Characterizing larval breeding sites: bacterial community
composition
In addition to microbiome density, we performed 16s-rRNA gene amplicon
sequencing to explore the bacterial community composition in most larval
breeding sites (Table 1), inspired by previous studies that suggested
different bacteria between habitats (Dickson et al., 2017). The sample
processing and sequencing library preparation are described in the
Appendix. In short, we collected cells from the water by centrifuge or
filtering, extracted DNA, and amplified the 16s-rRNA gene V4 region
using primers reported in Kozich
et al. (2013). The amplicons from multiple samples were multiplexed and
sequenced on Illumina MiSeq
(Illumina, USA) at the
Yale Center for Genome Analysis.
We conducted amplicon sequencing for La Lopé and Rabai samples
separately.
We demultiplexed the sequencing reads using USEARCH v10.0.240 (Edgar,
2010) and followed the pipeline of DADA2 (v1.8.0) (Callahan et al.,
2016) to determine the bacterial community composition. DADA2 estimates
sequencing errors and infers the exact sequence variants (i.e., amplicon
sequence variants, or ASVs), which are analog to the conventional
operational taxonomic unit (OTU). ASVs were blasted to the Ribosomal
Database Project (RDP) 16s-rRNA gene reference database (RDP trainset 16
and RDP species assignment 16) (Cole et al., 2014) for taxonomic
assignment.
Using R package phyloseq (McMurdie & Holmes, 2013), we first
calculated the alpha diversity of the bacteria community in each larval
breeding site indicated by the Shannon index (Shannon, 1948), using raw
read counts. We then compared the index across larval breeding site
groups, habitats, and between Ae. aegypti present and absent
sites. The community compositions were summarized by non-metric
multidimensional scaling (NMDS) with the Bray-Curtis distance matrix.
Similar to PCA, NMDS summarizes multivariate data (each bacterial taxa
as one variable) but is more appropriate for bacterial composition data
(Ramette, 2007). Before NMDS analysis, we first removed samples with
fewer than 5000 reads to avoid low-quality samples, and thinned each
sample proportionally to the lowest read depth of all samples to control
for uneven sequencing depth. Bacterial communities may show different
assembly patterns at different taxonomic levels (Goldford et al., 2018).
Therefore, we calculated the Shannon index and performed NMDS at four
taxonomic levels: ASV, species, genus, and family. We also demonstrated
the major bacterial groups at the family level using bar plots. Lastly,
we used R package DESeq2 to identify bacterial families that are
most differentiated between habitats (Love et al., 2014).
To estimate the temporal stability of bacterial communities, we
collected water samples more than once for five larval breeding sites in
each habitat. The average interval between two consecutive collections
ranges from 3 to 21 days, with an average of 8.4 days in La Lopé and 17
days in Rabai. All temporal samples were sequenced, but only the
first-day samples were included in the analyses described above. We
performed a separate NMDS analysis to examine variation between temporal
samples.