Journal of Nematology 22(3):386-394. 1990. © The Society of Nematologists 1990. Phylogenetic Implications of Phasmid Absence in Males of Three Genera in Heteroderinae 1 L. K. CARTA2 AND J. G. BALDWINs Abstract: Absence of the phasmid was demonstrated with the transmission electron microscope in immature third-stage (M3) and fourth-stage (M4) males and mature fifth-stage males (M5) of Heterodera schachtii, M3 and M4 of Verutus volvingentis, and M5 of Cactodera eremica. This absence was supported by the lack of phasmid staining with Coomassie blue and cobalt sulfide. All phasmid structures, except the canal and ampulla, were absent in the postpenetration second-stagejuvenile (]2) of H. schachtii. The prepenetration V. volvingentis J2 differs from H. schachtii by having only a canal remnant and no ampulla. This and parsimonious evidence suggest that these two types of phasmids probably evolved in parallel, although ampulla and receptor cavity shape are similar. Absence of the male phasmid throughout development might be associated with an amphimictic mode of reproduction. Phasmid function is discussed, and female pheromone reception ruled out. Variations in ampulla shape are evaluated as phylogenetic character states within the Heteroderinae and putative phylogenetic outgroup Hoplolaimidae. Key words: anaphimixis, ampulla, cell death, Cactodera eremica, Heterodera schachtii, Heteroderinae, parallel evolution, parthenogenesis, phasmid, phylogeny, ultrastructure, Verutus volvingentis. Phasmid sensory organs on the tails of phasmid openings in the males of most gen- secernentean nematodes are sometimes era within the plant-parasitic Heteroderi- notoriously difficult to locate with the light nae, except Meloidodera (24) and perhaps microscope (18). Because the assignment Cryphodera (10) and Zelandodera (43). of uncertain genera to classes Secernentea Recent transmission electron micro- or Adenophorea depends in part on this scope (TEM) observations of Meloidodera taxonomically important character, phas- males showed phasmids to be present in all mid obscurity can make this decision dif- stages, and their variability was described ficult. In addition, the phasmid may be sec- (7). Because sufficient reliable characters ondarily lost altogether in some species are lacking for phylogenetic evaluation in which, on the basis of other characters, Heteroderinae (5), further research is clearly belong to Secernentea (22,33). needed to determine the variability of the Where phasmids are clearly present in ju- phasmid within the family and specifically veniles and often in females of a species, confirm the absence of male phasmids with phasmids have sometimes been reported to TEM. be absent in males of the same species (9). Phasmids were not observed on males of This was supported by use of scanning elec- Heterodera schachtii Schmidt, 1871 with SEM tron microscopy (SEM) on the cockroach (24), but phasmids have been described in parasite, Hammerschmidtiella diesingi Chit- the literature (16). Males of Verutus volvin- wood, 1932, where phasmid openings are gentis Esser, 1981 and Cactodera eremica absent in males (44). The SEM did not show Baldwin & Bell, 1985 do not have phasmids visible with SEM (24). Verutus represents a genus of uncertain position in the subfam- Received for publication 4 August 1988. ily because the mixture of some ancestral 1 This research was supported in part by National Science Foundation grant BSR-84-15627 to the second author. This and other highly derived characters con- paper represents a portion of the first author's Ph.D. disser- founds parsimonious accommodation to tation. Mention of a product or trade name does not consti- tute an endorsement of that product. phylogenetic schemes (5). 2 Research Fellow, Division of Biology, California Institute There may be intermediate phasmid of Technology, Pasadena, CA 91125. 3 Associate Professor, Department of Nematology, Uni- character states throughout development versity of California, Riverside, CA 92521. forming a transition series within the Het- The authors thank D. T. Kaplan, USDA, Orlando, Florida, for greenhouse cultures of Verutus volvingentis, and the late eroderinae. Any evidence for functional A. R. Stone, Rothamsted Experimental Station, Harpenden, associations with stages in development Herts., England, for the English population of Heterodera schachtii. should be considered in evaluating reli- 386 Phasmid Absence in Heteroderinae: Carta, Baldwin 387 ability and homology of the potential char- Nematodes were fixed, infiltrated, sec- acters, including phasmid loss. There is a tioned, and stained for TEM by previously need for study, not only of the presence or reported methods (7). The following spec- absence of the phasmid, but of the mor- imens were sectioned: H. schachtii--eight phology of the characteristic phasmid M5, three M4, two M3, and one postpene- structures such as the ampulla, neuron, tration J2 (English population); V. volvin- socket and sheath cell. gentis--four M5, four M4, and three J2; C. eremica--two M5. MATERIALS AND METHODS Two procedures were used to stain adult Verutus volvingentis second-stage juve- male phasmids of V. volvingentis and H. niles (]2), fourth-stage males (M4), and ma- schachtii for light microscopy (LM). One ture adult males (M5) were extracted with was an adaptation of a cobalt sulfide-al- a warm mist from buttonweed (Diodea vir- kaline phosphatase method for insect sen- giniana) roots. The culture was maintained sory organs using an acetone dip followed in the University of California, Riverside by cobalt chloride and ammonium sulfide greenhouse from specimens supplied from (30). The second method used Coomassie Orlando, Florida, by D. T. Kaplan. The blue in acetic acid-methanol, where the posthatch stage of Verutus is considered a specimens were subjected to butyl acetate J2 for comparison with other heteroder- in a ring of Zut (26). Both procedures were ines, although Esser (14) was unable to de- lethal to the nematodes. tect a molt within the egg. The different stages were identified by superimposed ec- RESULTS dysed cuticles as well as by the tail and No phasmid or any part of its structure general body morphology (13,14). could be seen with TEM in M3, M4, and Heterodera schachtii sugar beet popula- M5 specimens of either population of H. tions from Santa Maria, California, and schachtii, in M4 and M5 of V. volvingentis, Great Barton, England, were maintained or in M5 ofC. eremica. Both phasmid stains on sugar beet in the greenhouse. The En- for LM gave negative results for the J2 and glish population, established from cysts M5 stages ofH. schachtii and V. volvingentis. provided by A. R. Stone, was believed to Phasmids in the J2 and M5 of Meloidodera be from similar populations used by Frank- floridensis and Meloidogyne incognita, which lin (16) in illustrating a male phasmid. were used as controls, were distinctly Nematodes were inoculated to sterile sugar stained. beet (Beta vulgaris) or rape seed (Brassica Heterodera schachtii: The postpenetra- napus) plants in Gamborg's medium (pH tionJ2 has a flask-shaped ampullar opening 6.5) and Gelrite supplemented with vita- (Figs. 1B, 3G); a slightly curved, ladle- mins (28). Plants were grown under stan- shaped canal (Fig. 1A); and remnants of dard Gro-lux lamps at 400 lumens for ap- the socket cells, canals, and sheath cells (Fig. proximately 1 week before being inoculated 1A, B). There is no indication of a neuron with nematodes. Nematodes were hand or cell nuclei. The cellular retraction from picked into Beem capsule baskets contain- the cuticle indicates that molting has be- ing previously boiled tap water, placed in gun (Fig. 2A, B). 0.0004% HgCI~ (23) for 3-20 hours, and Verutus volvingentis young J2 has a bottle- transferred to one-half strength saturated shaped ampulla opening (Figs. 2B, 3F) with rifampicin (6) for 10 minutes. Nematodes a plug of dense secretion traversing the were then directly pipetted onto the cul- outer pore to the outside of the body wall ture medium. cuticle (Fig. 2B). A canal is visible within a Heterodera schachtii stages were dissected single socket cell (Fig. 2A, C). The distal from the roots with a scalpel and needle, region of the sheath cell surrounds a nar- and the stages were identified by body mor- row, ladle-shaped end apparatus (canal ex- phology and surrounding cuticles (27). tension), which merges into the beginning 388 Journal of Nematology, Volume 22, No. 3, July 1990 Phasmid Absence in Heteroderinae: Carta, Baldwin 389 of a small circular receptor cavity. Periph- to the proposal by Ferris (15). This is fur- eral to the center circle of the receptor ther supported by characters unique to Ve- cavity, approximately eight long, curved rutus, including highly derived lip patterns, lamellae surround the dendrite (Fig. 2A). diminutive host syncytium, and multiple- The lamellae have dense deposits near the B-layer of the body wall (5). Consideration central cavity, and the sheath cell contains of 19 reliable characters in a computerized moderately dense glycogen deposits. The phylogenetic analysis placed Verutus as an socket cell has less glycogen than the sheath outgroup of all other Heteroderinae (5). It cell and a few mitochondria. has been proposed that Verutus is part of Older J2 specimens (Fig. 2D) exhibit ex- the Rotylenchulidae (Hoplolaimidae) (35), tensive cellular degeneration similar in ap- a hypothesis that will require further test- pearance to the degenerating tails in older ing (4). H. schachtii J2. Only an inflated canal and Loss of the phasmid in H. schachtii after possibly some of the condensed lamellar hatching clarifies the developmental po- membranes from the degenerate sheath cell larity of the two forms of the J2 phasmid are visible. Neither an ampulla nor den- originally reported in this species. Type A drite is apparent. is a relatively rare, well-developed phas- mid; type B is a poorly developed phasmid DISCUSSION (2). We have shown that the phasmid The sheath cell of Verutus has a receptor changes with time of development and that cavity and lamellar form very much like the type B phasmid represents an inter- those of Heterodera (2).