The microbiomes of blowflies and houseflies as bacterial transmission reservoirs

Blowflies and houseflies are mechanical vectors inhabiting synanthropic environments around the world. They feed and breed in fecal and decaying organic matter, but the microbiome they harbour and transport is largely uncharacterized. We sampled 116 individual houseflies and blowflies from varying habitats on three continents and subjected them to high-coverage, whole-genome shotgun sequencing. This allowed for genomic and metagenomic analyses of the host-associated microbiome at the species level. Both fly host species segregate based on principal coordinate analysis of their microbial communities, but they also show an overlapping core microbiome. Legs and wings displayed the largest microbial diversity and were shown to be an important route for microbial dispersion. The environmental sequencing approach presented here detected a stochastic distribution of human pathogens, such as Helicobacter pylori, thereby demonstrating the potential of flies as proxies for environmental and public health surveillance.


Supplementary
. Categories of reads assigned to Eukaryotes domain using rapsearch2 against the non-redundant NCBI database, showing that most matched to fly species of the order Diptera, thus revealing the reads that passed host-genome filtering. Blowflies are highlighted in blue and houseflies in yellow.
Supplementary Figure S2. The most frequently identified twenty viruses assigned from metagenomics datasets of 116 blowflies and houseflies using the rapsearch2 against the non-redundant NCBI database. Blowflies are highlighted in blue and houseflies in yellow. The bubble size is log-scaled and refers to the normalized number of reads.
Supplementary Figure S3. Phylum-level assignment of normalized metagenomic datasets with different bioinformatics methods. (A) reads assigned against the complete bacterial genomes database from NCBI using in-house developed script dbAssign. (B) reads assigned using specI clusters of 40 single universal genes. Proteobacteria, Bacteroidetes and Firmicutes are the most abundant phyla described in the blowfly Chrysomya megacephala (blue shadow) and in the housefly Musca domestica (yellow shadow).  Supplementary Tables S2 and S3. Different databases used for rapsearch2 (nr) and dbAssign (bacterial complete genomes) provided different bacterial diversity for blowflies and houseflies. dbAssign recovered less diversity (blowflies=1.69; houseflies= 3.15) than rapsearch2 (blowflies=5.18; houseflies= 5.04) due to different stringencies used for read mapping parameters and different databases. * * Supplementary Figure S5. Rarefaction curves of all blowflies (blue lines) and houseflies (yellow lines) analyzed with rapsearch2 (A) and dbAssign (B). The OTU discovery levels off around 25,000 reads sampled with rapsearch2 and around 500 reads for dbAssign, reflecting differences in databases used and alignment stringency. The boxplots in the right upper corners shows the OTU richness per host species, compiling the total number of observed bacterial species assigned for the two vectors. Solid line refers to median and dashed line to mean values. Whiskers represent minimum and maximum values. Individual points represent outliers. B A Supplementary Figure S6. Venn diagram indicating that more than 50% of the microbiome of houseflies and blowflies is shared.
Supplementary Figure S7. The core microbiome of C. megacephala and M. domestica (dbAssign taxa assignment output). All taxa listed refer to those present in more than 80% of flies analysed. The number of reads assigned to terminal nodes are inside the brackets. Bubble size indicates abundance of reads at different taxonomic levels (log-scaled) and the box describes all reads assigned to different taxonomic levels in the core microbiome.  Supplementary Figure 10. Comparison of methods used for OTU assignment. Venn diagram showing the number of OTUs assigned to species by three different bioinformatics approaches. Only high-confidence pairedend reads that were mapped above 97% identity are represented by the specI circle (total of 50 OTUs) and only OTUs with more than 500 mapped reads were considered for analysis with rapsearch2 (1,655 microbial species) and dbAssign (316 prokaryotic species) comparison. A total of 33 bacterial species were identified by all 3 methods and are listed in Table S7.
Supplementary Figure S11. Alpha-diversity of bacterial species assigned to the microbiome of four body parts of the blowfly (Shannon-Weaver diversity index). Legs+wings harbor the highest bacterial diversity, despite the least number of reads generated, compared to head, thorax and abdomen. This is particularly important in establishing the role of the outer body as the main route of bacterial dispersal by mechanical vectors. Fungi, archaea and viruses show a low diversity and were not detected in most of the flies analyzed, as well as in the four body parts.