. Proges- 70 7 terone inhibited early reproduction, but the 35 19 nematodes recovered reproductive capacity. 0 19 0 15 Inhibitory properties of some steroids were inversely correlated with the nutritional levels Yeast 700 -21 21 350 -18 of the medium. The culture fluid consisted 70 -13 of soy-peptone, yeast extract medium to 0 35 which 10% v/v of crude yeast supplement 0 11 was added (9). Each batch of crude yeast R Reproduction scoring system: 0 larvae/well ---- -1; 1-10 larvae -~ -}-1; 11-20 larvae ~--I-2; 21 or more larvae ----q-3. supplement differed in its ability to support

TARLE 2. Influence of mammalian sex hormones and analogues on growth of Caenorhabditis elegans.

Cpd. No. Compoundname Activity and dosage (/zg/ml) No. of tests a

1. Testosterone Inactive at 25, 50, 100, 250 2 2. Progesterone Inhibitory at 25, 50, 100, 200, 250 2 3. 17a-acetoxy-6-chloro-pregna- 4,6-diene-3,20-dione Inconsistent results 2 4. 17a-acetoxy-6-chloro-pregna- 1,4,6-triene-3,20-dione Inhibitory at 25, 50, 100, 200, 250 2 5. 11/~,17a,21-trihydroxypregna- 1,4-diene-3,20-dione Inhibitory at 50, 100; inactive at 25 and 250 1 6. Estradiol Inactive at 50, 100, 200, 250 2 7. 2a-methyldihydrotestosterone- 17-tetrahydropyranyl ether Inconsistent results, see Table 3. 2 8. 6=-fluoro-I lfl,16~-17c~-21-tetrahydroxy- pregna-1,4-diene-3,20-dione- 16,17 acetonide-21-acetate Inconsistent results 9. 17a-methyl-19-nor-testosterone Inactive at 50, 100, 200, 250 10. 17~-methyl-fl-nor-testosterone Inactive at 50, 100, 200 I 1. 17a-acetoxy-progesterone Inactive at 25, 50, 100 12. 6-fluoro-6-dehydro- 17~-acetoxy-progesterone Inactive at 25, 50, 100 13. 6-fluoro-6~,7a-difluoromethylene- 17a-acetoxy-progesterone Inactive at 25, 50, 100 14. 6-fluoro-6~,7a-difluoromethylene- 16- methylene- 17c~-acetoxy progesterone Inactive at 25, 50, 100 " Each test was a separate experiment with 21 replications per treatment. 352 Journal o/ Nematology, Vol. 3, No. 4, October 1971

TABLE 3. Nutrient level effect on the inhibition of Compound 2 ~200 jJg/ml growth of Caenorhabditis elegans by mammalian 20, sex hormones and analogues. 15. I0. Nutrient leveP' and hormone dosage (#g/ml) ~o. 5. High nutrient Low nutrient o, Conlpound a •LI~ 100 ~:~lc°nlroJ No. 250 100 50 25 200 100 50 -S.

2 D(' -- 1~0 li7 2~6 ai4 ~o 4 Hours after ;noculot;on

7 - 0 0 0 Compound 4 20, o*'**" :~ Compound-2 = progesterone; Compound-4 = 17a-acetoxy- 6-chloro-pregna-l,4,6-triene-3,20-dione; Compound-7 ~ 2~r- 15, methyldihydrotestosterone 17-tetrahydro pyranyl ether. 10. /' 200 1~ High nutrient = culture medium in which F1 larvae devel- 5. oped in 120 hr or less after transfer of 2nd stage larvae. Low nutrient = culture medium in which F1 larvae did not o. / .I- so develop until more than 120 hr after transfer. -s, " J. ~..,=,==~': -.,-~,oo ----- moderate inhibition; -- = strong inhibition; 0 = no effect. I~10 1.~7 226 254 360

nematode growth. Ft larvae were present in Compound 7 201 ~. F 100 microwells with the best crude yeast supple- "t /,-...... • . ment at about 90 hr after inoculation of 2nd 10 stage larvae. Such media are classed as high nutrient media. If development to F~ larvae required longer than 120 hr, the media were classed as low nutrient media. Table 3 shows ti130 157 226 254 360 that Compound-2 was moderately inhibitory F~. 1. Inhibition of production of F1 larvae by at both high and low nutrient levels, but Caenorhabditis elegans grown in nutrient medium Compounds 4 and 7 were strongly inhibitory containing vertebrate sex hormones. Compound-2 at low nutrient levels and much less in- = progesterone; Compound-4 = 17a-acetoxy-6- chloro-pregna- 1,4,6-triene-3,20-dione; Compound-7 hibitory at high levels. Two compounds (3 2 =-methyldihydrotestosterone- 17-tetrahydropy- and 8) were moderately inhibitory in one ranyl ether. (Reproduction scoring system: 0 lar- experiment at 25, 50 and 100 Fg/ml but vae/microwe]l = -1; 1-10 larvae = +I; 11-20 inactive at 25, 100 and 250 in another. larvae z +2; ~20 larvae = +3.) Nutrient levels for both assays were high. Fig. 1 presents plots of larval production Calbiochemical Corp.) inhibited growth of in typical bioassays at low nutrient levels. C. elegans at 63 to 250 Fg/ml. We therefore The strongly inhibitory compounds (4 and examined the inhibitory properties of a series 7) continued to depress reproduction for of insect juvenile hormones and analogues. more than 7 days after the controls had Table 4 summarizes results of this series. reached maximum scores of 21 per set of In general, the inhibition by sesquiter- seven wells. The moderately active Com- penoids was greater than by the steroids. pound-2 delayed reproduction about 1 day. Three compounds (Nos. 19, 20 and 22) were INSECT HORMONES, ANALOGUES AND strongly inhibitory at 50 Fg/ml. Fig. 2A MIMICS: A mixture of synthetic sesqui- shows larval production by C. elegans in terpenoid derivatives which showed a broad microwells with nutrient medium containing spectrum of activity in insect hormone assays various concentrations of Compound-22. At ("Juvenile Hormone, synthetic, B-grade," 25 kLg/ml, reproduction was delayed, but GROWTH INHIBITION BY STEROIDS AND TERPENOIDS • Dropkin, et al. 353

eventually F2 larvae were produced. At 50 and 100 ~g/ml, no F., larvae appeared, and F-2 A at 100 /zg/ml, the production of F1 larvae 20 was severely curtailed. Nematodes remained sco,~TM 10. smaller than their counterparts in medium 5° without the compound. Larvae isolated by O° sand filtration measured 400 -+ 17 /~m in -5. ./ ././' "~'~. 1OO length. Lengths (/xm) of C. elegans after 1~! 186 2&8 2~.2 2;;6 w 364 10 days in medium with Compound-22 were Hours after inoculation as follows: unamended medium, 1245 -+ 15;

50 /zg/ml, 1017 -+- 35; 100 /zg/ml, 721 --- 20, B 32; 200 /xg/ml, 607 + 25. At the highest 15, . concentration the nematodes grew very little. ~°1~0 ` . With decreasing concentration, nematode O, 22 " growth approached the normal rate. -5 The three most inhibitory compounds are compared in Fig. 2B with one of slight 8 2"s s'o 16o Dose in pg/ml activity. Reproductive scores were recorded when the control series first reached maxi- FIG. 2. Inhibition of production of F1 larvae by mum. The assay was sensitive enough to Caenorhabditis elegans growing in nutrient medium distinguish between 25 and 50 /zg/ml of an amended with: A. An insect juvenile hormone mimic (Compound-22), 3'-methyl-7'-ethylnona-2', inhibitory compound. 6'-dienyl 3,4-methylenedioxyphenyl ether at 25, 50, Five sesquiterpenoid compounds were and 100 ug/ml compared with untreated check tested at both high and low nutrient levels; (dashed line), or B. Synthetic insect juvenile nutrient level did not affect the degree of hormone (Compound-19) and three mimics rated activity. when control score was maximum. (Reproduction scoring system: 0 larvae/microwell ~- -1; 1-10 A number of insecticide synergists have larvae ~ +l; 11-20 larvae ~ +2; >20 larvae been reported to mimic the action of juvenile +3.)

TABLE 4. Influence of insect juvenile hormones and analogues on growth of Caenorhabditis e/egans.

Cpd. Dosage (/zg/ml) and Number No. Compound name relative activity of tests

15. 2-(3,4-methylenedioxyphenoxy)-3,6,9-trioxaundecane (Sesamex) Inactive at 25-200 2 16. Methyl 4-[l',5'-dimethyl-3'-oxo] hexyl cyclohex-l-enoate (Juvabione) Inactive at 25-200 2 17. Ethyl 7,11-dichloro-3,7,11-trimethyldodec-2-(trans)-enoate Inhibitory at 50--200, inactive at 25 2 18. Trans, trans, cis methyl 10,11-epoxy-7-ethyl-3,1 l-dimethyl- Inactive at 25-200 2 2,6-tridecadienoate (Cecropia JH-I) 19. Trans, trans, cis methyl 10,11-epoxy-3,7,11-trimethyl- Inhibitory at 25-100, 2,6-tridecadienoate (Cecropia J H-II) inactive at 0.008-5 2 20. 6',7'-epoxygeranyl 3,4-methylene-dioxyphenyl ether Inhibitory at 25-200 2 21. 3'-methyl-7'-ethylnona-2',6'-dienyl 4-nitrophenyl ether Inhibitory at 25-200 2 22. 3'-methyl-7'-ethylnona-2',6'-dienyl 3,4-methylene-dioxyphenyl ether Inhibitory at 25-200 2 23. Trans ethyl l 1-chloro-3,7,11-trimethyldodec-2-enoate Inhibitory at 50-200 1 24. Trans ethyl 3,7,7,11,11- pentamethyl-9-oxo-dodec-2-enoate Inhibitory at 50-200, inactive at 25 2 354 Journal o/ Nematology, Vol. 3, No. 4, October 1971

hormones in (1). We tested three We found no correlation between molec- such compounds (obtained from J. H. Fales, ular structure and growth inhibition. Many Pesticide Chemicals, U.S. Department of of the steroids tested were inactive but much Agriculture). Two of the three insecticide more work will be required to define the synergists with juvenile hormone mimetic particular configurations toxic to the test activity were inhibitory in the P. redivivus nematodes. The inhibitory activity of insect assay. Both propyl 2-propynyl phenyl phos- juvenile hormones and mimics did not paral- phonate and piperonyl butoxide prevented lel their activity as insect hormones. Com- reproduction at 7/~g/ml. The 2-(3,4-methyl- pound-19 is generally less active in insects enedioxyphenoxy)-3,6,9-trioxaundecane pre- than Compound-18, but the reverse was true vented reproduction at 670 /zg/ml but had in our assays. Some of the other inhibitory no effect at 67. Piperonyl butoxide strongly compounds are active as juvenile hormones inhibited elongation and reproduction of C. on Pyrrhocoris bugs but not on other insects; elegans at 5 /~g/ml, but was ineffective at however, Compound-22 (see Fig. 2) was in- 1 /zg/ml. Nematodes exposed to this chem- effective in Pyrrhocoris but effective in other ical in the nutrient medium for 7 days aver- insects (personal communication of G. B. aged 85 --- 3.3 /zm in length while the Staal). controls averaged 605 + 20.4/xm. That a variety of mammalian sex hor- Four phytoecdysones and one insect steroid mones and analogues, and insect juvenile were examined. Ponasterone A, cyasterone, hormones, analogues and mimics were non- ecdysone, and inokosterone (obtained from specifically inhibitory to nematode reproduc- T. Takemoto, Tohuku Univ., Japan) and tion suggested that further search would be /3-ecdysone were all inactive at 200 to 0.01 useful. Differences between our results and /~g/ml. those of other workers indicated that various species of nematodes react differently to DISCUSSION such compounds. The compounds tested were selected on The search for specific compounds in the basis of their activity as hormones or plants that act as growth regulators for mimics of hormonal action in vertebrates and parasites has hardly begun. Exogenous insects. Effective concentrations in our as- sterols are important for reproduction in says were in the range of 10 -4 M or higher. fungi (6) and are also required for the The most active compound, piperonyl butox- growth of some nematodes (2, 7). The ide, was effective at 10 -~ M. We concluded occurrence in plants of terpenoid regulators that the growth inhibition in assays on free- of insect growth (13) makes it probable that living nematodes was non-specific. This specific growth regulators of other pathogens contrasted with Johnson and Viglierchio's also exist in plants. It will therefore be useful finding that specific hypertrophy of the gonad to study plant resistance to nematodes from of Heterodera schachtii resulted from ex- this point of view. posure of larvae to farnesyl derivatives (8). LITERATURE CITED We observed no aberrations other than re- tarded growth and development. Our find- l. BOWERS, W.S. 1968. Juvenile hormone: activity of natural and synthetic synergists. ings also differ from those of Shanta and Science 161: 895-897. Meerovitch's report that low concentrations 2. COLE, R. J., AND S. R. DUTKY. 1969. A of farnesyl methyl ether inhibited develop- sterol requirement in Turbatrix aceti and Panagrellus redivivus. J. Nematol. 1: 72- ment of male copulatory appendages (12). 75. GROWTH INHIBITION BY STEROIDS AND TERPENOIDS • Dropkin, et al. 355

3. DAVEY, K. G. 1966. Neurosecretion and 9. LOWER, W. R., AND E. J. BUECHER, JR. 1970. molting in some parasitic nematodes. Amer. Axenic culturing of nematodes: an easily Zool. 6: 243-249. prepared medium containing yeast extract. 4. DOBSON, C. 1966. The effects of pregnancy Nematologica 16: 563-566. and treatment with progesterone on the 10. LOWER, W. R., E. HANSEN, AND E. A. YAR- host-parasite relationship of Amplicaecum WOOD. 1966. Assay of a proteinaceous robertsi Sprent & Mines, 1960, in the mouse. growth factor required for maturation of Parasitology 56: 417-424. the free-living nematode, Caenorhabditis 5. FENWICK, D. 1939. Studies on the saline briggsae. J. Exp. Zool. 161: 29-36. requirements of the larvae of Ascaris suum. I1. SAMOILOFF, M. R., AND J. PASTERNAK. 1968. J. Helminthol. 17: 211-228. Nematode morphogenesis: fine structure of 6. Hendrix, J.W. 1970. Sterols in growth and the cuticle of each stage of the nematode reproduction of fungi. Annu. Rev. Phy- Panagrellus silusiae (de Man ] 913) Goodey topathol. 8: 111-130. 1945. Can. J. Zool. 46: 1019-1022. 7. HIEB, W. F., AND M. ROTHSTEIN. 1968. 12. SHANTA, C. S., AND E. MEEROVITCH. 1970. Sterol requirement for reproduction of a Specific inhibition of morphogenesis in free-living nematode. Science 160: 778-780. Trichinella spiralis by insect juvenile hor- 8. JOHNSON, R. N., AND D. R. VIGLIERCHIO. mone mimics. Can. J. Zool. 48: 617-620. 1970. Heterodera schachtii responses to 13. SL~MA, K. 1969. Plants as a source of mate- exogenous hormones. Exp. Parasitol. 27: rials with insect hormone activity. Ent. Exp. 301-309. Appl. 12: 721-728.