Three species of Heterorhabditis were studied using scanning electron microscopy. In all three species, the sheath of the infective stage juvenile (the sheath is the cuticle of the second-stage) is tessellate anteriorly and bears longitudinal ridges throughout almost the entire body length. The infective juvenile, and the third stage which develops from it, have a dorsal tooth on the labial region. The fourth stage has low lips and labial papillae, and a rounded oral aperture. The hermaphrodite has massive lips with prominent labial papillae at the apex of the lips and a hexagonal mouth cavity.

The family Heterorhabditidae contains only one genus, Heterorhabditis, described by Poinar in 1975 (4). Currently, eight species have been described. The morphological characteristics of these species are similar, and they have been differentiated mostly based on morphometrics of the IJ (third-stage infective juvenile). Few studies have been done on the morphology of other juvenile stages or adults of this group of nematodes. Mracek et al. (1) published scanning electron microscopy (SEM) photographs of the anterior part of a J2 (second-stage juvenile), the labial region of a female (although the authors did not mention the type of female, we think it was the hermaphrodite), the excretory pore, and the tail of a female (probably the hermaphrodite). In a second paper, Mracek and Bednarek (2) reported on cuticular structures of the J2 and the IJ of Heterorhabditis spp.

In this paper, we report our observations on the morphology of life stages from the infective stage juvenile to the hermaphrodite for three species of Heterorhabditis.

MATERIALS AND METHODS

The three species studied were Heterorhabditisbacteriophora Poinar, 1975 from our collection; H. hawaiiensis Gardner et al., 1994; and H. megidis Poinar et al., 1987 from Dr. S. P. Stock, Dept. Nematology, Univ. of California, Davis. All three species were maintained in our laboratory by producing them on larvae of the greater wax moth, Galleria mellonella (L.) at 25^o C. Ten G. mellonella were exposed to about 10,000 IJs of each species in a petri dish (100 x 15 mm) containing a filter paper (90 mm). Each day after exposure until new IJs emerged, one insect cadaver infected with each nematode species was washed 3 times in tap water to remove any IJs on the exterior of the insect. Then the insect was dissected in a saline solution (1% NaCl in water) to collect nematodes. The nematodes were washed three times with saline solution, and then prepared for SEM observation by the method of Nguyen and Smart (3).

RESULTS AND DISCUSSION

Third-stage infective juvenile (IJ) before entering host(Fig. 1) : The IJ initially is encased in the cuticle of the second-stage juvenile (J2). The J2 cuticle is usually lost after storage or after entering the hemocoel of insects. The cuticle of the J2 of all three species has longitudinal ridges throughout most of the body length, and a tessellate pattern in the most anterior part of the body (Fig. 1A-C). The IJ of H. megidis has a prominent dorsal tooth associated with a distinct membranous ring around the oral aperture (Fig.1 D, E). The IJs of H. bacteriophora and H. hawaiiensis have a dorsal tooth, but the membranous ring is inconspicuous (Fig. 1F). The purpose of the membranous ring is not known, but it may play a role in penetrating the intestinal wall of an insect host. The two small subventral teeth described by Poinar and Georgis (5) were not observed in any of the three species studied. Amphids were circular and located on a protuberance (Fig. 1D-F). Neither lips, nor labial or cephalic papillae, were observed. The body was annulate. The lateral field began with one incisure anteriorly (Fig.1D), followed by three incisures posteriorly resulting in two ridges (Fig. 1G-I). The middle incisure appears deeper and wider than the two lateral ones. Except for the presence of the membranous ring in the labial region of H. megidis only, no other differences were found in the general morphology of the IJs for H. bacteriophora, H. hawaiiensis, and H. megidis.

Day one after entering host: After entering the insect body cavity, the IJ changed to a feeding third-stage juvenile (J3) and the body enlarged. The base of the dorsal tooth also enlarged and thickened to become well-sclerotized anteriorly (Fig. 2A-C). A few individuals of H. hawaiiensis began the molting process.

Day two after entering host: The J3 increased in size, and if the molting process had not begun at the end of day one, it began during day two. The cuticle loosened anteriorly, causing the labial region to collapse when specimens were prepared for SEM (Fig. 2D); the cause of labial collapse can be seen by light microscopy as the clear space between the two cuticles (Fig. 2E). Under the light microscope, a solid structure, in addition to the dorsal tooth, is present (Fig.2E). Since this structure does not appear in SEM micrographs it must be subcuticular. The J3 of H. bacteriophora and H. megidis had the same labial appearance (Fig. 2D), indicating similar growth rates to this point. H. hawaiiensis developed faster than the other two species, with most of the J3s molting to fourth-stage juveniles (J4s) (Fig. 2F). The labial region of the H. hawaiiensis J4 was similar to that of H. bacteriophora, as described below.

Day three after entering host (Fig. 3): Most of the J3s of H. bacteriophora molted to J4s by day three (Fig. 3A-C). The dorsal tooth was not present in the J4, and the cheilorhabdions appeared as a ring in the mouth. There were six lips, each with one indistinct papilla situated at a distance from the oral aperture; the amphids were less conspicuous than in the IJ and J3.

For H. megidis, both J4s and young hermaphrodites were present. The labial region of the J4 (Fig. 3D) was similar to that of H. bacteriophora (Fig. 3 A-C). The hermaphrodites of H. megidis (Fig. 3E, F) had prominent amphids, lips, and labial papillae. The lips were at the edge of or just in side the mouth cavity giving the oral aperture a hexagonal shape (Fig. 3E,F). By day 3, all juveniles of H. hawaiiensis had become young hermaphrodites with prominent lips and labial papillae, similar in shape and size to those of H. megidis but curved outward (Fig. 3G-I) .

Days four and five after entering host (Figs. 4): Four days after entry, most of the juveniles of all three species had become young hermaphrodites; by day 5 they became mature adults. The lips were large and elevated, sometimes curved outward, each with a labial papilla at its apex (Fig. 4 A-I). The ten cephalic papillae described by Poinar et al. (6) for H. megidis, if present, were not obvious on any of the three species that we studied. Sometimes one or two small dots were observed at the base of each lip, but they were not prominent in our preparations (Figs. 4).

The vulval area (Fig. 5 A-C) of the three species of Heterorhabditis is reminiscent of the perineal pattern of Meloidogyne spp. (7). The vulva of H. bacteriophora (Fig.5A) is characterized by an elliptical opening with rings around it which extend into the vagina. In H. megidis (Fig. 5B), the posterior vulval lip is depressed, the annules extend into the vagina, and the annules anterior and posterior to the vulva form acute angles at the lateral margins of the vulva. The vulva of H. hawaiiensis (Fig. 5C) is slit-like, the vulval lips are prominent, the annules do not extend into the vagina, and the annules around the vulva are wavy or broken. The tail of the hermaphrodite is conoid (Fig. 5D), and the anus, which appears to be curved, is on a protuberance.

CONCLUSIONS

SEM observations of three species of Heterorhabditis suggest several conclusions.

Based on morphological characters of the labial region, the rate of development of H. hawaiiensis is greater than that of H. megidis and H. bacteriophora.

The growth rate between the three species was different by days two and three after infection. For example, by day two after infection, most of the H. hawaiiensis juveniles had become J4s (Fig. 2F) whereas those of H. bacteriophora and H. megidis remained as J3s (Fig. 2A-E). By day three, most of the H. bacteriophora juveniles had become J4 (Fig. 3A-C), whereas H. megidis had a mixed population of J4 and young hermaphroditic females (Fig. 3D-F), and all juveniles of H. hawaiiensis had become young hermaphroditic females (Fig. 3G-I).

For all three species, the morphology of the labial region and the cuticle of the J2, IJ, J3, J4 and the adults are different. The oral aperture of the J2 is almost closed, the cuticle is tessellate anteriorly, and longitudinal ridges occur over almost the entire body posteriorly. The IJ, and the J3 which develops from it, have a dorsal tooth in the labial region. The body has annules and lateral fields with two ridges. Longitudinal ridges present in the J2 are absent. The J4 has low lips and labial papillae, and a rounded oral aperture. The lips of the hermaphrodite are massive with prominent, anteriorly directed labial papillae at their apex. The arrangement of the lips creates a hexagonal-shaped mouth cavity. All of the above characters of the different stages are consistent for the three species studied.

The structure of the vulval region of the three species seems different, and may or may not be different enough to use for species identification.

LITERATURE CITED

1. Mracek, Z., J. Weiser, and E. Arteaga 1984. Scanning electron microscope study of Heterorhabditis heliothidis (Nematoda: Heterorhabditidae). Nematologica 30:112- 114.

2. Mracek, Z., and A. Bednarek. 1992. Cuticular structures of J2 and J3 of Heterorhabditis. Nematologica 38:386-390.

3. Nguyen, K. B., and G. C. Smart, Jr. 1995. Scanning electron microscope studies of Steinernema glaseri (Nematoda: Steinernematidae). Nematologica 41:183-190.

4. Poinar, G. O., Jr. 1975. Description and biology of a new insect parasitic rhabditoid, Heterorhabditisbacteriophora n. gen. n. sp. (Rhabditida; Heterorhabditidae n. fam.). Nematologica 21:463-470.

5. Poinar, G. O., Jr., and R. Georgis. 1990. Charactization and field application of Heterorhabditis bacteriophora strain HP 88 (Heterorhabditidae: Rhabditida).
Revue de Nematologie 13:387-5

6. Poinar, G. O., Jr., T. Jackson, and M. Klein. 1987. Heterorhabditis megidis sp. n. (Heterorhabditidae: Rhabditida) parasitic in the Japanese beetle, Popillia japonica (Scarabaeidae: Coleoptera), in Ohio. Proc. Helminthological Soc. Washington 54:53-59.

7. Taylor, A. L., and J. N. Sasser. 1980. Biology, identification and control of root-knot nematodes (Meloidogyne species). North Carolina State Univ. Graphics, Raleigh, NC.