Species identification using morphological and molecular methods

Correct species identification is a prerequisite for virtually all nematode research. It is important not only for the understanding of nematode community structures, communication about the work, but also connection to relevant literature. The morphology-based and molecular-based methods are widely used to properly position nematodes under investigation within the taxonomic framework. Taking Baylisascaris procyonis as example, we discussed how the methods help to distinguish it from other nematode species.

The labial structure of adult B. procyonis was studied by scanning electron microscopy (SEM), and some morphological aspects which were not reported by light microscopy study were described: the evident three large lips of ascaridoid nematodes, with each lip’s apical part smooth and basal part reticulated (Fig 1); dome-shaped and smooth small papilla, and broad large papilla with a prominent central pore (Fig5); the externolateral papillae with lots of slits and creases (Fig 6); the internal papillae consisting of pits (Fig7); denticles from the apical edge of the inner labial surface (Fig8); the pit in cuticle in the central region of the denticular row of all lips (Fig 9).(Kazacos and Turek, 1982) New findings such as externolateral papillae and pits allow species differentiation within ascaridoid nematodes.

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The lip region is a useful identifying character in many species. Generally, nematode identification based on morphology requires special techniques to extract nematodes from animal or plant tissues, high-powered microscopes to observe minute morphological features, and special training in identification procedures. Specimen preparation and position of the specimen are critical factors for correct identification. Samples have to be undamaged, and any environmental cross-contamination of similar nematodes would make identification more difficult. Although some criteria such as presence or absence of certain structures could be evaluated in genus, recognition within genus is still considered difficult, since the degree of variation in morphology within genus was not that obvious, resulting in no definitive or conclusive diagnosis. Moreover, most identification based on morphology requires adults rather than eggs, which is not efficient regarding to early stage diagnosis.

With the development of molecular biology, polymerase chain reaction (PCR) assays were used for detection of the nematode eggs and larvae. A specific product of the mitochondrial cytochrome oxidase 2 (cox-2) gene was amplified to distinguish B. procyonis from other species. (Dangoudoubiyam et al, 2009) The figure shows the amplification of the 146 bp cox-2 product and 325 bp 18S rRNA gene product result. Lane 2 is the target species B. procyonis, with both 325 bp and 146 bp product. However, lane 9, the species of B. columnaris was not distinguished from B. procyonis.

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The PCR assays could detect eggs from the feces of a naturally patent animal, naturally contaminated soil samples, or larvae isolated from the brain tissue of an animal that was likely to have the nematode, that is, PCR assay samples would be not influenced by resource or environment. Also, the samples are not necessarily adults, they could be eggs or larvae. Eggs could be fully embryonated, unembryonated or in a morula stage, although the quality of DNA may be different. The effect of the quality of the template DNA would be a problem of PCR assay. If DNA was not isolated properly, the identification would not be clear and accurate.

For some genus, the use of morphology alone for species recognition is limited due to intraspecific variability. For example, morphological species recognition within the genus Panagrolaimus has long been considered difficult and morphological identification was strongly criticized in this genus (Williams, 1987). Furthermore, species that include both large and small individuals have been described in many species, implying that size may not be an important identifying character within genus. Therefore, based on established morphological and morphometrical criteria, some isolates of different species may belong to the same morphospecies.

It is difficult to find a species concept that has both a universal theoretical appeal and a practical value. Typically, nematode species are still diagnosed mainly on morphological characteristics. The use of morphological and morphometric characters in some groups has become difficult due to their within-population variability. Therefore, easier and faster methods of diagnosing species are being sought. So a more molecular-oriented taxonomy is adopted in nematology. Many nematode species descriptions have included molecular characterization. It was recommended that studies attempt to link morphological variation with molecular patterns for parthenogenetic species (Powers et al, 1997). The variability of the molecular markers was regarded as an important in taxonomic purposes. They should not be too variable to reliably infer relationships, nor too deeply conserved among all species in the genus. Previously described molecular methods of defining species or analyzing phylogeny have employed the intergenic spacer (IGS) region of the ribosomal repeats, the D2/D3 region of the large subunit rRNA gene, PCR-RFLP patterns for the internal transcribed spacer (ITS), sequencing the ITS region rDNA, NADH dehydrogenases subunit (NAD), or the mitochondrial cytochrome oxidase (COX), which was used for B. procyonis identification. The established markers are easy, fast, transferable, and applicable in nematode identification studies with the questions of biodiversity.  Some markers such as SSU rRNA sequences are informative for defining relationships among major lineages, and used for discrimination at order, family, genus and species level in phylogenetics.

Based on recent molecular methods, PCR assay now has a verified potential to contribute to solving some of the taxonomic problems within morphologically indistinguishable species and genera complexes within the nematode family. However, we do not propose that morphology is a less useful tool in taxonomy and systematics relative to molecular techniques. Even molecular method cannot tell apart B. procyonis from B. columnaris. It is likely that a combination of molecular and morphological approaches for species diagnosis and description is needed. It is still necessary to test the molecular efficacy of delimiting evolutionary species in other genus and other groups of Nematoda.

Post by Keyu Li

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