CaliVir

Characterisation of the invasive calico crayfish virome

Invasive non-native species are a primary driver of global biodiversity loss, especially in vulnerable freshwater ecosystems. Among the most successful invaders are North American crayfish. While their impact is often linked to the devastating "crayfish plague," a new frontier of research is emerging: the role of viruses.

Why do viruses matter?

Viruses are the most dominant entities in our biosphere, yet they remain the least studied microbial group in crayfish. Historically, research focused only on commercial aquaculture diseases. However, recent advances in RNA sequencing have revealed a world of novel, uncharacterized viral taxa. These discoveries raise critical question: How do viruses aid the success of an invasive species?

Why the Calico Crayfish (Faxonius immunis)?

Since its first sighting in the Rhine Valley in 1993, the calico crayfish has become a formidable invader. It spreads rapidly through interconnected waterways, decimating amphibians, destroying aquatic plants, and carrying the lethal crayfish plague. Remarkably, the Calico crayfish doesn't just outcompete native species—it can even displace other established North American invaders.

How do the smallest entities in our biosphere drive some of the largest changes in our environment?

The CaliVir project aims to characterise the virome of the Calico crayfish to understand how the hidden viral pathogens contribute to its extraordinary invasive success and impact on European biodiversity.

Project aim:

The overarching aim of CaliVir is to generate preliminary data on the virome of the calico crayfish from Rhine River. To this end, we will employ high-throughput RNA sequencing to identify DNA and RNA viruses associated with the calico crayfish. The identified virus sequences will be phylogenetically analysed with state-of-the-art bioinformatic tools. Furthermore, we will evaluate suitability of non-lethal methods for virus detection from the calico crayfish and develop novel qPCR assays for rapid assessment of the viral prevalence for selected viruses among calico crayfish populations.

Sampling
Preliminary sampling of the calico crayfish from the ponds in the area of Lauterbourg, (France) in cooperation with Dr. Jean-Yves Georges (University of Strasbourg) was conducted. The location from which the invasive calico crayfish are sampled is at the heart of the endangered European pond turtle reintroduction project, and co-existence of these two species is of high conservation relevance. For the preliminary virome analysis we collected calico crayfish individuals, and isolated different organs. For the evaluation of the non-lethal virome characterisation we collected cuticular swabs. Finally, we will evaluate of the rapid detection methods of the viruses from water and soil samples.
RNA extraction and sequencing
Total RNA will be isolated from the samples. Obtained total RNA will be pooled into sequencing libraries and sequenced on Illumina platform.
Data analysis
The obtained sequencing reads will be analysed using state-of-the art bioinformatic protocols which include: quality checking and trimming, read assembly, identification virus sequence identification, annotation and phylogenetic analysis. Based on the list of the viruses detected in the calico crayfish, we will construct specific qPCR primers for the selected viral species (based on the highest abundance), which will be utilised to evaluate the presence of the viruses in individual RNA isolates from the calico crayfish tissues, cuticular swabs, soil and water samples. This will be done to conduct preliminary evaluation of the potential viral tissue specificity, and prevalence within the crayfish samples and detection success rate for cuticular swabs, water filters and soil samples.