The surface coat of infective larvae of Trichinella spiralis

The surface coat of the infective larvae of the parasitic nematode Trichine lla spiralis was characterized with respect to its biophysical properties, morphology and composition. Labelling of larvae with the fluorescent surfac e probe PKH26 was lost after activation (by incubation in mammalian medium containing trypsin and bile), or following pronase treatment. Electron micr oscopical examination revealed that pronase treatment resulted in the loss of an amorphous surface layer only, further demonstrating the specificity o f PKH26 for the larval surface coat. Surface coat shedding was inhibited by sodium azide and carbonyl cyanide, or by incubation of larvae at 4 degrees C, suggesting the shedding process required metabolic energy. Pre-labelled , unactivated larvae demonstrated continuous slow surface coat shedding and could be re-labelled with PKH26, indicating that the shed coat is replaced in these parasites. However, pre-labelled larvae which were activated fail ed to re-label with the probe, suggesting that activation provides an irrev ersible trigger for surface changes. PKH26, therefore, is a useful marker f or larval activation. Examination of the shed coat material by scanning ele ctron microscopy revealed 2 types of morphologies; one comprising thin mult ilaminate sheets and the other of amorphous material with ridges producing a fingerprint-like motif. Western- and lectin-blotting of the shed coat mat erial demonstrated 2 prominent entities; a 90 kDa glycoprotein, which bound Datura stramonium agglutinin and was resistant to N- and O-glycanase treat ment and a 47-60 kDa set of protein(s). Analysis of the surface lipids by e lectrospray mass spectometry revealed the presence of lysophosphatidic acid (lysoPA, C14:2) and an unidentifiable component of 339.4 Da. These two lip ids constituted 36.9 %, and 36 % by mass of surface coat lipids respectivel y. The presence of lysoPA was confirmed by thin layer chromatography, which also detected phosphatidic acid (PA). The polar lipids detected in solvent rinses of intact parasites by electrospray mass spectrometry were PI (C48: 4), PE (C40:4 and C38:4), PS (C40:4), lysoPC (C20:2 and C18:2) and lysoPA ( C14:2). These observations are discussed with respect to the role of the su rface coat and its shedding in the T. spiralis host-parasite relationship.