Ecdysteroid-like substances have been identified from various nematodes (Hitcho and Thorson, 1971, J. Parasit. 57: 787-793; Horn et al., 1974, Experientia 30: 1109-1110; Dennis, 1977, Int. J. Parasit. 7: 181-188; Mendis et al., 1983, Mol. Biochem. Parasit. 9: 209-226). However, in all cases, the possibility exists that these substances were obtained from the host or environment and not synthesized by the nematode. This study reports ecdysteroid-like substances from infective third-stage larvae (L3) of Nippostrongylus brasiliensis which apparently were produced endogenously. The in vitro axenic cultivation of first stage larvae of N. brasiliensis to infective L3 is an established method (Bolla et al., 1972, Comp. Biochem. Physiol. 43B: 487-501) whereby the amount of exogenous sterol available to developing larvae in the culture medium can be strictly regulated. The medium was modified in that formalin-killed bacteria were added to Caenorhabditis briggsae Maintenance Medium (1: 1,v/v) (Buecher et al., 1966, Proc. Soc. exp. Biol. Med. 121: 390-393). Large numbers of axenic N. brasiliensis L3 were cultivated in this manner in which the only sterol available was cholesterol (Sigma Chemical Co., St. Louis, MO) dispersed in Tween 80 at a concentration of 10 ,ug cholesterol per ml medium. The cholesterol, analyzed by gas chromatography, was 99+% pure. After cultivation, the L3 were collected and extracted by a procedure specific for steroidal substances. Freeze-dried L3 were homogenized in 60% methanol, centrifuged and the supernatant was collected and filtered. The sediment was extracted similarly twice more and the 3 filtrates were combined with chloroform to precipitate any soluble proteins. The final mixture was filtered and the solvents removed by flash evaporation (Whisenton, 1980. Ph.D. Dissertation, University of Notre Dame, Notre Dame, Indiana, 169 p.). Extracts were divided into halves and taken to dryness under air. One half was hydrolyzed for 18 hr at 37 C in 50 mM acetate buffer (pH 5.9) containing 5,000 units/ml of sulfatase and 5,000 units/ml of glucuronidase (Helix pomatia, Sigma Chemical Co.) to release any conjugated ecdysteroids. These conjugates are the major steroid excretory products that are known from insects (Thompson et al., 1973. In Advances in lipid research, R. Paoletti and D. Kritchevsky (eds.). Academic Press, New York, pp. 219-265). Most Helix juices contain ecdysone immunoreactive compounds (Hoffman, 1980. Progress in ecdysone research. Elsevier, North Holland, 491 p.); therefore, enzyme without extract was assayed for RIA positive reactions and the results used for background correction. Six wk old L3 raised on charcoal-feces cultures and 13 day postinfection (PI) adults of N. brasiliensis were extracted similarly. Uninoculated culture medium was extracted also as a control for the axenically reared larvae. Radioimmunoassays were performed on these extracts to determine the presence of ecdysteroid-like substances by the method of Whisenton (1980, loc. cit.). The antibody used was generated against a 22-hemisuccinate ester of ecdysone conjugated to bovine thyroglobulin and exhibited specificity for ecdysone over 20-hydroxyecdysone (Gilbert et al., 1977. In International review of biochemistry: biochemistry of lipids II, pp. 1-50). The standard used in the assay was 20-hydroxyecdysone and the labeled ligand was 3H-ecdysone; thus activity is expressed in 20-hydroxyecdysone equivalents. In the axenic culture of N. brasiliensis, cholesterol was the only exogenous sterol available for possible conversion to ecdysteroids by the developing larvae. The detection of a significant amount of radioimmunoassay positive substance(s) (Table I) suggests that this nematode was capable of synthesizing ecdysteroids from sterol precursors. Cholesterol did not cross-react with 20-hydroxyecdysone for binding to the antibody at the tested concentrations. Interestingly, higher concentrations of non-conjugated ecdysteroid-like substances were detected in 4 day old axenic L3 than in 6 wk old charcoal/feces L3
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Aug 1, 1986
D Schulze 
Open Access