Application of Rose Bengal in Integrative Taxonomic Studies of Tardigrades

Tardigrades are microscopic invertebrates inhabiting a wide range of ecosystems worldwide. Their study increasingly relies on integrative taxonomy, combining morphological, molecular, and bioinformatic approaches. However, a major challenge in tardigrade research remains the time-consuming and labor-intensive process of manually isolating specimens from environmental samples. One potential solution is the application of Rose Bengal, a widely used biological stain that may facilitate specimen detection. This study aimed to evaluate the usefulness of Rose Bengal in tardigrade research and to determine its effects on molecular performance and preservation of morphological traits required for species identification.

Ninety specimens of Paramacrobiotus fairbanksi from laboratory culture were subjected to different preservation protocols, including water and ethanol (70% and 96%), with and without Rose Bengal treatment, and stored for varying periods (from 24 h to one month). The DNA extraction was performed using a standard non-destructive protocol enabling recovery of the exoskeleton for subsequent morphological examination. Effects of preservation conditions and Rose Bengal application on molecular performance and exoskeleton preservation were evaluated to identify the most suitable approach for integrative taxonomy.

Overall, Rose Bengal effectively stained tardigrade specimens, facilitating their detection in analyzed samples. Its application did not negatively affect DNA recovery or downstream molecular procedures. The optimized preservation protocol enabled successful recovery of high-quality nuclear and mitochondrial DNA markers while maintaining exoskeleton integrity for morphological analysis. Despite relatively low DNA concentration and reduced purity, PCR amplification and sequencing efficiency remained high (100% across all preservation variants). The obtained sequences enabled accurate molecular identification of Pam. fairbanksi and revealed intraspecific genetic variation.These results demonstrate that Rose Bengal is a valuable tool for integrative tardigrade research, facilitating efficient specimen isolation while remaining fully compatible with both morphological and molecular workflows.

This work was supported by grant no. UMO-2021/43/D/NZ8/00344 from the National Science Centre (Poland).