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Title: The use of mitochondrial DNA in the molecular systematics of malaria vectors
Author: Oshaghi, Mohammad Ali
ISNI:       0000 0001 3460 6252
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 1998
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The taxonomy of anopheline mosquitoes has been well characterised by cytological and morphological techniques. However, there are relatively few studies which deal with characters based on recently developed molecular techniques. This thesis describes the application of PCR techniques to the study of mitochondrial DNA in a number of groups of anopheline mosquitoes, so as to increase our understanding of the systematics and evolutionary relationships of the taxa concerned The molecular systematics of four important groups of malaria vectors was studied using sequence variation in mitochondrial DNA (mtDNA). In this study, up to one third of the mtDNA molecule was investigated (including COI, COI, ND2, ND4, NDS subunits, 12S and seven tRNAs genes, and the A-T rich region) in the An. gambiae, An. culicifacies, An. fluviatilis complexes, and An. stephensi. RFLP and sequence data were obtained by PCR and direct sequencing techniques, and used in phylogenetic analysis. Restriction enzyme digests revealed considerable differentiation between and within species. Thirteen enzymes revealed four haplotypes within the An. culicifacies complex. Species A was quite distinct from species B, but species B and C showed identical RFLP patterns in a portion of the NDS region. Within species B, a low level of genetic variation was seen between two populations from Sri-Lanka but both of these were highly divergent from an Indian population. These differences are reflected in differences in vectorial capacity between populations of species B from these two countries. The restriction enzymes Alul, EcoRV, Hinfl, EcoRI, and Dral gave species-specific RFLP profiles for four members of the An. gambiae complex but An. gambiae and An. arabiensis were identical in this RFLP analysis, possibly reflecting considerable gene flow between these two species. Ten strains of An. stephensi were identical at eighteen restriction enzyme sites but Dral enzyme revealed two haplotypes. Direct sequencing of mtDNA from a number of species revealed a high bias towards adenine and thymine, a bias which was mainly at the third position in the codon. The transition:transversion ratio was high among lineages exhibiting low divergences, but declined with increasing sequence divergence. Inferred amino acid sequence divergence in the ND sub-units was more than twice that found in the COI subunit, indicating different functional constraints in the two genes. The results showed that the COI subunit is the most variable region within the molecule, whereas the other protein coding regions studied, (COII and ND4 and NDS) showed less variation. The A-T rich region, ribosomal RNA and tRNAs genes were highly conserved within the anopheline mosquitoes studied. The COI subunit is a good candidate for studies of population genetics and systematics at the level of the species or species complex whereas other genes are more useful in studies of phylogeny at deeper levels of evolution. Rates of evolution inferred from phylogenetic analysis of mtDNA appear to differ from species to species, with the highest rate in the An. culicifacies complex, and decreasing rates occurring from the An. gambiae complex, through the An. fluviatilis complex to An. stephensi. This could be explained if the latter three were the most recently diverged species. Phylogenetic analysis of sequence data for each species consistently revealed high resolution within and between species. For the An. culicifacies complex results revealed two main clades: 1) species A, 2) species B and C. For the An. gambiae complex the COI-II tree revealed three main clades: 1) gambiae-arabiensis-quadriannulatus (Q2) 2) merus-quadriannulatus (Q1) and 3) melas-bwambae which is consistent with other molecular-based studies and with ecological and epidemiological data. The apparently anomalous position of An. quadriannulatus is discussed. Except for the implied relationship between gambiae and arabiensis, these findings are in agreement with chromosomal trees. For the An. fluviatilis complex, the results suggest that there may be two species within populations originating from Iran. A phylogenetic analysis for thirteen species of the genus Anopheles using the results of this study in combination with all published data revealed molecular trees which are supported strongly by morphological and cytological criteria. The inheritance of mtDNA was tested in An. stephensi using a series of backcrosses and a haplotypespecific PCR-RFLP analysis. Only maternal inheritance of the molecule could be demonstrated, suggesting that in An. stephensi paternal inheritance is either rare or absent.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Genetics