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Background: Two mitochondrial DNA clades have been described in Anopheles funestus populations from southern Africa. Clade I is common across the continent while clade II is known only from Mozambique and Madagascar. The specific biological status of these clades is at present unknown. We investigated the possible role that each clade might play in the transmission of Plasmodium falciparum and the insecticide resistance status of An. funestus from Zimbabwe and Zambia., Methods: Mosquitoes were collected inside houses from Nchelenge District, Zambia and Honde Valley, Zimbabwe in 2013 and 2014. WHO susceptibility tests, synergist assays and resistance intensity tests were conducted on wild females and progeny of wild females. ELISA was used to detect Plasmodium falciparum circumsporozoite protein. Specimens were identified to species and mtDNA clades using standard molecular methods., Results: The Zimbabwean samples were all clade I while the Zambian population comprised 80% clade I and 20% clade II in both years of collection. ELISA tests gave an overall infection rate of 2.3% and 2.1% in 2013, and 3.5% and 9.2% in 2014 for Zimbabwe and Zambia respectively. No significant difference was observed between the clades. All populations were resistant to pyrethroids and carbamates but susceptible to organochlorines and organophosphates. Synergist assays indicated that pyrethroid resistance is mediated by cytochrome P450 mono-oxygenases. Resistance intensity tests showed high survival rates after 8-hrs continuous exposure to pyrethroids but exposure to bendiocarb gave the same results as the susceptible control., Conclusions: This is the first record of An. funestus mtDNA clade II occurring in Zambia. No evidence was found to suggest that the clades are markers of biologically separate populations. The ability of An. funestus to withstand prolonged exposure to pyrethroids has serious implications for the use of these insecticides, either through LLINs or IRS, in southern Africa in general and resistance management strategies should be urgently implemented.

Background: Recent studies presented two clades (clades I and II) within the major malaria vector, Anopheles funestus s.s. on the mitochondrial DNA. We describe a hydrolysis probe analysis (TaqMan assay) method for the rapid identification of these two clades., Findings: A total of 53 An. funestus s.s. from Malawi and Mozambique were tested for detection of clade types using the hydrolysis probe analysis. Results were compared to DNA sequence analysis to verify the accuracy of the probes TaqMan assay for this vector species. Analysis using the hydrolysis probe revealed that there were 21 individuals from Malawi and 13 individuals from Mozambique for clade I, and 19 individuals from Mozambique for clade II. The results were consistent with the results of DNA sequences. A field sample from northern Zambia revealed the presence of both clade types., Conclusion: A diagnostic method using the hydrolysis probe analysis was developed to identify clade types within An. funestus s.s. This assay will be useful for screening clade types of field-collected An. funestus specimens accurately and efficiently in malaria vector research and control studies.

Background: Anopheles longipalpis is morphologically similar to the major African malaria vector Anopheles funestus at the adult stage although it is very different at the larval stage. Despite the development of the species-specific multiplex PCR assay for the An. funestus group, the genomic DNA of Anopheles longipalpis type C specimens can be amplified with the Anopheles vaneedeni and Anopheles parensis primers from this assay. The standard, species-specific An. funestus group PCR, results in the amplification of two fragments when An. longipalpis type C specimens are included in the analysis. This result can easily be misinterpreted as being a hybrid between An. vaneedeni and An. parensis. Anopheles longipalpis type C can be identified using a species-specific PCR assay but this assay is not reliable if other members of the An. funestus group, such as An. funestus, An. funestus-like and An. parensis, are included. The present study provides a multiplex assay that will identify An. longipalpis along with other common members of the African An. funestus group, including Anopheles leesoni., Methods: A total of 70 specimens from six species (An. funestus, An. funestus-like, An. parensis, Anopheles rivulorum, An. vaneedeni and An. leesoni) in the An. funestus group and An. longipalpis type C from Malawi, Mozambique, South Africa and Zambia were used for the study. A restriction fragment length polymorphism (RFLP) assay was designed based on the DNA sequence information in the GenBank database., Results: The enzyme, EcoRI digested only An. longipalpis type C and An. funestus-like after the species-specific An. funestus group PCR assay. The An. longipalpis and An. funestus-like digestion profiles were characterized by three fragments, 376 bp, 252 bp and 211 bp for An. longipalpis type C and two fragments, 375 bp and 15 bp for An. funestus-like., Conclusions: An RFLP method for the group was developed that is more accurate and efficient than those used before. Hence, this assay would be useful for field-collected adult specimens to be identified routinely in malaria vector research and control studies.