The research, published in the journal Ecology and Evolution, examined 116 free-ranging adult tortoises captured across 22 localities spanning roughly 2,600 square kilometers of coastal mountains between the provinces of Almería and Murcia. This region, a recognized biodiversity hotspot that has endured centuries of human intervention, presents a mosaic of land uses: Mediterranean shrubland still covers about 53 percent of the sampled area, traditional dryland crops account for 24 percent, intensified agriculture 10 percent, pine plantations 7 percent, and unproductive land such as urban zones and infrastructure the remaining 6 percent. The team searched each one-square-kilometer site for at least seven and a half hours with three searchers, capturing, marking, and measuring every tortoise they encountered.

Each animal was sexed, aged from the growth rings of its carapace, and measured for weight, height, plastron width, and carapace length. A growth rate score was then calculated using the von Bertalanffy model, a mathematical framework that assumes growth declines with age and reconstructs an individual’s growth trajectory from birth to the moment of capture. Freshly collected fecal samples were kept cool for 48 hours before adult nematodes were extracted, preserved in ethanol, cleared in Amann’s lactophenol, and identified to species level using established taxonomic keys. The result was an unusually complete picture of the nematode communities inhabiting wild tortoises, a topic that had previously been studied almost exclusively in captive animals, where oxyurid infections can become pathogenic and even life threatening.

The scale of infestation was striking: 68 percent of all sampled tortoises carried nematodes, with 43 of 66 females and 34 of 50 males infected. Individual worm counts ranged from zero to a remarkable 4,901 nematodes in a single female and up to 3,368 in a male. Yet despite these enormous loads, the mean infestation intensity did not differ significantly between the sexes, averaging about 183 worms per infected female and 130 per infected male. In total, the researchers identified 15 nematode species, all belonging to the order Oxyurida and the family Pharyngodonidae, the pinworms characteristic of herbivorous reptiles.

The critical discovery emerged when the researchers linked infestation to body traits and growth. For males, higher nematode loads were significantly associated with lower body weight and shorter carapace length, and growth rates told an even more nuanced story: at low infestation levels, males actually grew faster than females, but as worm burdens climbed, male growth slowed dramatically while female growth remained unaffected. Statistical models confirmed that the effect of infestation on growth depended on sex, and a principal components analysis, which explained 78.3 percent of the variation in the data, cleanly separated males from females, with growth rate and nematode infestation emerging as the variables contributing most to the divergence between the sexes.

Why would males suffer while females do not? The authors point to a familiar pattern in vertebrate biology: across birds and mammals, mature males typically carry heavier parasite loads and suffer greater consequences, a phenomenon attributed in part to the immunosuppressive effects of testosterone and the physiological stress of mating displays. In tortoises, the behavioral differences between the sexes may be equally important. Males roam over larger distances in search of mates, potentially increasing their exposure to infective stages of the worms, which are transmitted through orofecal routes, geophagy, and possibly sexual contact. Females, which are considerably larger and heavier than males, move within patches offering high-quality food and suitable nesting sites, and their greater food intake may simply accommodate the worms without measurable cost. The researchers also caution that causality could run in reverse: nematode communities might depend on the host’s developmental stage rather than shaping it, and they call for future work to disentangle these mechanisms.

The landscape itself added another layer to the story. Growth rates were positively associated with the proportion of unproductive, vegetation-free land in a tortoise’s home range, regardless of sex, while nematode infestation declined as unproductive land increased. This counterintuitive finding suggests that open, degraded terrain, harsh as it appears, may actually benefit tortoises by allowing more effective thermoregulation. Reptiles depend on external heat sources to optimize digestion, and with no shrub cover to shade the ground, tortoises in barren areas may digest more efficiently and allocate more resources to growth rather than immune defense. Moreover, bare ground lacking vegetation, litter, and exposed soil offers fewer refuges for the free-living stages of nematodes, which spend part of their life cycles in the environment. Previous work by the same team had shown that the tortoise-nematode relationship flips from mutualistic in preserved habitats to negative in disturbed ones, but the new study is the first to show that these effects are filtered through sex.

The nematode communities themselves differed subtly between the sexes. Using coverage-based rarefaction methods that standardize diversity estimates regardless of sample size, the researchers found that species richness, the effective number of equally common species, and the effective number of dominant species were all significantly higher in females than in males, and female worm communities were slightly more even in their relative abundances. Compositional dissimilarity between the sexes, however, was very low at just 6 percent, with no species replacement detected at all; the same suite of worms infects both sexes, and only a single female harbored a unique species, Thaparia thapari, previously recorded only in captive tortoises. This suggests that female hindguts can sustain a richer, more balanced community of endosymbionts, possibly because the larger female gut provides more resources, or because competition among worm species plays out differently under the two sexes’ distinct physiological conditions.

Perhaps the most innovative part of the analysis was the construction of individual-based ecological networks, treating each tortoise as a node connected to the nematode species it carries. Both male and female networks were significantly nested, meaning that worms infecting few tortoises tended to be subsets of the worms found in heavily infected hosts, a structure that can facilitate coinfections spreading through the population. Female networks showed greater nestedness and higher interaction diversity than male networks, hinting that they may be more stable. Two species, Tachygonetria dentata and Tachygonetria longicollis, emerged as core species in both sexes, and the authors speculate that these abundant worms may even play a mutualistic role, contributing nitrogenous excretions that enhance their host’s digestion of tough plant matter.

For a species listed as threatened across its entire Mediterranean range, these findings carry real conservation weight. Negative effects of parasites on individual fitness are most concerning for vulnerable chelonians, and habitat degradation could exacerbate parasite impacts on wildlife communities. The study’s central message is that sex cannot be ignored: males and females of the same species, living in the same landscapes and carrying similar worm burdens, can experience fundamentally different biological outcomes. Conservation management of spur-thighed tortoises, and likely of other sexually dimorphic reptiles, will need to account for sex-specific susceptibility to parasites and for the quality of the habitats that sustain not just the animals themselves, but the intricate communities of symbionts they carry.

Subject of Research: Sex-biased interactions between spur-thighed tortoises and their hindgut nematode communities in Mediterranean landscapes

Article Title: Sex‐Biased Interactions Between Spur‐Thighed Tortoises (Testudo graeca) and Hindgut Nematodes

Article References: Benítez‐Malvido, J., Graciá, E., Giménez, A., Ávila‐Eulogio, I., Méndez, D. M., Rodríguez‐Caro, R. C., Ybáñez, R. R.-D., & Traveset, A. (2026). Sex‐Biased Interactions Between Spur‐Thighed Tortoises ( Testudo graeca ) and Hindgut Nematodes. Ecology and Evolution, 16(10), Article e74377. https://doi.org/10.1002/ece3.74377

Image Credits: AI Generated

DOI: 10.1002/ece3.74377

Keywords: spur-thighed tortoise, Testudo graeca, nematodes, parasitology, sex-biased parasitism, host-parasite interactions, ecological networks, habitat loss, Mediterranean shrubland, growth rates, conservation, oxyurid pinworms

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