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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doaj-8948aaa28b2c4b22967bf9e41db7bbd02020-12-08T02:09:26ZengNature Publishing GroupScientific Reports2045-23222017-06-017111310.1038/s41598-017-03487-1Preferential suppression of Anopheles gambiae host sequences allows detection of the mosquito eukaryotic microbiomeEugeni Belda0Boubacar Coulibaly1Abdrahamane Fofana2Abdoul H. Beavogui3Sekou F. Traore4Daryl M. Gohl5Kenneth D. Vernick6Michelle M. Riehle7Department of Parasites and Insect Vectors, Unit of Genetics and Genomics of Insect Vectors, Institut PasteurMalaria Research and Training Centre (MRTC), Faculty of Medicine and Dentistry, University of MaliMalaria Research and Training Centre (MRTC), Faculty of Medicine and Dentistry, University of MaliCentre de Formation et de Recherche en Santé Rurale de MafèrinyahMalaria Research and Training Centre (MRTC), Faculty of Medicine and Dentistry, University of MaliUniversity of Minnesota Genomics CenterDepartment of Parasites and Insect Vectors, Unit of Genetics and Genomics of Insect Vectors, Institut PasteurDepartment of Microbiology and Immunology, University of MinnesotaAbstract 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.https://doi.org/10.1038/s41598-017-03487-1 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Eugeni Belda Boubacar Coulibaly Abdrahamane Fofana Abdoul H. Beavogui Sekou F. Traore Daryl M. Gohl Kenneth D. Vernick Michelle M. Riehle |
spellingShingle |
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 Scientific Reports |
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 Publishing Group |
series |
Scientific Reports |
issn |
2045-2322 |
publishDate |
2017-06-01 |
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. |
url |
https://doi.org/10.1038/s41598-017-03487-1 |
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