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Effects of an azasteroid on growth, development and reproduction of the free-living nematodes Caenorhabditis briggsae and Panagrellus redivivus
Comp. Biochem. Physiol. Vol. 82B, No. 1, pp. 99-106, 1985 Printed in Great Britain
0305-0491/85 $3.00+ 0.00 © 1985 Pergamon Press Ltd
EFFECTS OF AN AZASTEROID ON GROWTH, DEVELOPMENT AND REPRODUCTION OF THE FREE-LIVING NEMATODES CAENORHABDITIS BRIGGSAE A N D PANAGRELLUS REDIVIVUS* KURT P. BOTTJERt and PAUL P. WEINSTEIN Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556, USA and MALCOLM J. THOMPSON Insect Physiology Laboratory, USDA-APS, Beltsville, MD 20705, USA (Received 6 December 1984) Abstract--1. The azasteroid, 25-azacoprostane (ASA-6), was evaluated for its effects on the growth,
development and reproduction of the free-living nematodes, Caenorhabditis briggsae and Panagrellus redivivus. 2. The axenic culture medium for either species of nematode consisted of Caenorhabditis briggsae Maintenance Medium (CbMM): formalin-killed Escherichia coli (1:1) with or without the addition of 5 #g cholesterol per ml and/or 25 pg ASA-6 per ml medium. All cultures also contained 50 #g Tween 80 per ml medium. 3. After two generations of growth in sterol-deficient media, both species displayed a decrease in mean length, a decrease in the percent development to the adult stage and an inhibition of reproductive capability. These effects were more apparent in the sterol-deficient medium containing ASA-6. 4. In the presence of cholesterol and ASA-6, growth and reproduction of C. briggsae, but not of P. redivivus, was inhibited after five generations. 5. Morphologic abnormalities of azasteroid-inhibited worms were similar to those shown by worms cultured in sterol-deficient medium. 6. These results suggest that different species of nematodes may exhibit different responses to azasteroid and that sterol utilization and metabolism may vary between nematode species. In addition, the similarities between the known effects of azasteroid inhibition in insects and those presented in this study on nematodes suggest a similar mechanism of action by the inhibitor in both groups of organisms.
INTRODUCTION
opment and reproduction (Rothstein, 1968; Bolla et al., 1972; Willet and Downey, 1974; Lu et al., 1977) it seemed that azasteroids might be a useful tool with which to study the role of sterol in nematode development. In axenic cultures of the free-living stages of the rodent-parasitic nematodes, Nippostrongylus brasiliensis and Nematospiroides dubius, azasteroids, especially ASA-6, were shown to decrease the percentage of newly-hatched first stage larvae (L1) that developed to the infective third stage (L3) as well as to decrease the mean length of L3 (Bottjer et al., 1984a). The in vitro cultivation of the free-living stages of these parasites is an established method by which the amount of exogenous sterol available to the developing larvae can be strictly controlled (Bolla et al., 1972; Mauro and Weinstein, 1979). However, the in vitro cultivation of N. brasiliensis and N. dubius from L3 to adult requires a complex medium containing chick embryo homogenate and serum of which the sterol content is undefined (Weinstein and Jones, 1959). Therefore, a model in vitro system for the study of the effects of sterol deficiency or sterol metabolism inhibitors on the parasitic cycle of nematode development to sexually mature adults is as yet unavailable.
Azasteroids are vertebrate hypocholesterolemic agents that have proved useful in the study of insect sterol metabolism. These compounds inhibit the delta24-sterol reductase system that is responsible for the conversion of dietary phytosterols to cholesterol (Svoboda and Robbins, 1967, 1968). Certain azasteroids, including 25-azacholestane (ASA-3) and 25-azacoprostane (ASA-6) disrupt development in insects whose test diets contain sufficient cholesterol to support normal growth and development, suggesting that azasteroids affect pathways of sterol metabolism other than those involved in cholesterol production (Svoboda and Robbins, 1971; Svoboda et al., 1972; Thompson et al., 1975). Subsequently, it has been demonstrated that azasteroids interfere also in the metabolism of insect ecdysteroids (Marks et al., 1978; Svoboda et al., 1978). Because nematodes, like insects, appear to require a dietary or exogenous source of sterol for continuous normal growth, devel*Supported in part by NIH grants AI07030 and AI09625 to PPW. tPresent Address: Regional Parasite Research Laboratory, USDA, ARS, P.O. Box 952, Auburn, AL 36830, USA. 99
KURT P. BOTTJER et al.
100
The sterol c o n t e n t in the m e d i u m used for the in vitro cultivation of Caenorhabdit& briggsae a n d Panagrellus redivivus to the adult stage, however, can be controlled. These free-living n e m a t o d e s can be grown axenically in vitro from egg to adult t h r o u g h successive generations in b o t h complex a n d chemically defined media ( R o t h s t e i n a n d Cook, 1965; Hieb and Rothstein, 1968; Lu et al., 1978). O u r objectives were to study the effects of a n inhibitor o f sterol metabolism upon nematode growth and development to the adult stage a n d on adult n e m a t o d e s by the addition of the azasteroid, 2 5 - a z a c o p r o s t a n e (ASA6), to axenic cultures of C. briggsae a n d P. redivivus. This c o m p o u n d has been reported to inhibit b o t h a delta24-sterol reductase a n d a deltaV-sterol isomerase (Chitwood et al., 1983). MATERIAL A N D M E T H O D S
Processing of glassware All glassware was processed as previously described (Bottjer et al., 1984a) to ensure the absence of sterols. The following procedures were conducted using aseptic techniques and sterile materials as necessary. Preparation of culture medium components Caenorhabditis briggsae Maintenance Medium (CbMM) (Buecher et al., 1966) was purchased from Grand Island Biological Co., Grand Island, NY, or else prepared in individual lots in our laboratory. The 2X CbMM was sealed and stored in the dark at 4°C until its use. Formalin-killed Escherichia coll" was prepared by a modification of the methods of Bolla et al. (1972) as previously described (Bottjer et al., 1984a). The bacterial preparation was refrigerated and was used within 24 hr in the formulation of media described below. Cholesterol (99~o+ ) was purchased from Sigma Chemical Co., St. Louis, MO. Tween 80 was obtained from ICI Americas Inc., Wilmington, DE. 25-Azacoprostane (99~ + ) was prepared as described by Svoboda et al. (1972). Cholesterol and ASA-6 were dissolved in minimal amounts of absolute ethanol and prepared as stock solutions of 500/~g/ml in Tween 80 (1~'/o; w/v in double-glass distilled water). These were steam-sterilized for 15 min at 121°C and stored in the dark at 4°C. Preparation of cultures Stock cultures of both C. briggsae and P. redivivus were prepared in 50ml screw-capped Erlenmeyer flasks, each containing 2 ml of medium. The stock culture medium consisted of 2X CbMM:formalin-killed suspension of E. coli (1:1, v/v) to which cholesterol in Tween 80 was added to achieve final concentrations of 5 # g cholesterol and 50/~ g Tween 80 per ml. Each flask was inoculated with a 0.1 ml aliquot from a pre-existing stock culture. These stock cultures were incubated at 25°C and served as the source of eggs (C. briggsae) or newly-laid larvae (P. redivivus) to be used for initiating experiments. Experiments testing the effects of ASA-6 on growth, development and reproduction in axenic cultures of C. briggsae and P. redivivus were carried out in Falcon 3047, 24-well tissue culture plates with lids (Becton Dickinson, Oxnard, CA). The four media used in the experiments were designated as CHOL, CHOL/ASA-6, ASA-6 and TWEEN. The media consisted of 2X CbMM:f0rmalin-killed suspension of E. coli (1 : 1, v/v) plus: (a) 5 #g cholesterol per ml medium (CHOL), (b) 5 # g cholesterol and 25#g ASA-6 per ml medium (CHOL/ASA-6) (c) 25 #g ASA-6 per ml medium (ASA-6), and (d) 50 #g Tween 80 per ml medium (TWEEN).
The same amount of Tween 80 was contained in each medium as in (d). To initiate an experiment, l ml of each medium was pipetted into each of six wells. For experiments with C. briggsae, eggs from a 5-day-old stock culture were washed three times in Krebs-Ringer phosphate buffer (KRP) and allowed to hatch in 1X CbMM. Newly-hatched larvae were washed in KRP and five larvae were placed in each well. Experiments with P. redivivus were begun with newlydeposited, washed L2 from 5-day-old stock cultures. Ten larvae were placed into each well. Each experiment was performed twice. All experimental cultures were incubated in the dark at 25°C, agitated gently twice a day and terminated after 5 days' growth. At termination, the nematodes in each well were evaluated visually under a dissecting microscope in terms of development of the adult stage. Reproduction by C. br~ggsae was scored as the mean number of eggs deposited per adult per well. Reproduction by P. redivivus was scored as the mean number of larvae (L2) deposited per adult female per well. The growth, development and reproduction of either species of nematode in CHOL medium served as the basis of reference for all experiments. After evaluation, eggs or larvae from wells in wl-ieh reproduction had occurred were removed, washed twice with KRP and used to initiate the next experiment as previously described. Hence, first generation eggs or larvae from a medium were sub-cultured into fresh medium as indicated and became the progenitors for a second generation, etc. After removal of eggs or larvae for initiation of the succeeding experiment, the adults in each well were removed, washed in KRP and examined under a microscope for morphological changes with respect to CHOL medium adults. The worms were then heat-fixed and their length determined. Some living adults, after washing, were immobilized in propylene phenoxytol (1~o in distilled water) and photographed.
Sterility tests Samples of stock cultures, formalin-killed E. coli, CbM M, solutions of test compounds and samples from each culture medium at the termination of an experiment were placed into tubes of thioglycollate broth and incubated at 37°C for 7 days. If any part of the culture system was positive for microbial contamination, the results were discarded. Data were evaluated by Student's t-test. The 0.05 probability level was considered significant. RESULTS
Effect o f A S A - 6 on growth and reproduction The effects of ASA-6 o n growth, development, a n d r e p r o d u c t i o n o f Caenorhabditis briggsae are listed in Table 1. Newly-hatched, washed L1 from stock cultures ( C H O L m e d i u m ) placed into the four media as previously described, after 5 days developed a n d deposited eggs in all media. The m e a n lengths of first generation adults which developed in the two media lacking cholesterol ( T W E E N a n d ASA-6) were n o t significantly different from the m e a n lengths of adults which developed in the two media c o n t a i n i n g cholesterol ( C H O L a n d C H O L / A S A - 6 ) . However, there was a significant decrease in egg deposition by adults which developed in ASA-6 m e d i u m as c o m p a r e d with adults in the other media. Eggs deposited by first generation adults were collected, allowed to h a t c h in 1X C b M M , washed two times with K R P a n d subcultured into fresh media. Larvae o b t a i n e d from C H O L a n d C H O L / ASA-6 media grew, developed a n d deposited eggs normally w h e n subcultured into the same media.
Azasteroid inhibition of free-living nematodes
101
Table 1. Effect of ASA-6 on growth, development and reproduction of Caenorhabditis briggsae* % development Medium
Mean l e n g t h
to adult
(am+_SEM)
Generation
1st
2nd
C~OL
100
100
IgEl~
100
ASA-6
100
CItOL/ASA-6
100
40 ~§
0 5§ 100
Generation
1st
Eggs d e p o s i t e d
per adultt
Generation
2nd
1st
2nd
1.19 ±0.08
1.24 ~0.08
8.2
8.5
0.906_+0.015
0.661±0.01~§
8.0
05§
0.823+-0.32
0 . 4 5 +-0.07~§
4.0~
0~§
1.135+-0.075
1.01 ±0.207
8.7
8.5
*Five newly-hatched LI from stock cultures (CHOL medium) were washed twice in KRP and placed into microwells containing l ml of medium. Eggs of the succeeding generation were transferred to IX CbMM and allowed to hatch. The L1 were washed twice in KRP and placed into fresh medium. All measurements were taken after 5 days' growth. Six wells per medium in each of two experiments. tThe mean of the total number of eggs deposited per well divided by the total number of adults per well in each of 12 wells on the fifth day of culture. ~Significant difference between generations in the same medium (P < 0.05 by Student's t-test). §Significant difference between CHOL medium and experimental medium within the same generation (P < 0.05 by Student's t-test).
O n l y 4 0 ~ o f the larvae f r o m T W E E N m e d i u m d e v e l o p e d to the a d u l t stage in the s e c o n d g e n e r a t i o n . T h e s e a d u l t s were significantly s h o r t e r t h a n C H O L a d u l t s a n d did n o t r e p r o d u c e . N o l a r v a e d e v e l o p e d to a d u l t s after a s e c o n d g e n e r a t i o n o f g r o w t h in A S A - 6 medium.
Caenorhabditis briggsae larvae f r o m eggs laid in C H O L / A S A - 6 m e d i u m w e r e t r a n s f e r r e d t h r o u g h five g e n e r a t i o n s ( T a b l e 3). A l t h o u g h d e v e l o p m e n t to the a d u l t stage w a s n o r m a l , the m e a n length o f a d u l t s w a s significantly d e c r e a s e d by the f o u r t h g e n e r a t i o n . E g g d e p o s i t i o n decreased significantly by the f o u r t h
Table 2. Effects of ASA-6 on growth, development and reproduction of Panagrellus redivivus * % development Medittm
Mean l e n g t h
to adult
(mm+SEN)
Generation
ist
2nd
Clt0L
100
100
TWEEIq
100
ASA-6
100
Ctl0L/ASA-6
100
L2 d e p o s i t e d per a d u l t
Generation
Generation
ist
2nd
2.21+0.1 1.76+-0.13
14.5
15.0
80 ~§ 92.01+0.07 d1.60+_-0.18
1,65+0.06 5§ 1.55+0.04
14.1
0 .O~§
80 ~§ 91.81+0.11 d1.66+0.28
1.51+0.183~§ 1.13+0.03 5§
14.9
0.0~§
2.01+0.07 1.60+0.14
15.0
100
1st
femalet
92.09+0.15 d1.77~0.08
91.98+_0.22 61.60+0.18
2nd
14.0
*Ten newly deposited L2 from stock cultures (CHOL medium) washed twice in KRP and placed into microwells containing 1 ml of medium. L2 of the succeeding generation washed twice in KRP dnd transferred into fresh medium. All measurements taken after 5 days' growth. Six wells per medium in each of two experiments. tThe mean of the total number of L2 deposited per well divided by the total number of adult females per well in each of 12 wells on the fifth day of culture. :~Significant difference between generations in the same medium (P <0.05 by Student's t-test). §Significant difference between CHOL medium and experimental medium within the same generation (P < 0.05 by Student's t-test).
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KURT P. BOTTJER et al. Table 3. Effect of continued transfer through CHOL/ASA-6 medium on development, growth and reproduction of Caenorhabditis' briggsae and Panagrellus redivivus* C. b g i e g s a e
Generation
P.
% develop.
Mean l e n g t h
Eggs
to adult
(n~+$EM)
per adult
doposited
redivivus
% develop.
Mean l e n g t h
1.2 d e p o s i t e d
to adult
(mm+SEM)
per a d u l t ~
1st
100
1.135+0o075 --
8.7
100
9 1.98+0.22 ~ 1.60+--0.18
15.0
2rid
i00
1.0
+0.207
8.5
100
9 2.01+0.07 1.60+--0.14
14.0
3rd
100
0.99 +-0.05
8.0
100
9 2.04+0.16 1.60+_--0.16
14.2
4th
100
0.89 Z0.06 t
5.2 t
i00
~ 1.92+0.15 d 1.61~_0.iI
14.0
5th
100
0.80 +-O.07t
0
i00
9 1.97+0.12 1.60+-0.09
14.4
t
*Medium, cultureconditionsand measurementsas describedin previoustables. tSignificantdifferencefrom first generationvalues(P < 0.05 by Student'st-test).
generation and was completely inhibited by the fifth generation. Thus, the presence of 5/*g of cholesterol per ml medium delayed the inhibition by ASA-6. Table 2 presents the effects of ASA-6 on the growth, development and reproduction of P. redivivus. Newly-deposited, washed L2 from stock cultures developed to adults and reproduced in all four media initially inoculated. As was observed for C. briggsae, cultivation and subculture of larvae through TWEEN medium or ASA-6 medium resulted in a complete inhibition of reproduction after the second transfer. In contrast to C. briggsae, 80~ of the P. redivivus larvae developed into adults after the second transfer through ASA-6 medium. The continuous cultivation and subculture of P. redivivus larvae in CHOL/ASA-6 medium (Table 3) resulted in normal
growth, development and larval deposition after five generations. Thus, the presence of 5/,g cholesterol/ ml medium prevented azasteroid inhibition in P. redivivus.
Morphology o f inhibited nematodes The abnormalities manifested in cholesteroldeficient and azasteroid-inhibited cultures of C. briggsae and P. redivivus were similar to those reported in N. brasiliensis and N. dubius cultures grown under similar conditions (Bottjer et al., 1984a). There was a degeneration of intestinal cells and an abnormal dispersion of lipid globules in both species of free-living nematodes. This is demonstrated in Figs 1-3 which compare a second generation C. briggsae adult from CHOL medium (Fig. 1) to one from
Fig. 1.
Azasteroid inhibition of free-living nematodes
103
Fig. 2.
Fig. 3. Figs 1-3. Comparative morphology of Caenorhabditis briggsae cultured for two generations in various media ( x 200). Fig. 1. Adult grown in CHOL medium showing normal development with many eggs in the uteri. Fig. 2. Adult grown in sterol-free TWEEN medium. Note inhibition of development of reproductive organs, degenerative changes and shorter body length compared to worm in Fig. 1. Fig. 3. Inhibited larva from ASA-6 medium. Only early development of the reproductive anlage has occurred. TWEEN medium (Fig. 2) and to a second generation larva from ASA-6 medium (Fig. 3). In both C. briggsae and P. redivivus, the morphology resulting from azasteroid inhibition was very similar to that arising from cholesterol deficiency. Cholesterol delayed azasteroid inhibition of reproduction in C. briggsae. First generation adults from CHOL medium displayed normal internal morphology and contained an average of 8-14 fully developed eggs. Fourth generation adults that developed in CHOL/ASA-6 medium displayed abnormal
lipid accumulation, a reduction in the number of developing ova as well as a decrease in the number of fully developed eggs. Adults which developed after one generation in ASA-6 medium showed degeneration of intestinal cells and reproductive organs. There were only a few developing ova and no fully developed eggs in these organisms. The effects of ASA-6 on reproduction in P. redivivus is demonstrated in Figures 4-7. Figures 4 and 6 show the morphologic features and differentiation of a normal adult female and male, respectively,
104
KURT P. BOTTJER et al.
Fig. 4.
Fig. 6.
Fig. 5.
Fig. 7.
Figs 4-7. Panagrellus redivivus adults cultured in the presence or absence of ASA-6 ( x 200). Fig. 4. First generation adult female cultured in CHOL medium. Uterus contains hatched larvae developing normally. Fig. 5. Second generation adult female cultured in ASA-6 medium. Note degenerating ova and absence of larval development. Fig. 6. First generation adult male (CHOL medium) containing a normal testis (arrow). Fig. 7. Second generation adult male cultured in ASA-6 medium. Arrow points to poorly developed and degenerating testis.
Azasteroid inhibition of free-living nematodes cultured for two generations in CHOL medium. In a female from a second generation ASA-6 culture (Fig. 5), there were no larvae present; only degenerating ova. The testis of a second generation male from ASA-6 medium (Fig. 7), was underdeveloped and displayed areas of degeneration. Adults which developed in CHOL/ASA-6 medium were comparable to CHOL controls with regard to internal development, morphology and reproduction. As in C. briggsae, the morphology of P. redivivus cultured in ASA-6 medium was similar to worms cultured in TWEEN medium. DISCUSSION By utilizing axenic cultures of C. briggsae and P. redivivus, the effects of ASA-6 on the growth and development from egg to adult worm, on reproductive biology, as well as on subsequent generations, was studied (Tables 1, 2, and 3) in medium in which the sterol content was strictly controlled. Inhibition of growth and reproduction due to cultivation in sterol-free medium has been demonstrated previously in C. briggsae (Hieb and Rothstein, 1968) and in P. redivivus (Cole and Dutky, 1969). The results of this study agree with these reports in that C. briggsae and P. redivivus were shown to develop into adults which reproduced normally for one generation but failed to do so in the second generation. Another free-living nematode, Turbatrix aceti, failed to reproduce normally after one generation's growth in sterol-free medium (Cole and Dutky, 1969). Mauro and Weinstein (1979) have suggested that differences in the amount of endogenous sterol incorporated by a nematode species into its eggs may be responsible for differences between species and individual organisms in the requirement for exogenous sterol for normal growth and development. This suggestion deserves further investigation as it offers an explanation for the different effects that cultivation in sterol-free medium has on growth and reproduction in nematodes. The experimental design, by necessity, has made the interpretation of results obtained from ASA-6 medium complex. In this medium which lacks cholesterol, the effects of the inhibitor (ASA-6) as well as the effects of the sterol deficiency must both be considered. The inhibitive effects of ASA-6, however, become apparent when one compares the results obtained from ASA-6 medium to those from the sterol-deficient medium without ASA-6 (TWEEN). After one generation, adults in ASA-6 medium had a shorter mean length and deposited fewer eggs than did T W E E N adults. After two generations, adult development was completely inhibited in ASA-6 medium, but not in TWEEN medium. The inhibitory effects on development and reproduction resulting from transfer and subculture of larvae through sterol-free medium or sterol-free medium containing ASA-6 were more apparent in C. briggsae than in P. redivivus. Although reproduction was completely inhibited by the second transfer of larvae of both species into these media, P. redivivus larvae were able to develop into adults in ASA-6 medium. The presence of cholesterol in the growth medium delayed but did not prevent inhibition by
105
ASA-6 in C. briggsae. However, the presence of cholesterol prevented azasteroid-inhibition in P. rediv&us under the experimental conditions used. Panagrellus redivivus may be able to overcome ASA-6 inhibition because the larvae hatch in utero, develop and leave the female as L2. Therefore, the larvae may be acquiring sufficient cholesterol from the mother or developing past a point necessary to ensure normal development when cultured in the presence of both exogenous cholesterol and ASA-6. It is also possible that P. redivivus may have the metabolic capability to degrade the inhibitor or select against its uptake from the culture medium. These observations are different from those reported for N. brasiliensis and N. dubius (Bottjer et al., 1984a) and may indicate that the free-living stages of animal parasitic nematodes are more sensitive to azasteroid inhibition than are some free-living nematodes. Culture of C. briggsae and P. redivivus in sterol-free medium with or without ASA6 resulted in inhibition of growth, development and reproduction and produced morphological abnormalities (Figs. 1-7) which were comparable to those seen in N. brasiliensis and N. dubius larvae grown in similar media (Bottjer et al., 1984a). In the presence of cholesterol, ASA-6 eventually inhibited growth and reproduction in C. briggsae. One mechanism to account for this inhibition may be a competition for uptake and utilization between azasteroid and cholesterol that results in a decrease in the amount of available sterol to a level below the requirement for growth and reproduction for C. briggsae but not for P. redivivus. Recently, Chitwood et al. (1984) have demonstrated an inhibition by ASA-6 of motility and reproduction of another species, C. elegans. The data presented and previous findings (Bottjer et al., 1984a) suggest that nematodes may exhibit a range of responses to azasteroids and that a variety of mechanisms to account for this may be involved. They also suggest that sterol utilization and metabolism may vary significantly between nematode species. Azasteroids are known to interfere with the metabolism of insect ecdysteroids (Marks et al., 1978; Svoboda et al., 1978). Ecdysteroid-like substances occur in nematodes (Dennis, 1976; Mendis et al., 1983) and evidence for their synthesis by larval stages reared axenically in the absence of ecdysteroids has been reported (Bottjer et al., 1984b). The nematodes in this study were affected by ASA-6 in a manner similar to and at concentrations lower than those affecting some insects. ASA-6 inhibited growth of certain insects at concentrations of 0.5 to 2.0 ppm, whereas it affected development of Blattella germanica and Tribolium confusum at a dietary concentration of 250-500ppm (Svoboda et al., 1972). Growth, development and egg production of the southwestern corn borer, Diatraea grandiosella, were inhibited at a dietary concentration of 150 ppm (Al-Izzi and Hopkins, 1982). In our study, C. briggsae and P. redivivus growth, development and reproduction were inhibited at a concentration of 25 ppm. In summary, the azasteroid, ASA-6, inhibits the growth, development and reproduction of C. briggsae after two generations of culture in sterol-deficient medium. The presence of exogenous cholesterol at the concentration tested prevented azasteroid inhibition in P. redivivus cultures, but not in C. brigg-
106
KURT P. BOTTJER et al.
sac. These results indicate t h a t different species o f n e m a t o d e s m a y exhibit different responses to azasteroid and also t h a t sterol utilization a n d m e t a b o lism m a y vary between species. T h e similarities between the k n o w n effects of azasteroids o n insects a n d those presented in this study o n n e m a t o d e s suggest like m e c h a n i s m s of action by the i n h i b i t o r in b o t h groups of organisms. Acknowledgements--The authors wish to acknowledge the technical assistance of Katherine Taylor and the secretarial assistance of Karen Aberli. REFERENCES
Al-lzzi M. A. J. and Hopkins T. L. (1982) Effects of 25-azasteroids on development and reproduction of the southwestern corn borer Diatraea grandiosella Dyar. J. Insect Physiol. 28, 267-271. Bolla R. I., Weinstein P. P. and Lou C. (1972) In vitro nutritional requirements of Nippostrongylus brasiliensis. I. Effects of sterols, sterol derivatives and heme compounds on the free-living stages. Comp. Biochem. Physiol. 43B, 487-501. Bottjer K. P., Weinstein P. P. and Thompson M. J. (1984a) Effects of azasteroids growth and development of the free-living stages of Nippostrongylus brasiliensis and Nematospiroides dubius. Comp. Biochem. Physiol. 78B, 805 811. Bottjer K. P., Whisenton L. R. and Weinstein P. P. (1984b) Ecdysteroid-like substances in Nippostrongylus brasiliensis. J. Parasitol. 70, 986-987. Buecher E. J., Hansen E. L. and Yarwood E. A. (1966) Ficoll activation of a protein essential for maturation of the free-living nematode, Caenorhabditis briggsae. Proc. Soc. exp. Biol. Med. 121, 390-393. Chitwood D. J., Lusby W. R., Lozano R., Thompson M. J. and Svoboda J. A. (1983) Novel nuclear methylation of sterols by the nematode Caenorhabditis elegans. Steroids 42, 311 319. Chitwood D. J., Lusby W. R., Lozano R. and Thompson M. J. (1984) Sterol metabolism in the nematode Caenorhabditis briggsae. Lipids 19, 500-506. Cole R. J. and Dutky S. R. (1969) A sterol requirement in Turbatrix aceti and Panagrellus redivivus. J. Nematol. 1, 72-75. Dennis R. D. W. (1976) Insect morphogenetic hormones and developmental mechanisms in the nematode, Nematospiroides dubius. Comp. Biochem. Physiol. 53A, 53-56. Hieb W. F. and Rothstein M. (1968) Sterol requirements for reproduction of a free-living nematode. Science 160, 778-780.
Lu N. C., Newton C. and Stokstad E. L. R. (1977) The requirement of sterol and various sterol precursors in free-living nematodes. Nematologica 23, 57-61. Lu N. C., Hugenberg Jr., G., Briggs M. and Stokstad E. L. R. (1978) The growth promoting activity of several lipid-related compounds in the free-living nematode Caenorhabditis briggsae. Proc. Soc. exp. Biol. Med. 158, 187-191. Marks E. P., Robbins W. E. and Thompson M. J. (1978) The effects of certain ecdysteroids and inhibitory amines and amides on the metabolism of 22,25-dideoxyecdysone in cockroach organ culture. Lipids 13, 259 263. Mauro N. A. and Weinstein P. P. (1979) Effects of sterols, rat hematin and coproporphyrin on the free-living stages of Nematospiroides dubius. Int. J. Parasitol. 9, 421~,27. Mendis A. H. W., Rose, M. E., Rees H. H. and Goodwin T. W. (1983) Ecdysteroids in adults of the nematode, Dirofilaria immitis. Molec. Biochem. Parasitol. 9, 209-226. Rothstein M. (1968) Nematode biochemistry. IX. Lack of sterol biosynthesis in free-living nematodes. Comp. Biochem. Physiol. 27, 309-317. Rothstein M. and Cook E. (1965) Nematode biochemistry--VI. Conditions for axenic culture of Turbatrix aceti, Panagrellus redivivus, Rhabditis anomala and Caenorhabditis briggsae. Comp. Biochem. Physiol. 17, 683~92. Svoboda J. A. and Robbins W. E. (1967) Conversion of beta sitosterol to cholesterol blocked in an insect by hypocholesterolemic agents. Science 156, 1634-1638. Svoboda J. A. and Robbins W. E. (1968) Desmosterol as a common intermediate in the conversion of a number of C28 and C29 plants sterols to cholesterol by the tobacco hornworm. Experientia 24, 1131-1139. Svoboda J. A. and Robbins W. E. (1971) The inhibitive effect of azasterols on steroid metabolism and growth and development in insects with special reference to the tobacco hornworm. Lipids 6, 113-119. Svoboda J. A., Thompson M. J. and Robbins W. E. (1972) Azasteroids: potent inhibitors of insect molting and metamorphosis. Lipids 7, 553-556. Svoboda J. A., Thompson M. J., Robbins W. E. and Kaplainis J. N. (1978) Insect steroid metabolism. Lipids 13, 742-753. Thompson M. J., Serban N. N., Robbins W. E., Svoboda J. A., Shortino T. J., Dutky S. R. and Cohen C. F. (1975) Inhibitive effects of structurally modified azasteroids and related nitrogen containing sterols on insect growth and development. Lipids 10, 615-622. Weinstein P. P. and Jones M. F. (1959) Development in vitro of some parasitic nematodes of vertebrates. Ann. N.Y. Acad. Sci. 77, 137-162. Willet J. D. and Downey W. L. (1974) Sterol biosynthesis in the free-living nematode PanagreHus redivivus. Biochem. J. 138, 233-237.
0305-0491/85 $3.00+ 0.00 © 1985 Pergamon Press Ltd
EFFECTS OF AN AZASTEROID ON GROWTH, DEVELOPMENT AND REPRODUCTION OF THE FREE-LIVING NEMATODES CAENORHABDITIS BRIGGSAE A N D PANAGRELLUS REDIVIVUS* KURT P. BOTTJERt and PAUL P. WEINSTEIN Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556, USA and MALCOLM J. THOMPSON Insect Physiology Laboratory, USDA-APS, Beltsville, MD 20705, USA (Received 6 December 1984) Abstract--1. The azasteroid, 25-azacoprostane (ASA-6), was evaluated for its effects on the growth,
development and reproduction of the free-living nematodes, Caenorhabditis briggsae and Panagrellus redivivus. 2. The axenic culture medium for either species of nematode consisted of Caenorhabditis briggsae Maintenance Medium (CbMM): formalin-killed Escherichia coli (1:1) with or without the addition of 5 #g cholesterol per ml and/or 25 pg ASA-6 per ml medium. All cultures also contained 50 #g Tween 80 per ml medium. 3. After two generations of growth in sterol-deficient media, both species displayed a decrease in mean length, a decrease in the percent development to the adult stage and an inhibition of reproductive capability. These effects were more apparent in the sterol-deficient medium containing ASA-6. 4. In the presence of cholesterol and ASA-6, growth and reproduction of C. briggsae, but not of P. redivivus, was inhibited after five generations. 5. Morphologic abnormalities of azasteroid-inhibited worms were similar to those shown by worms cultured in sterol-deficient medium. 6. These results suggest that different species of nematodes may exhibit different responses to azasteroid and that sterol utilization and metabolism may vary between nematode species. In addition, the similarities between the known effects of azasteroid inhibition in insects and those presented in this study on nematodes suggest a similar mechanism of action by the inhibitor in both groups of organisms.
INTRODUCTION
opment and reproduction (Rothstein, 1968; Bolla et al., 1972; Willet and Downey, 1974; Lu et al., 1977) it seemed that azasteroids might be a useful tool with which to study the role of sterol in nematode development. In axenic cultures of the free-living stages of the rodent-parasitic nematodes, Nippostrongylus brasiliensis and Nematospiroides dubius, azasteroids, especially ASA-6, were shown to decrease the percentage of newly-hatched first stage larvae (L1) that developed to the infective third stage (L3) as well as to decrease the mean length of L3 (Bottjer et al., 1984a). The in vitro cultivation of the free-living stages of these parasites is an established method by which the amount of exogenous sterol available to the developing larvae can be strictly controlled (Bolla et al., 1972; Mauro and Weinstein, 1979). However, the in vitro cultivation of N. brasiliensis and N. dubius from L3 to adult requires a complex medium containing chick embryo homogenate and serum of which the sterol content is undefined (Weinstein and Jones, 1959). Therefore, a model in vitro system for the study of the effects of sterol deficiency or sterol metabolism inhibitors on the parasitic cycle of nematode development to sexually mature adults is as yet unavailable.
Azasteroids are vertebrate hypocholesterolemic agents that have proved useful in the study of insect sterol metabolism. These compounds inhibit the delta24-sterol reductase system that is responsible for the conversion of dietary phytosterols to cholesterol (Svoboda and Robbins, 1967, 1968). Certain azasteroids, including 25-azacholestane (ASA-3) and 25-azacoprostane (ASA-6) disrupt development in insects whose test diets contain sufficient cholesterol to support normal growth and development, suggesting that azasteroids affect pathways of sterol metabolism other than those involved in cholesterol production (Svoboda and Robbins, 1971; Svoboda et al., 1972; Thompson et al., 1975). Subsequently, it has been demonstrated that azasteroids interfere also in the metabolism of insect ecdysteroids (Marks et al., 1978; Svoboda et al., 1978). Because nematodes, like insects, appear to require a dietary or exogenous source of sterol for continuous normal growth, devel*Supported in part by NIH grants AI07030 and AI09625 to PPW. tPresent Address: Regional Parasite Research Laboratory, USDA, ARS, P.O. Box 952, Auburn, AL 36830, USA. 99
KURT P. BOTTJER et al.
100
The sterol c o n t e n t in the m e d i u m used for the in vitro cultivation of Caenorhabdit& briggsae a n d Panagrellus redivivus to the adult stage, however, can be controlled. These free-living n e m a t o d e s can be grown axenically in vitro from egg to adult t h r o u g h successive generations in b o t h complex a n d chemically defined media ( R o t h s t e i n a n d Cook, 1965; Hieb and Rothstein, 1968; Lu et al., 1978). O u r objectives were to study the effects of a n inhibitor o f sterol metabolism upon nematode growth and development to the adult stage a n d on adult n e m a t o d e s by the addition of the azasteroid, 2 5 - a z a c o p r o s t a n e (ASA6), to axenic cultures of C. briggsae a n d P. redivivus. This c o m p o u n d has been reported to inhibit b o t h a delta24-sterol reductase a n d a deltaV-sterol isomerase (Chitwood et al., 1983). MATERIAL A N D M E T H O D S
Processing of glassware All glassware was processed as previously described (Bottjer et al., 1984a) to ensure the absence of sterols. The following procedures were conducted using aseptic techniques and sterile materials as necessary. Preparation of culture medium components Caenorhabditis briggsae Maintenance Medium (CbMM) (Buecher et al., 1966) was purchased from Grand Island Biological Co., Grand Island, NY, or else prepared in individual lots in our laboratory. The 2X CbMM was sealed and stored in the dark at 4°C until its use. Formalin-killed Escherichia coll" was prepared by a modification of the methods of Bolla et al. (1972) as previously described (Bottjer et al., 1984a). The bacterial preparation was refrigerated and was used within 24 hr in the formulation of media described below. Cholesterol (99~o+ ) was purchased from Sigma Chemical Co., St. Louis, MO. Tween 80 was obtained from ICI Americas Inc., Wilmington, DE. 25-Azacoprostane (99~ + ) was prepared as described by Svoboda et al. (1972). Cholesterol and ASA-6 were dissolved in minimal amounts of absolute ethanol and prepared as stock solutions of 500/~g/ml in Tween 80 (1~'/o; w/v in double-glass distilled water). These were steam-sterilized for 15 min at 121°C and stored in the dark at 4°C. Preparation of cultures Stock cultures of both C. briggsae and P. redivivus were prepared in 50ml screw-capped Erlenmeyer flasks, each containing 2 ml of medium. The stock culture medium consisted of 2X CbMM:formalin-killed suspension of E. coli (1:1, v/v) to which cholesterol in Tween 80 was added to achieve final concentrations of 5 # g cholesterol and 50/~ g Tween 80 per ml. Each flask was inoculated with a 0.1 ml aliquot from a pre-existing stock culture. These stock cultures were incubated at 25°C and served as the source of eggs (C. briggsae) or newly-laid larvae (P. redivivus) to be used for initiating experiments. Experiments testing the effects of ASA-6 on growth, development and reproduction in axenic cultures of C. briggsae and P. redivivus were carried out in Falcon 3047, 24-well tissue culture plates with lids (Becton Dickinson, Oxnard, CA). The four media used in the experiments were designated as CHOL, CHOL/ASA-6, ASA-6 and TWEEN. The media consisted of 2X CbMM:f0rmalin-killed suspension of E. coli (1 : 1, v/v) plus: (a) 5 #g cholesterol per ml medium (CHOL), (b) 5 # g cholesterol and 25#g ASA-6 per ml medium (CHOL/ASA-6) (c) 25 #g ASA-6 per ml medium (ASA-6), and (d) 50 #g Tween 80 per ml medium (TWEEN).
The same amount of Tween 80 was contained in each medium as in (d). To initiate an experiment, l ml of each medium was pipetted into each of six wells. For experiments with C. briggsae, eggs from a 5-day-old stock culture were washed three times in Krebs-Ringer phosphate buffer (KRP) and allowed to hatch in 1X CbMM. Newly-hatched larvae were washed in KRP and five larvae were placed in each well. Experiments with P. redivivus were begun with newlydeposited, washed L2 from 5-day-old stock cultures. Ten larvae were placed into each well. Each experiment was performed twice. All experimental cultures were incubated in the dark at 25°C, agitated gently twice a day and terminated after 5 days' growth. At termination, the nematodes in each well were evaluated visually under a dissecting microscope in terms of development of the adult stage. Reproduction by C. br~ggsae was scored as the mean number of eggs deposited per adult per well. Reproduction by P. redivivus was scored as the mean number of larvae (L2) deposited per adult female per well. The growth, development and reproduction of either species of nematode in CHOL medium served as the basis of reference for all experiments. After evaluation, eggs or larvae from wells in wl-ieh reproduction had occurred were removed, washed twice with KRP and used to initiate the next experiment as previously described. Hence, first generation eggs or larvae from a medium were sub-cultured into fresh medium as indicated and became the progenitors for a second generation, etc. After removal of eggs or larvae for initiation of the succeeding experiment, the adults in each well were removed, washed in KRP and examined under a microscope for morphological changes with respect to CHOL medium adults. The worms were then heat-fixed and their length determined. Some living adults, after washing, were immobilized in propylene phenoxytol (1~o in distilled water) and photographed.
Sterility tests Samples of stock cultures, formalin-killed E. coli, CbM M, solutions of test compounds and samples from each culture medium at the termination of an experiment were placed into tubes of thioglycollate broth and incubated at 37°C for 7 days. If any part of the culture system was positive for microbial contamination, the results were discarded. Data were evaluated by Student's t-test. The 0.05 probability level was considered significant. RESULTS
Effect o f A S A - 6 on growth and reproduction The effects of ASA-6 o n growth, development, a n d r e p r o d u c t i o n o f Caenorhabditis briggsae are listed in Table 1. Newly-hatched, washed L1 from stock cultures ( C H O L m e d i u m ) placed into the four media as previously described, after 5 days developed a n d deposited eggs in all media. The m e a n lengths of first generation adults which developed in the two media lacking cholesterol ( T W E E N a n d ASA-6) were n o t significantly different from the m e a n lengths of adults which developed in the two media c o n t a i n i n g cholesterol ( C H O L a n d C H O L / A S A - 6 ) . However, there was a significant decrease in egg deposition by adults which developed in ASA-6 m e d i u m as c o m p a r e d with adults in the other media. Eggs deposited by first generation adults were collected, allowed to h a t c h in 1X C b M M , washed two times with K R P a n d subcultured into fresh media. Larvae o b t a i n e d from C H O L a n d C H O L / ASA-6 media grew, developed a n d deposited eggs normally w h e n subcultured into the same media.
Azasteroid inhibition of free-living nematodes
101
Table 1. Effect of ASA-6 on growth, development and reproduction of Caenorhabditis briggsae* % development Medium
Mean l e n g t h
to adult
(am+_SEM)
Generation
1st
2nd
C~OL
100
100
IgEl~
100
ASA-6
100
CItOL/ASA-6
100
40 ~§
0 5§ 100
Generation
1st
Eggs d e p o s i t e d
per adultt
Generation
2nd
1st
2nd
1.19 ±0.08
1.24 ~0.08
8.2
8.5
0.906_+0.015
0.661±0.01~§
8.0
05§
0.823+-0.32
0 . 4 5 +-0.07~§
4.0~
0~§
1.135+-0.075
1.01 ±0.207
8.7
8.5
*Five newly-hatched LI from stock cultures (CHOL medium) were washed twice in KRP and placed into microwells containing l ml of medium. Eggs of the succeeding generation were transferred to IX CbMM and allowed to hatch. The L1 were washed twice in KRP and placed into fresh medium. All measurements were taken after 5 days' growth. Six wells per medium in each of two experiments. tThe mean of the total number of eggs deposited per well divided by the total number of adults per well in each of 12 wells on the fifth day of culture. ~Significant difference between generations in the same medium (P < 0.05 by Student's t-test). §Significant difference between CHOL medium and experimental medium within the same generation (P < 0.05 by Student's t-test).
O n l y 4 0 ~ o f the larvae f r o m T W E E N m e d i u m d e v e l o p e d to the a d u l t stage in the s e c o n d g e n e r a t i o n . T h e s e a d u l t s were significantly s h o r t e r t h a n C H O L a d u l t s a n d did n o t r e p r o d u c e . N o l a r v a e d e v e l o p e d to a d u l t s after a s e c o n d g e n e r a t i o n o f g r o w t h in A S A - 6 medium.
Caenorhabditis briggsae larvae f r o m eggs laid in C H O L / A S A - 6 m e d i u m w e r e t r a n s f e r r e d t h r o u g h five g e n e r a t i o n s ( T a b l e 3). A l t h o u g h d e v e l o p m e n t to the a d u l t stage w a s n o r m a l , the m e a n length o f a d u l t s w a s significantly d e c r e a s e d by the f o u r t h g e n e r a t i o n . E g g d e p o s i t i o n decreased significantly by the f o u r t h
Table 2. Effects of ASA-6 on growth, development and reproduction of Panagrellus redivivus * % development Medittm
Mean l e n g t h
to adult
(mm+SEN)
Generation
ist
2nd
Clt0L
100
100
TWEEIq
100
ASA-6
100
Ctl0L/ASA-6
100
L2 d e p o s i t e d per a d u l t
Generation
Generation
ist
2nd
2.21+0.1 1.76+-0.13
14.5
15.0
80 ~§ 92.01+0.07 d1.60+_-0.18
1,65+0.06 5§ 1.55+0.04
14.1
0 .O~§
80 ~§ 91.81+0.11 d1.66+0.28
1.51+0.183~§ 1.13+0.03 5§
14.9
0.0~§
2.01+0.07 1.60+0.14
15.0
100
1st
femalet
92.09+0.15 d1.77~0.08
91.98+_0.22 61.60+0.18
2nd
14.0
*Ten newly deposited L2 from stock cultures (CHOL medium) washed twice in KRP and placed into microwells containing 1 ml of medium. L2 of the succeeding generation washed twice in KRP dnd transferred into fresh medium. All measurements taken after 5 days' growth. Six wells per medium in each of two experiments. tThe mean of the total number of L2 deposited per well divided by the total number of adult females per well in each of 12 wells on the fifth day of culture. :~Significant difference between generations in the same medium (P <0.05 by Student's t-test). §Significant difference between CHOL medium and experimental medium within the same generation (P < 0.05 by Student's t-test).
102
KURT P. BOTTJER et al. Table 3. Effect of continued transfer through CHOL/ASA-6 medium on development, growth and reproduction of Caenorhabditis' briggsae and Panagrellus redivivus* C. b g i e g s a e
Generation
P.
% develop.
Mean l e n g t h
Eggs
to adult
(n~+$EM)
per adult
doposited
redivivus
% develop.
Mean l e n g t h
1.2 d e p o s i t e d
to adult
(mm+SEM)
per a d u l t ~
1st
100
1.135+0o075 --
8.7
100
9 1.98+0.22 ~ 1.60+--0.18
15.0
2rid
i00
1.0
+0.207
8.5
100
9 2.01+0.07 1.60+--0.14
14.0
3rd
100
0.99 +-0.05
8.0
100
9 2.04+0.16 1.60+_--0.16
14.2
4th
100
0.89 Z0.06 t
5.2 t
i00
~ 1.92+0.15 d 1.61~_0.iI
14.0
5th
100
0.80 +-O.07t
0
i00
9 1.97+0.12 1.60+-0.09
14.4
t
*Medium, cultureconditionsand measurementsas describedin previoustables. tSignificantdifferencefrom first generationvalues(P < 0.05 by Student'st-test).
generation and was completely inhibited by the fifth generation. Thus, the presence of 5/*g of cholesterol per ml medium delayed the inhibition by ASA-6. Table 2 presents the effects of ASA-6 on the growth, development and reproduction of P. redivivus. Newly-deposited, washed L2 from stock cultures developed to adults and reproduced in all four media initially inoculated. As was observed for C. briggsae, cultivation and subculture of larvae through TWEEN medium or ASA-6 medium resulted in a complete inhibition of reproduction after the second transfer. In contrast to C. briggsae, 80~ of the P. redivivus larvae developed into adults after the second transfer through ASA-6 medium. The continuous cultivation and subculture of P. redivivus larvae in CHOL/ASA-6 medium (Table 3) resulted in normal
growth, development and larval deposition after five generations. Thus, the presence of 5/,g cholesterol/ ml medium prevented azasteroid inhibition in P. redivivus.
Morphology o f inhibited nematodes The abnormalities manifested in cholesteroldeficient and azasteroid-inhibited cultures of C. briggsae and P. redivivus were similar to those reported in N. brasiliensis and N. dubius cultures grown under similar conditions (Bottjer et al., 1984a). There was a degeneration of intestinal cells and an abnormal dispersion of lipid globules in both species of free-living nematodes. This is demonstrated in Figs 1-3 which compare a second generation C. briggsae adult from CHOL medium (Fig. 1) to one from
Fig. 1.
Azasteroid inhibition of free-living nematodes
103
Fig. 2.
Fig. 3. Figs 1-3. Comparative morphology of Caenorhabditis briggsae cultured for two generations in various media ( x 200). Fig. 1. Adult grown in CHOL medium showing normal development with many eggs in the uteri. Fig. 2. Adult grown in sterol-free TWEEN medium. Note inhibition of development of reproductive organs, degenerative changes and shorter body length compared to worm in Fig. 1. Fig. 3. Inhibited larva from ASA-6 medium. Only early development of the reproductive anlage has occurred. TWEEN medium (Fig. 2) and to a second generation larva from ASA-6 medium (Fig. 3). In both C. briggsae and P. redivivus, the morphology resulting from azasteroid inhibition was very similar to that arising from cholesterol deficiency. Cholesterol delayed azasteroid inhibition of reproduction in C. briggsae. First generation adults from CHOL medium displayed normal internal morphology and contained an average of 8-14 fully developed eggs. Fourth generation adults that developed in CHOL/ASA-6 medium displayed abnormal
lipid accumulation, a reduction in the number of developing ova as well as a decrease in the number of fully developed eggs. Adults which developed after one generation in ASA-6 medium showed degeneration of intestinal cells and reproductive organs. There were only a few developing ova and no fully developed eggs in these organisms. The effects of ASA-6 on reproduction in P. redivivus is demonstrated in Figures 4-7. Figures 4 and 6 show the morphologic features and differentiation of a normal adult female and male, respectively,
104
KURT P. BOTTJER et al.
Fig. 4.
Fig. 6.
Fig. 5.
Fig. 7.
Figs 4-7. Panagrellus redivivus adults cultured in the presence or absence of ASA-6 ( x 200). Fig. 4. First generation adult female cultured in CHOL medium. Uterus contains hatched larvae developing normally. Fig. 5. Second generation adult female cultured in ASA-6 medium. Note degenerating ova and absence of larval development. Fig. 6. First generation adult male (CHOL medium) containing a normal testis (arrow). Fig. 7. Second generation adult male cultured in ASA-6 medium. Arrow points to poorly developed and degenerating testis.
Azasteroid inhibition of free-living nematodes cultured for two generations in CHOL medium. In a female from a second generation ASA-6 culture (Fig. 5), there were no larvae present; only degenerating ova. The testis of a second generation male from ASA-6 medium (Fig. 7), was underdeveloped and displayed areas of degeneration. Adults which developed in CHOL/ASA-6 medium were comparable to CHOL controls with regard to internal development, morphology and reproduction. As in C. briggsae, the morphology of P. redivivus cultured in ASA-6 medium was similar to worms cultured in TWEEN medium. DISCUSSION By utilizing axenic cultures of C. briggsae and P. redivivus, the effects of ASA-6 on the growth and development from egg to adult worm, on reproductive biology, as well as on subsequent generations, was studied (Tables 1, 2, and 3) in medium in which the sterol content was strictly controlled. Inhibition of growth and reproduction due to cultivation in sterol-free medium has been demonstrated previously in C. briggsae (Hieb and Rothstein, 1968) and in P. redivivus (Cole and Dutky, 1969). The results of this study agree with these reports in that C. briggsae and P. redivivus were shown to develop into adults which reproduced normally for one generation but failed to do so in the second generation. Another free-living nematode, Turbatrix aceti, failed to reproduce normally after one generation's growth in sterol-free medium (Cole and Dutky, 1969). Mauro and Weinstein (1979) have suggested that differences in the amount of endogenous sterol incorporated by a nematode species into its eggs may be responsible for differences between species and individual organisms in the requirement for exogenous sterol for normal growth and development. This suggestion deserves further investigation as it offers an explanation for the different effects that cultivation in sterol-free medium has on growth and reproduction in nematodes. The experimental design, by necessity, has made the interpretation of results obtained from ASA-6 medium complex. In this medium which lacks cholesterol, the effects of the inhibitor (ASA-6) as well as the effects of the sterol deficiency must both be considered. The inhibitive effects of ASA-6, however, become apparent when one compares the results obtained from ASA-6 medium to those from the sterol-deficient medium without ASA-6 (TWEEN). After one generation, adults in ASA-6 medium had a shorter mean length and deposited fewer eggs than did T W E E N adults. After two generations, adult development was completely inhibited in ASA-6 medium, but not in TWEEN medium. The inhibitory effects on development and reproduction resulting from transfer and subculture of larvae through sterol-free medium or sterol-free medium containing ASA-6 were more apparent in C. briggsae than in P. redivivus. Although reproduction was completely inhibited by the second transfer of larvae of both species into these media, P. redivivus larvae were able to develop into adults in ASA-6 medium. The presence of cholesterol in the growth medium delayed but did not prevent inhibition by
105
ASA-6 in C. briggsae. However, the presence of cholesterol prevented azasteroid-inhibition in P. rediv&us under the experimental conditions used. Panagrellus redivivus may be able to overcome ASA-6 inhibition because the larvae hatch in utero, develop and leave the female as L2. Therefore, the larvae may be acquiring sufficient cholesterol from the mother or developing past a point necessary to ensure normal development when cultured in the presence of both exogenous cholesterol and ASA-6. It is also possible that P. redivivus may have the metabolic capability to degrade the inhibitor or select against its uptake from the culture medium. These observations are different from those reported for N. brasiliensis and N. dubius (Bottjer et al., 1984a) and may indicate that the free-living stages of animal parasitic nematodes are more sensitive to azasteroid inhibition than are some free-living nematodes. Culture of C. briggsae and P. redivivus in sterol-free medium with or without ASA6 resulted in inhibition of growth, development and reproduction and produced morphological abnormalities (Figs. 1-7) which were comparable to those seen in N. brasiliensis and N. dubius larvae grown in similar media (Bottjer et al., 1984a). In the presence of cholesterol, ASA-6 eventually inhibited growth and reproduction in C. briggsae. One mechanism to account for this inhibition may be a competition for uptake and utilization between azasteroid and cholesterol that results in a decrease in the amount of available sterol to a level below the requirement for growth and reproduction for C. briggsae but not for P. redivivus. Recently, Chitwood et al. (1984) have demonstrated an inhibition by ASA-6 of motility and reproduction of another species, C. elegans. The data presented and previous findings (Bottjer et al., 1984a) suggest that nematodes may exhibit a range of responses to azasteroids and that a variety of mechanisms to account for this may be involved. They also suggest that sterol utilization and metabolism may vary significantly between nematode species. Azasteroids are known to interfere with the metabolism of insect ecdysteroids (Marks et al., 1978; Svoboda et al., 1978). Ecdysteroid-like substances occur in nematodes (Dennis, 1976; Mendis et al., 1983) and evidence for their synthesis by larval stages reared axenically in the absence of ecdysteroids has been reported (Bottjer et al., 1984b). The nematodes in this study were affected by ASA-6 in a manner similar to and at concentrations lower than those affecting some insects. ASA-6 inhibited growth of certain insects at concentrations of 0.5 to 2.0 ppm, whereas it affected development of Blattella germanica and Tribolium confusum at a dietary concentration of 250-500ppm (Svoboda et al., 1972). Growth, development and egg production of the southwestern corn borer, Diatraea grandiosella, were inhibited at a dietary concentration of 150 ppm (Al-Izzi and Hopkins, 1982). In our study, C. briggsae and P. redivivus growth, development and reproduction were inhibited at a concentration of 25 ppm. In summary, the azasteroid, ASA-6, inhibits the growth, development and reproduction of C. briggsae after two generations of culture in sterol-deficient medium. The presence of exogenous cholesterol at the concentration tested prevented azasteroid inhibition in P. redivivus cultures, but not in C. brigg-
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sac. These results indicate t h a t different species o f n e m a t o d e s m a y exhibit different responses to azasteroid and also t h a t sterol utilization a n d m e t a b o lism m a y vary between species. T h e similarities between the k n o w n effects of azasteroids o n insects a n d those presented in this study o n n e m a t o d e s suggest like m e c h a n i s m s of action by the i n h i b i t o r in b o t h groups of organisms. Acknowledgements--The authors wish to acknowledge the technical assistance of Katherine Taylor and the secretarial assistance of Karen Aberli. REFERENCES
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