Miseq sequencing data
There were no PCR amplification bands in the negative control, while the total of 57 samples were successfully sequenced (Table 1), resulting in 3,017,655 trimmed reads, and the number of reads per sample ranged from 30,242 to 87, 211 (Table S1, Additional files). Rarefaction curves of the Shannon index of all samples can reach a plateau, suggesting sequencing coverage was sufficient and can be used in subsequent analyses (Fig. S1, Additional files). This result was also supported by an adequate coverage (Good’s coverage from 99.7% to 100%) estimate for each sample.
OTU and alpha diversity analyses
In total, 1,283 distinct OTUs were observed in these samples (Table S2, Additional files). Fed male had only one sample, which was excluded from alpha diversity comparison. Fed female had the highest bacterial richness, followed by the fed larva and female, and the difference between fed female and fed larva was not significant (ANOVA F=2.759, df = 8, P=0.177). By comparison, the bacterial richness in other samples was significantly lower (ANOVA F=8.76, df =24, P<0.05) (Table 1). The bacterial richness in bitten blood was higher than that in blood, without significant difference (t = 1.697, df = 3, P= 0.188) (Table 1). Four organs of fed female had similar levels of bacterial richness (ANOVA F = 0.3228, df = 13, P = 0.809) (Table 1). Shannon index was used to evaluate the bacterial diversity in these samples. Fed larva and fed nymph had the highest and lowest bacterial diversity, respectively. Five samples (Egg 1d, Egg 10d, Larva, Nymph, and Male) had intermediate levels of bacterial diversity, and their diversity was significantly different from the above two samples (ANOVA F = 14.45, df = 36, P<0.0001). Female had relatively lower diversity compared to the five samples (Table 1). The bacterial diversity did not differ between blood and bitten blood (t = 3.158, df = 3, P= 0.051) (Table 1). In the fed female, the lowest bacterial diversity was observed in OV, and the difference between it and SG and MG was significant (ANOVA F = 5.302, df = 3, 8, P = 0.026) (Table 1).
Bacterial microbiota composition
The bacterial microbiota was further assigned to 28 phyla, 67 classes, 165 orders, 274 families, 545 genera, and 836 species. At the phylum level, four phyla (Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes) had high relative abundances in all samples. Bacterial microbiota communities varied along with tick development. The relative abundances of Proteobacteria, Firmicutes, and Actinobacteria displayed a decrease or an increase with tick development, while Bacteroidetes was mostly detected in fed ticks. Blood and bitten blood had similar bacterial communities at the phylum level. The relative abundance of bacteria differed between tick surface and tick habitat samples. Additionally, four organs from fed female had distinct bacterial communities. It is worth noting that some phyla with low relative abundance were generally present in the blood, fed tick, and organ samples (Fig. 2A, Table S2, Additional files).
At the genus level, Staphylococcus was present in all samples, and in tick samples, its relative abundance firstly increased and then decreased along with tick development. Fed nymph had the highest relative abundance of Staphylococcus. Also, this genus showed higher relative abundance in fed ones than unfed ones in immature ticks, while a contrast trend was observed in adult ticks. Staphylococcus also had high relative abundance in environmental samples and bitten blood. Egg and larva had a higher relative abundance of Pseudomonas than other life stages; blood meals seemed to decrease its abundance. The relative abundance of Brevibacterium increased along with tick development with the exception of fed ticks, in which abundances were very low. Brevibacterium was also detected from environmental samples. In addition, the relative abundance of Kocuria was high in environmental samples, and this genus was also present in almost all tick samples. In the fed female, Coxiella was the dominant bacterium, and its proportion reached to 77.29%. Alcaligenes and Corynebacterium were mainly present in immature ticks, including egg, larva, and nymph. Many genera, such as Faecalibacterium, Bacteroides, Kosakonia, Parabacteroides, Bifidobacterium, Agathobacter, Alistipes, Lachnospira, and Blautia, were solely present or had higher abundances in fed male. Similarly, high abundances of Enterococcus, Olsenella and Prevotella were detected in fed larva. Bacterial microbiota communities did not differ between blood and bitten blood at the genus level. Bacterial microbiota communities also varied across tick organs, and bacterial abundances differed between them. As a prominent bacterium in the fed female, Coxiella was present in four organs and showed abundant in the OV and the MT (Fig. 2B).
In total of 28 OTUs were shared by fed ticks and blood samples (Fig. 3A), suggesting that ticks can acquire bacteria from blood meals, and few shared bacteria can occupy and transmit through tick development as they were present in tick eggs (Fig. 2B, Table S2, Additional files). The most shared OTUs between tick and environmental samples are genera of Staphylococcus, Pseudomonas, Brevibacterium, Enterobacter, Acinetobacer and Stenotrophomonas (Fig. 3B), some of them are also present in tick organ, suggesting that these environmental bacteria cannot be completely washed away and can be acquired by ticks.
The variation of bacterial communities in blood, tick, environmental and organ samples were further evaluated by the PCoA and ANOSIM analyses. The unweighted UniFrac PCoA (which does not account for abundance data) explained 16.02% (PC1) and 10.1% (PC2) of the variation among blood, tick and environmental samples, and blood samples clustered together and were clearly segregated from other samples, however, environmental samples clustered with tick samples. Tick samples were separated according to their developmental stages (Fig. 4A). In addition, the ANOSIM result (R=0.6106, P=0.001) also showed significant differences in bacterial composition between samples. The weighted UniFrac PCoA and ANOSIM (R=0.5863, P=0.001) obtained a similar result considering the abundance of bacteria (Fig. 4B). For organ samples, bacterial composition showed significant differences between organs when bacterial abundance was considered in the weighted UniFrac PCoA, which explained 52.77% (PC1) and 22.01% (PC2) of the variation (Fig. 4C, R=0.655, P=0.002). However, no distinct clustering was observed for each organin unweighted UniFrac PCoA, explaining 18.61% (PC1) and 13.41% (PC2) of the variation (Fig. 4D, R=0.1637, P=0.097).