Rapid and high throughput molecular identification of diverse mosquito species by high resolution melting analysis

Ajamma, Yvonne Ukamaka; Mararo, Enock; Omondi, David; Onchuru, Thomas Ogao; Muigai, Anne W. T.; Masiga, Daniel K.; Villinger, Jandouwe

doi:10.12688/f1000research.9224.1

Cited by 26 publications

(20 citation statements)

References 32 publications

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“…and An. gambiae mosquitoes involved polymerase chain reaction (PCR) amplification and sequencing of the cytochrome oxidase subunit 1 ( COI ) region [ 26 ], polymorphic ITS2 region of ribosomal DNA [ 27 , 28 ] and analysing melt curve differences of 165 base pairs (bp) intergenic spacer region ( IGS ) gene amplicons obtained using IGS gene primers [ 29 , 30 ]. Colony-reared, sugar-fed An.…”

Section: Methodsmentioning

confidence: 99%

Composition of Anopheles mosquitoes, their blood-meal hosts, and Plasmodium falciparum infection rates in three islands with disparate bed net coverage in Lake Victoria, Kenya

Ogola

Villinger

Mabuka

et al. 2017

Malar J

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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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Section: Methodsmentioning

confidence: 99%

Composition of Anopheles mosquitoes, their blood-meal hosts, and Plasmodium falciparum infection rates in three islands with disparate bed net coverage in Lake Victoria, Kenya

Ogola

Villinger

Mabuka

et al. 2017

Malar J

Self Cite

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“…There are a number of assays one can employ for SNP genotyping to facilitate species identification, e.g. TaqMan real-time PCR (Dhami et al 2016;Zhang et al 2016;Linck et al 2017), High-Resolution Melt (HRM) real-time PCR (Dhami et al 2014;Ajamma et al 2016), species-specific PCR (Sint et al 2016), KASP genotyping (Middlesex, UK), and SNP microarrays. We chose to adopt the Agena MassARRAY platform in combination with iPLEX chemistry (Gabriel et al 2009) to maximize the number of SNP markers we can multiplex in a single assay to reduce false positive rate and increase rigor of species identification.…”

Section: Discussionmentioning

confidence: 99%

Sequencing of Tuta absoluta genome to develop SNP genotyping assays for species identification

et al. 2019

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Tuta absoluta is one of the most devastating pests of fresh market and processing tomatoes. Native to South America, its detection was confined to that continent until 2006 when it was identified in Spain. It has now spread to almost every continent, threatening countries whose economies rely heavily on tomatoes. This insect causes damage to all developmental stages of its host plant, leading to crop losses as high as 80 to 100%. Although T. absoluta has yet to be found in the U.S. and China, which makes up a large portion of the tomato production in the world, computer models project a high likelihood of invasion. To halt the continued spread of T. absoluta and limit economic loss associated with tomato supply chain, it is necessary to develop accurate and efficient methods to identify T. absoluta and strengthen surveillance programs. Current identification of T. absoluta relies on examination of morphology and assessment of host plant damage, which are difficult to differentiate from that of native tomato pests. To address this need, we sequenced the genomes of T. absoluta and two closely related Gelechiidae, Keiferia lycopersicella, and Phthorimaea operculella, and developed a bioinformatic pipeline to design a panel of 21 SNP markers for species identification. The accuracy of the SNP panel was validated in a multiplex format using the iPLEX chemistry of Agena MassARRAY system. Finally, the new T. absoluta genomic resources we generated can be leveraged to study T. absoluta biology and develop species-specific management strategies. Key Message  Surveillance and identification of Tuta absoluta are challenging because it is morphologically similar to closely related species, e.g. Keiferia lycopersicella and Phthorimaea operculella.  We generated new genomic sequences for these three species and identified single nucleotide polymorphisms (SNPs) to facilitate species identification.  We validated a multiplex genotyping panel of 21 SNPs using the iPLEX MassARRAY platform and confirmed its accuracy for species identification.  We generated new molecular tools and genome resources to aid Tuta absoluta management. leaves, where the larvae will emerge from eggs and begin mining host tissues. Larvae can also enter the stems through the buds, and feed within the tomato fruit, leaving them unmarketable. Its distribution was largely confined to South America until it was first detected in Spain in 2006 (Desneux et al. 2010; Guillemaud et al. 2015). Since then T. absoluta has spread rapidly and is now

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“…vittatus and provide the first genetic characterization of the barcoding region of specimens of this species from Europe. Hitherto, sequences from this species were only available from China [ 15 ], India [ 16 ] and Kenya [ 17 ]. In addition, we review here available information on the potential role of this species in the transmission of virus of public health concern.…”

Section: Introductionmentioning

confidence: 99%

Aedes vittatus in Spain: current distribution, barcoding characterization and potential role as a vector of human diseases

et al. 2018

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BackgroundAedes vittatus is currently found in Africa, Asia and Europe, where it acts as a vector of pathogens causing animal and human diseases (e.g. chikungunya, Zika and dengue). Like other Aedes species, Ae. vittatus is able to breed in artificial containers. The ECDC has recently highlighted the need for molecular tools (i.e. barcoding characterization) that enable Aedes species to be identified in entomological surveys.ResultsWe sampled mosquito larvae and adults in southern Spain and used a molecular approach to amplify and sequence a fragment of the cytochrome c oxidase subunit 1 gene (barcoding region) of the mosquitoes. The blast comparison of the mosquito sequences isolated from Spain with those deposited in public databases provided a ≥ 99% similarity with sequences for two Aedes mosquitoes, Ae. vittatus and Ae. cogilli, while similarities with other Aedes species were ≤ 94%. Aedes cogilli is only present in India and there are no records of this species from Europe.ConclusionsDue to the low genetic differences between Ae. vittatus and Ae. cogilli, the barcoding region should not be used as the only method for identifying Ae. vittatus, especially in areas where both of these Aedes species are present. This type of analysis should thus be combined with morphological identification using available keys and/or the characterization of other molecular markers. In addition, further entomological surveys should be conducted in order to identify the fine-scale distribution of this mosquito species in Europe.

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Rapid and high throughput molecular identification of diverse mosquito species by high resolution melting analysis

Cited by 26 publications

References 32 publications

Composition of Anopheles mosquitoes, their blood-meal hosts, and Plasmodium falciparum infection rates in three islands with disparate bed net coverage in Lake Victoria, Kenya

Composition of Anopheles mosquitoes, their blood-meal hosts, and Plasmodium falciparum infection rates in three islands with disparate bed net coverage in Lake Victoria, Kenya

Sequencing of Tuta absoluta genome to develop SNP genotyping assays for species identification

Aedes vittatus in Spain: current distribution, barcoding characterization and potential role as a vector of human diseases

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