Dataset Information

Detection of clade types (clades I and II) within Anopheles funestus sensu stricto by the hydrolysis probe analysis (TaqMan assay).

ABSTRACT: 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.

SUBMITTER: Choi KS

PROVIDER: S-EPMC3691741 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Detection of clade types (clades I and II) within Anopheles funestus sensu stricto by the hydrolysis probe analysis (TaqMan assay).

Choi Kwang Shik KS Coetzee Maureen M Koekemoer Lizette L LL

Parasites & vectors 20130612

<h4>Background</h4>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.<h4>Findings</h4>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 pr ...[more]

PMID: 23758959

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Project description:BackgroundUnderstanding the mechanisms used by Anopheles mosquitoes to survive insecticide exposure is key to manage existing insecticide resistance and develop more suitable insecticide-based malaria vector control interventions as well as other alternative integrated tools. To this regard, the molecular basis of permethrin, DDT and dieldrin resistance in Anopheles funestus (sensu stricto) at Akaka-Remo was investigated.MethodsBioassays were conducted on 3-5-day-old adult An. funestus (s.s.) mosquitoes for permethrin, DDT and dieldrin susceptibility test. The molecular mechanisms of mosquito resistance to these insecticides were investigated using microarray and reverse transcriptase PCR techniques. The voltage-gated sodium channel region of mosquitoes was also screened for the presence of knockdown resistance mutations (kdr west and east) by sequencing method.ResultsAnopheles funestus (s.s.) population was resistant to permethrin (mortality rate of 68%), DDT (mortality rate of 10%) and dieldrin (mortality rate of 8%) insecticides. Microarray and RT-PCR analyses revealed the overexpression of glutathione S-transferase genes, cytochrome P450s, esterase, trypsin and cuticle proteins in resistant mosquitoes compared to control. The GSTe2 was the most upregulated detoxification gene in permethrin-resistant (FC = 44.89), DDT-resistant (FC = 57.39) and dieldrin-resistant (FC = 41.10) mosquitoes compared to control population (FC = 22.34). The cytochrome P450 gene, CYP6P9b was also upregulated in both permethrin- and DDT-resistant mosquitoes. The digestive enzyme, trypsin (hydrolytic processes) and the cuticle proteins (inducing cuticle thickening leading to reduced insecticides penetration) also showed high involvement in insecticide resistance, through their overexpression in resistant mosquitoes compared to control. The kdr east and west were absent in all mosquitoes analysed, suggesting their non-involvement in the observed mosquito resistance.ConclusionsThe upregulation of metabolic genes, especially the GSTe2 and trypsin, as well as the cuticle proteins is driving insecticide resistance of An. funestus (s.s.) population. However, additional molecular analyses, including functional metabolic assays of these genes as well as screening for a possible higher cuticular hydrocarbon and lipid contents, and increased procuticle thickness in resistant mosquitoes are needed to further describe their distinct roles in mosquito resistance.

Project description:BackgroundUnderstanding the ecology and behaviour of disease vectors, including the olfactory cues used to orient and select hosts and egg-laying sites, are essential for the development of novel, insecticide-free control tools. Selected graminoid plants have been shown to release volatile chemicals attracting malaria vectors; however, whether the attraction is selective to individual plants or more general across genera and families is still unclear.MethodsTo contribute to the current evidence, we implemented bioassays in two-port airflow olfactometers and in large field cages with four live graminoid plant species commonly found associated with malaria vector breeding sites in western Kenya: Cyperus rotundus and C. exaltatus of the Cyperaceae family, and Panicum repens and Cynodon dactylon of the Poaceae family. Additionally, we tested one Poaceae species, Cenchrus setaceus, not usually associated with water. The volatile compounds released in the headspace of the plants were identified using gas chromatography/mass spectrometry.ResultsAll five plants attracted gravid vectors, with the odds of a mosquito orienting towards the choice-chamber with the plant in an olfactometer being 2-5 times higher than when no plant was present. This attraction was maintained when tested with free-flying mosquitoes over a longer distance in large field cages, though at lower strength, with the odds of attracting a female 1.5-2.5 times higher when live plants were present than when only water was present in the trap. Cyperus rotundus, previously implicated in connection with an oviposition attractant, consistently elicited the strongest response from gravid vectors. Volatiles regularly detected were limonene, β-pinene, β-elemene and β-caryophyllene, among other common plant compounds previously described in association with odour-orientation of gravid and unfed malaria vectors.ConclusionsThe present study confirms that gravid Anopheles gambiae sensu stricto use chemical cues released from graminoid plants to orientate. These cues are released from a variety of graminoid plant species in both the Cyperaceae and Poaceae family. Given the general nature of these cues, it appears unlikely that they are exclusively used for the location of suitable oviposition sites. The utilization of these chemical cues for attract-and-kill trapping strategies must be explored under natural conditions to investigate their efficiency when in competition with complex interacting natural cues.

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Dataset Information

Detection of clade types (clades I and II) within Anopheles funestus sensu stricto by the hydrolysis probe analysis (TaqMan assay).

Publications

Detection of clade types (clades I and II) within Anopheles funestus sensu stricto by the hydrolysis probe analysis (TaqMan assay).

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