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 (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.

Validation of a universal set of primers to study animal‐associated microeukaryotic communities

“…It is true that sequencing to greater depth can still allow access to eukaryome diversity, but this is inefficient and expensive (Wilcox and Hollocher, 2018). Second, blocking primers have been successfully used to access the eukaryome, but there is no universal blocking primer that will eliminate all or even most animals, so blocking primers tend to be specific to a particular study (Belda et al, 2017). The UNonMet-PCR primers can be used with most animals, except sponges and perhaps ctenophores, and we show in vivo are effective with corals (cnidarian) and humans (bilaterians), two groups that are far apart in the animal tree of life (Dunn et al, 2014).…”

Exposure of Anopheles mosquitoes to trypanosomes reduces reproductive fitness and enhances susceptibility to Plasmodium

“…The vector microbiome is a major factor likely influencing parasite transmission through different mechanisms [3,47,50]. The presence of antibiotics in the mosquito midgut could increase its susceptibility to Plasmodium infection, highlighting beneficial effects of the microbiota on the mosquito resistance to Plasmodium infection [29,51].…”

“…In areas with the highest malaria burden, such as in Sub-Saharan Africa, Plasmodium falciparum transmission depends on the complex ecological determinants (biotic and abiotic factors) that drive population dynamics of the primary African vectors Anopheles gambiae s.l. and A. funestus but also mosquito intrinsic factors for pathogen development [2][3][4][5][6][7]. Eukaryotic microbes, including microsporidia, gregarines and trypanosomatids, have been identified in mosquitoes.…”

“…Eukaryotic microbes, including microsporidia, gregarines and trypanosomatids, have been identified in mosquitoes. However, relatively little attention has been paid to Anopheles vector interactions with eukaryotic microbes and their potential impact on Plasmodium development in the mosquito [3].…”

A universal set of primers to study animal associated microeukaryotic communities

“…One solution to this problem is to use blocking primers in the PCR reaction, which are chemically modified primers (with a C3 spacer) that target the host 18S rRNA and will prevent the extension during the PCR when using universal primers [15]. But because metazoans are so diverse, blocking primers have to be specifically designed case by case for each host [16][17][18][19][20], so this might work for a subject of intensive study like humans, but not for any broader study of animal microbiome diversity. Another option is to use specific primers for eukaryotes that avoid a particular host [21], this approach seems to be easier than the use of blocking primers, but once again requires a specific set of primers for every metazoan group.…”