Development of Oral Structure in Salmonema emphemeridarum (Nematoda: Spirurida: Cystidicolidae)

Abstract

The morphology of the oral region is important in the identification of adult and larval parasitic nematodes. This is nowhere more true than in the assignment of cystidicolid nematodes of fishes to one of approximately 23 genera. However, the small size of the anterior end of these worms has complicated identification of worms (Moravec 2007) and resulted in a poor understanding of the morphology of oral structures, including the oral structure of infective/third stage larvae/juveniles, which are found in crustacean and insects. The purpose of this study is to utilize Scanning Electron Microscopy (SEM) to document the morphogenesis of oral structures of the cystidicolid nematode Salmonema emphemeridarum (Linstow 1872) and to provide information on the certainty of the identification of third stage larva to genus and species. Adult female worms were collected from brook trout, Salvelinus fontinalis (Mitchell 1814), captured in Rocky Saugeen River, Ontario, Canada in June 1987. Larvated eggs were fed to larval mayflies (Ephemeroptera) collected from the Eramosa River where it passes under Stone Road, Guelph Ontario (43.547363, -80.1997499). Mayflies successfully infected included Stenonema ithaca, Stenonema sp. and Isonychia sp. No nematode larvae were found in 60 mayflies collected at this locality, and no salmonids are present in this portion of the Eramosa River (Osmond 1971). Mayflies were held in an aquarium at 21oC and examined for parasite larvae at intervals for 62 days. Adult worms used for SEM were from natural infection of the brook trout. Worms processed for SEM were cleaned in saline preserved in 1.25% gluteraldehyde, post-fixed in 1% osmium tetroxide, dehydrated in an ethanol series, critical point dried using CO2, mounted on stubs, coated with gold palladium alloy and viewed with a Jeol (Model JSM 35-C) scanning electron microscope. General terminology follows Anderson et al. (2009) and terminology of the oral morphology of third-stage and adult worms follows Appy (1981). First stage larvae, present one to five days post infection (dpi), possessed an oral opening, a pore, and a hooked tooth (Fig. 1A). Second stage larvae (11 to 17 dpi) possessed a smooth circular oral opening, with presumptive lateral amphids (Fig. 1B). Oral structures of the third stage larva (>17 dpi) are visible inside the oral opening of some molting second stage larvae. The oral structure of third stage larvae consisted of a dorsoventrally elongate oral opening with broad, relatively flat pseudolabia emanating from the cuticular lining of the buccal cavity (stoma) (Fig. 1C). The sublabia appear contiguous ventrally and dorsally and their apical surface appears smooth. Four cephalic papillae and amphidial openings are visible. The oral morphology of adult worms is similar to third stage larvae except that in adults the sublabia appear disconnected ventrally and dorsally and have an indentation forming two lobes (Fig. 1D). While SEM has become more common in the descriptions of adult cystidicolids (Appy 1981; Ko 1986; Moravec 2007) there are only a few cases where SEM has been used to describe third stage larva (Appy and Dadswell 1983; Moravec et al. 2003) and no studies have depicted firstand second-stage larvae. The boring tooth in first-stage larva was previously identified in light microscopic studies as a refractile body (Moravec 1967; Appy and Dadswell 1983) and is presumably used to penetrate the gut wall and gain access to the body cavity and migrate into the