Differentiation of Species and Life Cycle Stages of Brugia spp. by Isoenzyme Analysis

Flockhart, Hilary A.; Denham, D. A.

doi:10.2307/3281567

Cited by 16 publications

(3 citation statements)

References 11 publications

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“…Similarly, empirical studies of parasite population genetics were only beginning in the 1980s, and few studies investigating the genetics of geographic variation were published. Although systematic applications within parasitology were relatively few at this time, it was widely understood that molecular data had great potential for phylogenetic reconstruction, confirming the conspecificity of different life-cycle stages (Flockhart and Denham, 1984;Andrews et al 1988) and identifying cryptic species (Nadler, 1990).…”

Section: R Y P T I C S P E C I E S D E L I M I T a T I O N : A L T mentioning

confidence: 99%

Integrating molecular and morphological approaches for characterizing parasite cryptic species: implications for parasitology

2011

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Herein we review theoretical and methodological considerations important for finding and delimiting cryptic species of parasites (species that are difficult to recognize using traditional systematic methods). Applications of molecular data in empirical investigations of cryptic species are discussed from an historical perspective, and we evaluate advantages and disadvantages of approaches that have been used to date. Developments concerning the theory and practice of species delimitation are emphasized because theory is critical to interpretation of data. The advantages and disadvantages of different molecular methodologies, including the number and kind of loci, are discussed relative to tree-based approaches for detecting and delimiting cryptic species. We conclude by discussing some implications that cryptic species have for research programmes in parasitology, emphasizing that careful attention to the theory and operational practices involved in finding, delimiting, and describing new species (including cryptic species) is essential, not only for fully characterizing parasite biodiversity and broader aspects of comparative biology such as systematics, evolution, ecology and biogeography, but to applied research efforts that strive to improve development and understanding of epidemiology, diagnostics, control and potential eradication of parasitic diseases.

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Section: R Y P T I C S P E C I E S D E L I M I T a T I O N : A L T mentioning

confidence: 99%

Integrating molecular and morphological approaches for characterizing parasite cryptic species: implications for parasitology

2011

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“…During the present study, differential expression of certain enzyme loci was observed between larvae and adults of E. overstreeti. Recently, differential expression of enzyme loci has been shown to occur between third-stage larva and adults of the nematode Brugia patei (see FLOCKHART & DENHAM, 1984) and has also been documented for developmental stages in mammals (for examples see HARRIS & HOPKINSON, 1976). The non-expression of enzyme loci between life cycle stages presumably reflect different metabolic requirements of parasitic stages, particularly where they occur in different hosts.…”

Section: Resultsmentioning

confidence: 97%

Identification of life cycle stages of the nematodeEchinocephalus overstreetiby allozyme electrophoresis

et al. 1988

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Data presented in this study highlight the potential of allozyme electrophoresis in providing unequivocal genetic evidence for the identification of life cycle stages, particularly where species have complex life cycles. Adults of the nematode Echinocephalus overstreeti parasitize the elasmobranch Heterodontus portusjacksoni. The putative larval form which is morphologically dissimilar is found in two species of marine molluscs, Chlamys bifrons and Pecten albus. Electrophoretic analysis indicated that the adult and larval forms shared alleles at all of the 34 enzyme loci established. Furthermore, there were no fixed allelic differences between larval forms from different mollusc species.

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“…However, in the case of B. malayi and B. pahangi, the circulating microfilariae are difficult to distinguish morphologically (24). Differences have been observed in the electrophoretic mobility of isozymes from B. malayi and B. pahangi (25,26 (Fig. 5).…”

Section: Discussionmentioning

confidence: 94%

Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal parasite Brugia pahangi.

McReynolds¹,

DeSimone²,

Williams³

1986

Proc. Natl. Acad. Sci. U.S.A.

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A 320-base-pair repeated sequence was observed when DNA samples from the filarial parasites Brugia malayi and Brugia pahangi were digested with the restriction endonuclease Hha I. A 640-base-pair dimer of the repeated sequence from B. malayi was inserted into the plasmid pBR322. When dot hybridization was used, the copy number of the repeat in B. malayi was found to be about 30,000. The 320-base-pair Hha I repeated sequences are arranged in direct tandem arrays and comprise about 12% of the genome. B. pahangi has a related repeated sequence that cross-hybridizes with the cloned B. malayi Hha I repeat. Dot hybridization with the cloned repeat shows that the sequence is present in B. malayi and in B. pahangi but not in four other species of filarial parasites. The cloned repeated DNA sequence is an extremely sensitive probe for detection of Brugia in blood samples. Hybridization with the cloned repeat permits the detection of DNA isolated from a single parasite in an aliquot of blood from animals infected with B. malayi. There are differences in the restriction sites present in the repeated sequences that can be used to differentiate between the two Brugia species. The B. malayi repeated DNA sequence is cleaved byAlu I and Rsa I but the B. pahangi sequence is not. A comparison of repeated sequences between the two species by DNA sequence analysis indicates that some regions of individual repeats are over 95% homologous, while other short regions are only 60-65% ho mologous. These differences in DNA sequence will allow the construction of species-specific hybridization probes. (4,5). DNA was isolated from frozen adult female worms or from microfilariae in whole blood by the proteinase K-digestion method of Emmons et al. (6) followed by phenol extraction. For the Southern blots and cloning experiments, the DNA was further purified by ethidium bromide/CsCl centrifugation (7), which removed a band of white material, possibly from the cuticle, that was more dense than the DNA.Cloning of Repeated DNA from B. malayi. All restriction endonucleases, methylases, ligases, linkers, and 35S-labeled dideoxy sequencing reagents used in these experiments were prepared at New England Biolabs and used as described by the supplier. B. malayi DNA was cleaved with Alu I, protected with EcoRI methylase, and then ligated to 5'-phosphorylated EcoRI linkers with T4 DNA ligase. Cohesive EcoRI ends were generated by digestion with EcoRI. The linkers were separated from the Brugia DNA by electrophoresis on a 1.5% low melt agarose gel stained with ethidium bromide. The 320-base-pair (bp) and 640-bp restriction fragments were cut out and the DNA isolated (8). In two separate reactions, either the 640-bp or the 320-bp fragments were ligated to EcoRI cleaved pBR322 that had been dephosphorylated by treatment with bacterial alkaline phosphatase (9).Escherichia coli RRI cells were transformed with the recombinant plasmids, selected for growth on ampicillin, and screened for repeated sequences by hybridization with radioactively labeled B....

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Differentiation of Species and Life Cycle Stages of Brugia spp. by Isoenzyme Analysis

Cited by 16 publications

References 11 publications

Integrating molecular and morphological approaches for characterizing parasite cryptic species: implications for parasitology

Integrating molecular and morphological approaches for characterizing parasite cryptic species: implications for parasitology

Identification of life cycle stages of the nematodeEchinocephalus overstreetiby allozyme electrophoresis

Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal parasite Brugia pahangi.

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