The research, led by Mariachiara Vardeu and Elena Ghiorzi of the University of Padua together with colleagues at the University of Pavia, was conceived around a central weakness in the existing literature: most tick microbiome studies choose a decontamination method by convention rather than by evidence. Ethanol washes, bleach treatments, and commercial DNA-degrading reagents all appear in published workflows, but until now there has been no direct, controlled comparison of how effectively each removes surface-associated bacteria while preserving the tick’s internal microbial community. Without such a benchmark, differences between laboratories could reflect cleaning chemistry as much as genuine biology, undermining efforts to compare microbiome data across studies, regions, or tick populations.

To build that benchmark, the team designed an elegant experimental trick. Rather than relying on the unknown and variable bacteria naturally present on wild-caught ticks, they deliberately contaminated adult Ixodes ricinus ticks and nymphs with a defined bacterial mock community of known composition. This meant that the researchers knew exactly which bacterial sequences represented surface contamination and could track, with precision, how much of that signal survived each cleaning treatment. Any mock-community reads remaining after decontamination were, by definition, residual surface contamination; any reduction in those reads measured the effectiveness of the protocol. It is a simple idea in principle, but one that transforms decontamination from a matter of faith into a measurable quantity.

The contaminated ticks were then subjected to five decontamination regimens: a wash in 70% ethanol, the traditional mainstay of tick microbiome studies; a 1% bleach treatment; DNA/RNA Zap, a commercial reagent that degrades nucleic acids on external surfaces; and two combination protocols in which a lysozyme pre-treatment, designed to weaken bacterial cell walls, was followed by either bleach or DNA/RNA Zap. Mock-inoculated ticks that received no decontamination served as controls. After cleaning, the researchers characterized the microbial communities using Oxford Nanopore full-length 16S rRNA gene sequencing, a platform that reads the entire bacterial barcoding gene rather than short fragments, providing higher taxonomic resolution for identifying which bacteria survived which treatment.

The results were striking in their clarity. Ethanol, the most widely used decontamination agent in the field, consistently showed the weakest effect, leaving substantial mock-community signal intact and suggesting that many published tick microbiome profiles built on ethanol-washed specimens may carry a meaningful contribution from surface bacteria. By contrast, both bleach and DNA/RNA Zap, whether applied alone or preceded by lysozyme, produced near-complete depletion of the mock-community reads. In other words, the two harsher chemical approaches came closest to erasing the experimental contamination signal, while the gentler ethanol wash barely dented it. Perhaps unexpectedly, the enzymatic lysozyme pre-treatment conferred no measurable advantage over bleach or DNA/RNA Zap used on their own, a finding that simplifies protocol design and removes an unnecessary step from future workflows.

Effectiveness at removing contaminants is only half the story, however. An ideal decontamination protocol must also preserve the tick’s genuine internal microbiota, including endosymbionts that play roles in tick nutrition and reproduction, and any pathogens the tick may harbor. To probe this dimension, the team compared sequencing results from tick legs, which are dominated by surface-associated microbes, with leg-free bodies, which contain the internal organs and their resident communities. This anatomical dissection revealed that Brevibacterium, a bacterial genus frequently reported in tick studies, was predominantly surface-associated in this system, raising the possibility that some taxa previously interpreted as tick symbionts may in fact be exoskeletal passengers. Meanwhile, the endosymbiont Candidatus Midichloria, a bacterium closely associated with Ixodes ticks, remained readily detectable in leg-free bodies even after decontamination, indicating that the effective treatments stripped the surface without erasing the tick’s true internal signature.

The preservation of pathogen signals was equally reassuring. In nymphs that had been experimentally infected with Borrelia, the causative agent of Lyme disease, Borrelia-associated sequences remained readily detectable following surface treatment. This matters enormously for disease surveillance, because ticks are routinely screened for Borrelia and other pathogens using molecular methods, and a decontamination protocol aggressive enough to destroy pathogen DNA would compromise those assays. The study’s findings suggest that bleach- and DNA/RNA Zap-based protocols occupy a valuable middle ground: strong enough to eliminate exoskeletal contamination, yet gentle enough to leave internal microbial and pathogen signals intact and quantifiable.

Beyond the head-to-head protocol comparison, the study also explored how decontamination choice shapes community-level diversity analyses, the statistical frameworks ecologists use to compare microbiomes across samples. These analyses suggested treatment-associated differences in the bacterial profiles recovered from the ticks, hinting that laboratories using different cleaning methods may not be sampling quite the same biological reality. The authors are careful to frame these diversity patterns as exploratory rather than definitive, but the implication is clear: decontamination is not a neutral technical detail but an active variable that can shift the apparent composition of a tick’s microbiome, potentially confounding meta-analyses that pool data from studies using different protocols.

The authors are equally candid about the limits of their benchmark. The mock-community approach, while powerful, was tested under controlled laboratory conditions with a defined and relatively simple surface community. Real ticks collected in the field carry more diverse and unevenly distributed microbial loads, and different tick species present different exoskeleton chemistries and sizes that could influence how reagents penetrate and perform. Validation using field-collected ticks, additional tick species, more complex surface-associated microbial communities, and independent laboratory settings will be required before the community can propose broader methodological standardization. The researchers also emphasize that the optimal decontamination strategy may depend on the biological compartment and microbial fraction of interest, meaning that a one-size-fits-all protocol may never exist.

Nevertheless, the study delivers an immediately actionable message for anyone working on tick microbiomes or tick-borne pathogen surveillance. Under the conditions tested, bleach and DNA/RNA Zap produced the strongest reduction in experimentally introduced surface bacteria while preserving dominant tick-associated taxa and pathogen sequences, whereas ethanol, the field’s default choice, performed worst. Lysozyme pre-treatment added nothing measurable. For a research community racing to understand how tick microbiomes influence vector competence and disease transmission across a changing European landscape, this quantitative framework arrives at an opportune moment. By making surface decontamination a benchmarked, comparable step rather than an unexamined habit, the Padua-led team has taken a meaningful step toward microbiome data that can be trusted, compared, and built upon, and toward a clearer picture of what really lives inside one of Europe’s most medically important arthropods.

Subject of Research: Benchmarking surface decontamination protocols for tick microbiome profiling using 16S rRNA sequencing

Article Title: Benchmarking surface decontamination methods for reliable Ixodes ricinus microbiome profiling

Article References: Benchmarking surface decontamination methods for reliable Ixodes ricinus microbiome profiling. (n.d.). https://doi.org/10.1186/s13071-026-07726-1

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07726-1

Keywords: Ixodes ricinus, tick microbiome, surface decontamination, 16S rRNA sequencing, Oxford Nanopore, mock community, Borrelia, Midichloria, Brevibacterium, Lyme disease, vector competence, Parasites & Vectors

The study focused on Niğde province, a region of central Türkiye where tortoises roam freely and their parasites follow. The research team, led by Bilge Karatepe and Mustafa Karatepe of the Department of Biotechnology, together with Fatma Mutlu and Zeynep Ergüleş, examined 88 spur-thighed tortoises, Testudo graeca, a species whose conservation status makes every detail of its parasite burden relevant. From these animals they collected 184 adult ticks, 167 males and just 17 females, preserving each specimen in sealed bottles of 70 percent alcohol before carrying them to the laboratory for identification under a stereo microscope.

Every single tick turned out to be Hyalomma aegyptium, confirming the reputation of this species as the quintessential tortoise tick. The infestation pattern was itself informative. Ticks were found on 62 of the 88 tortoises examined, an infestation rate of 70.4 percent. When the researchers broke the data down by month, they discovered that infestation rates varied significantly across the season, a statistically robust pattern with a p value below 0.05. The sex distribution of ticks on infested hosts, however, showed no significant variation, suggesting that male and female ticks exploit their reptile hosts in broadly similar ways even though males vastly outnumbered females in the sample.

With the ticks identified and counted, the team turned to the molecular heart of the study. Rather than testing each tick individually, they organized the 184 specimens into 68 pools, with separate pools for male and female ticks collected from each tortoise. This pooling strategy is a standard and efficient approach in tick microbiology, allowing researchers to screen large numbers of arthropods for rare infections without running hundreds of separate reactions. DNA was extracted from each pool and subjected to polymerase chain reaction, or PCR, using primers specifically designed to amplify marker genes from two bacterial genera of great interest to tick biologists.

The first target was Wolbachia, arguably the most widespread endosymbiotic bacterium on Earth. Wolbachia infects an enormous range of arthropods, manipulating the reproduction of its hosts in ways that can skew sex ratios, kill males, or enable reproductive parasitism. Because of these dramatic effects, Wolbachia has become a darling of both evolutionary biology and pest control, engineered into mosquito populations to block the transmission of dengue and other viruses. The researchers targeted the wsp gene, a surface protein gene of about 610 base pairs that serves as a reliable molecular fingerprint for the bacterium. The outcome was unambiguous: not one of the 68 tick pools produced a Wolbachia amplification product. Only the positive control, supplied by Serap Aksoy of the Yale School of Public Health, yielded the expected 610 base pair band, confirming that the assay worked perfectly and that the negative results were real biology rather than technical failure.

The second target told a different story. The team used primers directed at the 16S ribosomal RNA gene of Spiroplasma, a roughly 1000 base pair region that serves as the gold standard barcode for identifying bacteria. Spiroplasma is a fascinating genus of spiral-shaped, wall-less bacteria in the class Mollicutes, relatives of Mycoplasma. These microbes are best known from insects and plants, where they can act as pathogens, mutualists, or reproductive manipulators. In some insect species, Spiroplasma strains are male killers, selectively destroying male embryos to favor their own transmission through infected females, a phenomenon documented in organisms such as the small brown planthopper. In ticks, Spiroplasma has been reported sporadically, and its ecological role remains poorly understood.

When the researchers ran their Spiroplasma assays, four of the 68 pools, or 5.8 percent, came back positive for Spiroplasma DNA. Every one of the positive pools originated from male ticks, an intriguing observation given that the sample was overwhelmingly male to begin with. The amplified 16S rDNA fragments were analyzed with molecular evolutionary software, allowing the team to characterize the endosymbionts genetically and confirm their identity as Spiroplasma species. This detection represents the first molecular evidence of Spiroplasma in Hyalomma aegyptium ticks in Central Anatolia, a region where tick microbiology has been studied far less intensively than in coastal or western parts of the country.

The absence of Wolbachia is arguably as interesting as the presence of Spiroplasma. The authors note that their null result is consistent with several recent studies reporting low or absent Wolbachia prevalence in ixodid, or hard, ticks. This pattern contrasts sharply with the situation in soft ticks and in many insects, where Wolbachia is common. The reasons remain debated. Some researchers suggest that the physiology of hard ticks, including their long feeding intervals and unique reproductive biology, may simply be inhospitable to Wolbachia colonization. Others point to the possibility that competitive exclusion by other endosymbionts, such as Coxiella-like or Francisella-like bacteria that frequently dominate tick microbiomes, keeps Wolbachia out. Whatever the mechanism, the growing consensus is that hard ticks are not the Wolbachia reservoirs that earlier assumptions based on insect data might have predicted.

The tortoise tick itself deserves attention as a microbial vehicle. Hyalomma aegyptium has been implicated as a long-term carrier of Coxiella burnetii, the agent of Q fever, and previous work in Türkiye and neighboring countries has detected Borrelia turcica, Rickettsia species, Ehrlichia, and the endosymbiont Candidatus Midichloria mitochondrii in these ticks. Because tortoise ticks feed intensively on reptiles and occasionally attach to other animals, understanding their endosymbiont community helps distinguish true pathogens from harmless or even beneficial residents. Endosymbionts can also shape vector competence, the ability of a tick to acquire, maintain, and transmit disease agents, so mapping which microbes are present is a prerequisite for assessing public health risk. The Niğde survey contributes to a broader effort, spanning studies from Algeria and Qatar to Israel and the Balkans, to catalog the microorganisms associated with this single tick species across its vast range.

For the tortoises themselves, the findings carry conservation implications. Testudo graeca is under pressure across its range from habitat loss and collection, and heavy tick burdens can impose physiological costs on already vulnerable populations. Epidemiological studies of tick infestation in spur-thighed tortoises have begun to unravel the demographic consequences of parasitism, and knowing which microbes circulate in the ticks adds a layer of understanding about disease dynamics in these reptile communities. The Niğde study, supported by the Scientific Research Projects Coordination Unit of Niğde Ömer Halisdemir University, also demonstrates the value of regional molecular surveillance. A single season of fieldwork in one Anatolian province produced a novel regional record for Spiroplasma and a robust negative result for Wolbachia, both of which refine the emerging global picture of tick microbiomes. As climate trends continue to alter the distribution and activity of ticks worldwide, baseline data like these will become ever more valuable for predicting where tick-borne agents may emerge next, and for separating the genuine threats from the hidden symbionts that simply ride along.

Subject of Research: Molecular detection and characterization of Spiroplasma and Wolbachia endosymbionts in Hyalomma aegyptium ticks infesting spur-thighed tortoises in Central Anatolia

Article Title: Molecular Characterization of Spiroplasma Endosymbionts in Ticks on Tortoises in Niğde Province, Türkiye

Article References: Karatepe, B., Karatepe, M., Mutlu, F., & Ergüleş, Z. (2026). Molecular Characterization of Spiroplasma Endosymbionts in Ticks on Tortoises in Niğde Province, Türkiye. Acta Parasitologica, 71(5), Article 224. https://doi.org/10.1007/s11686-026-01410-1

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01410-1

Keywords: Hyalomma aegyptium, Spiroplasma, Wolbachia, tick endosymbionts, Testudo graeca, tortoise ticks, Niğde, Türkiye, PCR, 16S rDNA, Central Anatolia, tick microbiome