Caudal Polymorphism and Cephalic Morphology among First-Stage Larvae of Parelaphostrongylus odocoilei (Protostrongylidae: Elaphostrongylinae) in Dallâ•Žs 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 3 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 6 single circumoral papillae of the inner circle, 10 papillae of the outer circle (4 paired and 2 single), and 2 lateral amphids. Our’s 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 firststage larvae for species, genera, and subfamilies within Protostrongylidae. In North American ungulates, species among 5 genera of protostrongylids, i.e., Elaphostrongylus Cameron, 1931, Parelaphostrongylus Boev and Shul’ts, 1950, Muellerius Cameron, 1927, Umingmakstrongylus Hoberg, Polley, Gunn and Nishi, 1995, and Varestrongylus Bhalerao, 1932, produce morphologically similar first-stage larvae (L1) with a characteristic dorsal spine (DSL) (Boev, 1975; Mason, 1995; Carreno and Hoberg, 1999). Other species of protostrongylids in the Nearctic (e.g., species of Protostrongylus Kamensky, 1905, and Orthostrongylus Dougherty and Gobel, 1946) produce spike-tailed larvae clearly distinguishable from such DSL (Boev, 1975; Mason, 1995; Carreno and Hoberg, 1999). Although relative and absolute lengths for L1 and third-stage larvae (L3) among a diversity of protostrongyles has been applied in species-level identification, measurements may overlap among congeners or among larvae representing otherwise divergent genera (Platt, 1978; Kralka and Samuel, 1984; Pybus and Shave, 1984; Pybus et al., 1984; Gray et al., 1985). Intraspecific variation in length for L1 has commonly been recognized among a diversity of protostrongylids in ungulate hosts, including Cystocaulus ocreatus (Railliet and Henry, 1907), Muellerius capillaris (Mueller, 1889), and Protostrongylus tauricus Schul’ts and Kadenazii, 1949 (Boev, 1975); among Elaphostrongylinae, such has been observed in Parelaphostrongylus odocoilei (Hobmaier and Hobmaier, 1934), P. andersoni Prestwood, 1972, and Elaphostrongylus alces Stéen, Chabaud and Rehbinder, 1989, from isolated geographic localities (e.g., Kontrimavichus et al., 1976; Platt, 1978; Lankester and Hauta, 1989; Lankester et al., 1998). Consequently, definitive identification of L1 among genera or Received 18 January 2005; revised 18 April 2005; accepted 18 April 2005. * Department of Veterinary Microbiology, Western College of Veterinary Medicine, 52 Campus Dr., University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B4. † Beltsville Electron Microscopy Unit, Soybean Genomics and Improvement Laboratory, US Department of Agriculture, Agricultural Research Service, Beltsville Area Research Center East, Bldg. 465, Entomology Road, Beltsville, Maryland 20705. species based on either morphological or meristic data is often problematic due to extensive intraspecific variation (length) or minimal interspecific variation manifested in multiple genera with structurally similar larvae (Jenkins et al., 2005). Structural polymorphism among L1 representing single species is considered uncommon among the Protostrongylidae (Boev, 1975; Kontrimavichus et al., 1976; Lankester, 2001), although it has been recognized, but not extensively described, in the structure of the tails and tail spikes among DSL of some Elaphostrongylinae, such as P. odocoilei and P. andersoni (Lankester et al., 1976; Pybus and Shave, 1984; Kutz et al., 2001). Pybus and Shave (1984) reported extensive variation in caudal structure among populations of DSL in fecal samples from mule deer, Odocoileus hemionus (Rafinesque), and mountain goats, Oreamnos americanus (de Blainville), infected with adult P. odocoilei from Alberta. Subsequently, Kutz et al. (2001) briefly described 2 distinctive forms of L1 based on specimens in fecal samples from Dall’s sheep, Ovis dalli dalli Nelson, with patent infections of P. odocoilei from the Mackenzie Mountains, Northwest Territories. They suggested that 1 of these morphotypes could represent an undescribed species with adult nematodes sequestered in an unknown site, perhaps in the vasculature. Meristic differences, however, were not demonstrated between morphotypes of larvae, and adult nematodes were all consistent with P. odocoilei (Platt and Samuel, 1978; Kutz et al., 2001); other elaphostrongylines were not discovered in the vasculature or central nervous system of definitive hosts. General observations of morphological variation in L1 of various protostrongylids and the specific reports of structurally divergent larvae associated with patent infections of P. odocoilei emphasize the challenges involved in identification and diagnostics for these nematodes (e.g., Boev, 1975; Kontrimavichus et al., 1976; Lankester et al., 1976; Platt, 1978; Pybus and Shave, 1984; Pybus et al., 1984). In the current study, we applied molecular methods and multilocus sequence analyses of nuclear (ITS-2) and mitochondrial (COX-II) DNA in a comparison of known adults and distinct morphotypes of DSL representing putative P. odocoilei. Our protocols paralleled those HOBERG ET AL.—NEMATODE LARVAL POLYMORPHISM 1319 developed by Jenkins et al. (2005), in which ITS-2 sequences for unknown larvae extracted from feces were validated against those from definitively identified adults of P. odocoilei. We examine the hypothesis that these distinct forms do not represent separate species, but are representative of dimorphic phenotypes. Additionally, we provide new information for structural characteristics of protostrongylid L1 based on scanning electron microscopy (SEM), as advocated by Carreno and Hoberg (1999). MATERIALS AND METHODS