A multiplex PCR assay for the identification of five species of the Anopheles barbirostris complex in Thailand

Brosseau, Laura; Udom, Chanya; Sukkanon, Chutipong; Chareonviriyaphap, Theeraphap; Bangs, Michael J.; Saeung, Atiporn; Manguin, Sylvie

doi:10.1186/s13071-019-3494-8

Cited by 38 publications

(42 citation statements)

References 28 publications

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“…barbirostris . The ITS2 sequence was compared to voucher sequences and primer sites used in Brosseau et al [ 17 ], as the highest homology was to these sequences. Although most similar to An.…”

Section: Resultsmentioning

confidence: 99%

“…Consensus sequences of each sequence group were compared (BLASTn) to the NCBI nr and BOLD databases to identify species. Sequences mapping to the Funestus Group, Hyrcanus Group, Barbirostris Complex, Sundaicus Complex and Maculatus Group were compared to voucher sequences and primers used in diagnostic PCR [ 17 , 33 – 37 ].…”

Section: Methodsmentioning

confidence: 99%

“…subpictus ( s.l .) [ 12 , 15 – 17 ]. There is documentation for multiple species in some complexes, such as the Barbirostris Complex [ 16 , 18 – 21 ].…”

Section: Introductionmentioning

confidence: 99%

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Molecular analysis reveals a high diversity of Anopheles species in Karama, West Sulawesi, Indonesia

et al. 2020

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Background Understanding local Anopheles species compositions and bionomic traits are vital for an effective malaria vector intervention strategy. Though eight malaria vectors, including species complexes, have been documented across the island of Sulawesi, Indonesia, a comprehensive survey linking morphological and molecular species identification has not been conducted in this global hotspot of biodiversity. Results Eighteen distinct species of Anopheles were molecularly identified in a 1 km 2 area in Karama village, West Mamuju Province, Sulawesi. Known species included An. aconitus , An. karwari , An. peditaeniatus , An. vagus , An. barbirostris , An. tessellatus , An. nigerrimus , An. crawfordi , An. maculatus, An. flavirostris and An. kochi . Of the 18 distinct sequence groups identified through both ribosomal DNA internal transcribed spacer region 2, and mitochondrial DNA cytochrome c oxidase subunit 1 loci, 8 could not be identified to species through comparison to published sequences. The comparison of morphological and molecular identities determined that interpretations of local species compositions for primary and expected species in Karama ( An. barbirostris and An. vagus ) had the highest rate of accuracy (92.1% and 87.6%, respectively) when compared to molecular analysis. However, the remaining distinct sequences molecularly identified to species were identified correctly by morphological methods less frequently, from 0 to 83%. Conclusions Karama, Indonesia has a high diversity of Anopheles spp. The unexpected high number of Anopheles species in a small area points to possible complex transmission dynamics and limitations with vector control based on possible varying behaviors and interactions with both humans and interventions. Morphological identification of Anopheles spp. in this study was more accurate for primary and expected species than secondary or unexpected species. Finally, the inability to identify seven sequence groups to species with consensus sequences implies that future studies employing sequencing are required to clarify species compositions in the Nigerrimus Subgroup, among others, as well as their distribution and vector status. Use of molecular methods in conjunction with morphological investigations for analysis of species composition, population dynamics and bionomic characteristics is directly implicated in understanding drivers of malaria transmission, intervention effectiveness, and the pursuit...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Molecular analysis reveals a high diversity of Anopheles species in Karama, West Sulawesi, Indonesia

et al. 2020

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“…barbirostris s.l. [59] could bind to the ITSII sequences generated in our study, and they amplified a 404-bp fragment (Additional File…”

Section: The Itsii Region and Coi Gene [See Additional Files 3 And 4 Fomentioning

confidence: 97%

“…barbirostris s. l., including those generated in this study, were also aligned without trimming using the parameters stated above. Primer binding sites for the multiplex PCR assay developed by Brosseau et al [58] to identify the members of the Barbirostris Complex were then identified from the alignment. Amplicon size(s) resulting from the multiplex PCR assay was also determined for the ITSII sequences generated from this study.…”

Section: Analysis Of Primer Binding Sites In the Itsii Sequence Of Anmentioning

confidence: 99%

New vectors in northern Sarawak, Malaysian Borneo, for the zoonotic malaria parasite, Plasmodium knowlesi

Ang

Kadir

Mohamad

et al. 2020

Preprint

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Background: The vectors for Plasmodium knowlesi, a significant cause of human malaria in Southeast Asia, identified previously in nature all belong to the Anopheles Leucosphyrus Group. Only one study has been previously undertaken in Sarawak, Malaysian Borneo, to identify vectors of P. knowlesi , where Anopheles latens was incriminated as the vector in Kapit, central Sarawak. A study was therefore undertaken to identify malaria vectors in a different location in Sarawak. Methods: Mosquitoes found landing on humans and resting on leaves over a 5-day period at two sites in the Lawas District of northern Sarawak were collected and identified. DNA samples extracted from salivary glands of Anopheles mosquitoes were subjected to nested PCR malaria-detection assays. The small sub-unit ribosomal RNA (SSUrRNA) genes of Plasmodium , and the internal transcribed spacer 2 (ITSII) and mitochondrial cytochrome c oxidase subunit 1 (COI) sequences of the mosquitoes were derived from the Plasmodium -positive samples for phylogenetic analyses. Results: A total of 65 anophelines and 127 culicines were collected. By PCR, six An. balabacensis and five An. barbirostris Clade VI were found to have single P. knowlesi infecions while three other An. balabacensis had either single, double or triple infections with P. inui, P. fieldi, P. cynomolgi and P. knowlesi . Phylogenetic analyses of the Plasmodium SSUrRNA genes confirmed 3 An. barbirostris Clade VI and 3 An. balabacensis with single P. knowlesi infections, while 3 other An. balabacensis had two or more Plasmodium species of P. inui, P. knowlesi, P. cynomolgi and possibly novel species of Plasmodium . Phylogenies inferred from the ITSII and COI sequences of An. balabacensis and An. barbirostris Clade VI indicate that the former is genetically indistinguishable from An. balabacensis in Borneo while the latter is a novel sibling species belonging to the Anopheles Barbirostris Subgroup. Conclusions: New vectors for P. knowlesi in Sarawak were identified, including An. barbirostris Clade VI , which is a species that does not belong to the Anopheles Leucosphyrus Group.

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Using capillary electrophoresis to identify Anopheline species in routine sampling sites

Chabanol,

Romoli,

Talaga

et al. 2024

Ecology and Evolution

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In the Anopheles genus, various mosquito species are able to transmit the Plasmodium parasites responsible for malaria, while others are non‐vectors. In an effort to better understand the biology of Anopheles species and to quantify transmission risk in an area, the identification of mosquito species collected in the field is an essential but problematic task. Morphological identification requires expertise and cannot be checked after processing samples in a destructive treatment, while sequencing of numerous samples is costly. Here, we introduce a method of Species identification via Simple Observation Coupled with Capillary Electrophoresis Technology (SOCCET). This molecular technique of species identification is based on precise determination of ITS2 length combined with a simple visual observation, the colour of mosquito hindleg tip. DNA extracted from field‐collected Anopheles mosquitoes was amplified with universal Anopheles ITS2 primers and analysed with a capillary electrophoresis device, which precisely determines the size of the fragments. We defined windows of amplicon sizes combined with fifth hind tarsus colour, which allows discrimination of the major Anopheles species found in our collections. We validated our parameters via Sanger sequencing of ITS2 amplicons. Using the SOCCET method, we characterised the composition of Anopheles populations in five locations of French Guiana, where we detected a total of nine species. Anopheles braziliensis and Anopheles darlingi were detected in four locations each and represented 13 and 67% of our samples, respectively. The SOCCET method can be particularly useful when working with routine sampling sites with a moderate species diversity, that is, when the number of local species is too high to define species‐specific primers but low enough to avoid individual ITS2 sequencing. This tool will be of interest to evaluate local malaria transmission risk and this approach may be further implemented for other mosquito genera.

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A multiplex PCR assay for the identification of five species of the Anopheles barbirostris complex in Thailand

Cited by 38 publications

References 28 publications

Molecular analysis reveals a high diversity of Anopheles species in Karama, West Sulawesi, Indonesia

Molecular analysis reveals a high diversity of Anopheles species in Karama, West Sulawesi, Indonesia

New vectors in northern Sarawak, Malaysian Borneo, for the zoonotic malaria parasite, Plasmodium knowlesi

Using capillary electrophoresis to identify Anopheline species in routine sampling sites

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