Integument ultrastructure of Oestrus ovis (L.) (Diptera:Oestridae) larvae: host immune response to various cuticular...

The nasal bot fly, Oestrus ovis, was investigated to establish which specific cuticular component is most immunogenic to infested sheep and how larval cuticle attains a protective role, if any, against the host immune system. To accomplish these goals, larval cuticle was extracted by a variety of agents and tested against immune sera from infested sheep and experimentally immunized rabbits. The cuticle substructure remaining after extraction was examined to localize various immunogenic components. O. ovis larval integument comprises an inner cellular layer, the epidermis, and an overlying cuticle layer. In 3rd instar larvae, the cuticle comprises 2 additional layers: the procuticle with numerous pore canals and the epicuticle which includes the wax canals. Three additional layers, altogether comprising the cuticulin layer, are present external to the epicuticle. The epicuticle is completed by apposition of an amorphous electrondense material extending for up to 1 micron in thickness. When fixed with ruthenium red, cuticle becomes heavily stained all along the epicuticular surface in larvae of all developmental stages. However, in 3rd instar larvae, ruthenium red deposits are restricted to the cuticulin layer alone. By gel electrophoresis, 3rd instar larval cuticle is shown to contain a number of polypeptides ranging in molecular weight from 180 to 4.5 kDa. The number and relative concentration of low molecular weight polypeptides was shown to vary in relation to the extraction media employed. Cuticular fragments examined after extraction exhibit an altered ultrastructure. When tested by immunoblotting, the cuticular polypeptides most reactive against sheep antisera are in the range of 180-56 kDa. A similar reaction was also detected with sera from rabbits infested experimentally with O. ovis larvae. Results are interpreted in relation to differential polypeptide distribution within the larval cuticle and to accessibility of the host immune system.