Surface sterilization methods impact measures of internal microbial diversity in ticks

Abstract

Background: Ticks are obligate blood feeders transmitting major pathogens worldwide. Over the past few years, considerable research efforts have focused on the diversity, distribution and impact of gut and intracellular bacterial symbionts on tick development and tick-borne pathogen transmission. The study of this internal microbiome requires the use of a sterilization method to remove external (i.e. cuticular) microbes present on the tick's surface and to avoid any further contamination. Several sterilization methods exist, including ethanol- or bleach-based treatments that are both effective in killing microbes but with different potential effects on DNA denaturation.

Methods: We examined how these different sterilization methods impact the measure of internal microbial diversity hosted by the Cayenne tick Amblyomma cajennense (sensu stricto). Bacterial barcoding investigations based on 16S rRNA gene sequences were conducted on two batches of 50 individuals each: Ticks of the first batch were sterilized with bleach diluted at 1% and the second batch with 70% ethanol. Tick external microbiome was also determined from cuticle smearing and water samples used for tick washing.

Results: Bacterial barcoding investigations showed major differences between ethanol- and bleach-treated specimens. Both methods led to the detection of major intracellular bacteria associated with A. cajennense (s.s.) but ethanol-treated ticks always harbored a higher bacterial diversity than bleach-treated ticks. Further examinations of tick gut and tick external microbiome revealed that ethanol-based surface sterilization method is inefficient to eliminate the DNA of external bacteria.

Conclusions: We herein provide evidence that studies investigating the internal microbiome of ticks should consider bleach as the gold standard to efficiently remove cuticular bacterial DNA. Indeed, this method does not impact the internal bacterial diversity hosted by ticks and is thus a better method than the ethanol-based one for studying the internal microbiome.

Keywords: 16S rRNA; Amblyomma; Bacterial communities; Metabarcoding; Tick microbiome.

Fig. 1

Effect of the sterilization treatment on bacterial diversity of whole ticks. a Bar plots of the relative abundance of the 10 most abundant bacterial families in each sample. Each bar represents a sample; ethanol samples on the left and bleach samples on the right. b NMDS plot of generalized Unifrac (α = 0.5) distances between treatments; blue dots correspond to bleach samples, red dots to ethanol samples. c Heatmap of the diversity and abundance of OTUs between bleach (on the right) and ethanol (on the left) samples with the different samples on the X axis and OTUs on the Y axis. The most common OTU, Coxiella-LE of the family Coxiellaceae, is shared by all samples and is delineated by the ‘red line’ across the heatmap

Fig. 2

Effect of the sterilization treatment on the internal microbiome of ticks. a Bar plots of the relative abundance of the 10 most abundant bacterial families in each sample regrouped by sample categories; EthCarcass corresponds to ethanol-treated carcass ticks, EthGut to ethanol midguts, BleCarcass to bleached carcass ticks, and BleGut to bleached tick midguts. b NMDS plot of generalized Unifrac (α = 0.5) distances between all categories with ellipses encompassing normal confidence range for each category

Fig. 3

Comparison of microbial communities between cuticle smears, external washes, and ethanol-treated whole bodies. a Bar plots of the relative abundance of the 12 most abundant families in each sample regrouped by samples category (ethanol for whole bodies, swab for cuticle smears, and wash). b NMDS plot of generalized Unifrac (α = 0.5) distances between samples categories. c Heatmap of the diversity and abundance of OTUs between samples categories