Ultrastructural study of anther parasitism of Ficus laevigata by Ficophagus laevigatus (Aphelenchoididae)

Late phase C Ficus laevigata figs were collected from a canal adjacent to Palm Avenue, Florida City, Dade County, Florida on March 7, 1994. Each fig was cut in half, with one half being chopped up and placed in sterilized water for >20 minutes to determine nematode presence or absence. Once determined, the other half of the fig was dissected, and whole anthers were removed for ultrastructural comparisons. Nematode-infested or uninfested (control) anthers were separately placed into 2% formaldehyde (prepared from paraformaldehyde) and 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.2 and fixed overnight at 4°C. After repeated rinsing in buffer, anthers were postfixed in 2% OsO₄ in 0.1 M cacodylate buffer at pH 7.2 for 3.5 h at 22°C. Anthers were then rinsed in water, fixed with 1% aqueous uranyl acetate, slowly dehydrated through a graded ethanol series into 100% ethanol and then into 100% acetone, and infiltrated with Spurr’s epoxy resin. Resin blocks were sectioned on a MC® MT-6000-XL ultra-microtome, and sections with silver refraction (75 nm) were picked up on copper grids with a 0.35% Formvar coating reinforced with a light carbon film. Sections were post-stained with 5% aqueous uranyl acetate and lead citrate before being viewed on a Zeiss EM-10CA® transmission electron microscope at 80 kV.

One nematode-infested anther from a nematode-infested fig and three un-infested (control) anthers from an un-infested (control) fig were examined. The anthers were sagittally (or near sagittally) sectioned, observed, and photographed for comparisons through the anther, anther filament, endothecium, and pollen tubes. Photographs were taken at 2.5K magnification in an overlapping linear series starting at the ventral side (along the anther endothecium) and running completely through to the dorsal side of the anther filament, from ensheathing perianth lobe to ensheathing perianth lobe. In all cases, the orientation of the series was standardized just below the vertical midline of the anther, where the anther filament connected to the anther, allowing for examination of the anther epidermal cells connecting to the ventral anther filament epidermal cells, because nematode-associated pathology was consistently observed in this region in LM sections (Giblin-Davis et al., 1995). A total of 90 photographs were taken using Kodak EM film 4489, including some at magnifications up to 50K for better discernment of specific targets.

The photographs were edited with Photoshop Elements 9 (Adobe) for contrast and brightness adjustments and to construct the figures. The original captures are available upon request.

The endothecium in the healthy anthers appeared as described by Center et al. (1999) with LM (Fig. 1A; Suppl. Fig. S1) as a two-to-three-cell layer of highly vacuolated cells with cellular components, mostly relegated to the periphery near the cell walls, and surrounding an electron lucent vacuole (Fig. 3). The cells in the endothecium of the nematode parasitized anther in this study were similar in size and cytoplasmic density to healthy anthers (Fig. 4). Thus, the effect of nematodes occurring in the space between the anther filament and the back of the anther on the endothecium was relatively minor. Center et al. (1999) observed that the effects on the endothecium were relatively minor when compared to the anther epidermal and anther filament epidermal cells that were in direct or close contact with the nematodes/exudate; however, the endothecium lost cell integrity as the densities of nematodes increased (Fig. 2; Suppl. Figs. S1, S2).

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Sagittal sections of the healthy anther epidermis comprised a single layer of cells with large central storage vacuoles filled with very electron-dense and darkly staining material and cellular components that were mostly arranged on the periphery near the cell walls (Fig. 5A; Suppl. Figs. S3,S4). This epidermal layer bounded a lightly staining multilayer of parenchymal cells that surrounded the pollentubes/tapetum. In the nematode-infested anther, this single layer of epidermal cells was hypertrophied and 2 to 5 times the size of the cells in the healthy epidermis with thickened cell walls (Figs. 2, 5B). Each hypertrophied cell had an enlarged and often irregularly shaped nucleus with an enlarged nucleolus, and were metabolically active as indicated by increased numbers of mitochondria and what appeared to be pro-plastids as well as endoplasmic reticulum. The darkly stained central storage vacuole had broken up into many smaller darkly stained globs, or less electron-dense granular cytoplasm of much higher volume in the enlarged cells (Figs. 6, 7; Suppl. Fig. S5). The bounded parenchymal cells were also slightly enlarged and had slightly thickened cell walls (Fig. 5B; Suppl. Fig. S6).

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Sagittal sections of healthy ventral anther filament epidermis near the connection to the backside of the anther showed a single to double (rarely triple) layer of cells that were smaller or near to the same size as the adjacent anther epidermal cells with similarly large central storage vacuoles, filled with very electron-dense and darkly stained material, and with cellular components arranged mostly at the periphery near the cell walls (Fig. 8A; Suppl. Figs. S3,S4). This layer of cells bounded a light-staining multilayer of parenchymal cells that was marbled with short runs of 3-7 larger, more squared-looking cells with similarly large central storage vacuoles, filled with very electron-dense and darkly stained material, and with cellular components mostly present at the periphery near the cell walls (Fig. 1; Suppl. Fig. S3) (these latter cells were equivalent to the safranin-rich red-staining cells in the filaments of healthy and nematode-infested anthers observed with LM by Giblin-Davis et al.,1995). Ventral anther filament epidermal cells near the propagating nematodes were similar to the adjacent anther epidermal cells with hypertrophy of the single or multiple layers with increases in cell size of 2-5x, a decentralization and dissipation of the dense material in the storage vacuoles creating a more granular-looking cytoplasm, enlargement of the irregularly bounded nucleus and spherical nucleolus, thickening of cell walls, and an apparent increase in cell metabolism (Figs. 8B-10). Mobilization of electron-dense vacuolar material appeared to be occurring in some epidermal cells (Figs. 9B, 10A).

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We also observed a few strands of fungal mycelium in the area between the anther and anther filament but found nothing that could reasonably have nourished the population of propagating nematodes or that could explain induction of the pathological effects observed in association with the nematodes (Fig. 11).

Figure 11:

Healthy pollen was observed in pollen tubes in each of the three un-infested anthers that were sectioned and photographed (Fig. 3). In addition, relatively healthy pollen was observed in one of the two pollen tubes observed in the nematode-infested anther (Fig. 4). The pollen tube expressing the most pathology was closest to the hypertrophied anther epidermal cells nearest nematodes in the sectioned anther (Fig. 2), suggesting a proximity-based systemic reaction to the nematode infestation. This supports the observations of Giblin-Davis et al. (1995) from LM observations of paraffin-sectioned material where “pollen production often appeared normal in one or more pollen sacs of lightly infested male florets. However, in heavily infested male florets, the four pollen sacs were completely deformed. The tapetum was sometimes necrotic and (or) disorganized.”

We arbitrarily sectioned through more than ten F. laevigatus in the nematode-infested anther between the anther epidermal cells and the ventral anther filament epidermal cells, revealing several ultrastructural snapshots of their morphology. Going from anterior to posterior of F. laevigatus, we observed: 1) two near transverse sections of the head showing the stylet shaft tip with a lateral aperture (with secretion?) within the vestibule (or vestibule extension?) which was ringed by an electron lucent material (or space) and the six equally spaced inner labial sensilla nerve (ilsn) pairs through the base of the cephalic framework (Fig. 12); 2) one near transverse section of the anterior region showing the procorpus, procorpus lumen, and secretory excretory cells (Fig. 13A); 3) one near mid-sagittal section of the anterior region showing the procorpus and metacorpus (Fig. 13B); and 4) two near transverse sections of the female (?) midbody showing the intestinal syncytium with a close-up of microvilli and the digested food within as well as the gonad (Fig. 14).

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The ultrastructure of the perianth lobes was very similar in both healthy and infested anthers (Fig. 1).