Changes Associated with Parasitism in Nematodes. V. Ultrastructure of the Stylet Exudation and Dorsal Esophageal Gland Contents of Female Meloidogyne javanica

Abstract

The fine structure of the contents of the dorsal esophageal gland and the material exuded from it through the nematode stylet has been examined. The exudate appears to be derived from the breakdown of granules in the ampulla of the dorsal esophageal gland. These granules are synthesized from rough endoplasmic reticulum and Golgi bodies in the dorsal esophageal gland and passed forward to the ampulla. The exudate consists of a meshwork of particles which coalesce at the periphery of the exudate in water and become granular. The volume of the exudate is considered in relation to normal cells and to the giant cells into which it is injected. The stylet exudate produced by females of M. javanica has been shown (Bird, 1968b) to consist of basic protein containing carbohydrate. It does not appear to contain either enzymes or nucleic acids. Thus the giant cells which are produced and maintained in the roots of plants by these exudations (Bird, 1961, 1962) are thought to have their metabolism controlled by these histonelike proteins rather than by the direct action of enzymes or messenger RNA. However, as has been stated (Bird, 1968b), the exudate may contain material which could synthesize enzymes when placed into the cytoplasm of the host. In this paper the ultrastructure of both the stylet exudate and the contents of the dorsal esophageal gland are described. MATERIAL AND METHODS Females were dissected from galls in tomato roots and examined with a stereo microscope. Those nematodes that showed stylet activity were removed and either fixed in cold (5 C) 4% phosphate-buffered formaldehyde (pH 7.3) as described for larvae (Bird, 1968a) or left in shallow distilled water in petri dishes for periods of up to 18 hr to permit the formation of a stylet exudate. Great care was taken not to dislodge this exudate. The whole nematode and stylet exudate were fixed in the cold 4% buffered formaldehyde for 24 hr at 5 C, washed and postfixed in 1% Zetterqvist's osmium tetroxide as described previously (Bird, 1968a), and embedded intact. This led to poor fixation of the nematode (Fig. 2) due to impermeability of the cuticle. This had to be Received for publication 22 October 1968. done, however, because disturbances such as cutting off the head region, even after fixation, were enough to cause the exudate to become detached and lost. Females freshly dissected from galls and showing stylet activity were fixed for 30 min, after which their heads were severed and they were fixed for another 30 min as described previously for larvae (Bird, 1968a). Fixation, dehydration, embedding, and sectioning were also done in an exactly similar way. In some cases, however, the initial fixation was with osmium and the postsectional staining was with a 1:1 solution of 1% potassium permanganate: saturated uranyl acetate instead of with Reynold's lead citrate. Living material was examined mounted in distilled water in a sitting drop slide by means of a long focal length condenser and a X 40 water immersion objective (NA 0.75). Photographs were taken with electronic flash using Ilford HP4 film. The sections were examined with a Siemens Elmiskop 1A electron microscope operated at 80 kv and photographs were taken on Ilford special lantern contrasty plates.