Preferential suppression of Anopheles gambiae host sequences allows detection of the mosquito eukaryotic microbiome

Abstract Anopheles mosquitoes are vectors of the human malaria parasite, Plasmodium falciparum. The vector microbiota is a likely factor influencing parasite transmission. The prokaryotic microbiota of mosquitoes is efficiently surveyed by sequencing of hypervariable regions of the 16s ribosomal RNA...

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Autores principales:	Eugeni Belda, Boubacar Coulibaly, Abdrahamane Fofana, Abdoul H. Beavogui, Sekou F. Traore, Daryl M. Gohl, Kenneth D. Vernick, Michelle M. Riehle
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/8948aaa28b2c4b22967bf9e41db7bbd0
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spelling	oai:doaj.org-article:8948aaa28b2c4b22967bf9e41db7bbd02021-12-02T11:40:42ZPreferential suppression of Anopheles gambiae host sequences allows detection of the mosquito eukaryotic microbiome10.1038/s41598-017-03487-12045-2322https://doaj.org/article/8948aaa28b2c4b22967bf9e41db7bbd02017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-03487-1https://doaj.org/toc/2045-2322Abstract Anopheles mosquitoes are vectors of the human malaria parasite, Plasmodium falciparum. The vector microbiota is a likely factor influencing parasite transmission. The prokaryotic microbiota of mosquitoes is efficiently surveyed by sequencing of hypervariable regions of the 16s ribosomal RNA (rRNA) gene. However, identification of the eukaryotic microbiota by targeting the 18s rRNA gene is challenging due to simultaneous amplification of the abundant 18s rRNA gene target in the mosquito host. Consequently, the eukaryotic microbial diversity of mosquitoes is vastly underexplored. An efficient methodology is needed to identify this component of the microbiota, expected to include relatives of Plasmodium. Here, we use defined panels of Anopheles samples from West Africa to test two experimental PCR clamp approaches to maximize the specific amplification of 18s rRNA gene hypervariable regions from eukaryotic microbes: anneal-inhibiting blocking primers and peptide-nucleic acid (PNA) oligonucleotide blockers. Of the two, PNA blockers were the only efficient blocking strategy, allowing a reduction of mosquito 18s rRNA gene sequences by more than 80% for the V4 hypervariable region. These PNA blockers will facilitate taxonomic profiling of the eukaryotic microbiota of the A. gambiae species complex, and contribute to a better understanding of microbial influence upon immunity and pathogen infection.Eugeni BeldaBoubacar CoulibalyAbdrahamane FofanaAbdoul H. BeavoguiSekou F. TraoreDaryl M. GohlKenneth D. VernickMichelle M. RiehleNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-13 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Eugeni Belda Boubacar Coulibaly Abdrahamane Fofana Abdoul H. Beavogui Sekou F. Traore Daryl M. Gohl Kenneth D. Vernick Michelle M. Riehle Preferential suppression of Anopheles gambiae host sequences allows detection of the mosquito eukaryotic microbiome
description	Abstract Anopheles mosquitoes are vectors of the human malaria parasite, Plasmodium falciparum. The vector microbiota is a likely factor influencing parasite transmission. The prokaryotic microbiota of mosquitoes is efficiently surveyed by sequencing of hypervariable regions of the 16s ribosomal RNA (rRNA) gene. However, identification of the eukaryotic microbiota by targeting the 18s rRNA gene is challenging due to simultaneous amplification of the abundant 18s rRNA gene target in the mosquito host. Consequently, the eukaryotic microbial diversity of mosquitoes is vastly underexplored. An efficient methodology is needed to identify this component of the microbiota, expected to include relatives of Plasmodium. Here, we use defined panels of Anopheles samples from West Africa to test two experimental PCR clamp approaches to maximize the specific amplification of 18s rRNA gene hypervariable regions from eukaryotic microbes: anneal-inhibiting blocking primers and peptide-nucleic acid (PNA) oligonucleotide blockers. Of the two, PNA blockers were the only efficient blocking strategy, allowing a reduction of mosquito 18s rRNA gene sequences by more than 80% for the V4 hypervariable region. These PNA blockers will facilitate taxonomic profiling of the eukaryotic microbiota of the A. gambiae species complex, and contribute to a better understanding of microbial influence upon immunity and pathogen infection.
format	article
author	Eugeni Belda Boubacar Coulibaly Abdrahamane Fofana Abdoul H. Beavogui Sekou F. Traore Daryl M. Gohl Kenneth D. Vernick Michelle M. Riehle
author_facet	Eugeni Belda Boubacar Coulibaly Abdrahamane Fofana Abdoul H. Beavogui Sekou F. Traore Daryl M. Gohl Kenneth D. Vernick Michelle M. Riehle
author_sort	Eugeni Belda
title	Preferential suppression of Anopheles gambiae host sequences allows detection of the mosquito eukaryotic microbiome
title_short	Preferential suppression of Anopheles gambiae host sequences allows detection of the mosquito eukaryotic microbiome
title_full	Preferential suppression of Anopheles gambiae host sequences allows detection of the mosquito eukaryotic microbiome
title_fullStr	Preferential suppression of Anopheles gambiae host sequences allows detection of the mosquito eukaryotic microbiome
title_full_unstemmed	Preferential suppression of Anopheles gambiae host sequences allows detection of the mosquito eukaryotic microbiome
title_sort	preferential suppression of anopheles gambiae host sequences allows detection of the mosquito eukaryotic microbiome
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/8948aaa28b2c4b22967bf9e41db7bbd0
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Preferential suppression of Anopheles gambiae host sequences allows detection of the mosquito eukaryotic microbiome

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