Exsheathment Phenomenon in the Infective-Stage Larvae of Haemonchus contortus

Abstract

The nature of exsheathment of 3rd-stage larvae of Haemonchus contortus was investigated further using a new simplified technique for the study of cuticular ring formation. The presence of a second ring on the 2nd-stage larval cuticle, 8 to 9 u from the anterior, is described. Exsheathing activity was observed to occur in 4 consecutive steps on isolated sheaths of H. contortus. Metabolic fluid and somatic extract of Ascaris suum and Caenorhabditis briggsae were investigated for their ability to stimulate exsheathment in H. contortus. Scanning electron microscopy revealed some aspects of the fine structure of cuticle. Exsheathing factor(s) was found to be thermostable, to require no metal ion activation, and to be active over a broad range of pH. Temperature appeared to be an important factor for the exsheathment activity. Refractile ring formation at 19 to 20 tL from the anterior end of larval Haemonchus contortus cuticles, as a criterion for exsheathing activity, was first reported by Lapage (1935). Empty sheaths with their caps still attached were also described by Veglia (1916). Sommerville (1957) described the presence of exsheathing fluid and its action on cuticles. Some properties of exsheathing fluid and its relation to the exsheathment phenomenon were investigated by Rogers and Sommerville (1957, 1960). The presence of leucine aminopeptidase in exsheathing fluid was first reported by Rogers (1963, 1965), and its role as an exsheathing enzyme was postulated by Rogers and Sommerville (1968). However, data of Ozerol and Silverman (1969) indicated that leucine aminopeptidase is not the exsheathing enzyme, and they pointed out the possible involvement of other factors in this phenomenon. The possible function of leucine aminopeptidase in exsheathing fluid was reemphasized by Rogers (1970); however, Slocombe and Whitlock (1971a, b) have recently presented additional data in support of Ozerol and Silverman's (1969) conclusions. The present studies were undertaken to investigate further the nature of the exsheathment phenomenon in infective larvae of Haemonchus contortus. MATERIALS AND METHODS Infective juvenile of H. contortus (L3) were used for all the experiments. Larvae, which were Received for publication 27 July 1971. * This work supported by Grant AI 05910, NIH, U. S. Public Health Service. kept f r several weeks at 10 C 1, were used for the isolation of the sheaths and preparation of the metabolic fluids (XL3F, XL4,5F) and somatic extracts (L3Fp, XLsFp, XL4.sFp) (Ozerol and Silverman, 1969). Preparation of metabolic fluid Metabolic fluid from third-stage (XLsF) and from fourth-fifth stage larvae (XL,.sF) were prepared in accordance with methods described by Silverman, Alger, and Hansen (1966), and Alger (1968). The fluid was separated by sedimentation of the larvae, withdrawn by syringe, and filtered through Whatman No. 1 paper containing some glass wool. Metabolic fluids from both stages of larvae were concentrated by lyophilization and subsequently fractionated as described by Ozerol and Silverman (1969, 1970). Concentration of the metabolic fluid In addition to lyophilization, the metabolic fluids were also concentrated by the following techniques in order to prevent the possible loss of biological and enzymic activities. (a) Lyphogel: Lyphogel, a polyacrylamide hydrogel in pellet form (Gelman Instrument Co., Ann Arbor, Mich.), was used for the concentration of fresh metabolic fluid from the third larval stage. One gram of lyphogel generally absorbs 5.4 ml of water. Approximately 10 ml fresh dialyzed metabolic fluid was placed in a beaker containing 1.7 g of lyphogel pellets. Concentration was carried out at 5 C + 1 for several hours. (b) Amicon membrane filter (Centriflo): Centriflo membrane ultrafilter cones (obtained from Amicon Corp., Lexington, Mass.) retains molecules above 5,000 MW. They are laminated to a tough, inert substrate which improves the handling qualities. Fresh, dialyzed metabolic fluid of third-stage larvae was filtered under gravitational force and the concentrate was recovered by pipette without damaging the membrane.