Hyponeustonic species, living at the interface between the atmosphere and the oceans, comprise one of the most understudied and vast critical marine biotope, constantly exposed to high concentrations of anthropogenic contaminants and microplastic (MPs). Copepods of the Pontellidae family represent the primary components of this biotic assemblage worldwide, and their interaction with MPs is still unknown. We studied this interaction for the first time in Pontella mediterranea, assessing the ingestion of anthropogenic particles (APs) in 2793 individuals collected by manta trawl in the Northern Alboran Sea. We observed P. mediterranea abundances ranging from 41.67 to 1174.83 ind/m3, with a mean ingestion of 0.11 APs/ind, predominantly composed of MPs. These results confirm the low ingestion values observed for other copepod taxa. However, given its abundance, this species could retain an average of 45.15 and a maximum of 220 APs per m3 of seawater (APs/m3), mostly composed of cellulose acetate and cotton fibers. The abundances of APs were evaluated in surface (0-12 cm) and sub-surface waters (5 m depth) by combining manta trawl and Continuous Underway Fish Egg Sampler sampling. The AP abundances found (surface waters: 0.67 ± 1.04 APs/m3; subsurface waters: 3.85 ± 2.67 APs/m3) were consistent with those previously observed in the Mediterranean basin, confirming that the ingestion values observed in P. mediterranea are not due to a local accumulation of environmental MPs. Results highlight how this neustonic copepod could represent one of the largest reservoirs of APs within the upper layers of the oceans, representing an entry point for these particles within food webs. Considering the worldwide distribution and abundance of this family of copepods, the results are of environmental concern.

Photomotor response assay (PMR) is very useful in an ecotoxicological context because it allows evaluation of behavioral response to potential toxic compounds. However, a lack of procedure standardization makes results comparison difficult between labs and organisms. Here, we aimed to propose five different tools to standardize the PMR procedure so that it may be applied to all model species, regarding: (1) the minimum total sample size, (2) the acclimation period, (3) the number and duration of light and dark phases alternation, (4) the measured behavior, and (5) the statistical analysis. As an example of procedure application, we analyzed the effect of an exposure to the antibiotic gentamicin on the locomotion behavior during PMR in an invertebrate species: the asexual freshwater planaria Schmidtea mediterranea. We encourage future studies using PMR to follow these five tools to improve data analysis and results comparability.

The flatworm planarian, Schmidtea mediterranea (Smed) is a master at regenerating and rebuilding whole animals from fragments. A full understanding of Smed\'s regenerative capabilities requires a high-resolution characterization of organs, tissues, and the adult stem cells necessary for regeneration in their native environment. Here, we describe a serial block face scanning electron microscopy (SBF-SEM) protocol, optimized for Smed specifically, for visualizing the ultrastructure of membranes and condensed chromosomes in this model organism.

Planarians have become a powerful model system for stem cell research and regeneration. While the tool kit for mechanistic investigations has been steadily expanding over the last decade, robust genetic tools for transgene expression are still lacking. We describe here methods for in vivo and in vitro mRNA transfection of the planarian species Schmidtea mediterranea. These methods utilize the commercially available TransIT-mRNA transfection reagent to efficiently deliver mRNA encoding a synthetic nanoluciferase reporter. Using a luminescent reporter overcomes the bright autofluorescent background of planarian tissues and allows quantitative measurements of protein expression levels. Collectively, our methods provide the means for heterologous reporter expression in planarian cells and the basis for future development of transgenic techniques.

The use of flow cytometry and fluorescence-activated cell sorting to roughly separate subpopulations of cells in Schmidtea mediterranea is long established. In this chapter, we describe a method for the immunostaining-either single or double-of live planarian cells, using mouse monoclonal antibodies reactive against S. mediterranea plasma membrane antigens. This protocol allows to sort live cells according to their membrane signature, offering the possibility to further characterize the cell populations in S. mediterranea in a variety of downstream applications, like transcriptomics and cell transplantation, also at the single-cell level.

The need of highly viable cells dissociated from Schmidtea mediterranea is constantly growing. In this chapter, we describe a cell dissociation method based on papain (papaya peptidase I). This enzyme, often used to dissociate cells with complex morphology, is a cysteine protease with a broad specificity and increases both the yield and the viability of the dissociated cell suspension. The papain dissociation is preceded by a pretreatment for mucus removal, as this was shown to greatly improve the yield of cell dissociation, regardless of the method used. Papain-dissociated cells are suitable for a variety of downstream applications, like live immunostaining, flow cytometry, cell sorting, transcriptomics, and cell transplantation, also at the single-cell level.

Whole-mount in situ hybridization (WISH), colorimetric or fluorescent (FISH), allows for the visualization of endogenous RNA. For planarians, robust WISH protocols exist for small-sized animals (>5 mm) of the model species Schmidtea mediterranea and Dugesia japonica. However, the sexual strain of Schmidtea mediterranea studied for germline development and function reaches much larger body sizes in excess of 2 cm. The existing whole-mount WISH protocols are not optimal for such large specimens, owing to insufficient tissue permeabilization. Here, we describe a robust WISH protocol for 12-16 mm long sexually mature Schmidtea mediterranea individuals that could serve as a starting point for adapting WISH to other large planarian species.

RNA sequencing (RNA-seq) is a high-throughput technology that provides in-depth information on transcriptome. The advancement and dropping costs of RNA sequencing, accompanied by more available reference genomes for different species, make transcriptome analysis in non-model organisms possible. Current obstacles in analyzing RNA-seq data include a lack of functional annotation, which may complicate the process of linking genes to corresponding functions. Here, we provide a one-stop RNA-seq analysis pipeline, PipeOne-NM, for transcriptome functional annotation, non-coding RNA identification, and transcripts alternative splicing analysis of non-model organisms, intended for use with Illumina platform-based RNA-seq data. We performed PipeOne-NM on 237 Schmidtea mediterranea RNA-seq runs and assembled a transcriptome with 84,827 sequences from 49,320 genes, identifying 64,582 mRNA from 35,485 genes, 20,217 lncRNA from 17,084 genes, and 3481 circRNAs from 1103 genes. In addition, we performed a co-expression analysis of lncRNA and mRNA and identified that 1319 lncRNA co-express with at least one mRNA. Further analysis of samples from S. mediterranea sexual and asexual strains revealed the role of sexual reproduction in gene expression profiles. Samples from different parts of asexual S. mediterranea revealed that differential expression profiles of different body parts correlated with the function of conduction of nerve impulses. In conclusion, PipeOne-NM has the potential to provide comprehensive transcriptome information for non-model organisms on a single platform.

The objective of the present study was to develop a model of avoidance learning and its extinction in planarians (Schmidtea mediterranea). Based on previous experiments showing conditioned place preference, we developed a procedure to investigate conditioned place avoidance (CPA) using shock as an unconditioned stimulus (US) and an automated tracking system to record the animals\' behaviour. In Experiment 1, we assessed the unconditioned properties of different shock intensities by measuring post shock activity. In two subsequent experiments we investigated CPA using different designs, surfaces as conditioned stimuli (CSs; rough and smooth), and different US intensities (5 V and 10 V). In general, we observed the successful development of CPA. However, CPA was stronger with higher shock intensities, and we found that, in our preparation, a rough surface is best at entering into an association with the shock than a smooth surface. Finally, we also observed extinction of CPA. The evidence of CPA and its extinction in flatworms validates the planaria as a pre-clinical model for the study of avoidance learning, a hallmark of anxiety disorders.

Cilia and flagella are evolutionarily conserved organelles that form protrusions on the surface of many growth-arrested or differentiated eukaryotic cells. Due to the structural and functional differences, cilia can be roughly classified as motile and non-motile (primary). Genetically determined dysfunction of motile cilia is the basis of primary ciliary dyskinesia (PCD), a heterogeneous ciliopathy affecting respiratory airways, fertility, and laterality. In the face of the still incomplete knowledge of PCD genetics and phenotype-genotype relations in PCD and the spectrum of PCD-like diseases, a continuous search for new causative genes is required. The use of model organisms has been a great part of the advances in understanding molecular mechanisms and the genetic basis of human diseases; the PCD spectrum is not different in this respect. The planarian model (Schmidtea mediterranea) has been intensely used to study regeneration processes, and-in the context of cilia-their evolution, assembly, and role in cell signaling. However, relatively little attention has been paid to the use of this simple and accessible model for studying the genetics of PCD and related diseases. The recent rapid development of the available planarian databases with detailed genomic and functional annotations prompted us to review the potential of the S. mediterranea model for studying human motile ciliopathies.