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.
Highlights
Much of the eukaryotic microbial diversity in natural environments remains to be characterized, despite the critical importance of these lineages as pathogens, symbionts and commensals, environmental quality indicators, and markers of past environmental changes
Annealing blockers were designed as dual priming oligonucleotides with the 5′ segment overlapping the universal amplification primer and the 3′ segment specific to the non-desired 18s ribosomal RNA (rRNA) gene sequence
For the V4 hypervariable region, annealing blockers were designed that separately target A. coluzzii and mammal sequences respectively, whereas for the V9 region, an annealing blocker was designed targeting A. coluzzii, while for mammalian sequences the mammal blocker of the Earth Microbiome Project (EMP) was used
Summary
Much of the eukaryotic microbial diversity in natural environments remains to be characterized, despite the critical importance of these lineages as pathogens, symbionts and commensals, environmental quality indicators, and markers of past environmental changes. Exposure of mosquitoes and their ancestors to the numerous eukaryotic and prokaryotic microbes encountered in the aquatic environment, which include aquatic relatives of Plasmodium within the apicomplexan lineage, has probably been integral to shaping the evolution of the mosquito innate immune system. Mosquitoes of the Anopheles gambiae species complex are primary African vectors of the human malaria parasite, Plasmodium falciparum, which is responsible for extensive human morbidity and mortality. Multiple studies have catalogued the prokaryotic microbiota composition of different Anopheles mosquito populations by sequencing of 16s rRNA hypervariable www.nature.com/scientificreports/. Microscopic examination and insect pathology studies have identified a handful of eukaryotic microbes, including microsporidia, gregarines and trypanosomatids, in different mosquitoes[14,15,16,17,18,19,20,21,22,23,24]. A correlation was found between components of the midgut microflora and Plasmodium infection status in mosquitoes from Cameroon[30]
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