Increasing Resolution and Versatility in Low Temperature Conventional and Field Emission Scanning Electron Microscopy

Studies were undertaken to expand the versatility and the resolution of low temperature conventional and field emission scanning electron microscopy (SEM). The results indicated that simple modified specimen holders, which could be used in conjunction with the commercial cryosystems, allowed one to store specimens for several weeks in liquid nitrogen, either before or after observation in a conventional SEM, without incurring degradation of the surface features. Other modified holders permitted one to move the specimen closer to the final lens or to use the upper secondary electron detector, which is available with some SEMs. Both of these procedures increased the resolution that was attainable with the standard holders. In conventional SEM (CSEM) and field emission SEM (FESEM), holders were also modified to allow one to obtain complementary images of fractured specimens. When a conventional vacuum evaporator equipped with a freeze-etch module was used in conjunction with these holders, specimens could be fractured, etched, shadowed with platinum and coated with carbon before the sample was transferred to the cryostage in the SEM. This procedure increased resolution beyond that obtained with the sputter units in two commercial cryosystems that were used on a CSEM and a FESEM, provided membrane particle resolution in the FESEM and produced a coating or replica that could be recovered and examined in a TEM. These results, which demonstrated how resolution of cryospecimens can be enhanced in CSEM and FESEM, indicated that coating specimens in a high vacuum evaporator provided an alternative procedure that could be used to obtain high resolution images in a FESEM

Caudal Polymorphism and Cephalic Morphology among First-Stage Larvae of \u3ci\u3eParelaphostrongylus odocoilei\u3c/i\u3e (Protostrongylidae: Elaphostrongylinae) in Dall’s Sheep from the Mackenzie Mountains, Canada

We demonstrate polymorphism in the structure of the tail among first-stage larvae of Parelaphostrongylus odocoilei (Protostrongylidae). Two distinct larvae, both with a characteristic dorsal spine, include (1) a morphotype with a kinked conical tail marked by three distinct transverse folds or joints and a symmetrical terminal tail spike and (2) a morphotype with a digitate terminal region lacking folds or joints and with an asymmetrical, subterminal tail spike. These divergent larval forms had been postulated as perhaps representing distinct species of elaphostrongyline nematodes. Application of a multilocus approach using ITS-2 sequences from the nuclear genome and COX-II sequences from the mitochondrial genome confirmed the identity of these larvae as P. odocoilei. Additionally, based on scanning electron microscopy (low-temperature field emission), the cephalic region of these larvae consisted of a cuticular triradiate stoma surrounded by six single circumoral papillae of the inner circle, ten papillae of the outer circle (four paired and two single), and two lateral amphids. Ours is the first demonstration of structural polymorphism among larval conspecifics in the Metastrongyloidea and Strongylida. The basis for this polymorphism remains undetermined, but such phenomena, if discovered to be more widespread, may contribute to continued confusion in discriminating among first-stage larvae for species, genera, and subfamilies within Protostrongylidae

Evaluation of Some Vulval Appendages in Nematode Taxonomy

A survey of the nature and phylogenetic distribution of nematode vulval appendages revealed 3 major classes based on composition, position, and orientation that included membranes, flaps, and epiptygmata. Minor classes included cuticular inflations, protruding vulvar appendages of extruded gonadal tissues, vulval ridges, and peri-vulval pits. Vulval membranes were found in Mermithida, Triplonchida, Chromadorida, Rhabditidae, Panagrolaimidae, Tylenchida, and Trichostrongylidae. Vulval flaps were found in Desmodoroidea, Mermithida, Oxyuroidea, Tylenchida, Rhabditida, and Trichostrongyloidea. Epiptygmata were present within Aphelenchida, Tylenchida, Rhabditida, including the diverged Steinernematidae, and Enoplida. Within the Rhabditida, vulval ridges occurred in Cervidellus, peri-vulval pits in Strongyloides, cuticular inflations in Trichostrongylidae, and vulval cuticular sacs in Myolaimus and Deleyia. Vulval membranes have been confused with persistent copulatory sacs deposited by males, and some putative appendages may be artifactual. Vulval appendages occurred almost exclusively in commensal or parasitic nematode taxa. Appendages were discussed based on their relative taxonomic reliability, ecological associations, and distribution in the context of recent 18S ribosomal DNA molecular phylogenetic trees for the nematodes. Characters were found to be distributed across subsets of terminal and phylogenetically distant taxa, demonstrating considerable homoplasy. Accurate definitions, terminology, and documentation of the taxonomic distribution of vulval appendages are important in evaluations of hypotheses for either parallelism and developmental constraint or convergence and adaptation

Caudal Polymorphism and Cephalic Morphology among First-Stage Larvae of \u3ci\u3eParelaphostrongylus odocoilei\u3c/i\u3e (Protostrongylidae: Elaphostrongylinae) in Dall’s Sheep from the Mackenzie Mountains, Canada

We demonstrate polymorphism in the structure of the tail among first-stage larvae of Parelaphostrongylus odocoilei (Protostrongylidae). Two distinct larvae, both with a characteristic dorsal spine, include (1) a morphotype with a kinked conical tail marked by three distinct transverse folds or joints and a symmetrical terminal tail spike and (2) a morphotype with a digitate terminal region lacking folds or joints and with an asymmetrical, subterminal tail spike. These divergent larval forms had been postulated as perhaps representing distinct species of elaphostrongyline nematodes. Application of a multilocus approach using ITS-2 sequences from the nuclear genome and COX-II sequences from the mitochondrial genome confirmed the identity of these larvae as P. odocoilei. Additionally, based on scanning electron microscopy (low-temperature field emission), the cephalic region of these larvae consisted of a cuticular triradiate stoma surrounded by six single circumoral papillae of the inner circle, ten papillae of the outer circle (four paired and two single), and two lateral amphids. Ours is the first demonstration of structural polymorphism among larval conspecifics in the Metastrongyloidea and Strongylida. The basis for this polymorphism remains undetermined, but such phenomena, if discovered to be more widespread, may contribute to continued confusion in discriminating among first-stage larvae for species, genera, and subfamilies within Protostrongylidae