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Ecdysteroids during embryonation of eggs of Ascaris suum
Camp. Biochem. Physiol. Vol. Printed in Great Britain
87A,No. 3, pp. 803-805, 1987
0300-9629/87 $3.00 + 0.00 0 1987 Pergamon Journals Ltd
ECDYSTEROIDS DURING EMBRYONATION EGGS OF ASCARIS SUUM
OF
MICHAELW. FLEMING Helminthic Diseases Laboratory, Animal Parasitology Institute, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA (Received 21 October 1986)
The optimal temperature for in vitro development of fertilized eggs of Ascaris suum was 24°C. Samples (2 x 10’ eggs) were obtained from in oirro embryonating cultures every 3 days for 4 weeks; lipids were extracted, partially purified, fractionated with HPLC and analyzed for ecdysteroids by
Abstract-l. 2.
radioimmunoassay. 3. Free ecdysone and 20-hydroxyecdysone (20-HE) were at low levels (~20 pg) in freshly excised eggs and rose to maximal values on day 6 of embryonation. 4. Conjugated ecdysone and conjugated 20-HE rose to maximal values on day 9. 5. Both free and conjugated ecdysteroids were undetectable from days 15 to 27 of cultivation. 6. These profiles indicate that ecdysteroids might have a selective role in nematode embryonation and/or tanning of the egg shell.
INTRODUCTION Molting in nematodes has been proposed to be regulated, in part, by steroidal hormones (Willett, 1980), analogous to the mechanism defined for arthropods (Karlson, 1980). In the intestinal roundworm of swine, Ascuris suum, the active metabolite of ecdysone, 20-hydroxyecdysone (20-HE), was isolated initially from whole worm extracts of adults (Horn et al., 1974). Both ecdysone and 20-HE were identified in extracts of third- and fourth-stage larvae of A. suum grown in vitro as well as in isolated reproductive tracts of adult female A. suum (Fleming, 1985a). Specific association of these hormones with ovarian, uterine or embryonic component was not determined. Brief exposure to low concentrations of exogenous ecdysteroids in vitro resulted in a dose-dependent enhancement of larval molting and growth in A. suum (Fleming, 1985b). Embryonation of A. suum to fully infective eggs under laboratory conditions requires approximately 30 days at room temperature; the first molt or second-stage larvae are released upon hatching (Urban et al., 1981). The present study was designed to define the profile of production of endogenous ecdysteroids during embryonation of the eggs of A. suum. However, to determine the optimal conditions for embryonation, a preliminary experiment was conducted to evaluate the rate of embryonation relative to incubation temperature.
MATERIALSAND
METHODS
Adult female A. suum were collected from a local abattoir, reproductive tracts were excised, and eggs were isolated by digestion of uterine tissue with 0.5 N NaOH (Costello et al., 1963). Quadruplicate cultures of 2 x lo4 eggs/40 ml were maintained at 4, 15, 24, 30 and 37°C in 0.1% formalin in water that was gassed continually with air. At 3-day inter-
vals, samples (2ml) of eggs were removed and fixed in absolute methanol. Eggs (2OO/sample) were classified microscopically as unfertilized, incompletely embryonated or fully embryonated (Alicata, 1935). At 3-day intervals a sample of 2 x 10’ eggs was obtained from a culture of eggs embryonating in 0.1% formalin at 24°C. Samples were centrifuged, the supematant aspirated, and lipids were extracted from eggs three times with 70% methanol at -20°C and three times with 100% methanol at -20°C (Mendis ef al., 1983). After evaporation to dryness under vacuum, samples were partitioned between countersaturated hexane and 70% aqueous methanol. The latter phase was evaporated to dryness. Free and conjugated ecdysteroids were separated on a silicic-acid column prepared in chloroform. Free ecdysteroids were eluted with 30% (v/v) methanol/chloroform, and conjugated ecdysteroids were eluted with 80% (v/v) methanol/chloroform. The free ecdysteroid fraction was evaporated and loaded onto a SEP-PAK Cl8 column (Waters Associated, Milford, Massachusetts) in 10% (v/v) methanol/water and eluted sequentially with 4ml of IO%, 4ml of 30% and 4ml of 60% methanol/water (free ecdysteroid fraction) (Mendis el al., 1983). The conjugated ecdysteroid fraction from the silicic acid column was evaporated to dryness under vacuum and incubated in 0.2 M 2(N-morpholino)ethanesulfonic acid (4 ml, pH 5.8) with 1 mg of crude Helix porn&u aryl sulphatase (Sigma, St. Louis, Missouri) for 24 hr at 37°C. After incubation, protein was precipitated with 1Oml of ethanol; free ecdysteroids were extracted with 70% methanol. This extract was purified partially on the SEP-PAK Cl9 columns as outlined previously (Mendis et al., 1983). Free ecdysteroids were separated on a radial compression Cl8 column (Waters Associates, Milford. Massachusetts) with 18% (v/v) acetonitrile/wateras the mobile phase. Pump rate was 7ml/min and 2-ml fractions were collected for 15 min after sample injections. All fractions were- assayed in a radioimmunoassay (Soumoff et al., 1981) with an antiserum which cross-reacted equally with ecdysone and 20-HE (provided by Dr John O’Connor, University of California, Berkeley). Sensitivity of the assay was 15 pg/tube; recovery of tritiated ecdysone from a tissue sample
803
averaged
64%.
MICHAELW. FLEMING
804 RESULTS
Approximately 12% of all eggs were unfertilized. Embryonic development was most rapid at 24°C; by day 16 of incubation, 65% of the eggs maintained at this temperature contained morphologically fully differentiated larvae (Table 1). The percentage of fully embryonated eggs by day 16 at each temperature remained constant until day 30 of incubation (data not shown). Levels of free ecdysone and free 20-HE were detectable, but at low concentrations in freshly recovered eggs. Concentrations of both steroids were maximal on day 6 (86 and 150 pg/2 x 10’ eggs, respectively); by day 15, these hormones were undetectable and remained so through day 27 (Fig. 1). The conjugated ecdysteroids had a similar pattern, however, the concentrations were three orders of magnitude higher than the free steroids. Levels of conjugated ecdysone and conjugated 20-HE were both maximal on day 9 (35 and 9 ng/2 x 10’ eggs, respectively). After day 15, the conjugates also were undetectable for the remainder of the incubation (Fig. 1). DISCUSSION
Embryonic development under laboratory conditions correlates well with the development of A. suum under field conditions in southern England (Connan, 1977). The summer months had the fastest rates of development; little or no development occurred during the winter. Utilizing a different incubation system, Alicata (1935) found incubations at 33°C to produce larvae in their first molt more quickly than at 24°C. Although almost 90% of the eggs recovered from the uteri of A. suum were fertilized, a third of these did not develop to motile larval stage by day 30 of incubation, and this percentage remained constant over the final 2 weeks. Apparently a significant number of eggs undergo post-fertilization mortality. The criterion of morphologically developed, motile larvae as fully embryonated does not correlate completely with physiological responses. Maximum infectivity of eggs from similar cultures maintained at 24°C does not occur until day 28 (Dr Joseph Urban, Jr, personal communication). Levels of both ecdysteroids and their conjugates increased temporarily while in cultures indicating synthesis and metabolism of these steroids by the embryonic tissues. The maximum levels occurred during a phase of morphogenesis and differentiation, implicating a steroidal role in these processes.
4
0
I
3
I,-
-_
9
12
DAY
OF
6
15
(“0 4 I5 24 30 36 Each percentage
5
1
O/86 O/88 O/87 0188 o;s9
O/87 O/87 O/87 0186 0186
O/87 O/89 o/90 4185 Oj8S
24
27
0
Fig. I. Concentrations of ecdysone (a) and 20-hydroxyecdysone (0) as free (-) or conjugated (- - - - -) forms from embryonating eggs of Ascaris suurn. Note differences in scales.
Ecdysteroids have been identified in a variety of insects and crustaceans (Karlson, 1974). Furthermore, these same hormones have been isolated from a variety of other invertebrates including a species of snail (Romer, 1979), trematode (Torpier et al., 1982), cestode (Mendis et al., 1984) and nematode (Fleming, 1984a) although their functions remain speculative, particularly in those taxa which do not molt. The initial selective recovery of ecdysone and 20-HE from the reproductive tract of adult female A. suum (Fleming, 1985a) suggested potential roles in gametogenesis, fertilization, and/or embryogenesis. Through the incubation of isolated egg preparations, the profiles of free and conjugated ecdysteroids support a role during the first 2 weeks of embryonation when major morphological development occurs. If ecdysteroid functions in nematode are analogous to functions identified in arthropods, these steroids might serve as the hormonal trigger for a process analogous to sclerotization (Karlson, 1980). Chitin is component of Ascaris eggs and is produced by the fertilized egg, not the uterus of the female (Barrett, 1981). Dopamine, a potential sclerotizing agent, was identified in greatest concentrations in the intact reproductive tract of adult female A. lumbricoides (Mishra et al., 1984). The browning of the egg shell might be analogous to the tanning of the insect cuticle, and ecdysteroids might modulate this process in nematodes. Ecdysone and 20-HE, both as conjugates and as free steroids, were produced in similar profiles
Days of 3
21
INCUBATION
Table I. Percentage of completely embryonated eggs/percentage of partially embryonated eggs of Ascaris ~tlum incubated at different temperatures in 0.1% formalin
Temperature
I 18
IO o/g7 o/91 2189 18171 Ii86
incubation I2 o/92 o/91 20/64 22165 I)86
is the mean of four samples of 200 eggs.
I4
I6
O/89 O/87 36155 23165 2/88
O/89 o/90 65127 27167 Ii90
Ecdysteroids in Ascaris eggs
throughout embryonation of A. suum eggs. Maximal values of all compounds were attained between 6 and 9 days of incubation suggesting this period as a critical phase for hormonal function. This function might be related directly to larval morphogenesis andfor to egg-shell browning. Acknawledgemen6s-The
author thanks Dr John O’Connor for the generous gift of antiserum and Dr Joseph Urban, Jr for the tutorial in Ascaris cultivation. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, nor does it imply its approval to the exclusion of other products that also may be suitable.
805
parasitic larvae of Ascaris suum: Validation of a bioassay. J. exp. Zool. ZU, 229-233. Horn D. H. S., Wilkie J. S. and Thomson J. A. (1974) Isolation of @-ecdysone (20-hydroxyecdysone) from the parasitic nematode Ascaris lumbricoides. Experientiu 30, 1109.
Karlson P. (1974) Mode of action of ecdysone In Inver6ebrate Endocrinology and Hormonal HeferophyBy (Edited by Burdett W. J.), pp. 4354. Springer, New York. Karlson P. (1980) Ecdysone in retrospect and prospect. In Progress in Ecdysone Research (Edited by Hoffman J. A.), Developments in Endocrinology, Vol. 7, pp. l-l 1. Efsevier/North Holland Biomedical, Amsterdam. Mendis A. H. W., Rees H. H. and Goodwin T. W. (1984) The occurrence of ecdysteroids in the c&ode, Monienzia expansa. Molec. Biochem. Parasitoi. 10, 123-138.
Mendis A. H. W.. Rose M. E.. Rees H. H. and Goodwin T. W. (1983) &&steroids in adults of the nematode. Dirofilaria immitis.Molec. Biochem. Parasitol. 9,209-226:
RETRENCH
Mishra S. K.. Ramanui S. and Ghatak S. c1984) Ascaris hanbricoides and Asiaridia galli: Biogedic amines in adults and deveIopmenta1 stages. Expl Parasitol. 57,
Ahcata J. E. (1935) Early developmental stages of nematodes occurring in swine. U.S.D.A. Technical Bulletin No.
Romer F. (I 979) Ecdysteroids in snails. Naturwissenschuften 665,471.
489.
Barrett J. (1981) Biochemistry of Parasitic Helminths. University Park Press, Baltimore. Connan R. R. (1977) Ascariasis: The development of eggs Asearis suum under the conditions prevailing in a pig house. Vet. Rec. 100, 421422. Costello L. C., Oya H. and Smith W. (1963) The comparative biochemistry of developing Ascaris eggs. I. Substrate oxidation and the cytochrome system in embryonated and unembryonated eggs. Archs Biochem. Biophys. 103, 345-35
34-99.
1.
Fleming M. W. (1985af Ascaris suum: Role of ecdysteroids in molting. Expl Par~j~o~. 69, 207-Z 10. Fleming M. W. (I 985b) Steroidaf enhancement of growth in
Soumoff C., Horn D. H. S. and O’Connor J. D. (1981) Production of a new antiserum to arthropod molting hormone and comparison with two other antisera. J. Srer. Bio~hem. 14, 429435.
Torpier G., Him M., Nirde P., De Reggi M. and Capron A. (1982) Detection of ecdysteroids in the human trematode, Schistosoma mansoni. Parasitology 84, 123-130. Urban J. F., Jr., Douvres F. W. and Tromba F. G. (1981) A rapid method for hatching Ascaris suum eggs in vitro. Proc. helminth. Sot. Wash. 48, 241-243.
Willett J. D. (1980) Control mechanisms in nematodes. In ~emafodes as Biological Models (Edited by Zuckerman B. M.), pp. 197-225. Academic Press, New York.
87A,No. 3, pp. 803-805, 1987
0300-9629/87 $3.00 + 0.00 0 1987 Pergamon Journals Ltd
ECDYSTEROIDS DURING EMBRYONATION EGGS OF ASCARIS SUUM
OF
MICHAELW. FLEMING Helminthic Diseases Laboratory, Animal Parasitology Institute, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA (Received 21 October 1986)
The optimal temperature for in vitro development of fertilized eggs of Ascaris suum was 24°C. Samples (2 x 10’ eggs) were obtained from in oirro embryonating cultures every 3 days for 4 weeks; lipids were extracted, partially purified, fractionated with HPLC and analyzed for ecdysteroids by
Abstract-l. 2.
radioimmunoassay. 3. Free ecdysone and 20-hydroxyecdysone (20-HE) were at low levels (~20 pg) in freshly excised eggs and rose to maximal values on day 6 of embryonation. 4. Conjugated ecdysone and conjugated 20-HE rose to maximal values on day 9. 5. Both free and conjugated ecdysteroids were undetectable from days 15 to 27 of cultivation. 6. These profiles indicate that ecdysteroids might have a selective role in nematode embryonation and/or tanning of the egg shell.
INTRODUCTION Molting in nematodes has been proposed to be regulated, in part, by steroidal hormones (Willett, 1980), analogous to the mechanism defined for arthropods (Karlson, 1980). In the intestinal roundworm of swine, Ascuris suum, the active metabolite of ecdysone, 20-hydroxyecdysone (20-HE), was isolated initially from whole worm extracts of adults (Horn et al., 1974). Both ecdysone and 20-HE were identified in extracts of third- and fourth-stage larvae of A. suum grown in vitro as well as in isolated reproductive tracts of adult female A. suum (Fleming, 1985a). Specific association of these hormones with ovarian, uterine or embryonic component was not determined. Brief exposure to low concentrations of exogenous ecdysteroids in vitro resulted in a dose-dependent enhancement of larval molting and growth in A. suum (Fleming, 1985b). Embryonation of A. suum to fully infective eggs under laboratory conditions requires approximately 30 days at room temperature; the first molt or second-stage larvae are released upon hatching (Urban et al., 1981). The present study was designed to define the profile of production of endogenous ecdysteroids during embryonation of the eggs of A. suum. However, to determine the optimal conditions for embryonation, a preliminary experiment was conducted to evaluate the rate of embryonation relative to incubation temperature.
MATERIALSAND
METHODS
Adult female A. suum were collected from a local abattoir, reproductive tracts were excised, and eggs were isolated by digestion of uterine tissue with 0.5 N NaOH (Costello et al., 1963). Quadruplicate cultures of 2 x lo4 eggs/40 ml were maintained at 4, 15, 24, 30 and 37°C in 0.1% formalin in water that was gassed continually with air. At 3-day inter-
vals, samples (2ml) of eggs were removed and fixed in absolute methanol. Eggs (2OO/sample) were classified microscopically as unfertilized, incompletely embryonated or fully embryonated (Alicata, 1935). At 3-day intervals a sample of 2 x 10’ eggs was obtained from a culture of eggs embryonating in 0.1% formalin at 24°C. Samples were centrifuged, the supematant aspirated, and lipids were extracted from eggs three times with 70% methanol at -20°C and three times with 100% methanol at -20°C (Mendis ef al., 1983). After evaporation to dryness under vacuum, samples were partitioned between countersaturated hexane and 70% aqueous methanol. The latter phase was evaporated to dryness. Free and conjugated ecdysteroids were separated on a silicic-acid column prepared in chloroform. Free ecdysteroids were eluted with 30% (v/v) methanol/chloroform, and conjugated ecdysteroids were eluted with 80% (v/v) methanol/chloroform. The free ecdysteroid fraction was evaporated and loaded onto a SEP-PAK Cl8 column (Waters Associated, Milford, Massachusetts) in 10% (v/v) methanol/water and eluted sequentially with 4ml of IO%, 4ml of 30% and 4ml of 60% methanol/water (free ecdysteroid fraction) (Mendis el al., 1983). The conjugated ecdysteroid fraction from the silicic acid column was evaporated to dryness under vacuum and incubated in 0.2 M 2(N-morpholino)ethanesulfonic acid (4 ml, pH 5.8) with 1 mg of crude Helix porn&u aryl sulphatase (Sigma, St. Louis, Missouri) for 24 hr at 37°C. After incubation, protein was precipitated with 1Oml of ethanol; free ecdysteroids were extracted with 70% methanol. This extract was purified partially on the SEP-PAK Cl9 columns as outlined previously (Mendis et al., 1983). Free ecdysteroids were separated on a radial compression Cl8 column (Waters Associates, Milford. Massachusetts) with 18% (v/v) acetonitrile/wateras the mobile phase. Pump rate was 7ml/min and 2-ml fractions were collected for 15 min after sample injections. All fractions were- assayed in a radioimmunoassay (Soumoff et al., 1981) with an antiserum which cross-reacted equally with ecdysone and 20-HE (provided by Dr John O’Connor, University of California, Berkeley). Sensitivity of the assay was 15 pg/tube; recovery of tritiated ecdysone from a tissue sample
803
averaged
64%.
MICHAELW. FLEMING
804 RESULTS
Approximately 12% of all eggs were unfertilized. Embryonic development was most rapid at 24°C; by day 16 of incubation, 65% of the eggs maintained at this temperature contained morphologically fully differentiated larvae (Table 1). The percentage of fully embryonated eggs by day 16 at each temperature remained constant until day 30 of incubation (data not shown). Levels of free ecdysone and free 20-HE were detectable, but at low concentrations in freshly recovered eggs. Concentrations of both steroids were maximal on day 6 (86 and 150 pg/2 x 10’ eggs, respectively); by day 15, these hormones were undetectable and remained so through day 27 (Fig. 1). The conjugated ecdysteroids had a similar pattern, however, the concentrations were three orders of magnitude higher than the free steroids. Levels of conjugated ecdysone and conjugated 20-HE were both maximal on day 9 (35 and 9 ng/2 x 10’ eggs, respectively). After day 15, the conjugates also were undetectable for the remainder of the incubation (Fig. 1). DISCUSSION
Embryonic development under laboratory conditions correlates well with the development of A. suum under field conditions in southern England (Connan, 1977). The summer months had the fastest rates of development; little or no development occurred during the winter. Utilizing a different incubation system, Alicata (1935) found incubations at 33°C to produce larvae in their first molt more quickly than at 24°C. Although almost 90% of the eggs recovered from the uteri of A. suum were fertilized, a third of these did not develop to motile larval stage by day 30 of incubation, and this percentage remained constant over the final 2 weeks. Apparently a significant number of eggs undergo post-fertilization mortality. The criterion of morphologically developed, motile larvae as fully embryonated does not correlate completely with physiological responses. Maximum infectivity of eggs from similar cultures maintained at 24°C does not occur until day 28 (Dr Joseph Urban, Jr, personal communication). Levels of both ecdysteroids and their conjugates increased temporarily while in cultures indicating synthesis and metabolism of these steroids by the embryonic tissues. The maximum levels occurred during a phase of morphogenesis and differentiation, implicating a steroidal role in these processes.
4
0
I
3
I,-
-_
9
12
DAY
OF
6
15
(“0 4 I5 24 30 36 Each percentage
5
1
O/86 O/88 O/87 0188 o;s9
O/87 O/87 O/87 0186 0186
O/87 O/89 o/90 4185 Oj8S
24
27
0
Fig. I. Concentrations of ecdysone (a) and 20-hydroxyecdysone (0) as free (-) or conjugated (- - - - -) forms from embryonating eggs of Ascaris suurn. Note differences in scales.
Ecdysteroids have been identified in a variety of insects and crustaceans (Karlson, 1974). Furthermore, these same hormones have been isolated from a variety of other invertebrates including a species of snail (Romer, 1979), trematode (Torpier et al., 1982), cestode (Mendis et al., 1984) and nematode (Fleming, 1984a) although their functions remain speculative, particularly in those taxa which do not molt. The initial selective recovery of ecdysone and 20-HE from the reproductive tract of adult female A. suum (Fleming, 1985a) suggested potential roles in gametogenesis, fertilization, and/or embryogenesis. Through the incubation of isolated egg preparations, the profiles of free and conjugated ecdysteroids support a role during the first 2 weeks of embryonation when major morphological development occurs. If ecdysteroid functions in nematode are analogous to functions identified in arthropods, these steroids might serve as the hormonal trigger for a process analogous to sclerotization (Karlson, 1980). Chitin is component of Ascaris eggs and is produced by the fertilized egg, not the uterus of the female (Barrett, 1981). Dopamine, a potential sclerotizing agent, was identified in greatest concentrations in the intact reproductive tract of adult female A. lumbricoides (Mishra et al., 1984). The browning of the egg shell might be analogous to the tanning of the insect cuticle, and ecdysteroids might modulate this process in nematodes. Ecdysone and 20-HE, both as conjugates and as free steroids, were produced in similar profiles
Days of 3
21
INCUBATION
Table I. Percentage of completely embryonated eggs/percentage of partially embryonated eggs of Ascaris ~tlum incubated at different temperatures in 0.1% formalin
Temperature
I 18
IO o/g7 o/91 2189 18171 Ii86
incubation I2 o/92 o/91 20/64 22165 I)86
is the mean of four samples of 200 eggs.
I4
I6
O/89 O/87 36155 23165 2/88
O/89 o/90 65127 27167 Ii90
Ecdysteroids in Ascaris eggs
throughout embryonation of A. suum eggs. Maximal values of all compounds were attained between 6 and 9 days of incubation suggesting this period as a critical phase for hormonal function. This function might be related directly to larval morphogenesis andfor to egg-shell browning. Acknawledgemen6s-The
author thanks Dr John O’Connor for the generous gift of antiserum and Dr Joseph Urban, Jr for the tutorial in Ascaris cultivation. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, nor does it imply its approval to the exclusion of other products that also may be suitable.
805
parasitic larvae of Ascaris suum: Validation of a bioassay. J. exp. Zool. ZU, 229-233. Horn D. H. S., Wilkie J. S. and Thomson J. A. (1974) Isolation of @-ecdysone (20-hydroxyecdysone) from the parasitic nematode Ascaris lumbricoides. Experientiu 30, 1109.
Karlson P. (1974) Mode of action of ecdysone In Inver6ebrate Endocrinology and Hormonal HeferophyBy (Edited by Burdett W. J.), pp. 4354. Springer, New York. Karlson P. (1980) Ecdysone in retrospect and prospect. In Progress in Ecdysone Research (Edited by Hoffman J. A.), Developments in Endocrinology, Vol. 7, pp. l-l 1. Efsevier/North Holland Biomedical, Amsterdam. Mendis A. H. W., Rees H. H. and Goodwin T. W. (1984) The occurrence of ecdysteroids in the c&ode, Monienzia expansa. Molec. Biochem. Parasitoi. 10, 123-138.
Mendis A. H. W.. Rose M. E.. Rees H. H. and Goodwin T. W. (1983) &&steroids in adults of the nematode. Dirofilaria immitis.Molec. Biochem. Parasitol. 9,209-226:
RETRENCH
Mishra S. K.. Ramanui S. and Ghatak S. c1984) Ascaris hanbricoides and Asiaridia galli: Biogedic amines in adults and deveIopmenta1 stages. Expl Parasitol. 57,
Ahcata J. E. (1935) Early developmental stages of nematodes occurring in swine. U.S.D.A. Technical Bulletin No.
Romer F. (I 979) Ecdysteroids in snails. Naturwissenschuften 665,471.
489.
Barrett J. (1981) Biochemistry of Parasitic Helminths. University Park Press, Baltimore. Connan R. R. (1977) Ascariasis: The development of eggs Asearis suum under the conditions prevailing in a pig house. Vet. Rec. 100, 421422. Costello L. C., Oya H. and Smith W. (1963) The comparative biochemistry of developing Ascaris eggs. I. Substrate oxidation and the cytochrome system in embryonated and unembryonated eggs. Archs Biochem. Biophys. 103, 345-35
34-99.
1.
Fleming M. W. (1985af Ascaris suum: Role of ecdysteroids in molting. Expl Par~j~o~. 69, 207-Z 10. Fleming M. W. (I 985b) Steroidaf enhancement of growth in
Soumoff C., Horn D. H. S. and O’Connor J. D. (1981) Production of a new antiserum to arthropod molting hormone and comparison with two other antisera. J. Srer. Bio~hem. 14, 429435.
Torpier G., Him M., Nirde P., De Reggi M. and Capron A. (1982) Detection of ecdysteroids in the human trematode, Schistosoma mansoni. Parasitology 84, 123-130. Urban J. F., Jr., Douvres F. W. and Tromba F. G. (1981) A rapid method for hatching Ascaris suum eggs in vitro. Proc. helminth. Sot. Wash. 48, 241-243.
Willett J. D. (1980) Control mechanisms in nematodes. In ~emafodes as Biological Models (Edited by Zuckerman B. M.), pp. 197-225. Academic Press, New York.