Changes Associated with Parasitism in Nematodes. IV. Cytochemical Studies on the Ampulla of the Dorsal Esophageal Gland of Meloidogyne javanica and on Exudations from the Buccal Stylet

Abstract

The ampulla of the dorsal esophageal gland and exudations from the buccal stylet were examined in both living and fixed nematodes by various cytological techniques. A pronounced ampulla is found only in females. It is packed with granules which are approximately 0.4 to 0.6 u in diameter. By means of histochemical tests and examination with a UV microspectrograph they have been shown to consist predominantly of protein. Living females, when dissected from roots and placed in distilled water, produce a stylet exudate within a few hours. The use of a perfusion slide enabled this precipitate to be tested with various chemical reagents including stains and enzymes and it was shown to consist of basic proteins containing carbohydrates. Neither lipids, nucleic acids nor enzymes were detected. The possible role of these basic proteins in the host-parasite relationship is discussed. Previous papers in this series (Bird, 1967; Bird and Saurer, 1967; Bird, 1968) have been concerned with changes taking place in the subventral esophageal glands during the initial stages of parasitic development. No changes were detected in the dorsal esophageal gland or its ampulla near the base of the spear during this transition. It is clear, however, from the work of earlier investigators (Linford, 1937; Rhoades and Linford, 1961; Hechler, 1962; Anderson, 1964; Doncaster, 1966; and Nickle, 1967) that the ampulla of the dorsal esophageal gland is not only closely associated with feeding in stylet bearing nematodes but that its granular contents appear to be morphologically similar among widely divergent genera. As yet there is little information on the chemical nature of either the material in the ampulla or the oral precipitate which exudes from the stylets of plant parasitic nematodes. However, in Meloidogyne javanica, Bird (1964) has shown that these oral exudates are antigenic and are thought to consist of a number of proteins. Further information on the chemical nature of these structures is required if their role in the establishment, development and maintenance of the tumorlike giant cells on which these nematodes feed (Bird, 1961; 1962) is to be understood. In this paper the chemical compositions of the contents of the ampulla of the dorsal esophReceived for publication 14 May 1968. ageal gland and the stylet exudate are examined by various techniques in an effort to clarify at least some aspects of M. javanica's complex host-parasite relationship. MATERIALS AND METHODS Preparasitic and parasitic larvae were obtained by methods described previously (Bird, 1967). Females were dissected from galls in tomato roots using a stereo microscope and males were collected in tubes by sedimentation after they had migrated from tomato roots in a thermally controlled mist chamber. Living larvae and males were photographed at high resolution under anesthetic (Bird 1967). However, living females were not anesthetized because of their relative immobility but were examined with the coverslip raised slightly by means of a fine brass ring to prevent crushing. All specimens were photographed with the aid of electronic flash using either oil or water immersion objectives with normal transmitted light optics or phase contrast. Observations on both the material exuded from the tip of the buccal stylet in living females and the influence of various chemicals on these precipitates were made possible by the use of a perfusion chamber (Paul, 1957) and phase contrast optics incorporating a long-focal-length condenser. Pepsin (Calbiochem) was made up as a 0.1% solution in 0.1 N hydrochloric acid, trypsin (both BDH and Mann Research Labs) as a 0.1% solution in phosphate buffer (both pH 7.3 and 8.0) and papain (Calbiochem) as a 0.1% solution in phosphate buffer (pH 6.0). After fixation in 4% buffered formaldehyde (Bird and Saurer, 1967) both larvae and adults were examined and photographed under a Leitz microspectrograph, the spectra being recorded and scanned as previously described (Bird and Saurer, 1967).