Abstract
BackgroundTicks 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.MethodsWe 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.ResultsBacterial 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.ConclusionsWe 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.
Highlights
Ticks are obligate blood feeders transmitting major pathogens worldwide
Additional 16S rRNA gene sequences were generated from 27 swabs used for cuticle smearing and 25 water samples used for tick washing
After filtration of false-positives operational taxonomic unit (OTU), 4,504,538 reads distributed in 320 OTUs were obtained in the data sets of the 50 whole ticks and 6,097,046 reads distributed in 373 OTUs were obtained in the 100 tick organs samples (50 guts and 50 carcasses of ticks)
Summary
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. Ticks feed exclusively on blood at all stages in their development and exhibit a unique internal microbiome with a diversity of non-pathogenic extracellular and intracellular bacteria [21,22,23,24,25,26,27,28,29,30]. A deeper investigation of the biodiversity of the internal microbes of ticks is ongoing, as shown by the increasing number of metagenomics studies using high-throughput sequencing [23, 34,35,36]
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