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Probe-based multiplex qPCR identifies blood-meal hosts in Anopheles mosquitoes from Papua New Guinea.

ABSTRACT: BACKGROUND:Determination of blood-meal hosts in blood-fed female Anopheles mosquitoes is important for evaluating vectorial capacity of vector populations and assessing effectiveness of vector control measures. Sensitive molecular methods are needed to detect traces of host blood in mosquito samples, to differentiate hosts, and to detect mixed host blood meals. This paper describes a molecular probe-based quantitative PCR for identifying blood-meal hosts in Anopheles malaria vectors from Papua New Guinea. METHODS:TaqMan oligonucleotide probes targeting specific regions of mitochondrial or nuclear DNA of the three primary Anopheles blood-meal hosts, humans, pigs and dogs, were incorporated into a multiplex, quantitative PCR which was optimized for sensitivity and specificity. RESULTS:Amplification of serially diluted DNA showed that the quantitative PCR detected as low as 10-5 ng/?l of host DNA. Application to field-collected, blood-fed Anopheles showed that the quantitative PCR identified the vertebrate hosts for 89% (335/375) of mosquitoes whereas only 55% (104/188) of blood-meal samples tested in a conventional PCR were identified. Of the 104 blood-fed Anopheles that were positive in both PCR methods, 16 (15.4%) were identified as mixed blood meals by the quantitative PCR whereas only 3 (2.9%) were mixed blood meals by the conventional PCR. CONCLUSIONS:The multiplex quantitative PCR described here is sensitive at detecting low DNA concentration and mixed host DNA in samples and useful for blood-meal analysis of field mosquitoes, in particular mixed-host blood meals.

SUBMITTER: Keven JB

PROVIDER: S-EPMC7048118 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Probe-based multiplex qPCR identifies blood-meal hosts in Anopheles mosquitoes from Papua New Guinea.

Keven John B JB Artzberger Georgia G Gillies Mary L ML Mbewe Rex B RB Walker Edward D ED

Parasites & vectors 20200228 1

<h4>Background</h4>Determination of blood-meal hosts in blood-fed female Anopheles mosquitoes is important for evaluating vectorial capacity of vector populations and assessing effectiveness of vector control measures. Sensitive molecular methods are needed to detect traces of host blood in mosquito samples, to differentiate hosts, and to detect mixed host blood meals. This paper describes a molecular probe-based quantitative PCR for identifying blood-meal hosts in Anopheles malaria vectors from ...[more]

PMID: 32111232

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Project description:BackgroundAedes aegypti and Ae. albopictus are important vectors of infectious diseases, especially those caused by arboviruses such as dengue, chikungunya and Zika. Aedes aegypti is very well adapted to urban environments, whereas Ae. albopictus inhabits more rural settings. Pyrethroid resistance is widespread in these vectors, but limited data exist from the Southwest Pacific Region, especially from Melanesia. While Aedes vector ecology is well documented in Australia, where incursion of Ae. albopictus and pyrethroid resistance have so far been prevented, almost nothing is known about Aedes populations in neighbouring Papua New Guinea (PNG). With pyrethroid resistance documented in parts of Indonesia but not in Australia, it is important to determine the distribution of susceptible and resistant Aedes populations in this region.MethodsThe present study was aimed at assessing Aedes populations for insecticide resistance in Madang and Port Moresby, located on the north and south coasts of PNG, respectively. Mosquitoes were collected using ovitraps and reared in an insectary. Standard WHO bioassays using insecticide-treated filter papers were conducted on a total of 253 Ae. aegypti and 768 Ae. albopictus adult mosquitoes. Subsets of samples from both species (55 Ae. aegypti and 48 Ae. albopictus) were screened for knockdown resistance mutations in the voltage-sensitive sodium channel (Vssc) gene, the target site of pyrethroid insecticides.ResultsHigh levels of resistance against pyrethroids were identified in Ae. aegypti from Madang and Port Moresby. Aedes albopictus exhibited susceptibility to pyrethroids, but moderate levels of resistance to DDT. Mutations associated with pyrethroid resistance were detected in all Ae. aegypti samples screened. Some genotypes found in the present study had been observed previously in Indonesia. No Vssc mutations associated with pyrethroid resistance were found in the Ae. albopictus samples.ConclusionsTo our knowledge, this is the first report of pyrethroid resistance in Ae. aegypti mosquitoes in PNG. Interestingly, usage of insecticides in PNG is low, apart from long-lasting insecticidal nets distributed for malaria control. Further investigations on how these resistant Ae. aegypti mosquito populations arose in PNG and how they are being sustained are warranted.

Project description:BackgroundSmall islands serve as potential malaria reservoirs through which new infections might come to the mainland and may be important targets in malaria elimination efforts. This study investigated malaria vector species diversity, blood-meal hosts, Plasmodium infection rates, and long-lasting insecticidal net (LLIN) coverage on Mageta, Magare and Ngodhe Islands of Lake Victoria in western Kenya, a region where extensive vector control is implemented on the mainland.ResultsFrom trapping for six consecutive nights per month (November 2012 to March 2015) using CDC light traps, pyrethrum spray catches and backpack aspiration, 1868 Anopheles mosquitoes were collected. Based on their cytochrome oxidase I (COI) and intergenic spacer region PCR and sequencing, Anopheles gambiae s.l. (68.52%), Anopheles coustani (19.81%) and Anopheles funestus s.l. (11.67%) mosquitoes were differentiated. The mean abundance of Anopheles mosquitoes per building per trap was significantly higher (p < 0.001) in Mageta than in Magare and Ngodhe. Mageta was also the most populated island (n = 6487) with low LLIN coverage of 62.35% compared to Ngodhe (n = 484; 88.31%) and Magare (n = 250; 98.59%). Overall, 416 (22.27%) engorged Anopheles mosquitoes were analysed, of which 41 tested positive for Plasmodium falciparum infection by high-resolution melting (HRM) analysis of 18S rRNA and cytochrome b PCR products. Plasmodium falciparum infection rates were 10.00, 11.76, 0, and 18.75% among blood-fed An. gambiae s.s. (n = 320), Anopheles arabiensis (n = 51), An. funestus s.s. (n = 29), and An. coustani (n = 16), respectively. Based on HRM analysis of vertebrate cytochrome b, 16S rRNA and COI PCR products, humans (72.36%) were the prominent blood-meal hosts of malaria vectors, but 20.91% of blood-meals were from non-human vertebrate hosts.ConclusionsThese findings demonstrate high Plasmodium infection rates among the primary malaria vectors An. gambiae s.s. and An. arabiensis, as well as in An. coustani for the first time in the region, and that non-human blood-meal sources play an important role in their ecology. Further, the higher Anopheles mosquito abundances on the only low LLIN coverage island of Mageta suggests that high LLIN coverage has been effective in reducing malaria vector populations on Magare and Ngodhe Islands.

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Probe-based multiplex qPCR identifies blood-meal hosts in Anopheles mosquitoes from Papua New Guinea.

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Probe-based multiplex qPCR identifies blood-meal hosts in Anopheles mosquitoes from Papua New Guinea.

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