Orientia, Rickettsia, and the microbiome in rodent attached chiggers in North Carolina, USA

Abstract

Chiggers are larval mites that pose a significant health risk globally via the spread of scrub typhus. However, fundamental studies into the bacterial microbiome in North America have never been considered. In this investigation, chiggers were collected in the wild from two locally common rodent host species (i.e., Sigmodon hispidus and Peromyscus leucopus) in three different ecoregions of North Carolina (NC), United States to investigate the composition of their bacterial communities, including potential pathogens. DNA was extracted from the chiggers, and the V3-V4 regions of the bacterial 16S rRNA gene were sequenced using next-generation sequencing (NGS). Alpha diversity metrics revealed significant differences in bacterial diversity among different collection counties. Beta diversity metrics also revealed that bacterial communities across counties were significantly different, suggesting changes in the microbiome as the environment changed. Specifically, we saw that the two western NC collection counties had similar bacterial composition as did the two eastern collection counties. In addition, we found that the chigger microbiome bacterial diversity and composition differed between rodent host species. The 16S rRNA sequence reads were assigned to 64 phyla, 106 orders, 199 families, and 359 genera. The major bacterial phylum was Actinobacteria. The most abundant species were in the genera Corynebacterium, Propionibacterium, class ZB2, and Methylobacterium. Sequences derived from potential pathogens within the genera Orientia and Rickettsia were also detected. Our findings provide the first insights into the ecology of chigger microbiomes in the US. Further research is required to determine if the potential pathogens found detected in chiggers are a threat to humans and wildlife.

Fig 1. Map of collection sites where rodent-associated chiggers were sampled.

The three ecoregions of North Carolina are indicated in different colors. The red dots represent the geographic location where chiggers were collected off live rodents. The acronyms in uppercase letters below the red dots represent the different chigger collection sites: SMGL—South Mountains Game Lands, LNSP—Lake Norman State Park, MMSP—Morrow Mountain State Park, and CNF—Croatan National Forest. The county names are indicated in black text cases above the red diamond shape. The hispid cotton rat is represented by the black rodent symbol and the white-footed mouse by the white rodent symbol. The genus of chigger collected is indicated in the bottom key next to the rodent type. The chiggers collected from the white-footed mouse were morphologically identified to be Leptotrombidium spp. This map was created using a free, open-source quantum geographic information system (QGIS, version 3.34.6). The North Carolina shapefile was modified from the USA shapefile from gadm.org (GADM data, version 4.1).

Fig 2. Rarefaction curves of the mean number of observed features (ASVs) (amplicon sequence variants) (y-axis) in North Carolina, USA, where chiggers were collected off rodents.

Error bars represent the standard error of the mean. The hispid cotton rat is represented by the black rodent symbol and the white-footed mouse by the white rodent symbol.

Fig 3. Alpha diversity measures of the microbiome of chiggers collected from rodents in Rutherford, Iredell, Stanly, and Craven counties, North Carolina, USA.

(a) Shannon diversity, (b) Faiths phylogenetic diversity, and (c) Observed (ASVs). Letters A and B are shown above each box to show significant differences. The horizontal lines on the bottom of box plots denoting the lower interquartile value, the middle line is the median, and the top line is represented by the upper interquartile value. Filled circles outside of the line are outliers. The hispid cotton rat is represented by the black rodent symbol and the white-footed mouse by the white rodent symbol.

Fig 4. Principal coordinate analysis (PCoA) of bacterial composition in chiggers collected from rodents in Rutherford, Iredell, Stanly, and Craven counties, North Carolina, USA.

The analysis was based on the weighted UniFrac metric and visualized using Emperor. The circle points out clustering among the collection counties. The key shows the color used to represent each county and the rodent type that was collected in that county.

Fig 5. Comparison of unique and shared bacterial taxa found in chiggers collected in different counties in North Carolina, USA.

Venn diagram shows the percentages of the common and different predicted bacterial species level taxa (level 7) found in the microbiome of chiggers. The hispid cotton rat is represented by the black rodent symbol and the white-footed mouse by the white rodent symbol.

Fig 6. Relative abundances of major bacterial taxa at the genus level compared for chiggers collected from rodents in Rutherford, Iredell, Stanly, and Craven counties, North Carolina, USA.

The ‘Other’ category represents all taxa with relative abundance below 2%. Some taxa were only able to be identified up to the genus level. In the legend, the “f_” represents family; “p_” represents phylum, and “c_” represents class. The hispid cotton rat is represented by the black rodent symbol and the white-footed mouse by the white rodent symbol.

Fig 7. Relative abundances of major bacterial taxa at the genus level compared among the two host types (hispid cotton rat and white-footed mouse).

The ‘Other’ category represents all taxa with relative abundance below 2%. Each stacked bar graph represents the mean sample composition for both host types. Some taxa were only able to be identified up to the genus level. “f_” represents family; “p_” represents phylum, and “c_” represents class.

Fig 8. Maximum likelihood phylogenetic analysis of Orientia 16S rRNA gene amplicon sequence variants (ASVs).

ASVs identified in this study are shown in red, sequences from study on free living chiggers in blue (and *), and closest related species black. Numbers at nodes represent bootstrap values (%), which were obtained from 1000 replicates; values <50 % are not shown. Bar, 0.01 substitutions per nucleotide position. Each ASV has an identification label in parentheses next to it. Rickettsia parkeri strain (NR029156) was included as an outgroup.

Fig 9. Phylogenetic analysis of Rickettsia amplicon sequence variants (ASVs).

ASVs identified in this study (in bold red) and other known Rickettsial species (black) are shown. The phylogenetic tree was constructed by the maximum likelihood method using 16S rRNA gene sequences. The percentage of replicate trees with 50% cutoff value where the associated taxa clustered together in the bootstrap test (1000 replicates) are shown below the branches. Each ASV has an identification label in parentheses next to it. Orientia tsutsugamushi strain (NR025860) was included as an outgroup.