Fig 1.
Sampling locations of environmental water.
(A) Representative agro-ecological regions of Sri Lanka are shown by color shading: dark green, light green, and brown indicating wet, intermediate, and dry zones, respectively. K01 to K10, Kandy sampling sites; and GK01 to GK10, Girandurukotte sampling sites, respectively. Satellite imagery were obtained from Google Maps (https://www.google.com/maps/); data providers of the satellite imagery are Google, Data SIO, NOAA, U.S. Navy, NGA, GEBCO, Maxar Technologies, CNES/Airbus, Landsat/Copernicus, and TerraMetrics. Adobe Illustrator CS6 was used to create the map with satellite imagery. B) Representative landscape of paddy fields in a mountainous region of Kandy. C) Representative landscape of a paddy field in a flat region of Girandurukotte.
Table 1.
Tailed PCR primers used for environmental DNA metabarcoding sequencing by Illumina MiSeq platform in this study.
Fig 2.
Environmental detection of leptospiral 16S rRNA gene and vertebrate mitochondrial 12S rRNA gene.
The number of sequence reads detected in each sample are shown with colored matrices in red shading, where the red color intensity is relative to the number of sequence reads. Color gradation from light red to dark red represents low number to high number of sequence reads, respectively. More than thousand sequence reads are denoted in dark red cells and white bold numbers. P1 and P2 denote phylogenetic subclades of Leptospira. Green numbers above the scientific name of animals indicate the number of times of appearance of the species in total of 20 water samples. K and GK indicate sampling locations Kandy and Girandurukotte, respectively. K01−K10 and GK01−GK10 denote sample names. K-nega and GK-nega indicate negative control samples (RNase free water).
Fig 3.
Molecular phylogenetic tree of leptospiral flaB genes.
In total 432 nucleotide sites of flaB genes determined from environmental DNA analysis of the present study (shown in gray shading) were aligned and analyzed against known flaB sequences of representative Leptospira species. The GenBank accession numbers of the reference sequences were shown within sequence names. Maximum-likelihood phylogenetic analysis was performed with GTR + G + I model of nucleotide substitution. Numbers on the tree indicate support values for the nodes estimated from 100 bootstrap replications. K03 denotes the sample name collected from Kandy.
Fig 4.
Correlation of Leptospira and vertebrate animals.
Pearson’s correlation coefficients between detected read numbers of Leptospira (results from leptospiral 16S rRNA) and those of the vertebrates (results from mitochondrial 12S rRNA) were indicated in red (positive value) to blue (negative value) shading. The vertebrate species that showed positive correlation of r > 0.3 with at least one Leptospira OTU were shown. Asterisks indicate vertebrates that exhibited the significant correlation after Benjamini–Hochberg correction of false discovery rate at q < 0.01 in either row. P1 and P2 denote phylogenetic subclades of Leptospira. Numbers in the parenthesis above the scientific name of animals indicate number of times of appearance of the species in total of 10 water samples from the respective locations.
Fig 5.
Correlation of Leptospira and other bacteria in environmental water.
Pearson’s correlation coefficients between detected read numbers of Leptospira (results from leptospiral 16S rRNA) and those of bacteria (results from 16S rRNA V4 region) are indicated in red (positive value) to blue (negative value) shading. These bacteria are the top 25 species ordered by positive correlation values with summed counts of P1 Leptospira in respective regions. Asterisks indicate significant correlation after Benjamini–Hochberg correction of false discovery rate at q < 0.01. Underlines denote the bacteria which showed significant correlation with at least one of the columns of Leptospira OTUs or those of summed counts. P1 and P2 denote phylogenetic subclades of Leptospira. Numbers colored in green indicate number of times of appearance of the species in total of 10 water samples of respective regions.