mec13187-sup-0001
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1 Supplemental Material 2 3 Methods 4 Field Sampling 5 Both collection sites (Supplemental Table 1) were characterized by a patchwork of successional 6 grassland and young forest. Two 50x50m grids were established in forested and open, grassy 7 habitats at each site. Sixty Sherman live traps (3x3.5x9 in, H.B. Sherman Traps, Tallahassee FL, 8 USA) were placed in each grid for a total of 120 traps per site and sampled for three consecutive 9 days. Traps were baited each evening before dusk with millet seeds, puffed corn peanut butter 10 snack (Bamba ©) and cotton batting, and were checked early the next morning. 11 12 Sequencing Bioinformatics 13 One run was performed with all tick samples in one flowcell partition and blood samples in 14 another (Supplemental Table 5). Therefore any comparisons between ticks and blood are also 15 comparing between libraries. 16 17 Samples that underwent PCR on 14 November 2012 contained significantly higher amounts of 18 Bartonella than other all other samples, indicative of contamination and were removed. Samples 19 with fewer than 500 sequences were also excluded. These are described in Supplemental Table 6. 20 OTUs were removed if they were not significantly higher in samples compared to negative 21 controls, if they were matched to Eukaryotic taxa, or if there was no taxa match (Supplemental 22 Table 5). The remaining OTUs were re-ranked in order of abundance, weighted by how many 23 sequences came from each sample type due to uneven sample sizes. The original OTU numbers 24 are given in Supplemental Table 2. 25 26 All data on the samples can be found in the materials uploaded to Dryad. The following OTUs 27 are not included in the SRA submission only, due to the quality of their representative sequence 28 not meeting the standards for GenBank: 21, 28, 45, 56, 60, 67, 74, 88, 93, 99, 103, 108, 110, 111. 29 30 Results 31 Bacterial richness and diversity 32 Host blood had significantly lower bacterial OTU richness than either tick species (zDv=5.53, zIs= 33 2.26, p < 0.001 both) but there was not a significant difference between tick species. D. 34 variabilis had a higher Shannon Diversity Index than host blood (z = 3.33, p = 0.029) but there 35 was not a significant difference between I. scapularis and the other sample types. D. variabilis 36 had higher evenness than host blood (z =2.07, p < 0.001) but was not different from I. scapularis 37 (Supplemental Table 4). 38 39 We predicted that nymphs would have higher bacterial diversity than larvae, which are expected 40 to only be infected with vertically-transmitted bacteria or with bacteria acquired from the 41 environment. We predicted that more highly engorged ticks would have higher bacterial 42 diversity due to the higher volume of blood. There was a significant difference in OTU richness 43 and diversity between larvae and nymphs. However, in both tick species larvae had higher OTU 44 richness (z = -2.24, p = 0.025) than nymphs, opposite of what was predicted. There were no 45 significant differences in Shannon Diversity Index or Evenness between tick life stages. There 46 was a significant difference between tick engorgement levels with more highly-engorged ticks 47 having lower richness (z = -2.22, p = 0.026), also opposite of our predictions. 48 49 References 50 Gotelli, N. J. 2000. Null model analysis of species co-occurrence patterns. Ecology 81:2606- 51 2621. 52 53 54 Figures a b 55 56 Supplemental Figure 1. NMDS plot for D. variabilis (a) and I. scapularis (b) labeled by life 57 stage (larva or nymph) and engorgement level (0, 0.5, 1). Ellipses drawn one standard deviation 58 from the centroid of each group using the function ordiellipse (solid lines = life stages, dashed 59 lines = engorgement levels). 60 61 62 63 64 Supplemental Figure 2. Proportion of rarefied sequences from each of the top 5 OTUs in each 65 tick species, life stage, and engorgement level. The genus match for each OTU cluster is given 66 with OTU number in ( ). Bartonella sequences were found in all of these categories. 67 68 69 Supplemental Figure 3. Regression between the total rarefied sequences from Bartonella of a 70 host sample and the total Bartonella sequences from all ticks collected from that host. The 71 regression was non-significant (t = 0.240, p = 0.812). 72 73 Tables 74 Supplemental Table 1. Locations of the two sites where rodents and ticks were collected. Site Location 1 S. Snow Road, Bloomington IN 2 W. Popcorn Rd, Springville IN Latitude 39 0 22.6 N 39 0 13.01 N Longitude 86 39 6.74 W 86 35 18.25 W 75 76 77 Supplemental Table 2. The 118 OTUs used in the microbiome analysis with total rarefied 78 sequences and proportion of each in the three sample types. The taxonomic matches for each 79 OTU to species (if possible) are presented. The Percent Identity of the consensus sequence for 80 each OTU is also given to describe the match to the sequence in the SILVA database, with most 81 being above 97%. 82 83 Supplemental Table 3. Taxonomic matches of OTUs removed from analysis. OTUs were 84 removed due to being higher or no different from abundance in negative controls, being matched 85 to Eukaryotic taxa or having no match found. 86 87 Supplemental Table 4. Mean Richness, Shannon Diversity Index, Evenness, and number of 88 Bartonella sequences (± SE) for all sample types (from rarefied data). OTU Richness Shannon Diversity OTU Evenness Bartonella Sequences Host blood 5.98 ± 0.70 0.11 ± 0.04 0.05 ± 0.01 4.89.33 ± 4.84 D. variabilis 12.45 ± 1.07 0.55 ± 0.06 0.22 ± 0.02 4.16 ± 0.69 I. scapularis 9.47 ± 2.52 0.20 ± 0.07 0.08 ± 0.02 2.84 ± 1.14 89 90 91 Supplemental Table 5. Proportion of rarefied sequences of each OTU from each individual 92 sample. This table also included select metadata for each sample (“SampleID”), including the 93 sequencing library associated with each sample (“Library”); one for ticks and one for host blood 94 samples. The host from which each tick or blood sample was collected is “Rodent ID” while 95 “HostID_Date” included the collection date as well as host ID due to some hosts being captured 96 more than once. 97 98 99 100 101 102 103 104 Supplemental Table 6. Tick and blood samples that were removed from analyses. Sample 15 69 71 72 73 97 190 198 199 202 203 207 215 216 219 250 273 283 286 301 303 356 358 361 362 367 378 380 385 388 389 395 402 433 455 530 550 607 648 650 Library tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick tick SampleType IS DV DV IS DV DV DV DV IS IS DV DV DV IS DV DV DV DV DV IS DV DV DV DV DV IS IS DV DV DV IS DV DV DV DV DV DV IS IS DV PrimerName XLR_534R_v2bBar23L XLR_534R_v2bBar174L XLR_534R_v2bBar602L XLR_534R_v2bBar212L XLR_534R_v2bBar25L XLR_534R_v2bBar600L XLR_534R_v2bBar144L XLR_534R_v2bBar48L XLR_534R_v2bBar166L XLR_534R_v2bBar613L XLR_534R_v2bBar560L XLR_534R_v2bBar741L XLR_534R_v2bBar441L XLR_534R_v2bBar1174L XLR_534R_v2bBar209L XLR_534R_v2bBar298L XLR_534R_v2bBar146L XLR_534R_v2bBar554L XLR_534R_v2bBar646L XLR_534R_v2bBar481L XLR_534R_v2bBar419L XLR_534R_v2bBar1379L XLR_534R_v2bBar208L XLR_534R_v2bBar1267L XLR_534R_v2bBar637L XLR_534R_v2bBar435L XLR_534R_v2bBar1202L XLR_534R_v2bBar413L XLR_534R_v2bBar289L XLR_534R_v2bBar433L XLR_534R_v2bBar121L XLR_534R_v2bBar669L XLR_534R_v2bBar49L XLR_534R_v2bBar161L XLR_534R_v2bBar233L XLR_534R_v2bBar1225L XLR_534R_v2bBar693L XLR_534R_MID55 XLR_534R_MID74 XLR_534R_MID75 Why Removed Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Possible contamination Possible contamination Possible contamination Possible contamination Possible contamination Possible contamination Possible contamination Possible contamination Possible contamination Possible contamination Possible contamination Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences 664 530_5.11 533_5.12 534_5.12 534_5.25 535_5.12 536_5.19 536_6.12 538_5.19 538_5.31 539_6.10 539_7.05 539_9.28 542_5.25 545_5.25 545_6.07 547_5.25 549_5.26 549_6.07 549_6.30 549_9.21 550_5.26 550_6.07 553_5.26 554_5.27 555_5.27 559_7.05 563_6.02 564_6.02 566_6.29 566_9.20 567_6.07 569_6.08 570A_6.29 570B_6.29 573_6.12 573_7.05 576_6.29 578_6.29 579_6.30 580_7.01 581_7.06 587_9.21 tick blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood IS blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood blood XLR_534R_MID79 XLR_534R_v2bBar8L XLR_534R_v2bBar1173L XLR_534R_v2bBar602L XLR_534R_v2bBar212L XLR_534R_v2bBar599L XLR_534R_v2bBar25L XLR_534R_v2bBar622L XLR_534R_v2bBar600L XLR_534R_v2bBar559L XLR_534R_v2bBar144L XLR_534R_v2bBar575L XLR_534R_v2bBar48L XLR_534R_v2bBar580L XLR_534R_v2bBar31L XLR_534R_v2bBar551L XLR_534R_v2bBar184L XLR_534R_v2bBar228L XLR_534R_v2bBar1273L XLR_534R_v2bBar441L XLR_534R_v2bBar1174L XLR_534R_v2bBar78L XLR_534R_v2bBar555L XLR_534R_v2bBar364L XLR_534R_v2bBar393L XLR_534R_v2bBar1196L XLR_534R_v2bBar481L XLR_534R_v2bBar1031L XLR_534R_v2bBar76L XLR_534R_v2bBar141L XLR_534R_v2bBar119L XLR_534R_v2bBar99L XLR_534R_v2bBar1225L XLR_534R_v2bBar637L XLR_534R_v2bBar1202L XLR_534R_v2bBar433L XLR_534R_v2bBar121L XLR_534R_v2bBar731L XLR_534R_v2bBar438L XLR_534R_v2bBar693L XLR_534R_v2bBar672L XLR_534R_v2bBar355L XLR_534R_MID35 Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences 588_9.22 589_9.22 590_9.27 594_9.27 596_9.29 NC H2O NC H2O NC_H2O NC_H2O NC1 NC1 NC10 NC10 NC11 NC12 NC2 NC2 NC3 NC3 NC4 NC4 NC5 NC5 NC5 NC6 NC6 NC7 NC7 NC8 NC8 NC9 NC9 NCH2O 105 blood blood blood blood blood tick tick blood blood tick blood blood tick tick tick blood tick blood tick blood tick tick blood tick blood tick blood tick blood tick blood tick blood blood blood blood blood blood NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC XLR_534R_MID36 XLR_534R_MID37 XLR_534R_MID39 XLR_534R_MID43 XLR_534R_MID45 XLR_534R_v2bBar807L IU30Aug2010-MID14 XLR_534R_v2bBar208L XLR_534R_v2bBar1164L XLR_534R_v2bBar31L XLR_534R_v2bBar1149L XLR_534R_MID48 XLR_534R_MID56 XLR_534R_MID68 IU30Aug2010-MID13 XLR_534R_v2bBar807L XLR_534R_v2bBar228L XLR_534R_v2bBar207L XLR_534R_v2bBar207L XLR_534R_v2bBar1379L XLR_534R_v2bBar119L XLR_534R_v2bBar1156L XLR_534R_v2bBar281L XLR_534R_v2bBar1156L XLR_534R_v2bBar350L XLR_534R_v2bBar364L XLR_534R_v2bBar628L XLR_534R_v2bBar99L XLR_534R_MID38 XLR_534R_v2bBar162L XLR_534R_MID47 XLR_534R_MID44 XLR_534R_MID49 Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Insufficient sequences Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Possible contamination Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control Negative Control
