Snakes are famously dependent on chemical communication. With limited visual capabilities, most species use tongue-flicking to sample chemical cues for trailing conspecifics, locating hibernation sites, and finding mates during the breeding season. Decades of research on the red-sided garter snake (Thamnophis sirtalis parietalis) established that snake sex pheromones are typically complex mixtures of heavy, non-volatile, long-chained methyl ketones embedded in skin lipids. Yet this paradigm does not hold universally: the brown tree snake (Boiga irregularis), for example, lacks methyl ketones in female samples entirely, and several studies suggest airborne compounds may also participate in sexual signaling. Because chemical profiles alone cannot confirm pheromone function, behavioral assays are essential.

Previous chemical analyses of Vipera ammodytes skin extracts identified 2-pentacosanone (a C25 ketone) and 2-heptacosanone (a C27 ketone) as the two most frequently occurring heavy, long-chained methyl ketones in the species. Intriguingly, both males and females produce these compounds in similar concentrations, raising the question of what role they actually play. The new study, led by researchers working with a population near the village of Karlukovo in northwest Bulgaria, set out to determine whether these ketones elicit courtship-related behavior and whether responses differ between the sexes.

The team collected eight adult males and seven adult females in April 2022 and April 2023, keeping them in captivity for no more than two months before releasing them at their exact capture sites. The vipers were housed individually in terraria with controlled temperature gradients, fed every ten to fifteen days, and given a week to acclimate before testing began. All experiments ran from early May to the first ten days of June, coinciding with the species’ natural mating period. In total, the researchers conducted 270 behavioral assays: 144 with males and 126 with females.

Methodologically, the study broke with convention in several ways. Initial trials with Y-shaped mazes proved unproductive, as males displayed mainly exploratory behavior and females rarely moved at all. The vipers also became visibly stressed when moved to unfamiliar spaces, and they tended to remain still or defensive in the presence of humans. To minimize interference, the researchers abandoned the commonly used cotton swab test and direct observation. Instead, they placed paper towels soaked with test compounds directly into each viper’s home terrarium and recorded responses with a video camera positioned in front of the enclosure, with no human present in the room. Each towel was left for ten minutes, and each compound was tested three times per individual, with no more than four tests per day per animal.

The compounds tested included synthetic 2-pentacosanone, 2-heptacosanone, an equal-parts mixture of the two, the straight-chain alkane heptacosane (C27H56), the solvent n-hexane, and a blank control. Solid compounds were dissolved in 99% purity n-hexane at a concentration of 1 mg/ml, and between 0.20 and 0.25 ml of solution was dripped onto each towel. The researchers transcribed full ethograms from every video, counting tongue-flicks to calculate general tongue-flick rates (total flicks divided by experiment duration) and specific tongue-flick rates (total flicks divided by the time between first and last flick). They also recorded whether individuals showed directed interest in the towel for at least ten seconds and whether they performed chin-rubbing, a behavior typical of courting male vipers, for more than ten consecutive seconds.

Statistical analysis relied on Bayesian multivariate generalized linear mixed-effects models fitted in the R package brms, with sex and compound as predictors and individual identity as a random effect. Because many variables contained large proportions of zero values—interest in the compound was absent in 81.85% of tests, and chin-rubbing in 92.96%—the team compared standard, zero-inflated, and hurdle models using the Watanabe-Akaike information criterion and leave-one-out cross-validation. Hurdle gamma models best fit the tongue-flick rate data, while a zero-inflated Poisson model best captured chin-rubbing counts. On average, vipers took 291 seconds to make their first tongue-flick, underscoring how long recordings must run to capture meaningful behavior in these sedentary ambush predators.

The results revealed a clear sexual divide. Males showed significantly higher levels of interest in the ketones than females did, and chin-rubbing behavior was observed exclusively in males, with the most active individuals responding to the ketone treatments. In pairwise comparisons, male interest was significantly stronger toward the C25 ketone and the C27 ketone than toward the C27 alkane, and the ketone mixture elicited significantly more chin-rubbing than the alkane, the hexane solvent, or the control. When compounds were grouped, males responded significantly more to ketones than to alkanes for both interest and chin-rubbing. Females, by contrast, showed no significant differences in interest or chin-rubbing for any compound, though their tongue-flick rates were modestly elevated toward the C27 ketone and the ketone mixture relative to controls—patterns the authors interpret cautiously due to convergence issues and the overwhelming number of zero responses.

The authors conclude that 2-pentacosanone and 2-heptacosanone do indeed provoke sexual attraction in male nose-horned vipers and are likely involved in intraspecific communication, potentially contributing to the female sex pheromone. Notably, the combination of the two ketones proved more attractive than either compound alone, echoing findings from garter snake studies. However, the response was modest rather than overwhelming, leading the researchers to hypothesize that additional compounds—possibly volatile, airborne molecules of a different chemical nature—complete the pheromone blend. They also note that the absence of visual stimuli, such as an actual female, may have dampened male responses, since mating signals in snakes often integrate multiple sensory channels.

Perhaps the most provocative implication concerns why males produce the same ketones as females. In red-sided garter snakes, so-called she-males emit female-like pheromones to deceive rival males, gaining a mating advantage in the species’ chaotic mating balls. Nose-horned vipers, which engage in ritualized male-to-male combat rather than mating balls, might use a different strategy: males could deposit female-like ketones—heavy, non-volatile molecules well suited to substrate marking—to distract rivals or lure them into exhausting fights. Alternatively, the authors suggest that females lacking sufficient energy reserves for reproduction, which in European vipers reproduce only once every one to three years, may simply skip ketone production, rendering themselves unattractive to males. Both hypotheses await direct testing, as does the possibility that body condition and hormone levels modulate the behavioral responses observed here. What is already clear is that chemical communication in European vipers is more nuanced than a single compound can explain, and that the male nose-horned viper’s tongue holds answers still being uncovered.

Subject of Research: Behavioral responses of the nose-horned viper to long-chained ketones as candidate sex pheromone components

Article Title: Behavioral effects of long-chained ketones in Vipera ammodytes : an experimental approach

Article References: Andonov, K., Dyugmedzhiev, A., Lukanov, S., Vacheva, E., Popgeorgiev, G., Nedeltcheva-Antonova, D., & Naumov, B. (2025). Behavioral effects of long-chained ketones in Vipera ammodytes: an experimental approach. Herpetozoa, 38, 161-171. https://doi.org/10.3897/herpetozoa.38.e151358

Image Credits: AI Generated

DOI: 10.3897/herpetozoa.38.e151358

Keywords: Vipera ammodytes, pheromones, long-chained ketones, chemical communication, snake behavior, courtship, 2-pentacosanone, 2-heptacosanone, tongue-flicking, vipers, Bulgaria, chemical ecology

Caryospora species are single-celled parasites belonging to the phylum Apicomplexa, the same broad group that includes the malaria parasite and Toxoplasma. These organisms belong to the family Eimeriidae and are unusual among coccidia for their distinctive oocysts, the tough environmental transmission stages shed in host feces. Caryospora oocysts are monosporocystic and octozoic, meaning each contains a single sporocyst housing eight infectious sporozoites, and each sporocyst bears a Stieda body, a specialized plug used during excystation inside a new host. The genus is also remarkable for its life cycle: transmission to snakes occurs either directly through the fecal-oral route or indirectly when a snake preys upon a rodent harboring caryocysts, dormant parasite stages embedded in the prey’s tissues.

That second route involves one of the more striking examples of host manipulation in the parasite world. Caryospora species induce cyst formation in the oral musculature of rodents, impairing the animals’ feeding and agility and thereby making them easier prey for snakes. This contrasts sharply with their closest relatives, the raptor parasites now assigned to the genus Eumonospora, which form cysts in the skin and connective tissue of their rodent hosts, preserving mobility while keeping the prey visible to hunting birds of prey. The two strategies reflect deep evolutionary commitment to two entirely different predator-prey pathways, and they underscore why Caryospora species that parasitize snakes are considered to have a particularly robust taxonomic foundation.

Until recently, the genus had been poorly explored at the molecular level. Since the last comprehensive review, the number of Caryospora species recorded in snakes has grown from 18 to 56, yet only seven nominal species have been documented in vipers, the family that includes rattlesnakes, lanceheads, and true vipers. The new study focused on two of Brazil’s most important venomous snakes: the Jararaca, Bothrops jararaca, and the Cascabel Rattlesnake, Crotalus durissus. Researchers collected fecal samples from five individuals of each species that had been rescued from urban environments in the municipality of Miguel Pereira and temporarily housed at a municipal zoonosis control facility.

The results were striking. Oocysts were detected in 60 percent of the Jararacas and 80 percent of the rattlesnakes, an overall prevalence of 70 percent. Under the microscope, the oocysts were subspheroidal, measuring roughly 14 by 14 micrometres, with a bi-layered wall, no micropyle, and a polar granule present in only about half of the specimens. Each broadly ellipsoidal sporocyst carried a flattened, knob-like Stieda body and a rounded to trapezoidal sub-Stieda body. These features placed the parasites squarely within the morphotype of the genus’s type species, Caryospora simplex, first described from the Asp Viper in Europe more than a century ago. But morphology alone could not settle the identification, because the quantitative differences among related species proved unreliable.

To break the deadlock, the team turned to DNA. Individual oocysts, each photographed and documented before isolation, were subjected to repeated freeze-thaw cycles to rupture their walls, followed by proteinase K digestion and column-based DNA extraction. The researchers then amplified four non-overlapping genetic loci: three mitochondrial regions, designated MAVCOXI, MARI, and MACOIII, targeting cytochrome c oxidase subunits 1 and 3 and rDNA fragments, plus a nuclear locus spanning the 18S ribosomal RNA gene. Sequences from both host species were essentially identical to one another, differing at a single locus by just 0.4 percent, or three nucleotides out of 748. The concatenated four-locus dataset spanned 2,438 base pairs and was analyzed under a General Time Reversible model with gamma-distributed rate variation, using both Bayesian inference and maximum likelihood phylogenetics.

The molecular comparisons delivered a clear verdict. The Brazilian parasites matched most closely with Caryospora bigenetica, the only other Caryospora species from a viper to have been sequenced previously, isolated from the Massasauga rattlesnake in North America. Identity scores reached 100 percent at the nuclear 18S locus and between 97.7 and 99.3 percent at the mitochondrial loci, yielding 99 percent identity across the full concatenated dataset. In the phylogenetic trees, the two species formed a highly supported monophyletic clade, with posterior probability of 0.99 and bootstrap support of 100 percent. On this combined morphological and molecular evidence, the team confirmed the parasites as Caryospora jararacae, originally described from Jararacas in 1939, and reidentified earlier Brazilian records attributed to C. bigenetica as belonging to jararacae instead. This makes C. jararacae the sole Caryospora species known from Neotropical vipers and only the second genotypically characterized species from the entire viper family worldwide.

The broader taxonomic review that accompanied the sequencing led to the formal definition of the Caryospora simplex species complex, encompassing five nominal species that are morphologically indistinguishable: C. simplex from Eurasia, C. bigenetica from North America, C. jararacae from South America, C. maculatus from Africa, and C. bothriechis from Central America. Each occupies a different continent, effectively representing five geographic variants, or geotypes, of what appears to be a single young lineage. The researchers argue that these geotypes likely share a relatively recent common ancestor, and that their near-identical morphology and genetics reflect insufficient evolutionary time for structural divergence rather than ongoing genetic exchange across oceans. As a secondary hypothesis, they note that broad-ranging or migratory hosts, and even the international wildlife trade, could theoretically bridge geographic barriers, a possibility they flag for future population-level study.

The team deliberately retained all five names rather than collapsing them into a single species, because an integrative framework combining biogeography, host records, and molecular data allows each geotype to be delimited meaningfully. They also rejected a subspecies-based scheme, noting that such nomenclature is exceedingly rare within the Eimeriidae and would destabilize established names. Two additional viper parasites, C. matatu and C. saudiarabiensis, remain morphologically distinguishable from the complex and await molecular characterization. The researchers emphasize that sequencing C. simplex itself, ideally from its type host, will be essential to consolidate the emerging picture. For now, the study stands as a compelling demonstration that even among microscopic parasites, continents tell stories, and that some of evolution’s newest chapters are written in the feces of venomous snakes.

Subject of Research: Integrative taxonomy and first molecular characterization of the coccidian parasite Caryospora jararacae and the Caryospora simplex species complex in viperid snakes

Article Title: Integrative Taxonomy of the Caryospora simplex Species Complex (Apicomplexa: Eimeriidae) From Vipers (Squamata: Viperidae), With Redescription and First Molecular Characterization of Caryospora jararacae

Article References: da Silva, A. M. M., Ortúzar-Ferreira, C. N., Oliveira, M. D. S., Dorna-Santos, L., Ferreira, V. D. S., de Lima, V. M., & Berto, B. P. (2026). Integrative Taxonomy of the Caryospora simplex Species Complex (Apicomplexa: Eimeriidae) From Vipers (Squamata: Viperidae), With Redescription and First Molecular Characterization of Caryospora jararacae. Acta Parasitologica, 71(5), Article 229. https://doi.org/10.1007/s11686-026-01411-0

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01411-0

Keywords: Caryospora, Apicomplexa, Eimeriidae, vipers, Bothrops jararaca, Crotalus durissus, integrative taxonomy, molecular phylogeny, coccidia, species complex, host specificity, Neotropical region