Crayfish plague is a water mold, an oomycete pathogen native to southeastern North America, where local crayfish species tolerate it thanks to millions of years of coevolution. When North American crayfish were shipped to Europe for aquaculture and stocking, they brought the fungus with them as an invisible passenger. The pathogen reproduces asexually by releasing zoospores, microscopic swimming spores that home in on new hosts using chemical cues. For European native crayfish, which never evolved defenses against it, infection is usually catastrophic: the fungus invades the cuticle, melanization responses fail, and mass mortality follows within days to weeks.

The consequences for Europe’s freshwater ecosystems have been devastating. Native crayfish are keystone species and ecosystem engineers, large omnivores that structure food webs and shape streambeds. Crayfish plague swept across Europe in two major waves, the first beginning in northern Italy in 1859 and the second in the late 1980s, driven by introduced signal crayfish (Pacifastacus leniusculus) and spinycheek crayfish (Faxonius limosus). Populations of the white-clawed crayfish have declined by an estimated 50 to 80 percent since the 2000s. Five major genotype groups of A. astaci (A through E) have been described, with group A representing the oldest European lineage and the others associated with different North American carriers, each differing in virulence and transmission efficiency.

Croatia’s history with the pathogen stretches back to the late nineteenth century, when the first plague-related mass mortalities were recorded, with another outbreak documented in the late 1960s. Molecular confirmation came only in 2016, and that initial survey covered a limited number of localities using earlier qPCR assays with known specificity issues. To fill the gap, the new study turned to a remarkable resource: archived crayfish specimens preserved in 96 percent ethanol, collected over 27 years from both the Black Sea and Adriatic Sea drainages. The researchers analyzed 238 specimens representing all seven crayfish species present in the country, four native and three invasive.

The technical approach was meticulous. Tissue pools of eyes, abdominal cuticle, and appendage setae were dissected under bleach-decontaminated conditions to prevent cross-contamination. DNA was extracted with a modified insect kit protocol, tripling reagent volumes to fully submerge the tissue. Detection relied on a TaqMan qPCR assay targeting a 131-base-pair fragment of the pathogen’s internal transcribed spacer region, with synthetic gBlocks gene fragments serving as positive controls and contamination sentinels. Every presumptive positive was confirmed by Sanger sequencing against the GenBank database, and an exogenous internal positive control flagged any PCR inhibition from the decades-old ethanol-preserved samples.

The headline result: 91 of 238 samples, 38.2 percent, tested positive for A. astaci. The pathogen was detected in both major drainages, in all seven host species, and throughout the entire sampling period. Positives ranged from 33.8 percent in stone crayfish (Austropotamobius torrentium) to 42.3 percent in narrow-clawed crayfish (Pontastacus leptodactylus) among native species, and reached 50 percent in signal crayfish among invasives. Metadata accompanying the archived material also revealed four suspected plague outbreaks: Kotluša Stream in 2019 and the Krupa River in 2020, both involving stone crayfish; the Bregana River in 2025, involving stone crayfish again; and the Otuča River in 2019, involving noble crayfish (Astacus astacus).

Genotyping proved harder than detection, as expected. The researchers integrated three methods: a pentaplex qPCR assay distinguishing genotype groups A through E using five fluorescently labeled probes, mitochondrial haplotyping of the rnnS and rnnL ribosomal genes, and microsatellite genotyping. Only 11 of the 91 positive samples yielded interpretable consensus genotype profiles, because genotyping requires substantially higher pathogen loads than species-level detection, and archived specimens were mostly collected from apparently healthy populations where pathogen loads were low. DNA degradation from years in ethanol further reduced amplification success.

Those 11 profiles told a striking story. Genotype group A, the old European lineage, appeared in six samples across three native species and both drainages, including two outbreak localities, with the earliest record extending back to 2007. Group A is generally considered less virulent, but the study shows it can still trigger mass mortalities when hosts are stressed by pollution or elevated water temperatures. Genotype group B, typically carried by signal crayfish, was confirmed in two native species, noble crayfish and stone crayfish, including a 2025 outbreak in the Bregana River, a locality outside the signal crayfish’s known invasion range. Two previously unreported profiles also emerged: an SSR-UP-like genotype in a signal crayfish from the Drava River, the first such record in an invasive host, and a putatively novel genotype in a narrow-clawed crayfish from the Danube River that yielded discordant results across methods.

The detection of group B in native crayfish far from any known signal crayfish population is particularly alarming. Similar patterns, where NICS-associated genotypes appear in native populations years before their carriers are identified, have been reported in Switzerland, the Czech Republic, and Ireland. The researchers suggest two explanations: spillover from undetected invasive crayfish upstream, or transfer via contaminated fishing gear. Either way, the finding implies that A. astaci strains can reach highly susceptible native populations through pathways that current monitoring does not capture. The marbled crayfish (Procambarus virginalis), present in Croatia since 2013, tested positive in two samples from Šoderica Lake, but their genotypes could not be resolved, leaving open the question of whether genotype group D lurks undetected. Similarly, several positive spinycheek crayfish samples could not be genotyped, so genotype group E may also be present but invisible to current methods.

The study’s authors argue that Croatia needs a coordinated, genotype-resolved national monitoring framework, modeled on systems in Norway, Switzerland, and Ireland. Although crayfish plague has been legally notifiable in Croatia since 1997, practical reporting of wild crayfish mortality events remains limited, hampered by field observers who are not trained to recognize outbreaks and by diagnostic pathways that depend on authorized veterinarians visiting sites before carcasses degrade. The researchers call for targeted training of field personnel, integration of samples from protected-area staff and researchers into the official diagnostic pathway, continuous biosafety education for fishermen, and the adoption of environmental DNA screening as a non-invasive early-warning tool. Priority zones should include invasion fronts and contact zones between native and invasive crayfish, such as the Drava–Mura system and the Korana–Mrežnica area. As this study demonstrates, the pathogen’s true distribution may still be broader than documented, and only sustained, genotype-informed surveillance can reveal where it will strike next.

Subject of Research: Time capsules of infection: Updating the distribution of Aphanomyces astaci in Croatia using archived crayfish specimens

Article Title: Time capsules of infection: Updating the distribution of Aphanomyces astaci in Croatia using archived crayfish specimens

Article References: Škugor, T., Baudry, T., Maguire, I., Hudina, S., Jemmi, E., Delaunay, C., Pisano, S. R. R., Temunović, M., Schmidt-Posthaus, H., & Bielen, A. (2026). Time capsules of infection: Updating the distribution of Aphanomyces astaci in Croatia using archived crayfish specimens. NeoBiota, 109, 247-270. https://doi.org/10.3897/neobiota.109.200821

Image Credits: AI Generated

DOI: 10.3897/neobiota.109.200821

Keywords: Time, capsules, infection, Updating, distribution, Aphanomyces, astaci, Croatia, archived, crayfish, specimens, scientific research