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To fully comprehend host-microorganism interactions, it is crucial to understand the composition and diversity of the microbiome, as well as the factors that shape these characteristics. We investigated microbiome variation using the freshwater planarian Schmidtea mediterranea, an invertebrate model in regeneration biology and (eco-)toxicology, by exposing the organisms to various controlled conditions. The microbiome composition exhibited high variability, with most of the bacteria belonging to the Betaproteobacteria. Among the diverse microbial communities, a few genera, such as Curvibacter, were consistently present, but exhibited significant alterations in response to changing conditions. The relative abundance of Curvibacter fluctuated during the regeneration process, initially increasing before returning to a composition similar to the beginning situation. After applying external stress, the relative abundance of Curvibacter and other genera decreased. Variation over time, between different origin laboratories and between individuals, showed that additional, yet to-be-identified, factors of variation are present. Taking all results together, our study provides a solid basis for future research focusing on bacterial functionality in planarians and other invertebrates., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Imaging of living animals allows the study of metabolic processes in relation to cellular structures or larger functional entities. To enable in vivo imaging during long-term time-lapses in planarians, we combined and optimized existing protocols, resulting in an easily reproducible and inexpensive procedure. Immobilization with low-melting-point agarose eliminates the use of anesthetics, avoids interfering with the animal during imaging-functionally or physically-and allows recovering the organisms after the imaging procedure. As an example, we used the immobilization workflow to image the highly dynamic and fast-changing reactive oxygen species (ROS) in living animals. These reactive signaling molecules can only be studied in vivo and mapping their location and dynamics during different physiological conditions is crucial to understand their role in developmental processes and regeneration. In the current protocol, we describe both the immobilization and ROS detection procedure. We used the intensity of the signals together with pharmacological inhibitors to validate the signal specificity and to distinguish it from the autofluorescent nature of the planarian., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A strict coordination between pro- and antioxidative molecules is needed for normal animal physiology, although their exact function and dynamics during regeneration and development remains largely unknown. Via in vivo imaging, we were able to locate and discriminate between reactive oxygen species (ROS) in real-time during different physiological stages of the highly regenerative planarian Schmidtea mediterranea . All ROS signals were strong enough to overcome the detected autofluorescence. Combined with an in situ characterisation and quantification of the transcription of several antioxidant genes, our data showed that the planarian gut and epidermis have a well-equipped redox system. Pharmacological inhibition or RNA interference of either side of the redox balance resulted in alterations in the regeneration process, characterised by decreased blastema sizes and delayed neurodevelopment, thereby affecting tails more than heads. Focusing on glutathione, a central component in the redox balance, we found that it is highly present in planarians and that a significant reduction in glutathione content led to regenerative failure with tissue lesions, characterised by underlying stem cell alterations. This exploratory study indicates that ROS and antioxidants are tightly intertwined and should be studied as a whole to fully comprehend the function of the redox balance in animal physiology.

Silver nanoparticles (AgNPs) are widely incorporated in household, consumer and medical products. Their unintentional release via wastewaters raises concerns on their environmental impact, particularly for aquatic organisms and their associated bacterial communities. It is known that the microbiome plays an important role in its host's health and physiology, e.g. by producing essential nutrients and providing protection against pathogens. A thorough understanding of the effects of AgNPs on bacterial communities and on their interactions with the host is crucial to fully assess AgNP toxicity on aquatic organisms. Our results indicate that the microbiome of the invertebrate Schmidtea mediterranea, a freshwater planarian, is affected by AgNP exposure at the tested 10 μg/ml concentration. Using targeted amplification of the bacterial 16S rRNA gene V3-V4 region, two independent experiments on the microbiomes of adult worms revealed a consistent decrease in Betaproteobacteriales after AgNP exposure, mainly attributed to a decrease in Curvibacter and Undibacterium. Although developing tissues and organisms are known to be more sensitive to toxic compounds, three independent experiments in regenerating worms showed a less pronounced effect of AgNP exposure on the microbiome, possibly because underlying bacterial community changes during development mask the AgNP induced effect. The presence of a polyvinyl-pyrrolidone (PVP) coating did not significantly alter the outcome of the experiments compared to those with uncoated particles. The observed variation between the different experiments underlines the highly variable nature of microbiomes and emphasises the need to repeat microbiome experiments, within and between physiological states of the animal., (Copyright © 2020 Elsevier B.V. All rights reserved.)