Internal Repetition and Intraindividual Variation in the rDNA ITS1 of the Anopheles punctulatus Group (Diptera: Culicidae): Multiple Units and Rates of Turnover

Bower, James E.; Cooper, Robert D.; Beebe, Nigel W.

doi:10.1007/s00239-008-9188-z

Cited by 38 publications

(34 citation statements)

References 49 publications

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“…However, it appears there is a limit to this process, given that An. annulipes has a larger ITS2 region (601 bp, Bower et al 2009) but does not assemble into a single contig.…”

Section: Resultsmentioning

confidence: 99%

Using Next-Generation Sequencing for DNA Barcoding: Capturing Allelic Variation in ITS2

Batovska

Cogan

Lynch

et al. 2017

G3 Genes|Genomes|Genetics

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Internal Transcribed Spacer 2 (ITS2) is a popular DNA barcoding marker; however, in some animal species it is hypervariable and therefore difficult to sequence with traditional methods. With next-generation sequencing (NGS) it is possible to sequence all gene variants despite the presence of single nucleotide polymorphisms (SNPs), insertions/deletions (indels), homopolymeric regions, and microsatellites. Our aim was to compare the performance of Sanger sequencing and NGS amplicon sequencing in characterizing ITS2 in 26 mosquito species represented by 88 samples. The suitability of ITS2 as a DNA barcoding marker for mosquitoes, and its allelic diversity in individuals and species, was also assessed. Compared to Sanger sequencing, NGS was able to characterize the ITS2 region to a greater extent, with resolution within and between individuals and species that was previously not possible. A total of 382 unique sequences (alleles) were generated from the 88 mosquito specimens, demonstrating the diversity present that has been overlooked by traditional sequencing methods. Multiple indels and microsatellites were present in the ITS2 alleles, which were often specific to species or genera, causing variation in sequence length. As a barcoding marker, ITS2 was able to separate all of the species, apart from members of the Culex pipiens complex, providing the same resolution as the commonly used Cytochrome Oxidase I (COI). The ability to cost-effectively sequence hypervariable markers makes NGS an invaluable tool with many applications in the DNA barcoding field, and provides insights into the limitations of previous studies and techniques.

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“…However, it appears there is a limit to this process, given that An. annulipes has a larger ITS2 region (601 bp, Bower et al 2009) but does not assemble into a single contig.…”

Section: Resultsmentioning

confidence: 99%

Using Next-Generation Sequencing for DNA Barcoding: Capturing Allelic Variation in ITS2

Batovska

Cogan

Lynch

et al. 2017

G3 Genes|Genomes|Genetics

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“…punctulatus species group species considered to be the most significant vectors of malaria in PNG. 59,[64][65][66][67] Additionally, some polymorphisms within species, namely An. farauti s.s. and An.…”

Section: Resultsmentioning

confidence: 99%

Multiplex Assay for Species Identification and Monitoring of Insecticide Resistance in Anopheles punctulatus Group Populations of Papua New Guinea

Halldin

Nadesakumaran

Keven

et al. 2012

The American Society of Tropical Medicine and Hygiene

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Abstract. Anopheles punctulatus sibling species ( An. punctulatus s.s., Anopheles koliensis , and Anopheles farauti species complex [eight cryptic species]) are principal vectors of malaria and filariasis in the Southwest Pacific. Given significant effort to reduce malaria and filariasis transmission through insecticide-treated net distribution in the region, effective strategies to monitor evolution of insecticide resistance among An . punctulatus sibling species is essential. Mutations in the voltage-gated sodium channel ( VGSC ) gene have been associated with knock-down resistance ( kdr ) to pyrethroids and DDT in malarious regions. By examining VGSC sequence polymorphism we developed a multiplex assay to differentiate wild-type versus kdr alleles and query intron-based polymorphisms that enable simultaneous species identification. A survey including mosquitoes from seven Papua New Guinea Provinces detected no kdr alleles in any An. punctulatus species. Absence of VGSC sequence introgression between species and evidence of geographic separation within species suggests that kdr must be monitored in each An . punctulatus species independently. 41 Presently, the Global Fund to Fight AIDS, Tuberculosis and Malaria (GFATM) has provided support for the distribution of over 2.5 million long-lasting insecticide-treated nets (LLIN; deltamethrin) at no cost to individual families. 42 Introduction of LLINs to all of PNG's 20 Provinces represents the first attempt at a comprehensive plan for mosquito-based malaria control. Of importance, our recently published study observed no phenotypic evidence of resistance to the synthetic pyrethroid insecticides among Anopheles mosquitoes collected at five different PNG malaria-endemic sites. 43 In response to intensive insecticide exposure, malaria vectors in other regions of the world have developed resistance to a number of different classes of insecticide compounds. Because several insect species have shown evidence for development of specific polymorphisms in the voltage-gated sodium channel ( VGSC ) after prolonged exposure to DDT and pyrethroid insecticides, 44, 45 a number of studies have focused on sequence encoding the S4-S6 region of VGSC domain II. 46,47 In Anopheles mosquitoes, knockdown resistance ( kdr ) has been associated with an A → T mutation (L1014F) 46 or a T → C mutation (L1014S) 48 in VGSC . These single nucleotide polymorphisms (SNPs) have been found in African, Indian, and Asian malaria vectors, 49-54 and most recently in neighboring Indonesia, 55 showing the capacity of these mutations to develop independently in different Anopheles species . Another point mutation in VGSC associated with very high levels of DDT resistance, super-kdr (M918T), has been described in many insects, however this point mutation has not yet been detected in Anopheles . 44,45,56 To date, Southwest Pacific malaria vectors have not been surveyed for any mutations associated with insecticide resistance. Assuming validity of An. punctulatus sibling species definitions, and the hetero...

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“…The presence of a high GC ratio would stabilize dsDNA in both PCR products and intramolecular secondary structures, and could inhibit the PCR. Moreover Bower et al (2009) reported that the GC ratio ITS2 from An. punctulatus group varied between 64-71.3%.…”

Section: Resultsmentioning

confidence: 99%

The effect of DMSO on ITS2 amplification in the molecular identification of Anopheles farauti Laveran (Diptera: Culicidae), from a colony established in the laboratory

et al. 2015

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Sibling species identification is very important in the understanding of malaria epidemiology. Morphological criteria are usually used in the identification of anopheline species, but this fails when sibling or cryptic species occur. Analysis by PCR-RFLP of rDNA ITS2 is currently the most reliable and sensitive method for distinguishing between members of the Anopheles punctulatus group. The objective of this study was to investigate the effect of DMSO concentration on ITS2 amplification of An. farauti from the colony maintained at BATAN Jakarta using PCR-RFLP based on the rDNA ITS2. The results showed that the addition of 6 % and 7 % DMSO produced ITS2 amplification products in the size 750 bp. DMSO could be used in PCR to relieve secondary structures when amplifying high GC templates. Molecular identification of An. farauti is found to be Anopheles farauti sensu stricto.Key words: molecular identification, sibling species, PCR-RFLP, rDNA ITS2, secondary structure ABSTRAK Identifikasi spesies sibling sangat penting untuk mempelajari epidemiologi malaria. Ciri morfologi secara umum digunakan dalam identifikasi spesies Anopheles, namun tidak sesuai ketika digunakan untuk spesies sibling dan spesies kriptik. Analisis dengan menggunakan PCR-RFLP dari ITS2 rDNA pada saat ini merupakan metode yang paling cocok dan sensitif untuk membedakan spesies Anopheles punctulatus group. Tujuan penelitian ini adalah untuk mengetahui pengaruh konsentrasi DMSO terhadap amplifikasi ITS2 dalam mengidentifikasi An. farauti dari koloni di BATAN Jakarta dengan PCR-RFLP berdasarkan ITS2 rDNA. Hasil penelitian menunjukkan bahwa penambahan DMSO dengan konsentrasi 6% dan 7% menghasilkan produk amplifikasi ITS2 dengan ukuran 750 pb. DMSO dapat digunakan dalam PCR untuk mencegah terbentuknya struktur sekunder *Penulis korespondensi: Ign.

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Internal Repetition and Intraindividual Variation in the rDNA ITS1 of the Anopheles punctulatus Group (Diptera: Culicidae): Multiple Units and Rates of Turnover

Cited by 38 publications

References 49 publications

Using Next-Generation Sequencing for DNA Barcoding: Capturing Allelic Variation in ITS2

Using Next-Generation Sequencing for DNA Barcoding: Capturing Allelic Variation in ITS2

Multiplex Assay for Species Identification and Monitoring of Insecticide Resistance in Anopheles punctulatus Group Populations of Papua New Guinea

The effect of DMSO on ITS2 amplification in the molecular identification of Anopheles farauti Laveran (Diptera: Culicidae), from a colony established in the laboratory

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