Towards the molecular characterisation of parasitic nematode assemblages: An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis

• Published in: Experimental parasitology 2014-09, Vol.144, p.76-83
• Main Authors: Lott, M.J., Hose, G.C., Power, M.L.
• Format: Article
• Language: English
• Subjects: Amplified Fragment Length Polymorphism Analysis - methods; Amplified Fragment Length Polymorphism Analysis - veterinary; Animals; Base Sequence; Cloning, Molecular; CO1 mtDNA; DNA, Helminth - chemistry; DNA, Helminth - isolation & purification; DNA, Ribosomal Spacer - chemistry; DNA, Ribosomal Spacer - isolation & purification; Electron Transport Complex IV - genetics; ITS rDNA; Macropodidae - parasitology; Molecular ecology; Molecular Sequence Data; Nematoda; Nematoda - classification; Nematoda - genetics; Nematoda - isolation & purification; Nematode Infections - parasitology; Nematode Infections - veterinary; Phylogeny; Polymorphism, Restriction Fragment Length; RNA, Ribosomal, 5.8S - genetics; Sequence Alignment - veterinary; T-RFLP
• ISSN: 0014-4894; 1090-2449
• DOI: 10.1016/j.exppara.2014.06.011

[Display omitted] •ITS+ and CO1 sequence data were obtained for 18–20 nematode species.•Twelve of the thirteen ITS+ derived T-RF peak profiles were species specific.•5–7 Species of Cloacina shared an identical ITS+ T-RF peak profile.•The CO1 derived T-RF peak profiles were taxonomically inconsistent.•Several ITS+ T-RF peak profiles showed unexpected secondary peaks. Identifying factors which regulate temporal and regional structuring within parasite assemblages requires the development of non-invasive techniques which facilitate both the rapid discrimination of individual parasites and the capacity to monitor entire parasite communities across time and space. To this end, we have developed and evaluated a rapid fluorescence-based method, terminal restriction fragment length polymorphism (T-RFLP) analysis, for the characterisation of parasitic nematode assemblages in macropodid marsupials. The accuracy with which T-RFLP was capable of distinguishing between the constituent taxa of a parasite community was assessed by comparing sequence data from two loci (the ITS+ region of nuclear ribosomal DNA and the mitochondrial CO1) across ∼20 species of nematodes (suborder Strongylida). Our results demonstrate that with fluorescent labelling of the forward and reverse terminal restriction fragments (T-RFs) of the ITS+ region, the restriction enzyme Hinf1 was capable of generating species specific T-RFLP profiles. A notable exception was within the genus Cloacina, in which closely related species often shared identical T-RFs. This may be a consequence of the group’s comparatively recent evolutionary radiation. While the CO1 displayed higher sequence diversity than the ITS+, the subsequent T-RFLP profiles were taxonomically inconsistent and could not be used to further differentiate species within Cloacina. Additionally, several of the ITS+ derived T-RFLP profiles exhibited unexpected secondary peaks, possibly as a consequence of the restriction enzymes inability to cleave partially single stranded amplicons. These data suggest that the question of T-RFLPs utility in monitoring parasite communities cannot be addressed without considering the ecology and unique evolutionary history of the constituent taxa.