Systematic study of the new Anopheles funestus-like species from Malawi

Vezenegho, Samuel Bumuh
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Morphological similarity between malaria vectors and non-vectors occurring in sympatry has serious consequences if the killer diseases have to be controlled. Malaria in Malawi is transmitted by Anopheles gambiae, An. arabiensis and An. funestus. This vector diversity is further complicated by the recently discovered An. funestus-like species which is morphologically similar to An. funestus, and found in association with humans. Currently there is no single assay available that differentiates An. funestus-like from the other African members in the An. funestus group. The objective of this study was to investigate the biology and behavior of the newly discovered An. funestus-like species and its possible role in malaria transmission. This information will assist in the implementation of vector control programs. In addition to this, the study investigated the development of a DNA based assay to differentiate between the members of the An. funestus group and to morphologically described An. funestus-like species. Anopheles mosquitoes were collected resting indoors and outdoors from Karonga in Malawi. Specimens were identified morphologically and molecularly using chain reaction PCR. Identified samples were analyzed by ELISA for blood meal source and Plasmodium sporozoite infection. Anopheles funestus-like was morphologically compared with An. funestus. Real time based PCR was developed and compared to the current multiplex or allele-specific PCR (AS-PCR) assay for sensitivity and performance. The IGS region of the rDNA gene was investigated for development of AS-PCR. Phylogenetic relationship of mosquitoes was constructed from ITS2 and D3 sequences. Adult An. funestus mosquitoes (n = 391) were collected during April and September, 2010. Karonga contributed 63.9% and Likoma Island 36.1%. Of the identified specimens (n = 347) An. funestus-like comprised 10.4%, An. rivulorum 31.7%, An. funestus 57.3% and An. parensis 0.6%. Most of the An. funestus-like species were collected resting indoors 91.7% (33/36) compared to outdoors 8.3% (3/36). The species was predominant during the dry season 63.9% (23/36) compared to the wet season. A total of 19 An. funestus-like females were analyzed for blood meal source. Mixed blood meal from goat and bovine was found in 7 specimens and a single blood meal from goat in 3 specimens.. The rest of the An. funestus-like was negative for blood meal. An overall dry season infection rate of An. funestus-like species by Plasmodium vivax was 5% (1/20) in this study and 3.1% (2/64) from samples collected in 2009 was found. However, the possibility of false positivity could not be excluded and further study is urgently needed to investigate this. Real-time PCR for the identification of members of the An. funestus group was found to be more sensitive (0.02ng/μl) than AS-PCR (0.04ng/μl) and had performance comparable to AS-PCR. AS-PCR developed from the intergenic spacer region of rDNA discriminates An. funestus, An. rivulorum, An. vaneedeni and An. parensis. Of all assays developed in this study, the hydrolysis probe assay is the most reliable assay for identifying members in the An. funestus group. This study confirmed the existence of An. funestus-like species in sympatry with An. funestus group members. An. funestus-like was predominantly found resting indoors (endophilic) but preferring animal over human blood (zoophilic). No consistent morphological characters were found to discriminate between An. funestus and An. funestus-like based on morphological data, An. funestus-like is very similar and closely related to An. funestus which is supported by phylogenetic analysis. However, Restriction Fragment Length Polymorphism separates the two species.
Thesis (Ph.D.(Virology))--University of the Witwatersrand, Faculty of Health Sciences, 2012