Cilia are microtubule-based organelles essential for motility, sensory signaling and development. In humans, motile cilia facilitate fluid movement, and their dysfunction causes ciliopathies, including infertility. We used RNAi-mediated knockdown of two human spermatid flagellar genes in Schmidtea mediterranea to assess effects on ciliary function and locomotion. Knockdown of Smed-ropn1l and Smed-tex9 significantly reduced planarian swim rate by 26.41% and 33.21% respectively and shortened cilia by 38.77% and 37.14% respectively. These findings highlight the critical roles of ROPN1L and TEX9 in cilia function and the use of planarians as a valuable model for studying ciliopathies.
","acknowledgements":"
We thank Kaleigh Powers and Dr. Jason Pellettieri (Keene State College) for their valuable guidance, Dr. Mark Scimone (University of New Hampshire) for assistance with DIC microscopy. We are also grateful to Dr. Megan Thompson (Oyster River High School) and Dr. A. Sanchez Alvarado (Stowers Institute) for generously providing the planaria used in this study.
","authors":[{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["dataCuration","formalAnalysis","investigation","validation","writing_originalDraft","visualization","writing_reviewEditing"],"email":"Rachel.Pitt@unh.edu","firstName":"Rachel ","lastName":"Pitt","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["dataCuration","formalAnalysis","investigation","validation","writing_originalDraft","visualization","writing_reviewEditing"],"email":"chayanika.gogoi@unh.edu","firstName":"Chayanika","lastName":"Gogoi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["investigation"],"email":"Angelina.Calnan@unh.edu","firstName":"Angelina","lastName":"Calnan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["investigation"],"email":"Lata.Patnaik@unh.edu","firstName":"Lata","lastName":"Patnaik","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["conceptualization","formalAnalysis","fundingAcquisition","investigation","methodology","project","resources","supervision","validation","writing_reviewEditing"],"email":"kristen.johnson@unh.edu","firstName":"Kristen C","lastName":"Johnson","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" https://orcid.org/0000-0002-1825-0265"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":null,"extendedData":[{"description":"20 sec swim video of neg.cont (RNAi) worms","doi":null,"resourceType":"Audiovisual","name":"Extended data video 1.mp4","url":"https://portal.micropublication.org/uploads/1dcb1441dd3d5a4e47d137ea62d079de.mp4"},{"description":"20 sec swim video of ropn1l(RNAi) worms","doi":null,"resourceType":"Audiovisual","name":"Extended data video 2.mp4","url":"https://portal.micropublication.org/uploads/81718a714afb503b091eb832cfdf7b83.mp4"},{"description":"20 sec swim video of tex9(RNAi) worms","doi":null,"resourceType":"Audiovisual","name":"Extended data video 3.mp4","url":"https://portal.micropublication.org/uploads/7f9e500c2994653f6c35b04a9f56bac3.mp4"},{"description":"Image showing bloating in the trunk region of RNAi worms","doi":null,"resourceType":"Image","name":"Extended data image 1.jpg","url":"https://portal.micropublication.org/uploads/508e8d1d856e446bb89fd09dac275950.jpg"}],"funding":"Student research participation and publication support were funded by the Cell Biology Education Consortium (CBEC): Path to Publication (Award ID #2316122) through the National Science Foundation (NSF) and NH-INBRE (NIH Award: P20GM103506).
","image":{"url":"https://portal.micropublication.org/uploads/aaa6e7e8b8e76c726e63dffe4ec947d6.png"},"imageCaption":"A) Pathway tracing of RNAi-mediated knockdown worms showing reduced distance traveled by gliding motility. Overlays of sequential frames (t=20s) from 20 second video segments, illustrating the distance travelled by individual flatworms in control (neg.ctrl.(RNAi)), ropn1l(RNAi) and tex9(RNAi) worms. Z-projection within FIJI/Image J software was used to perform the path tracing analysis. Scale bar: 10mm B) Differential interference microscopy (DIC) images showing reduced cilia length after gene knockdown. DIC images of planarian cilia showed that knockdown of ropn1l and tex9 resulted in visibly shorter cilia, with quantitative analysis confirming a reduction in length of 37.1% and 38.8% respectively. Scale bar 10µm. C) RNAi-mediated knockdown impairs planarian swim speed. Average swim speed for neg.ctrl.(RNAi) worms was 1.3 mm/s. ropn1l(RNAi) and tex9(RNAi) treated worms showed a 26.4% (0.96mm/s) and 33.2% (0.87mm/s) decrease in swim speed respectively (**** p<0.0001, n=30). Error bars indicate SEM. D) RNAi-mediated knockdown reduces cilia length in planarians. Average cilia length in neg.ctrl.(RNAi) worms was 11.0μm. ropn1l(RNAi) and tex9(RNAi) treated worms exhibited a 37.1% and 38.8% reduction in length respectively (*** p<0.0001, n=10). Error bars indicate SEM. E) Single-cell RNA-Seq expression of ropn1l (dd_Smed_v6_4888_0) in planarian cell clusters. Expression of ropn1l is enriched in pharyngeal, epidermal, and specifical neuronal cells with lower expression in other lineages (Plass et al., 2018). F) Single-cell RNA-Seq expression of tex9 (dd_Smed_v6_7007_0) in planarian cell clusters. Expression of tex9 is strongly enriched in pharyngeal and neuronal cells with lower expression in other lineages (Plass et al., 2018).
","imageTitle":"Knockdown of Smed-ropn1l and Smed-tex9 reduces planarian gliding locomotion and cilia length
","methods":"Planarian maintenance and feeding protocol
Planarians (S. mediterranea asexual strain ClW4) were maintained in a dark environment at room temperature in vessels containing water prepared as follows: 1.6mM NaCl, 1.0mM CaCl2, 1.0mM MgSO4, 0.1mM KCl, 1.2mM NaHCO3 (Cebrià & Newmark, 2005). Worms were fed once weekly with organic beef liver paste. After two hours of feeding, any remaining food was removed using plastic transfer pipettes and a small volume of fresh planarian water was added to top off the vessel. A complete water change was performed every two days to remove accumulated excretory waste and maintain optimal water quality (Rompolas et al., 2013).
RNAi plasmid cloning and feeding
Following the methods established in our previous study (Gogoi et al., 2026), RNAi constructs were generated and administered in Schmidtea mediterranea with gene-specific modifications as described below. FASTA transcript sequences for ropn1l (dd_Smed_v6_4888_0) and tex9 (dd_Smed_v6_7007_0) were obtained from the PlanMine database, and Benchling was used to design primers generating 200–800 bp fragments with plasmid-homologous overhangs compatible with the pPR-T4P vector. A 543 bp fragment of ropn1l and a 615 bp fragment of tex9 were amplified from cDNA using gene-specific primers sharing identical pPR-T4P overhang sequences, with homology arms indicated in capital letters and gene-specific regions in lowercase (primer sequences available under Reagents). Total RNA was isolated from homogenized animals, reverse transcribed to cDNA, and target fragments were amplified using either Q5 or Taq polymerase, verified by gel electrophoresis, purified, and quantified to calculate a 2:1 insert-to-backbone ratio. Inserts were cloned into pPR-T4P using Gibson Assembly for ropn1l and InFusion assembly for tex9. Assembled constructs were transformed into E. coli DH5α, screened by PCR, purified, and sequence-verified by nanopore sequencing prior to RNAi food preparation. The C. elegans gene unc-22 with no known homolog in the planarian genome was used as a negative control neg.cont.(RNAi) (Wagner, 2012). Verified plasmids were subsequently transformed into E. coli HT115 for dsRNA production, and induced cultures were mixed with homogenized liver for feeding. Following a one-week starvation period, animals were fed six times over two weeks (every 2-3 days). For each RNAi condition, three experimental plates containing ten animals each were analyzed.
Seven days after the final RNAi feeding, phenotypic assays were performed as follows:
Cilia Phenotype Analysis:
Planarians were immobilized by incubation in a relaxant solution (1% HNO3, 0.8325% formaldehyde, and 50 mM MgSO4) for 5 minutes prior to mounting on glass slides. Specimens were imaged using differential interference contrast (DIC) optics on a Zeiss LSM 510 Meta confocal microscope with a tungsten halogen lamp using an EC Plan Neofluar 40x/1.3 oil immersion objective lens with pixel size of 0.11μm/px. Images were captured using a Teledyne Lumenera Infinity 3 camera with Infinity Analyze software. For each condition, five worms were analyzed. Images were captured from both the left and right sides of the head region, near the eyes, where cilia are more densely distributed. From each image, the lengths of 10 individual cilia were measured, resulting in 10 data points per image. Measurements were performed using Fiji/ImageJ software to calculate the average cilia length for both control and RNAi-treated planarians.
Gliding Assays:
Planarian locomotion was recorded over a 20-second period using an Andonstar AD246S-M HDMI Digital Microscope. Videos were analyzed in Fiji ImageJ, where z-projection was applied to generate tracks of individual worms. The lengths of these tracks were measured to calculate average swimming speed (mm/s).
Statistical Analysis:
Gliding velocity and cilia length were analyzed using FIJI/ImageJ software. Graph Pad Prism Software version 10.0 was used to analyze the data, employing one way ANOVA and post-hoc analysis using Dunnett’s test to analyze data variance with significance defined as p<0.05.
","reagents":"Category | Reagents | Use/Notes | Source of Reagent |
Plasmids & Vectors | pPRt4p plasmid | Expression vector backbone for cloning & RNAi experiments | Gifted by Jason Pellettieri |
Bacterial Strains | HT115 E. coli | RNAi feeding strain (RNase III deficient, used in dsRNA expression) | Gifted by Jason Pellettieri |
| NEB 5-alpha E. coli | High efficiency cloning strain for plasmid propagation | New England Biolabs |
Primers | ropn1l-fwd | CATTACCATCCCGaagctgccattcgtacccagcc | Invitrogen |
| ropn1l-rev | CCAATTCTACCCGtgcttggttgaacgaggccacc | Invitrogen |
| tex9-fwd | CATTACCATCCCGtccagcaaattccaacacgttcca | Invitrogen |
| tex9-rev | CCAATTCTACCCGgctttctgacgctctagccttctgc | Invitrogen |
Cilia are small hair-like structures that are highly evolutionarily conserved and found on the surface of most eukaryotic cells (Horani & Ferkol, 2021). There are two main types of cilia, primary (non-motile) and motile which include flagella and nodal cilia (Hyland & Brody, 2021). Motile cilia and related microtubule-based structures, such as flagella, are essential for a wide range of cellular and developmental processes across eukaryotes. Ciliopathies are a set of human diseases arising from genetic mutations that affect cilia structure or function. Many ciliopathies do not currently have a cure, although research is being carried out into gene therapies rather than just management of symptoms (Wee et al., 2024). Our understanding of these diseases is still not fully understood (Hyland & Brody, 2021). There are over 2000 genes that have been associated with cilia formation and function, and this list is growing (Yenisert & Kaplan, 2025). Despite their importance, many cilia-associated proteins remain poorly characterized. Rhophilin associated tail protein 1–like (ROPN1L) and testis-expressed protein 9 (TEX9) have been identified through proteomic and transcriptomic studies as candidate components of cilia or flagella in humans, mice, and flies, with some evidence suggesting roles in flagellar or sperm tail structure. However, direct functional analyses of these genes are limited. To address this gap, we investigated the roles of ROPN1L and TEX9 homologs in the planarian Schmidtea mediterranea using RNA interference (Newmark et al., 2003), a model system well suited for studying motile cilia, to test their contributions to cilia structure and function.
ROPN1L is a scaffold protein in the ROPN1/ROPN1L–Dependent 2 (R2D2) family that anchors protein kinase A (PKA) signaling complexes to cilia and flagella (Bontems et al., 2014). In human bronchial epithelial cells undergoing mucociliary differentiation, ROPN1L was identified as one of 37 key cilia-associated genes upregulated during this process, based on transcriptomic analysis (Ross et al., 2007). Proteomic and immunolocalization data from the same study further demonstrated that ROPN1L localizes to the ciliary axoneme, providing direct evidence of its association with cilia (Ross et al., 2007). Functional evidence for the role of ROPN1L in microtubule-based motility comes from mouse models, where knockout of Ropn1l or its paralog rhophilin associated tail protein 1 (Ropn1) resulted in moderately reduced sperm motility, while deletion of both genes caused complete sperm immotility, structural disruption of the fibrous sheath, and loss of A kinase anchoring protein 3 (AKAP3) in sperm flagella (Fiedler et al., 2013). Consistent with these findings, qPCR and RNA-seq analyses of germ cells from men with cryptorchidism a congenital condition in which one or both testes fail to descend into the scrotum, revealed ROPN1L as the most downregulated gene among several fertility-associated targets, including AKAP4, IZUMO1, and SPAG6, further supporting its role in human fertility (Sun et al., 2023).
Functional analysis of TEX9 using comparative genomics across eukaryotes initially suggested a role for TEX9 in cilia biology. Phylogenetic profiling across 100 eukaryotic genomes identified TEX9 as a candidate ciliary protein, and cross-species comparisons of flagellated and non-flagellated organisms placed TEX9 within a highly conserved subset of six proteins associated with cilia-related centriolar satellites (Wu et al., 2025). Consistent with these predictions, CilioGenics—an integrative platform combining single-cell RNA sequencing, protein–protein interactions, comparative genomics, transcription factor network analysis, and text mining—identified TEX9 among 256 candidate ciliary genes, including 89 previously annotated by CiliaCarta and 28 with experimental validation (Pir et al., 2024). Proteomic analyses further detected TEX9 in human testes, suggesting an association with sperm development and cilia or flagella formation (Vandenbrouck et al., 2020). Direct evidence for ciliary association was provided by immunofluorescence studies demonstrating localization of TEX9 to cilia and basal bodies in cultured human kidney proximal tubule epithelial (HK2) cells (Nevers et al., 2017). Functional support for a role in cilia and flagella came from loss-of-function studies, where RNAi knockdown of TEX9 in Drosophila caused sensory cilia defects and impaired sperm flagellar motility, and C. elegans mutants exhibited dye-fill defects indicative of structural or trafficking abnormalities in sensory cilia (Dobbelaere et al., 2023).
Schmidtea mediterranea (freshwater planaria) serves as an ideal model organism for the study of ciliary genes. Cilia are used in planarian locomotion and are external and visible under the microscope in live specimens (Rabiasz & Ziętkiewicz, 2023; Rompolas et al., 2009); Rompolas et al., 2010). The planarian is a low-cost, easily maintained laboratory model and is one of few multicellular organisms with an external multiciliated epithelium. Its ventral epithelial cells bear many cilia that beat against a secreted mucus layer, in coordination with muscle contraction, to drive locomotion—similar to mucus transport by cilia in the human airway, reproductive tract, and ventricles of the brain. Defects in cilia function can be expected to result in visible impaired locomotion due to impacts on the ciliated ventral epidermis. Widely studied for its regenerative abilities, S. mediterranea now has a sequenced genome and robust molecular tools, including RNAi for gene function analysis. Additionally, protonephridia, excretory and osmoregulatory structures, function by using the movement of cilia to filter waste products and excess water from the body fluid. Disruption of the cilia can impact the excretory system giving rise to fluid retention and planarian bloating (Reddien et al.,2005; Rink et al., 2009; Rink et al., 2011; Vu et al., 2015).
In our functional studies, RNAi-mediated knockdown of the planarian homologs of human ROPN1L and TEX9 in S. mediterranea resulted in clear phenotypic changes in swim rate when compared to neg.cont.(RNAi) (Fig. 1A, Extended data video 1 – neg.cont.(RNAi) 20 sec., Extended data video 2 – ropn1l(RNAi) 20 sec., Extended data video 3 – tex9(RNAi) 20 sec.). One week post-RNAi treatment, gliding locomotion was assayed using 20 second video segments, and distance of travel over that time was determined using z-projection in Fiji/Image J. Quantitative analysis (n=30) showed that knockdowns of Smed-ropn1l and Smed-tex9 led to impaired swim speed compared to the control: 26.4% and 33.2% reduction respectively (Fig 1C).
Differential interference contrast microscopy images of cilia located in the lateral region of planarians were taken, and these indicated reduced cilia length for ropn1l(RNAi) and tex9(RNAi) treated worms (Fig 1B). Quantitative analysis revealed reduction of cilia lengths of 37.1% and 38.8%, respectively. Additionally, uneven length of cilia was observed in the tex9(RNAi) knockdown. These images strongly suggest that functionally, these genes are important for ciliary integrity. In addition, qualitative observations showed bloating in the trunk region in some of the ropn1l(RNAi) and tex9(RNAi) treated worms that was not observed in the neg.cont.(RNAi) worms, indicating potential defects in fluid regulation in the flame cells of the protonephridia, which act as the planarian osmoregulatory system (Extended data image 1 – bloating in trunk of representative ropnl1(RNAi) and tex9(RNAi) planarians).
Single cell transcriptomic analysis of planarian by Plass and colleagues revealed that ropn1l and tex9 exhibit strongest expression in specific neuronal subtypes, epidermal populations, protonephridia, and a distinct pharyngeal cell type described as epidermal-related (Fig. 1E, F) (Plass et al., 2018). In addition, single-cell transcriptomic data from Fincher and colleagues indicates that some of the highest-expressing cells for both genes are found in the protonephridial cluster (cluster 29), further supporting a role in protonephridial ciliary function. This observation provides a link to the bloating phenotype observed in a subset of RNAi knockdown animals. The lineage diagrams (Fig 1E, F) also depict inferred differentiation trajectories, in which increased gene expression toward the distal tips reflects enrichment in terminally differentiated cell types. Because the formation of motile cilia is expected to occur late in differentiation, the localization of ropn1l and tex9 expression toward terminal branches is consistent with their proposed roles in motile ciliogenesis. Across major cell categories including secretory, muscle, gut, and parenchymal populations both genes show comparatively low expression, whereas enrichment is observed in specific neurons, epidermis, protonephridia, and pharyngeal lineages, all of which include tissues known to harbor abundant motile cilia (Fincher et al., 2018)
In summary, our findings demonstrate that ROPN1L and TEX9 are required for the maintenance of normal ciliary length and motile function in multiciliated cells in planarians. Integration of single-cell expression analyses with RNAi-mediated knockdown phenotypes—including impaired gliding motility, shortened cilia, and posterior abdominal swelling—provides compelling functional evidence that these genes are conserved components essential for proper motile cilia assembly and performance. These results extend prior proteomic and transcriptomic associations by establishing a direct role for ROPN1L and TEX9 in ciliary biology. Moreover, our data highlights the value of Schmidtea mediterranea as a good in vivo model for dissecting the functions of evolutionarily conserved ciliary genes. Given the established links between motile cilia dysfunction and human ciliopathies and reproductive disorders, elucidating the roles of ROPN1L and TEX9 may contribute to a deeper understanding of the molecular mechanisms underlying these conditions. Collectively, our findings identify ROPN1L and TEX9 as integral components of a conserved motile cilia program operating across tissues and species, consistent with their proposed roles in humans.
","references":[{"reference":"Bontems F, Fish RJ, Borlat I, Lembo Fdr, Chocu S, Chalmel Fdr, et al., Lane. 2014. C2orf62 and TTC17 Are Involved in Actin Organization and Ciliogenesis in Zebrafish and Human. PLoS ONE 9: e86476.
","pubmedId":"","doi":"10.1371/journal.pone.0086476"},{"reference":"Cebrià F, Newmark PA. 2005. Planarian homologs ofnetrinandnetrin receptorare required for proper regeneration of the central nervous system and the maintenance of nervous system architecture. Development 132: 3691-3703.
","pubmedId":"","doi":"10.1242/dev.01941"},{"reference":"Dobbelaere J, Su TY, Erdi B, Schleiffer A, Dammermann A. 2023. A phylogenetic profiling approach identifies novel ciliogenesis genes in\n Drosophila\n and\n C. elegans. The EMBO Journal 42: 10.15252/embj.2023113616.
","pubmedId":"","doi":"10.15252/embj.2023113616"},{"reference":"Fiedler SE, Dudiki T, Vijayaraghavan S, Carr DW. 2013. Loss of R2D2 Proteins ROPN1 and ROPN1L Causes Defects in Murine Sperm Motility, Phosphorylation, and Fibrous Sheath Integrity1. Biology of Reproduction 88: 10.1095/biolreprod.112.105262.
","pubmedId":"","doi":"10.1095/biolreprod.112.105262"},{"reference":"Fincher CT, Wurtzel O, de Hoog T, Kravarik KM, Reddien PW. 2018. Cell type transcriptome atlas for the planarian\n Schmidtea mediterranea. Science 360: 10.1126/science.aaq1736.
","pubmedId":"","doi":"10.1126/science.aaq1736"},{"reference":"Gogoi C, Pitt R, Mazur K, Naraharisetti R, Johnson K. 2026. undefined. microPublication Biology. 2026","pubmedId":"","doi":"10.17912/MICROPUB.BIOLOGY.001887"},{"reference":"Horani A, Ferkol TW. 2021. Understanding Primary Ciliary Dyskinesia and Other Ciliopathies. The Journal of Pediatrics 230: 15-22.e1.
","pubmedId":"","doi":"10.1016/j.jpeds.2020.11.040"},{"reference":"Hyland RM, Brody SL. 2021. Impact of Motile Ciliopathies on Human Development and Clinical Consequences in the Newborn. Cells 11: 125.
","pubmedId":"","doi":"10.3390/cells11010125"},{"reference":"Nevers Y, Prasad MK, Poidevin L, Chennen K, Allot A, Kress A, et al., Lecompte. 2017. Insights into Ciliary Genes and Evolution from Multi-Level Phylogenetic Profiling. Molecular Biology and Evolution 34: 2016-2034.
","pubmedId":"","doi":"10.1093/molbev/msx146"},{"reference":"Newmark PA, Reddien PW, Cebrià F, Alvarado ASn. 2003. Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians. Proceedings of the National Academy of Sciences 100: 11861-11865.
","pubmedId":"","doi":"10.1073/pnas.1834205100"},{"reference":"Pir MS, Begar E, Yenisert F, Demirci HC, Korkmaz ME, Karaman A, et al., Kaplan. 2024. CilioGenics: an integrated method and database for predicting novel ciliary genes. Nucleic Acids Research 52: 8127-8145.
","pubmedId":"","doi":"10.1093/nar/gkae554"},{"reference":"Plass M, Solana J, Wolf FA, Ayoub S, Misios A, Glažar P, et al., Rajewsky. 2018. Cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics. Science 360: 10.1126/science.aaq1723.
","pubmedId":"","doi":"10.1126/science.aaq1723"},{"reference":"Rabiasz A, Ziętkiewicz E. 2023. Schmidtea mediterranea as a Model Organism to Study the Molecular Background of Human Motile Ciliopathies. International Journal of Molecular Sciences 24: 4472.
","pubmedId":"","doi":"10.3390/ijms24054472"},{"reference":"Reddien PW, Bermange AL, Murfitt KJ, Jennings JR, Sánchez Alvarado A. 2005. Identification of Genes Needed for Regeneration, Stem Cell Function, and Tissue Homeostasis by Systematic Gene Perturbation in Planaria. Developmental Cell 8: 635-649.
","pubmedId":"","doi":"10.1016/j.devcel.2005.02.014"},{"reference":"Rink JC, Gurley KA, Elliott SA, Sánchez Alvarado A. 2009. Planarian Hh Signaling Regulates Regeneration Polarity and Links Hh Pathway Evolution to Cilia. Science 326: 1406-1410.
","pubmedId":"","doi":"10.1126/science.1178712"},{"reference":"Rink JC, Vu HTK, Alvarado ASn. 2011. The maintenance and regeneration of the planarian excretory system are regulated by EGFR signaling. Development 138: 3769-3780.
","pubmedId":"","doi":"10.1242/dev.066852"},{"reference":"Rompolas P, Azimzadeh J, Marshall WF, King SM. 2013. Analysis of Ciliary Assembly and Function in Planaria. Methods in Enzymology,Cilia, Part B : 245-264.
","pubmedId":"","doi":"10.1016/B978-0-12-397944-5.00012-2"},{"reference":"Rompolas P, Patel-King RS, King SM. 2009. Schmidtea mediterranea: a model system for analysis of motile cilia. Methods Cell Biol 93: 81-98.
","pubmedId":"","doi":"https://doi.org/10.1016/S0091-679X(08)93004-1"},{"reference":"Rompolas, P., Patel-King, R. S., & King, S. M. (2010). An outer arm Dynein conformational switch is required for metachronal synchrony of motile cilia in planaria. Molecular biology of the cell, 21(21), 3669–3679. https://doi.org/10.1091/mbc.E10-04-0373
","pubmedId":"","doi":""},{"reference":"Ross AJ, Dailey LA, Brighton LE, Devlin RB. 2007. Transcriptional Profiling of Mucociliary Differentiation in Human Airway Epithelial Cells. American Journal of Respiratory Cell and Molecular Biology 37: 169-185.
","pubmedId":"","doi":"10.1165/rcmb.2006-0466OC"},{"reference":"Sun W, Zhang X, Wang L, Ren G, Piao S, Yang C, Liu Z. 2023. RNA sequencing profiles reveals progressively reduced spermatogenesis with progression in adult cryptorchidism. Frontiers in Endocrinology 14: 10.3389/fendo.2023.1271724.
","pubmedId":"","doi":"10.3389/fendo.2023.1271724"},{"reference":"Thi-Kim Vu H, Rink JC, McKinney SA, McClain M, Lakshmanaperumal N, Alexander R, Sánchez Alvarado A. 2015. Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ. eLife 4: 10.7554/elife.07405.
","pubmedId":"","doi":"10.7554/eLife.07405"},{"reference":"Vandenbrouck Y, Pineau C, Lane L. 2020. The Functionally Unannotated Proteome of Human Male Tissues: A Shared Resource to Uncover New Protein Functions Associated with Reproductive Biology. Journal of Proteome Research 19: 4782-4794.
","pubmedId":"","doi":"10.1021/acs.jproteome.0c00516"},{"reference":"Wagner DE, Ho JJ, Reddien PW. 2012. Genetic Regulators of a Pluripotent Adult Stem Cell System in Planarians Identified by RNAi and Clonal Analysis. Cell Stem Cell 10: 299-311.
","pubmedId":"","doi":"10.1016/j.stem.2012.01.016"},{"reference":"Wee WB, Kinghorn B, Davis SD, Ferkol TW, Shapiro AJ. 2024. Primary Ciliary Dyskinesia. Pediatrics 153: 10.1542/peds.2023-063064.
","pubmedId":"","doi":"10.1542/peds.2023-063064"},{"reference":"Wu B, Long C, Liu J, Huang X, Ma S, Ma Y, et al., Li. 2025. A subset of evolutionarily conserved centriolar satellite core components is crucial for sperm flagellum biogenesis. Theranostics 15: 7025-7044.
","pubmedId":"","doi":"10.7150/thno.117118"},{"reference":"Yenisert F, Kaplan OI. 2025. Expanded Catalog of Gold Standard Ciliary Gene List: Integration of Novel Ciliary Genes into the Ciliome. : 10.1101/2025.08.22.671678.
","pubmedId":"","doi":"10.1101/2025.08.22.671678"}],"title":"Insights into Human Ciliopathies: Gene Silencing of ropn1l and tex9 in Schmidtea mediterranea Indicate Association with Ciliary Structure and Motility
","reviews":[{"reviewer":{"displayName":"Brett Mommer"},"openAcknowledgement":true,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"c6e7b084-dd1c-4422-afd9-a581685aba25","decision":"edit","abstract":"Cilia are microtubule-based organelles essential for motility, sensory signaling and development. In humans, motile cilia facilitate fluid movement, and their dysfunction causes ciliopathies, including infertility. We used RNAi-mediated knockdown of two human spermatid flagellar genes in Schmidtea mediterranea to assess effects on ciliary function and locomotion. Knockdown of Smed-ropn1l and Smed-tex9 significantly reduced planarian swim rate by 26.4% and 33.2% respectively and shortened cilia by 37.1% and 38.7% respectively. These findings highlight the critical roles of ROPN1L and TEX9 in cilia function and the use of planarians as a valuable model for studying ciliopathies.
","acknowledgements":"
We thank Kaleigh Powers and Dr. Jason Pellettieri (Keene State College) for their valuable guidance, Dr. Mark Scimone (University of New Hampshire) for assistance with DIC microscopy. We are also grateful to Dr. Megan Thompson (Oyster River High School) and Dr. A. Sanchez Alvarado (Stowers Institute) for generously providing the planaria used in this study.
","authors":[{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["dataCuration","formalAnalysis","investigation","validation","writing_originalDraft","visualization","writing_reviewEditing"],"email":"Rachel.Pitt@unh.edu","firstName":"Rachel ","lastName":"Pitt","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["dataCuration","formalAnalysis","investigation","validation","writing_originalDraft","visualization","writing_reviewEditing"],"email":"chayanika.gogoi@unh.edu","firstName":"Chayanika","lastName":"Gogoi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["investigation"],"email":"Angelina.Calnan@unh.edu","firstName":"Angelina","lastName":"Calnan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["investigation"],"email":"Lata.Patnaik@unh.edu","firstName":"Lata","lastName":"Patnaik","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["conceptualization","formalAnalysis","fundingAcquisition","investigation","methodology","project","resources","supervision","validation","writing_reviewEditing"],"email":"kristen.johnson@unh.edu","firstName":"Kristen C","lastName":"Johnson","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" https://orcid.org/0000-0002-1825-0265"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":null,"extendedData":[{"description":"20 sec swim video of neg.cont (RNAi) worms","doi":"10.22002/z5vja-gfv92","resourceType":"Audiovisual","name":"Extended data video 1.mp4","url":"https://portal.micropublication.org/uploads/1dcb1441dd3d5a4e47d137ea62d079de.mp4"},{"description":"20 sec swim video of ropn1l(RNAi) worms","doi":null,"resourceType":"Audiovisual","name":"Extended data video 2.mp4","url":"https://portal.micropublication.org/uploads/81718a714afb503b091eb832cfdf7b83.mp4"},{"description":"20 sec swim video of tex9(RNAi) worms","doi":null,"resourceType":"Audiovisual","name":"Extended data video 3.mp4","url":"https://portal.micropublication.org/uploads/7f9e500c2994653f6c35b04a9f56bac3.mp4"},{"description":"edema observations","doi":"10.22002/s8625-b8x92","resourceType":"Image","name":"Extended data image 1 - edema observations.jpg","url":"https://portal.micropublication.org/uploads/c462d033a0bdc6863d41653ffc1d9cd4.jpg"}],"funding":"Student research participation and publication support were funded by the Cell Biology Education Consortium (CBEC): Path to Publication (Award ID #2316122) through the National Science Foundation (NSF) and NH-INBRE (NIH Award: P20GM103506).
","image":{"url":"https://portal.micropublication.org/uploads/67b48af00eed41a196fcff43d7e36c1b.png"},"imageCaption":"A) Pathway tracing of RNAi-mediated knockdown worms shows reduced distance traveled by gliding motility. Overlays of sequential frames (t=20s) from 20 second video segments, illustrating the distance travelled by individual flatworms in control (neg.ctrl.(RNAi)), ropn1l(RNAi), and tex9(RNAi) worms. Z-projection within FIJI/Image J software was used to perform the path tracing analysis. Scale bar: 10mm B) Differential interference microscopy (DIC) images at 63X magnification show planarian cilia length. Gene knockdown of ropn1l and tex9 resulted in visibly shorter cilia. Scale bar = 10µm. C) RNAi-mediated knockdown impairs planarian swim speed. Mean swim speed (± SEM) for neg.ctrl.(RNAi) worms was 1.3 ± 0.05 mm/s. ropn1l(RNAi) and tex9(RNAi) worms displayed 0.96 ± 0.17 mm/s and 0.87 ± 0.09 mm/s swim speeds, respectively (****p<0.0001, one-way ANOVA, n=30). Error bars indicate SEM. D) RNAi-mediated knockdown reduces cilia length in planarians. Mean cilia length (± SEM) in neg.ctrl.(RNAi) worms was 11.04 ± 0.19 µm. ropn1l(RNAi) and tex9(RNAi) worms exhibited 6.94 ± 0.40 µm and 6.77 ± 0.30 µm cilia lengths, respectively (****p<0.0001, one-way ANOVA, n=5). Error bars indicate SEM. E) Single-cell RNA-Seq expression of ropn1l (dd_Smed_v6_4888_0) in planarian cell clusters. Expression of ropn1l is strongly enriched in pharyngeal, epidermal, and specific neuronal cells with lower expression in other lineages (lineage diagram created using Planaria Single Cell Database; https://shiny.mdc-berlin.de/psca/). F) Single-cell RNA-Seq expression of tex9 (dd_Smed_v6_7007_0) in planarian cell clusters. Expression of tex9 is strongly enriched in pharyngeal and neuronal cells with lower expression in other lineages (lineage diagram created using Planaria Single Cell Database; https://shiny.mdc-berlin.de/psca/).
","imageTitle":"Knockdown of Smed-ropn1l and Smed-tex9 reduces planarian gliding locomotion and cilia length.
","methods":"Planarian maintenance and feeding protocol
Planarians (S. mediterranea asexual strain ClW4) were maintained in a dark environment at room temperature in vessels containing water prepared as follows: 1.6mM NaCl, 1.0mM CaCl2, 1.0mM MgSO4, 0.1mM KCl, 1.2mM NaHCO3 (Cebrià & Newmark, 2005). Worms were fed once weekly with organic beef liver paste. After two hours of feeding, any remaining food was removed using plastic transfer pipettes and a small volume of fresh planarian water was added to top off the vessel. A complete water change was performed every two days to remove accumulated excretory waste and maintain optimal water quality (Rompolas et al., 2013).
RNAi plasmid cloning and feeding
Following the methods established in our previous study (Gogoi et al., 2026), RNAi constructs were generated and administered in Schmidtea mediterranea with gene-specific modifications as described below. FASTA transcript sequences for ropn1l (dd_Smed_v6_4888_0_1) and tex9 (dd_Smed_v6_7007_0_1) were obtained from the PlanMine database, and Benchling was used to design primers generating 200–800 bp fragments with plasmid-homologous overhangs compatible with the pPR-T4P vector. A 543 bp fragment of ropn1l and a 615 bp fragment of tex9 were amplified from cDNA using gene-specific primers sharing identical pPR-T4P overhang sequences, with homology arms indicated in capital letters and gene-specific regions in lowercase (primer sequences available under Reagents). Total RNA was isolated from homogenized animals, reverse transcribed to cDNA, and target fragments were amplified using either Q5 or Taq polymerase, verified by gel electrophoresis, purified, and quantified to calculate a 2:1 insert-to-backbone ratio. Inserts were cloned into pPR-T4P using Gibson Assembly for ropn1l and InFusion assembly for tex9. Assembled constructs were transformed into E. coli DH5α, screened by M13 PCR, purified, and sequence-verified by nanopore sequencing prior to RNAi food preparation. The C. elegans gene unc-22 with no known homolog in the planarian genome was used as a negative control neg.cont.(RNAi) (Wagner, 2012). Verified plasmids were subsequently transformed into E. coli HT115 for dsRNA production, and induced cultures were mixed with homogenized liver for feeding. Following a one-week starvation period, animals were fed six times over two weeks (every 2-3 days). For each RNAi condition, three experimental plates containing ten animals each were analyzed.
Seven days after the final RNAi feeding, phenotypic assays were performed as follows:
Cilia Phenotype Analysis:
Planarians were immobilized by incubation in a relaxant solution (1% HNO3, 0.8325% formaldehyde, and 50 mM MgSO4) for 5 minutes prior to mounting on glass slides. Specimens were imaged using differential interference contrast (DIC) optics on a Zeiss LSM 510 Meta confocal microscope with a tungsten halogen lamp using a Plan Apochromat 63x/1.4 oil immersion objective lens with pixel size of 0.07μm/px. Images were captured using a Teledyne Lumenera Infinity 3 camera with Infinity Analyze software. For each condition, five worms were analyzed. Images were captured from both the left and right sides of the head region, near the eyes, where cilia are more densely distributed. From each image, the lengths of 10 individual cilia were measured, resulting in 10 data points per image. Measurements were performed using Fiji/ImageJ software to calculate the mean cilia length for both control and RNAi-treated planarians.
Gliding Assays:
Planarian locomotion was recorded over a 20-second period using an Andonstar AD246S-M HDMI Digital Microscope. Videos were analyzed in Fiji ImageJ, where z-projection was applied to generate tracks of individual worms. The lengths of these tracks were measured to calculate average swimming speed (mm/s).
Statistical Analysis:
Gliding velocity and cilia length measurements were quantified using FIJI/ImageJ software. For gliding velocity analysis, 30 individual worms were analyzed per condition. For cilia length analysis, 5 worms were used per condition, and for each worm, images were acquired from both left and right sides of the animal. Ten cilia were measured per side, yielding a total of 100 measurements per condition, which were averaged to obtain a single mean value per worm for statistical analysis.
The normality of replicates was confirmed using the Shapiro–Wilk test. Both RNAi groups (ropn1l(RNAi) and tex9(RNAi)) were compared with the same negative control (RNAi) animals. Analyses were conducted using GraphPad Prism version 10.6.1. One-way ANOVA revealed significant differences among groups for both cilia length (df=4, p < 0.0001) and gliding velocity (df = 29, p < 0.0001), and post hoc analysis using Dunnett’s multiple comparisons test further confirmed that both ropn1l(RNAi) and tex9(RNAi) worms differed significantly from negative control animals (neg.cont.(RNAi)) in both gliding speed and length of cilia (p < 0.0001 for all comparisons).
","reagents":"Category | Reagents | Use/Notes | Source of Reagent |
Plasmids & Vectors | pPRt4p plasmid | Expression vector backbone for cloning & RNAi experiments | Gifted by Jason Pellettieri |
Bacterial Strains | HT115 E. coli | RNAi feeding strain (RNase III deficient, used in dsRNA expression) | Gifted by Jason Pellettieri |
| NEB 5-alpha E. coli | High efficiency cloning strain for plasmid propagation | New England Biolabs |
Primers | ropn1l-fwd | CATTACCATCCCGaagctgccattcgtacccagcc | Invitrogen |
| ropn1l-rev | CCAATTCTACCCGtgcttggttgaacgaggccacc | Invitrogen |
| tex9-fwd | CATTACCATCCCGtccagcaaattccaacacgttcca | Invitrogen |
| tex9-rev | CCAATTCTACCCGgctttctgacgctctagccttctgc | Invitrogen |
Cilia are small hair-like structures that are highly evolutionarily conserved and found on the surface of most eukaryotic cells (Horani & Ferkol, 2021). There are two main types of cilia, primary (non-motile) and motile which include flagella and nodal cilia (Hyland & Brody, 2021). Motile cilia and related microtubule-based structures, such as flagella, are essential for a wide range of cellular and developmental processes across eukaryotes. Ciliopathies are a set of human diseases arising from genetic mutations that affect cilia structure or function. Many ciliopathies currently lack curative treatments, although ongoing research is exploring gene therapy approaches that aim to address underlying causes rather than only managing symptoms (Wee et al., 2024). Yet, our understanding of these diseases is still not fully understood (Hyland & Brody, 2021).
There are over 2000 genes that have been associated with cilia formation and function, and this list is growing (Yenisert & Kaplan, 2025). Despite their importance, many cilia-associated proteins remain poorly characterized. Rhophilin associated tail protein 1–like (ROPN1L) and testis-expressed protein 9 (TEX9) have been identified through proteomic and transcriptomic studies as candidate components of cilia or flagella in humans, mice, and flies, with some evidence suggesting roles in flagellar or sperm tail structure. However, direct functional analyses of these genes are limited. To address this gap, we used RNA interference to examine the functions of Smed-ropn1l and Smed-tex9, planarian homologs of the human genes, in Schmidtea mediterranea, a model well suited for studying motile cilia, and evaluated their roles in ciliary structure and function.
ROPN1L is a scaffold protein in the ROPN1/ROPN1L–Dependent 2 (R2D2) family that anchors protein kinase A (PKA) signaling complexes to cilia and flagella (Bontems et al., 2014). In human bronchial epithelial cells, ROPN1L was identified as one of 37 key cilia-associated genes upregulated during mucociliary differentiation, based on transcriptomic analysis (Ross et al., 2007). Proteomic and immunolocalization data from the same study further demonstrates that ROPN1L localizes to the ciliary axoneme, providing direct evidence of its association with cilia (Ross et al., 2007). Functional evidence for the role of ROPN1L in microtubule-based motility comes from mouse models, where knockout of Ropn1l or its paralog rhophilin associated tail protein 1 (Ropn1) results in moderately reduced sperm motility, while deletion of both genes causes complete sperm immotility, structural disruption of the fibrous sheath, and loss of A kinase anchoring protein 3 (AKAP3) in sperm flagella (Fiedler et al., 2013). Consistent with these findings, qPCR and RNA-seq analyses of germ cells from men with cryptorchidism, a congenital condition in which one or both testes fail to descend into the scrotum, reveals ROPN1L as the most downregulated gene among several fertility-associated targets, including AKAP4, IZUMO1, and SPAG6, further supporting its role in human fertility (Sun et al., 2023).
Functional and comparative genomic analysis of TEX9 across eukaryotes suggests a role for TEX9 in cilia biology. Phylogenetic profiling across 100 eukaryotic genomes identified TEX9 as a candidate ciliary protein, and cross-species comparisons of flagellated and non-flagellated organisms placed TEX9 within a highly conserved subset of six proteins associated with cilia-related centriolar satellites (Wu et al., 2025). Consistent with these predictions, CilioGenics—an integrative platform combining single-cell RNA sequencing, protein–protein interactions, comparative genomics, transcription factor network analysis, and text mining—identified TEX9 among 256 candidate ciliary genes, including 89 previously annotated by CiliaCarta and 28 with experimental validation (Pir et al., 2024). Proteomic analyses detected TEX9 in human testes, suggesting an association with sperm development and cilia or flagella formation (Vandenbrouck et al., 2020). Direct evidence for ciliary association was provided by immunofluorescence studies demonstrating localization of TEX9 to ciliary basal bodies in cultured human kidney proximal tubule epithelial (HK2) cells (Nevers et al., 2017). Functional support for a role in cilia and flagella comes from loss-of-function studies, where RNAi knockdown of TEX9 in Drosophila causes sensory cilia defects and impaired sperm flagellar motility, and C. elegans mutants exhibit dye-fill defects indicative of structural or trafficking abnormalities in sensory cilia (Dobbelaere et al., 2023).
Widely studied for its regenerative abilities, S. mediterranea (freshwater planaria) has a fully sequenced genome and robust molecular tools, including RNAi for gene function analysis, making it an ideal model for studying ciliary genes. The planarian is a low-cost, easily maintained laboratory model and is one of few multicellular organisms with an external multiciliated epithelium. Cilia are used in planarian locomotion and are external and visible in live specimens under the microscope (Rabiasz & Ziętkiewicz, 2023; Rompolas et al., 2009; Rompolas et al., 2010). Ventral epithelial cells bear multiple cilia that beat against a secreted mucus layer, together with muscle contraction, to drive locomotion, similar to mucus transport in the human airway, reproductive tract, and brain ventricles. Defects in ciliary function are therefore expected to cause visibly impaired locomotion. Additionally, protonephridia depend on ciliary movement for waste filtration and osmoregulation, and ciliary disruption can impair this function, resulting in fluid retention and edema. (Reddien et al., 2005; Rink et al., 2009; Rink et al., 2011; Thi-Kim Vu et al., 2015).
RNAi-mediated knockdown of ropn1l and tex9 in S. mediterranea resulted in clear phenotypic changes in swim rate when compared to neg.cont.(RNAi) (Fig. 1A; Extended data video 1 – neg.cont.(RNAi) 20 sec., Extended data video 2 – ropn1l(RNAi) 20 sec., Extended data video 3 – tex9(RNAi) 20 sec.). One week post-RNAi treatment, gliding locomotion was assessed using 20 second video segments, and distance traveled over time was determined using z-projection in Fiji/Image J. Quantitative analysis (n = 30) demonstrated that control worms (neg.cont.(RNAi)) had a mean swim rate ± SEM of 1.30 ± 0.05 mm/s, whereas ropn1l(RNAi) and tex9(RNAi) worms had significantly reduced swim speeds. ropn1l(RNAi) mean swim rate was 0.96 ± 0.17 mm/s and tex9(RNAi) mean swim rate was 0.87 ± 0.09 mm/s, representing decreases of 26.4% and 33.2%, respectively. (Fig 1C).
Differential interference contrast microscopy images of cilia located in the lateral region of planarian heads were taken, and these indicated reduced cilia length for ropn1l(RNAi) and tex9(RNAi) treated worms (Fig 1B). Quantitative analysis demonstrated significant reductions in cilia length in ropn1l(RNAi) and tex9(RNAi) worms, corresponding to decreases of 37.1% and 38.7%, respectively (mean ± SEM: 6.94 ± 0.40 µm and 6.77 ± 0.30 µm), relative to negative control (neg.cont.(RNAi)) worms (11.04 ± 0.19 µm) (Fig 1D). These data strongly suggest that functionally, these genes are important for ciliary integrity. In addition, edema in the trunk region of a small number of the ropn1l(RNAi) and tex9(RNAi) worms was observed, indicating potential defects in fluid regulation by the flame cells of the protonephridia (Reddien et al.,2005; Rink et al., 2009; Rink et al., 2011; Thi-Kim Vu et al., 2015). This edema was not observed in any of our neg.cont.(RNAi) worms (Extended data image 1 – edema observations).
Single-cell transcriptomic analysis of ropn1l and tex9 obtained from Planaria Single Cell Database (https://shiny.mdc-berlin.de/psca/) revealed that these genes exhibit strongest expression in specific neuronal subtypes, epidermal populations, protonephridia, and a distinct pharyngeal cell type described as epidermal-related (Fig. 1E, F) (Plass et al., 2018). In addition, single-cell transcriptomic data from Planarian Digiworm (https://digiworm.wi.mit.edu/) indicates that some of the highest-expressing cells for both genes are found in the protonephridial cluster (cluster 29), further supporting a role in protonephridial ciliary function (Fincher et al., 2018). This observation provides a link to the edema phenotype observed in a subset of RNAi knockdown animals. The lineage diagrams (Fig 1E, F) also depict inferred differentiation trajectories, in which increased gene expression toward the distal tips reflects enrichment in terminally differentiated cell types. Because the formation of motile cilia is expected to occur late in differentiation, the localization of ropn1l and tex9 expression toward terminal branches is consistent with their proposed roles in motile ciliogenesis. Across major cell categories including secretory, muscle, gut, and parenchymal populations both genes show comparatively low expression, whereas enrichment is observed in specific neurons, epidermis, protonephridia, and pharyngeal lineages, all of which include tissues known to harbor abundant motile cilia (Fincher et al., 2018).
Our results show that Schmidtea mediterranea is a valuable in vivo model for studying conserved ciliary genes. Since defects in motile cilia are linked to human disease, analyzing ROPN1L and TEX9 helps clarify shared molecular mechanisms across tissues and species. Although ROPN1L and TEX9 function through distinct molecular pathways, both genes are strongly associated with spermatogenesis and sperm flagellar structure, suggesting a shared role in motile microtubule-based systems (Sharma et al., 2014). We therefore presented these genes together in an exploration of their potential involvement in ciliary structure and function. In summary, our findings demonstrate that ROPN1L and TEX9 are required for the maintenance of normal ciliary length and motile function in multiciliated cells in planarians. Integration of single-cell expression analyses with RNAi-mediated knockdown phenotypes—including impaired gliding motility, shortened cilia, and posterior abdominal edema—provides compelling functional evidence that these genes are conserved components essential for proper motile cilia assembly and performance. These results extend prior proteomic and transcriptomic associations by establishing a direct role for ROPN1L and TEX9 in ciliary biology.
","references":[{"reference":"Bontems F, Fish RJ, Borlat I, Lembo Fdr, Chocu S, Chalmel Fdr, et al., Lane. 2014. C2orf62 and TTC17 Are Involved in Actin Organization and Ciliogenesis in Zebrafish and Human. PLoS ONE 9: e86476.
","pubmedId":"","doi":"10.1371/journal.pone.0086476"},{"reference":"Cebrià F, Newmark PA. 2005. Planarian homologs ofnetrinandnetrin receptorare required for proper regeneration of the central nervous system and the maintenance of nervous system architecture. Development 132: 3691-3703.
","pubmedId":"","doi":"10.1242/dev.01941"},{"reference":"Dobbelaere J, Su TY, Erdi B, Schleiffer A, Dammermann A. 2023. A phylogenetic profiling approach identifies novel ciliogenesis genes in\n Drosophila\n and\n C. elegans. The EMBO Journal 42: 10.15252/embj.2023113616.
","pubmedId":"","doi":"10.15252/embj.2023113616"},{"reference":"Fiedler SE, Dudiki T, Vijayaraghavan S, Carr DW. 2013. Loss of R2D2 Proteins ROPN1 and ROPN1L Causes Defects in Murine Sperm Motility, Phosphorylation, and Fibrous Sheath Integrity1. Biology of Reproduction 88: 10.1095/biolreprod.112.105262.
","pubmedId":"","doi":"10.1095/biolreprod.112.105262"},{"reference":"Fincher CT, Wurtzel O, de Hoog T, Kravarik KM, Reddien PW. 2018. Cell type transcriptome atlas for the planarian\n Schmidtea mediterranea. Science 360: 10.1126/science.aaq1736.
","pubmedId":"","doi":"10.1126/science.aaq1736"},{"reference":"Gogoi C, Pitt R, Mazur K, Naraharisetti R, Johnson K. 2026. undefined. microPublication Biology. 2026","pubmedId":"","doi":"10.17912/MICROPUB.BIOLOGY.001887"},{"reference":"Horani A, Ferkol TW. 2021. Understanding Primary Ciliary Dyskinesia and Other Ciliopathies. The Journal of Pediatrics 230: 15-22.e1.
","pubmedId":"","doi":"10.1016/j.jpeds.2020.11.040"},{"reference":"Hyland RM, Brody SL. 2021. Impact of Motile Ciliopathies on Human Development and Clinical Consequences in the Newborn. Cells 11: 125.
","pubmedId":"","doi":"10.3390/cells11010125"},{"reference":"Nevers Y, Prasad MK, Poidevin L, Chennen K, Allot A, Kress A, et al., Lecompte. 2017. Insights into Ciliary Genes and Evolution from Multi-Level Phylogenetic Profiling. Molecular Biology and Evolution 34: 2016-2034.
","pubmedId":"","doi":"10.1093/molbev/msx146"},{"reference":"Newmark PA, Reddien PW, Cebrià F, Alvarado ASn. 2003. Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians. Proceedings of the National Academy of Sciences 100: 11861-11865.
","pubmedId":"","doi":"10.1073/pnas.1834205100"},{"reference":"Pir MS, Begar E, Yenisert F, Demirci HC, Korkmaz ME, Karaman A, et al., Kaplan. 2024. CilioGenics: an integrated method and database for predicting novel ciliary genes. Nucleic Acids Research 52: 8127-8145.
","pubmedId":"","doi":"10.1093/nar/gkae554"},{"reference":"Plass M, Solana J, Wolf FA, Ayoub S, Misios A, Glažar P, et al., Rajewsky. 2018. Cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics. Science 360: 10.1126/science.aaq1723.
","pubmedId":"","doi":"10.1126/science.aaq1723"},{"reference":"Rabiasz A, Ziętkiewicz E. 2023. Schmidtea mediterranea as a Model Organism to Study the Molecular Background of Human Motile Ciliopathies. International Journal of Molecular Sciences 24: 4472.
","pubmedId":"","doi":"10.3390/ijms24054472"},{"reference":"Reddien PW, Bermange AL, Murfitt KJ, Jennings JR, Sánchez Alvarado A. 2005. Identification of Genes Needed for Regeneration, Stem Cell Function, and Tissue Homeostasis by Systematic Gene Perturbation in Planaria. Developmental Cell 8: 635-649.
","pubmedId":"","doi":"10.1016/j.devcel.2005.02.014"},{"reference":"Rink JC, Gurley KA, Elliott SA, Sánchez Alvarado A. 2009. Planarian Hh Signaling Regulates Regeneration Polarity and Links Hh Pathway Evolution to Cilia. Science 326: 1406-1410.
","pubmedId":"","doi":"10.1126/science.1178712"},{"reference":"Rink JC, Vu HTK, Alvarado ASn. 2011. The maintenance and regeneration of the planarian excretory system are regulated by EGFR signaling. Development 138: 3769-3780.
","pubmedId":"","doi":"10.1242/dev.066852"},{"reference":"Rompolas P, Azimzadeh J, Marshall WF, King SM. 2013. Analysis of Ciliary Assembly and Function in Planaria. Methods in Enzymology,Cilia, Part B : 245-264.
","pubmedId":"","doi":"10.1016/B978-0-12-397944-5.00012-2"},{"reference":"Rompolas P, Patel-King RS, King SM. 2009. Schmidtea mediterranea: a model system for analysis of motile cilia. Methods Cell Biol 93: 81-98.
","pubmedId":"","doi":"https://doi.org/10.1016/S0091-679X(08)93004-1"},{"reference":"Rompolas, P., Patel-King, R. S., & King, S. M. (2010). An outer arm Dynein conformational switch is required for metachronal synchrony of motile cilia in planaria. Molecular biology of the cell, 21(21), 3669–3679. https://doi.org/10.1091/mbc.E10-04-0373
","pubmedId":"","doi":""},{"reference":"Ross AJ, Dailey LA, Brighton LE, Devlin RB. 2007. Transcriptional Profiling of Mucociliary Differentiation in Human Airway Epithelial Cells. American Journal of Respiratory Cell and Molecular Biology 37: 169-185.
","pubmedId":"","doi":"10.1165/rcmb.2006-0466OC"},{"reference":"Sharma E, Künstner A, Fraser BA, Zipprich G, Kottler VA, Henz SR, Weigel D, Dreyer C. 2014. Transcriptome assemblies for studying sex-biased gene expression in the guppy, Poecilia reticulata. BMC Genomics 15: 10.1186/1471-2164-15-400.
","pubmedId":"","doi":"doi.org/10.1186/1471-2164-15-400"},{"reference":"Sun W, Zhang X, Wang L, Ren G, Piao S, Yang C, Liu Z. 2023. RNA sequencing profiles reveals progressively reduced spermatogenesis with progression in adult cryptorchidism. Frontiers in Endocrinology 14: 10.3389/fendo.2023.1271724.
","pubmedId":"","doi":"10.3389/fendo.2023.1271724"},{"reference":"Thi-Kim Vu H, Rink JC, McKinney SA, McClain M, Lakshmanaperumal N, Alexander R, Sánchez Alvarado A. 2015. Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ. eLife 4: 10.7554/elife.07405.
","pubmedId":"","doi":"10.7554/eLife.07405"},{"reference":"Vandenbrouck Y, Pineau C, Lane L. 2020. The Functionally Unannotated Proteome of Human Male Tissues: A Shared Resource to Uncover New Protein Functions Associated with Reproductive Biology. Journal of Proteome Research 19: 4782-4794.
","pubmedId":"","doi":"10.1021/acs.jproteome.0c00516"},{"reference":"Wagner DE, Ho JJ, Reddien PW. 2012. Genetic Regulators of a Pluripotent Adult Stem Cell System in Planarians Identified by RNAi and Clonal Analysis. Cell Stem Cell 10: 299-311.
","pubmedId":"","doi":"10.1016/j.stem.2012.01.016"},{"reference":"Wee WB, Kinghorn B, Davis SD, Ferkol TW, Shapiro AJ. 2024. Primary Ciliary Dyskinesia. Pediatrics 153: 10.1542/peds.2023-063064.
","pubmedId":"","doi":"10.1542/peds.2023-063064"},{"reference":"Wu B, Long C, Liu J, Huang X, Ma S, Ma Y, et al., Li. 2025. A subset of evolutionarily conserved centriolar satellite core components is crucial for sperm flagellum biogenesis. Theranostics 15: 7025-7044.
","pubmedId":"","doi":"10.7150/thno.117118"},{"reference":"Yenisert F, Kaplan OI. 2025. Expanded Catalog of Gold Standard Ciliary Gene List: Integration of Novel Ciliary Genes into the Ciliome. : 10.1101/2025.08.22.671678.
","pubmedId":"","doi":"10.1101/2025.08.22.671678"}],"title":"Insights into Human Ciliopathies: Gene Silencing of ropn1l and tex9 in Schmidtea mediterranea Indicate Association with Ciliary Structure and Motility
","reviews":[{"reviewer":{"displayName":"Brett Mommer"},"openAcknowledgement":true,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"a7cd2bf4-e427-40e3-ba81-b4688de13a25","decision":"accept","abstract":"Cilia are microtubule-based organelles essential for motility, sensory signaling and development. In humans, motile cilia facilitate fluid movement, and their dysfunction causes ciliopathies, including infertility. We used RNAi-mediated knockdown of two human spermatid flagellar genes in Schmidtea mediterranea to assess effects on ciliary function and locomotion. Knockdown of Smed-ropn1l and Smed-tex9 significantly reduced planarian swim rate by 26.4% and 33.2% respectively and shortened cilia by 37.1% and 38.7% respectively. These findings highlight the critical roles of ROPN1L and TEX9 in cilia function and the use of planarians as a valuable model for studying ciliopathies.
","acknowledgements":"
We thank Kaleigh Powers and Dr. Jason Pellettieri (Keene State College) for their valuable guidance, Dr. Mark Scimone (University of New Hampshire) for assistance with DIC microscopy. We are also grateful to Dr. Megan Thompson (Oyster River High School) and Dr. A. Sanchez Alvarado (Stowers Institute) for generously providing the planaria used in this study.
","authors":[{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["dataCuration","formalAnalysis","investigation","validation","writing_originalDraft","visualization","writing_reviewEditing"],"email":"Rachel.Pitt@unh.edu","firstName":"Rachel ","lastName":"Pitt","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["dataCuration","formalAnalysis","investigation","validation","writing_originalDraft","visualization","writing_reviewEditing"],"email":"chayanika.gogoi@unh.edu","firstName":"Chayanika","lastName":"Gogoi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["investigation"],"email":"Angelina.Calnan@unh.edu","firstName":"Angelina","lastName":"Calnan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["investigation"],"email":"Lata.Patnaik@unh.edu","firstName":"Lata","lastName":"Patnaik","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["conceptualization","formalAnalysis","fundingAcquisition","investigation","methodology","project","resources","supervision","validation","writing_reviewEditing"],"email":"kristen.johnson@unh.edu","firstName":"Kristen C","lastName":"Johnson","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" https://orcid.org/0000-0002-1825-0265"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"20 sec swim video of neg.cont (RNAi) worms","doi":"10.22002/z5vja-gfv92","resourceType":"Audiovisual","name":"Extended data video 1.mp4","url":"https://portal.micropublication.org/uploads/1dcb1441dd3d5a4e47d137ea62d079de.mp4"},{"description":"20 sec swim video of ropn1l(RNAi) worms","doi":"10.22002/5m8tf-2aq36","resourceType":"Audiovisual","name":"Extended data video 2.mp4","url":"https://portal.micropublication.org/uploads/81718a714afb503b091eb832cfdf7b83.mp4"},{"description":"20 sec swim video of tex9(RNAi) worms","doi":"10.22002/f41a4-hgf75","resourceType":"Audiovisual","name":"Extended data video 3.mp4","url":"https://portal.micropublication.org/uploads/7f9e500c2994653f6c35b04a9f56bac3.mp4"},{"description":"edema observations","doi":"10.22002/s8625-b8x92","resourceType":"Image","name":"Extended data image 1 - edema observations.jpg","url":"https://portal.micropublication.org/uploads/c462d033a0bdc6863d41653ffc1d9cd4.jpg"}],"funding":"Student research participation and publication support were funded by the Cell Biology Education Consortium (CBEC): Path to Publication (Award ID #2316122) through the National Science Foundation (NSF) and NH-INBRE (NIH Award: P20GM103506).
","image":{"url":"https://portal.micropublication.org/uploads/67b48af00eed41a196fcff43d7e36c1b.png"},"imageCaption":"A) Pathway tracing of RNAi-mediated knockdown worms shows reduced distance traveled by gliding motility. Overlays of sequential frames (t=20s) from 20 second video segments, illustrating the distance travelled by individual flatworms in control (neg.ctrl.(RNAi)), ropn1l(RNAi), and tex9(RNAi) worms. Z-projection within FIJI/Image J software was used to perform the path tracing analysis. Scale bar: 10mm B) Differential interference microscopy (DIC) images at 63X magnification show planarian cilia length. Gene knockdown of ropn1l and tex9 resulted in visibly shorter cilia. Scale bar = 10µm. C) RNAi-mediated knockdown impairs planarian swim speed. Mean swim speed (± SEM) for neg.ctrl.(RNAi) worms was 1.3 ± 0.05 mm/s. ropn1l(RNAi) and tex9(RNAi) worms displayed 0.96 ± 0.17 mm/s and 0.87 ± 0.09 mm/s swim speeds, respectively (****p<0.0001, one-way ANOVA, n=30). Error bars indicate SEM. D) RNAi-mediated knockdown reduces cilia length in planarians. Mean cilia length (± SEM) in neg.ctrl.(RNAi) worms was 11.04 ± 0.19 µm. ropn1l(RNAi) and tex9(RNAi) worms exhibited 6.94 ± 0.40 µm and 6.77 ± 0.30 µm cilia lengths, respectively (****p<0.0001, one-way ANOVA, n=5). Error bars indicate SEM. E) Single-cell RNA-Seq expression of ropn1l (dd_Smed_v6_4888_0) in planarian cell clusters. Expression of ropn1l is strongly enriched in pharyngeal, epidermal, and specific neuronal cells with lower expression in other lineages (lineage diagram created using Planaria Single Cell Database; https://shiny.mdc-berlin.de/psca/). F) Single-cell RNA-Seq expression of tex9 (dd_Smed_v6_7007_0) in planarian cell clusters. Expression of tex9 is strongly enriched in pharyngeal and neuronal cells with lower expression in other lineages (lineage diagram created using Planaria Single Cell Database; https://shiny.mdc-berlin.de/psca/).
","imageTitle":"Knockdown of Smed-ropn1l and Smed-tex9 reduces planarian gliding locomotion and cilia length.
","methods":"Planarian maintenance and feeding protocol
Planarians (S. mediterranea asexual strain ClW4) were maintained in a dark environment at room temperature in vessels containing water prepared as follows: 1.6mM NaCl, 1.0mM CaCl2, 1.0mM MgSO4, 0.1mM KCl, 1.2mM NaHCO3 (Cebrià & Newmark, 2005). Worms were fed once weekly with organic beef liver paste. After two hours of feeding, any remaining food was removed using plastic transfer pipettes and a small volume of fresh planarian water was added to top off the vessel. A complete water change was performed every two days to remove accumulated excretory waste and maintain optimal water quality (Rompolas et al., 2013).
RNAi plasmid cloning and feeding
Following the methods established in our previous study (Gogoi et al., 2026), RNAi constructs were generated and administered in Schmidtea mediterranea with gene-specific modifications as described below. FASTA transcript sequences for ropn1l (dd_Smed_v6_4888_0_1) and tex9 (dd_Smed_v6_7007_0_1) were obtained from the PlanMine database, and Benchling was used to design primers generating 200–800 bp fragments with plasmid-homologous overhangs compatible with the pPR-T4P vector. A 543 bp fragment of ropn1l and a 615 bp fragment of tex9 were amplified from cDNA using gene-specific primers sharing identical pPR-T4P overhang sequences, with homology arms indicated in capital letters and gene-specific regions in lowercase (primer sequences available under Reagents). Total RNA was isolated from homogenized animals, reverse transcribed to cDNA, and target fragments were amplified using either Q5 or Taq polymerase, verified by gel electrophoresis, purified, and quantified to calculate a 2:1 insert-to-backbone ratio. Inserts were cloned into pPR-T4P using Gibson Assembly for ropn1l and InFusion assembly for tex9. Assembled constructs were transformed into E. coli DH5α, screened by M13 PCR, purified, and sequence-verified by nanopore sequencing prior to RNAi food preparation. The C. elegans gene unc-22 with no known homolog in the planarian genome was used as a negative control neg.cont.(RNAi) (Wagner, 2012). Verified plasmids were subsequently transformed into E. coli HT115 for dsRNA production, and induced cultures were mixed with homogenized liver for feeding. Following a one-week starvation period, animals were fed six times over two weeks (every 2-3 days). For each RNAi condition, three experimental plates containing ten animals each were analyzed.
Seven days after the final RNAi feeding, phenotypic assays were performed as follows:
Cilia Phenotype Analysis:
Planarians were immobilized by incubation in a relaxant solution (1% HNO3, 0.8325% formaldehyde, and 50 mM MgSO4) for 5 minutes prior to mounting on glass slides. Specimens were imaged using differential interference contrast (DIC) optics on a Zeiss LSM 510 Meta confocal microscope with a tungsten halogen lamp using a Plan Apochromat 63x/1.4 oil immersion objective lens with pixel size of 0.07μm/px. Images were captured using a Teledyne Lumenera Infinity 3 camera with Infinity Analyze software. For each condition, five worms were analyzed. Images were captured from both the left and right sides of the head region, near the eyes, where cilia are more densely distributed. From each image, the lengths of 10 individual cilia were measured, resulting in 10 data points per image. Measurements were performed using Fiji/ImageJ software to calculate the mean cilia length for both control and RNAi-treated planarians.
Gliding Assays:
Planarian locomotion was recorded over a 20-second period using an Andonstar AD246S-M HDMI Digital Microscope. Videos were analyzed in Fiji ImageJ, where z-projection was applied to generate tracks of individual worms. The lengths of these tracks were measured to calculate average swimming speed (mm/s).
Statistical Analysis:
Gliding velocity and cilia length measurements were quantified using FIJI/ImageJ software. For gliding velocity analysis, 30 individual worms were analyzed per condition. For cilia length analysis, 5 worms were used per condition, and for each worm, images were acquired from both left and right sides of the animal. Ten cilia were measured per side, yielding a total of 100 measurements per condition, which were averaged to obtain a single mean value per worm for statistical analysis.
The normality of replicates was confirmed using the Shapiro–Wilk test. Both RNAi groups (ropn1l(RNAi) and tex9(RNAi)) were compared with the same negative control (RNAi) animals. Analyses were conducted using GraphPad Prism version 10.6.1. One-way ANOVA revealed significant differences among groups for both cilia length (df=4, p < 0.0001) and gliding velocity (df = 29, p < 0.0001), and post hoc analysis using Dunnett’s multiple comparisons test further confirmed that both ropn1l(RNAi) and tex9(RNAi) worms differed significantly from negative control animals (neg.cont.(RNAi)) in both gliding speed and length of cilia (p < 0.0001 for all comparisons).
","reagents":"Category | Reagents | Use/Notes | Source of Reagent |
Plasmids & Vectors | pPRt4p plasmid | Expression vector backbone for cloning & RNAi experiments | Gifted by Jason Pellettieri |
Bacterial Strains | HT115 E. coli | RNAi feeding strain (RNase III deficient, used in dsRNA expression) | Gifted by Jason Pellettieri |
| NEB 5-alpha E. coli | High efficiency cloning strain for plasmid propagation | New England Biolabs |
Primers | ropn1l-fwd | CATTACCATCCCGaagctgccattcgtacccagcc | Invitrogen |
| ropn1l-rev | CCAATTCTACCCGtgcttggttgaacgaggccacc | Invitrogen |
| tex9-fwd | CATTACCATCCCGtccagcaaattccaacacgttcca | Invitrogen |
| tex9-rev | CCAATTCTACCCGgctttctgacgctctagccttctgc | Invitrogen |
Cilia are small hair-like structures that are highly evolutionarily conserved and found on the surface of most eukaryotic cells (Horani & Ferkol, 2021). There are two main types of cilia, primary (non-motile) and motile which include flagella and nodal cilia (Hyland & Brody, 2021). Motile cilia and related microtubule-based structures, such as flagella, are essential for a wide range of cellular and developmental processes across eukaryotes. Ciliopathies are a set of human diseases arising from genetic mutations that affect cilia structure or function. Many ciliopathies currently lack curative treatments, although ongoing research is exploring gene therapy approaches that aim to address underlying causes rather than only managing symptoms (Wee et al., 2024). Yet, our understanding of these diseases is still not fully understood (Hyland & Brody, 2021).
There are over 2000 genes that have been associated with cilia formation and function, and this list is growing (Yenisert & Kaplan, 2025). Despite their importance, many cilia-associated proteins remain poorly characterized. Rhophilin associated tail protein 1–like (ROPN1L) and testis-expressed protein 9 (TEX9) have been identified through proteomic and transcriptomic studies as candidate components of cilia or flagella in humans, mice, and flies, with some evidence suggesting roles in flagellar or sperm tail structure. However, direct functional analyses of these genes are limited. To address this gap, we used RNA interference to examine the functions of Smed-ropn1l and Smed-tex9, planarian homologs of the human genes, in Schmidtea mediterranea, a model well suited for studying motile cilia, and evaluated their roles in ciliary structure and function.
ROPN1L is a scaffold protein in the ROPN1/ROPN1L–Dependent 2 (R2D2) family that anchors protein kinase A (PKA) signaling complexes to cilia and flagella (Bontems et al., 2014). In human bronchial epithelial cells, ROPN1L was identified as one of 37 key cilia-associated genes upregulated during mucociliary differentiation, based on transcriptomic analysis (Ross et al., 2007). Proteomic and immunolocalization data from the same study further demonstrates that ROPN1L localizes to the ciliary axoneme, providing direct evidence of its association with cilia (Ross et al., 2007). Functional evidence for the role of ROPN1L in microtubule-based motility comes from mouse models, where knockout of Ropn1l or its paralog rhophilin associated tail protein 1 (Ropn1) results in moderately reduced sperm motility, while deletion of both genes causes complete sperm immotility, structural disruption of the fibrous sheath, and loss of A kinase anchoring protein 3 (AKAP3) in sperm flagella (Fiedler et al., 2013). Consistent with these findings, qPCR and RNA-seq analyses of germ cells from men with cryptorchidism, a congenital condition in which one or both testes fail to descend into the scrotum, reveals ROPN1L as the most downregulated gene among several fertility-associated targets, including AKAP4, IZUMO1, and SPAG6, further supporting its role in human fertility (Sun et al., 2023).
Functional and comparative genomic analysis of TEX9 across eukaryotes suggests a role for TEX9 in cilia biology. Phylogenetic profiling across 100 eukaryotic genomes identified TEX9 as a candidate ciliary protein, and cross-species comparisons of flagellated and non-flagellated organisms placed TEX9 within a highly conserved subset of six proteins associated with cilia-related centriolar satellites (Wu et al., 2025). Consistent with these predictions, CilioGenics—an integrative platform combining single-cell RNA sequencing, protein–protein interactions, comparative genomics, transcription factor network analysis, and text mining—identified TEX9 among 256 candidate ciliary genes, including 89 previously annotated by CiliaCarta and 28 with experimental validation (Pir et al., 2024). Proteomic analyses detected TEX9 in human testes, suggesting an association with sperm development and cilia or flagella formation (Vandenbrouck et al., 2020). Direct evidence for ciliary association was provided by immunofluorescence studies demonstrating localization of TEX9 to ciliary basal bodies in cultured human kidney proximal tubule epithelial (HK2) cells (Nevers et al., 2017). Functional support for a role in cilia and flagella comes from loss-of-function studies, where RNAi knockdown of TEX9 in Drosophila causes sensory cilia defects and impaired sperm flagellar motility, and C. elegans mutants exhibit dye-fill defects indicative of structural or trafficking abnormalities in sensory cilia (Dobbelaere et al., 2023).
Widely studied for its regenerative abilities, S. mediterranea (freshwater planaria) has a fully sequenced genome and robust molecular tools, including RNAi for gene function analysis, making it an ideal model for studying ciliary genes. The planarian is a low-cost, easily maintained laboratory model and is one of few multicellular organisms with an external multiciliated epithelium. Cilia are used in planarian locomotion and are external and visible in live specimens under the microscope (Rabiasz & Ziętkiewicz, 2023; Rompolas et al., 2009; Rompolas et al., 2010). Ventral epithelial cells bear multiple cilia that beat against a secreted mucus layer, together with muscle contraction, to drive locomotion, similar to mucus transport in the human airway, reproductive tract, and brain ventricles. Defects in ciliary function are therefore expected to cause visibly impaired locomotion. Additionally, protonephridia depend on ciliary movement for waste filtration and osmoregulation, and ciliary disruption can impair this function, resulting in fluid retention and edema. (Reddien et al., 2005; Rink et al., 2009; Rink et al., 2011; Vu et al., 2015).
RNAi-mediated knockdown of ropn1l and tex9 in S. mediterranea resulted in clear phenotypic changes in swim rate when compared to neg.cont.(RNAi) (Fig. 1A; Extended data video 1 – neg.cont.(RNAi) 20 sec., Extended data video 2 – ropn1l(RNAi) 20 sec., Extended data video 3 – tex9(RNAi) 20 sec.). One week post-RNAi treatment, gliding locomotion was assessed using 20 second video segments, and distance traveled over time was determined using z-projection in Fiji/Image J. Quantitative analysis (n = 30) demonstrated that control worms (neg.cont.(RNAi)) had a mean swim rate ± SEM of 1.30 ± 0.05 mm/s, whereas ropn1l(RNAi) and tex9(RNAi) worms had significantly reduced swim speeds. ropn1l(RNAi) mean swim rate was 0.96 ± 0.17 mm/s and tex9(RNAi) mean swim rate was 0.87 ± 0.09 mm/s, representing decreases of 26.4% and 33.2%, respectively. (Fig 1C).
Differential interference contrast microscopy images of cilia located in the lateral region of planarian heads were taken, and these indicated reduced cilia length for ropn1l(RNAi) and tex9(RNAi) treated worms (Fig 1B). Quantitative analysis demonstrated significant reductions in cilia length in ropn1l(RNAi) and tex9(RNAi) worms, corresponding to decreases of 37.1% and 38.7%, respectively (mean ± SEM: 6.94 ± 0.40 µm and 6.77 ± 0.30 µm), relative to negative control (neg.cont.(RNAi)) worms (11.04 ± 0.19 µm) (Fig 1D). These data strongly suggest that functionally, these genes are important for ciliary integrity. In addition, edema in the trunk region of a small number of the ropn1l(RNAi) and tex9(RNAi) worms was observed, indicating potential defects in fluid regulation by the flame cells of the protonephridia (Reddien et al.,2005; Rink et al., 2009; Rink et al., 2011; Vu et al., 2015). This edema was not observed in any of our neg.cont.(RNAi) worms (Extended data image 1 – edema observations).
Single-cell transcriptomic analysis of ropn1l and tex9 obtained from Planaria Single Cell Database (https://shiny.mdc-berlin.de/psca/) revealed that these genes exhibit strongest expression in specific neuronal subtypes, epidermal populations, protonephridia, and a distinct pharyngeal cell type described as epidermal-related (Fig. 1E, F) (Plass et al., 2018). In addition, single-cell transcriptomic data from Planarian Digiworm (https://digiworm.wi.mit.edu/) indicates that some of the highest-expressing cells for both genes are found in the protonephridial cluster (cluster 29), further supporting a role in protonephridial ciliary function (Fincher et al., 2018). This observation provides a link to the edema phenotype observed in a subset of RNAi knockdown animals. The lineage diagrams (Fig 1E, F) also depict inferred differentiation trajectories, in which increased gene expression toward the distal tips reflects enrichment in terminally differentiated cell types. Because the formation of motile cilia is expected to occur late in differentiation, the localization of ropn1l and tex9 expression toward terminal branches is consistent with their proposed roles in motile ciliogenesis. Across major cell categories including secretory, muscle, gut, and parenchymal populations both genes show comparatively low expression, whereas enrichment is observed in specific neurons, epidermis, protonephridia, and pharyngeal lineages, all of which include tissues known to harbor abundant motile cilia (Fincher et al., 2018).
Our results show that Schmidtea mediterranea is a valuable in vivo model for studying conserved ciliary genes. Since defects in motile cilia are linked to human disease, analyzing ROPN1L and TEX9 helps clarify shared molecular mechanisms across tissues and species. Although ROPN1L and TEX9 function through distinct molecular pathways, both genes are strongly associated with spermatogenesis and sperm flagellar structure, suggesting a shared role in motile microtubule-based systems (Sharma et al., 2014). We therefore presented these genes together in an exploration of their potential involvement in ciliary structure and function. In summary, our findings demonstrate that ROPN1L and TEX9 are required for the maintenance of normal ciliary length and motile function in multiciliated cells in planarians. Integration of single-cell expression analyses with RNAi-mediated knockdown phenotypes—including impaired gliding motility, shortened cilia, and posterior abdominal edema—provides compelling functional evidence that these genes are conserved components essential for proper motile cilia assembly and performance. These results extend prior proteomic and transcriptomic associations by establishing a direct role for ROPN1L and TEX9 in ciliary biology.
","references":[{"reference":"Bontems F, Fish RJ, Borlat I, Lembo Fdr, Chocu S, Chalmel Fdr, et al., Lane. 2014. C2orf62 and TTC17 Are Involved in Actin Organization and Ciliogenesis in Zebrafish and Human. PLoS ONE 9: e86476.
","pubmedId":"","doi":"10.1371/journal.pone.0086476"},{"reference":"Cebrià F, Newmark PA. 2005. Planarian homologs ofnetrinandnetrin receptorare required for proper regeneration of the central nervous system and the maintenance of nervous system architecture. Development 132: 3691-3703.
","pubmedId":"","doi":"10.1242/dev.01941"},{"reference":"Dobbelaere J, Su TY, Erdi B, Schleiffer A, Dammermann A. 2023. A phylogenetic profiling approach identifies novel ciliogenesis genes in\n Drosophila\n and\n C. elegans. The EMBO Journal 42: 10.15252/embj.2023113616.
","pubmedId":"","doi":"10.15252/embj.2023113616"},{"reference":"Fiedler SE, Dudiki T, Vijayaraghavan S, Carr DW. 2013. Loss of R2D2 Proteins ROPN1 and ROPN1L Causes Defects in Murine Sperm Motility, Phosphorylation, and Fibrous Sheath Integrity1. Biology of Reproduction 88: 10.1095/biolreprod.112.105262.
","pubmedId":"","doi":"10.1095/biolreprod.112.105262"},{"reference":"Fincher CT, Wurtzel O, de Hoog T, Kravarik KM, Reddien PW. 2018. Cell type transcriptome atlas for the planarian\n Schmidtea mediterranea. Science 360: 10.1126/science.aaq1736.
","pubmedId":"","doi":"10.1126/science.aaq1736"},{"reference":"Gogoi C, Pitt R, Mazur K, Naraharisetti R, Johnson K. 2026. undefined. microPublication Biology. 2026","pubmedId":"","doi":"10.17912/MICROPUB.BIOLOGY.001887"},{"reference":"Horani A, Ferkol TW. 2021. Understanding Primary Ciliary Dyskinesia and Other Ciliopathies. The Journal of Pediatrics 230: 15-22.e1.
","pubmedId":"","doi":"10.1016/j.jpeds.2020.11.040"},{"reference":"Hyland RM, Brody SL. 2021. Impact of Motile Ciliopathies on Human Development and Clinical Consequences in the Newborn. Cells 11: 125.
","pubmedId":"","doi":"10.3390/cells11010125"},{"reference":"Nevers Y, Prasad MK, Poidevin L, Chennen K, Allot A, Kress A, et al., Lecompte. 2017. Insights into Ciliary Genes and Evolution from Multi-Level Phylogenetic Profiling. Molecular Biology and Evolution 34: 2016-2034.
","pubmedId":"","doi":"10.1093/molbev/msx146"},{"reference":"Newmark PA, Reddien PW, Cebrià F, Alvarado ASn. 2003. Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians. Proceedings of the National Academy of Sciences 100: 11861-11865.
","pubmedId":"","doi":"10.1073/pnas.1834205100"},{"reference":"Pir MS, Begar E, Yenisert F, Demirci HC, Korkmaz ME, Karaman A, et al., Kaplan. 2024. CilioGenics: an integrated method and database for predicting novel ciliary genes. Nucleic Acids Research 52: 8127-8145.
","pubmedId":"","doi":"10.1093/nar/gkae554"},{"reference":"Plass M, Solana J, Wolf FA, Ayoub S, Misios A, Glažar P, et al., Rajewsky. 2018. Cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics. Science 360: 10.1126/science.aaq1723.
","pubmedId":"","doi":"10.1126/science.aaq1723"},{"reference":"Rabiasz A, Ziętkiewicz E. 2023. Schmidtea mediterranea as a Model Organism to Study the Molecular Background of Human Motile Ciliopathies. International Journal of Molecular Sciences 24: 4472.
","pubmedId":"","doi":"10.3390/ijms24054472"},{"reference":"Reddien PW, Bermange AL, Murfitt KJ, Jennings JR, Sánchez Alvarado A. 2005. Identification of Genes Needed for Regeneration, Stem Cell Function, and Tissue Homeostasis by Systematic Gene Perturbation in Planaria. Developmental Cell 8: 635-649.
","pubmedId":"","doi":"10.1016/j.devcel.2005.02.014"},{"reference":"Rink JC, Gurley KA, Elliott SA, Sánchez Alvarado A. 2009. Planarian Hh Signaling Regulates Regeneration Polarity and Links Hh Pathway Evolution to Cilia. Science 326: 1406-1410.
","pubmedId":"","doi":"10.1126/science.1178712"},{"reference":"Rink JC, Vu HTK, Alvarado ASn. 2011. The maintenance and regeneration of the planarian excretory system are regulated by EGFR signaling. Development 138: 3769-3780.
","pubmedId":"","doi":"10.1242/dev.066852"},{"reference":"Rompolas P, Azimzadeh J, Marshall WF, King SM. 2013. Analysis of Ciliary Assembly and Function in Planaria. Methods in Enzymology,Cilia, Part B : 245-264.
","pubmedId":"","doi":"10.1016/B978-0-12-397944-5.00012-2"},{"reference":"Rompolas P, Patel-King RS, King SM. 2009. Schmidtea mediterranea: a model system for analysis of motile cilia. Methods Cell Biol 93: 81-98.
","pubmedId":"","doi":"https://doi.org/10.1016/S0091-679X(08)93004-1"},{"reference":"Rompolas, P., Patel-King, R. S., & King, S. M. (2010). An outer arm Dynein conformational switch is required for metachronal synchrony of motile cilia in planaria. Molecular biology of the cell, 21(21), 3669–3679. https://doi.org/10.1091/mbc.E10-04-0373
","pubmedId":"","doi":""},{"reference":"Ross AJ, Dailey LA, Brighton LE, Devlin RB. 2007. Transcriptional Profiling of Mucociliary Differentiation in Human Airway Epithelial Cells. American Journal of Respiratory Cell and Molecular Biology 37: 169-185.
","pubmedId":"","doi":"10.1165/rcmb.2006-0466OC"},{"reference":"Sharma E, Künstner A, Fraser BA, Zipprich G, Kottler VA, Henz SR, Weigel D, Dreyer C. 2014. Transcriptome assemblies for studying sex-biased gene expression in the guppy, Poecilia reticulata. BMC Genomics 15: 10.1186/1471-2164-15-400.
","pubmedId":"","doi":"doi.org/10.1186/1471-2164-15-400"},{"reference":"Sun W, Zhang X, Wang L, Ren G, Piao S, Yang C, Liu Z. 2023. RNA sequencing profiles reveals progressively reduced spermatogenesis with progression in adult cryptorchidism. Frontiers in Endocrinology 14: 10.3389/fendo.2023.1271724.
","pubmedId":"","doi":"10.3389/fendo.2023.1271724"},{"reference":"Vu HTK, Rink JC, McKinney SA, McClain M, Lakshmanaperumal N, Alexander R, Sánchez Alvarado A. 2015. Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ. eLife 4: e07405.
","pubmedId":"","doi":""},{"reference":"Vandenbrouck Y, Pineau C, Lane L. 2020. The Functionally Unannotated Proteome of Human Male Tissues: A Shared Resource to Uncover New Protein Functions Associated with Reproductive Biology. Journal of Proteome Research 19: 4782-4794.
","pubmedId":"","doi":"10.1021/acs.jproteome.0c00516"},{"reference":"Wagner DE, Ho JJ, Reddien PW. 2012. Genetic Regulators of a Pluripotent Adult Stem Cell System in Planarians Identified by RNAi and Clonal Analysis. Cell Stem Cell 10: 299-311.
","pubmedId":"","doi":"10.1016/j.stem.2012.01.016"},{"reference":"Wee WB, Kinghorn B, Davis SD, Ferkol TW, Shapiro AJ. 2024. Primary Ciliary Dyskinesia. Pediatrics 153: 10.1542/peds.2023-063064.
","pubmedId":"","doi":"10.1542/peds.2023-063064"},{"reference":"Wu B, Long C, Liu J, Huang X, Ma S, Ma Y, et al., Li. 2025. A subset of evolutionarily conserved centriolar satellite core components is crucial for sperm flagellum biogenesis. Theranostics 15: 7025-7044.
","pubmedId":"","doi":"10.7150/thno.117118"},{"reference":"Yenisert F, Kaplan OI. 2025. Expanded Catalog of Gold Standard Ciliary Gene List: Integration of Novel Ciliary Genes into the Ciliome. : 10.1101/2025.08.22.671678.
","pubmedId":"","doi":"10.1101/2025.08.22.671678"}],"title":"Insights into Human Ciliopathies: Gene Silencing of ropn1l and tex9 in Schmidtea mediterranea Indicate Association with Ciliary Structure and Motility
","reviews":[],"curatorReviews":[]},{"id":"214f47f7-3673-4011-b3a0-4081d6696f67","decision":"edit","abstract":"Cilia are microtubule-based organelles essential for motility, sensory signaling and development. In humans, motile cilia facilitate fluid movement, and their dysfunction causes ciliopathies, including infertility. We used RNAi-mediated knockdown of two human spermatid flagellar genes in Schmidtea mediterranea to assess effects on ciliary function and locomotion. Knockdown of Smed-ropn1l and Smed-tex9 significantly reduced planarian swim rate by 26.4% and 33.2% respectively and shortened cilia by 37.1% and 38.7% respectively. These findings highlight the critical roles of ROPN1L and TEX9 in cilia function and the use of planarians as a valuable model for studying ciliopathies.
","acknowledgements":"
We thank Kaleigh Powers and Dr. Jason Pellettieri (Keene State College) for their valuable guidance, Dr. Mark Scimone (University of New Hampshire) for assistance with DIC microscopy. We are also grateful to Dr. Megan Thompson (Oyster River High School) and Dr. A. Sanchez Alvarado (Stowers Institute) for generously providing the planaria used in this study.
","authors":[{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["dataCuration","formalAnalysis","investigation","validation","writing_originalDraft","visualization","writing_reviewEditing"],"email":"Rachel.Pitt@unh.edu","firstName":"Rachel ","lastName":"Pitt","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["dataCuration","formalAnalysis","investigation","validation","writing_originalDraft","visualization","writing_reviewEditing"],"email":"chayanika.gogoi@unh.edu","firstName":"Chayanika","lastName":"Gogoi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["investigation"],"email":"Angelina.Calnan@unh.edu","firstName":"Angelina","lastName":"Calnan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["investigation"],"email":"Lata.Patnaik@unh.edu","firstName":"Lata","lastName":"Patnaik","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["conceptualization","formalAnalysis","fundingAcquisition","investigation","methodology","project","resources","supervision","validation","writing_reviewEditing"],"email":"kristen.johnson@unh.edu","firstName":"Kristen C","lastName":"Johnson","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" https://orcid.org/0000-0002-1825-0265"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"20 sec swim video of neg.cont (RNAi) worms","doi":"10.22002/z5vja-gfv92","resourceType":"Audiovisual","name":"Extended data video 1.mp4","url":"https://portal.micropublication.org/uploads/1dcb1441dd3d5a4e47d137ea62d079de.mp4"},{"description":"20 sec swim video of ropn1l(RNAi) worms","doi":"10.22002/5m8tf-2aq36","resourceType":"Audiovisual","name":"Extended data video 2.mp4","url":"https://portal.micropublication.org/uploads/81718a714afb503b091eb832cfdf7b83.mp4"},{"description":"20 sec swim video of tex9(RNAi) worms","doi":"10.22002/f41a4-hgf75","resourceType":"Audiovisual","name":"Extended data video 3.mp4","url":"https://portal.micropublication.org/uploads/7f9e500c2994653f6c35b04a9f56bac3.mp4"},{"description":"edema observations","doi":"10.22002/s8625-b8x92","resourceType":"Image","name":"Extended data image 1 - edema observations.jpg","url":"https://portal.micropublication.org/uploads/c462d033a0bdc6863d41653ffc1d9cd4.jpg"}],"funding":"Student research participation and publication support were funded by the Cell Biology Education Consortium (CBEC): Path to Publication (Award ID #2316122) through the National Science Foundation (NSF) and NH-INBRE (NIH Award: P20GM103506).
","image":{"url":"https://portal.micropublication.org/uploads/0ca3ef7d3ff1a8a06f822292e1bc7a8a.jpg"},"imageCaption":"A) Pathway tracing of RNAi-mediated knockdown worms shows reduced gliding motility. Overlays of sequential frames from 20 second video segments, illustrating the distance travelled by individual flatworms in control (neg.ctrl.(RNAi)), ropn1l(RNAi), and tex9(RNAi) worms. Z-projection within FIJI/Image J software was used to perform the path tracing analysis. Scale bar = 10mm B) Differential Interference Contrast Microscopy images at 63X magnification show planarian cilia length. Gene knockdown of ropn1l and tex9 resulted in visibly shorter cilia. Scale bar = 10µm. C) RNAi-mediated knockdown impairs planarian swim speed. Mean swim speed (± SEM) for neg.ctrl.(RNAi) worms was 1.3 ± 0.05 mm/s. ropn1l(RNAi) and tex9(RNAi) worms displayed 0.96 ± 0.17 mm/s and 0.87 ± 0.09 mm/s swim speeds, respectively (****p<0.0001, one-way ANOVA, n=30). D) RNAi-mediated knockdown reduces cilia length in planarians. Mean cilia length (± SEM) in neg.ctrl.(RNAi) worms was 11.04 ± 0.19 µm. ropn1l(RNAi) and tex9(RNAi) worms exhibited 6.94 ± 0.40 µm and 6.77 ± 0.30 µm cilia lengths, respectively (****p<0.0001, one-way ANOVA, n=5). E) Single-cell RNA-Seq expression of ropn1l (dd_Smed_v6_4888_0) in planarian cell clusters. Expression of ropn1l is strongly enriched in pharyngeal, epidermal, and specific neuronal cells with lower expression in other lineages (lineage diagram created using Planaria Single Cell Database; https://shiny.mdc-berlin.de/psca/). F) Single-cell RNA-Seq expression of tex9 (dd_Smed_v6_7007_0) in planarian cell clusters. Expression of tex9 is strongly enriched in pharyngeal and neuronal cells with lower expression in other lineages (lineage diagram created using Planaria Single Cell Database; https://shiny.mdc-berlin.de/psca/).
","imageTitle":"Knockdown of Smed-ropn1l and Smed-tex9 reduces planarian gliding locomotion and cilia length.
","methods":"Planarian maintenance and feeding protocol
Planarians (S. mediterranea asexual strain ClW4) were maintained in a dark environment at room temperature in vessels containing water prepared as follows: 1.6mM NaCl, 1.0mM CaCl2, 1.0mM MgSO4, 0.1mM KCl, 1.2mM NaHCO3 (Cebrià & Newmark, 2005). Worms were fed once weekly with organic beef liver paste. After two hours of feeding, any remaining food was removed using plastic transfer pipettes, and a small volume of fresh planarian water was added to top off the vessel. A complete water change was performed every two days to remove accumulated excretory waste and maintain optimal water quality (Rompolas et al., 2013).
RNAi plasmid cloning and feeding
Following the methods established in our previous study (Gogoi et al., 2026), RNAi constructs were generated and administered in Schmidtea mediterranea with gene-specific modifications as described below. FASTA transcript sequences for ropn1l (dd_Smed_v6_4888_0_1) and tex9 (dd_Smed_v6_7007_0_1) were obtained from the PlanMine database, and Benchling was used to design primers generating 200–800 bp fragments with plasmid-homologous overhangs compatible with the pPR-T4P vector. A 543 bp fragment of ropn1l and a 615 bp fragment of tex9 were amplified from cDNA using gene-specific primers with homology arms indicated in capital letters and gene-specific regions in lowercase (primer sequences available under Reagents). Total RNA was isolated from homogenized animals, reverse transcribed to cDNA, and target fragments were amplified using either Q5 or Taq polymerase, verified by gel electrophoresis, purified, and quantified to calculate a 2:1 insert-to-backbone ratio. Inserts were cloned into pPR-T4P using Gibson Assembly for ropn1l and InFusion assembly for tex9. Assembled constructs were transformed into E. coli DH5α, screened by M13 PCR, purified, and sequence-verified by nanopore sequencing prior to RNAi food preparation. The C. elegans gene unc-22 with no known homolog in the planarian genome was used as a negative control neg.cont.(RNAi) (Wagner, 2012). Verified plasmids were subsequently transformed into E. coli HT115 for dsRNA production, and induced cultures were mixed with homogenized liver for feeding. Following a one-week starvation period, animals were fed six times over two weeks (every 2-3 days). For each RNAi condition, three experimental dishes containing ten animals each were analyzed (n=30).
Seven days after the final RNAi feeding, phenotypic assays were performed as follows:
Cilia Phenotype Analysis:
Planarians were immobilized by incubation in a relaxant solution (1% HNO3, 0.8325% formaldehyde, and 50 mM MgSO4) for 5 minutes prior to mounting on glass slides. Specimens were imaged using differential interference contrast (DIC) optics on a Zeiss LSM 510 Meta confocal microscope with a tungsten halogen lamp using a Plan Apochromat 63x/1.4 oil immersion objective lens with pixel size of 0.07μm/px. Images were captured using a Teledyne Lumenera Infinity 3 camera with Infinity Analyze software. For each condition, five worms were analyzed. Images were captured from both the left and right sides of the head region, near the eyes, where cilia are more densely distributed. From each image, the lengths of 10 individual cilia were measured, resulting in 10 data points per image. Measurements were performed using Fiji/ImageJ software to calculate the mean cilia length for both control and RNAi-treated planarians.
Gliding Assays:
Planarian locomotion was recorded over a 20-second period using an Andonstar AD246S-M HDMI Digital Microscope. Videos were analyzed in Fiji ImageJ, where z-projection was applied to generate tracks of individual worms. The lengths of these tracks were measured to calculate average swimming speed (mm/s).
Statistical Analysis:
Gliding velocity and cilia length measurements were quantified using FIJI/ImageJ software. For gliding velocity analysis, 30 individual worms were analyzed per condition. For cilia length analysis, 5 worms were used per condition, and for each worm, images were acquired from both left and right sides of the animal. Ten cilia were measured per side, yielding a total of 100 measurements per condition, which were averaged to obtain a single mean value per worm for statistical analysis.
The normality of replicates was confirmed using the Shapiro–Wilk test. Both RNAi groups (ropn1l(RNAi) and tex9(RNAi)) were compared with the same negative control neg.cont.(RNAi) animals. Analyses were conducted using GraphPad Prism version 10.6.1. One-way ANOVA revealed significant differences among groups for both cilia length (df=4, p < 0.0001) and gliding velocity (df = 29, p < 0.0001), and post hoc analysis using Dunnett’s multiple comparisons test further confirmed that both ropn1l(RNAi) and tex9(RNAi) worms differed significantly from negative control animals (neg.cont.(RNAi)) in both gliding speed and length of cilia (p < 0.0001 for all comparisons).
","reagents":"Category | Reagents | Use/Notes | Source of Reagent |
Plasmids & Vectors | pPRt4p plasmid | Expression vector backbone for cloning & RNAi experiments | Gifted by Jason Pellettieri |
Bacterial Strains | HT115 E. coli | RNAi feeding strain (RNase III deficient, used in dsRNA expression) | Gifted by Jason Pellettieri |
| NEB 5-alpha E. coli | High efficiency cloning strain for plasmid propagation | New England Biolabs |
Primers | ropn1l-fwd | CATTACCATCCCGaagctgccattcgtacccagcc | Invitrogen |
| ropn1l-rev | CCAATTCTACCCGtgcttggttgaacgaggccacc | Invitrogen |
| tex9-fwd | CATTACCATCCCGtccagcaaattccaacacgttcca | Invitrogen |
| tex9-rev | CCAATTCTACCCGgctttctgacgctctagccttctgc | Invitrogen |
Cilia are small hair-like structures that are highly evolutionarily conserved and found on the surface of most eukaryotic cells (Horani & Ferkol, 2021). There are two main types of cilia, primary (non-motile) and motile which include flagella and nodal cilia (Hyland & Brody, 2021). Motile cilia and related microtubule-based structures, such as flagella, are essential for a wide range of cellular and developmental processes across eukaryotes. Ciliopathies are a set of human diseases arising from genetic mutations that affect cilia structure or function. Many ciliopathies currently lack curative treatments, although ongoing research is exploring gene therapy approaches that aim to address underlying causes rather than only managing symptoms (Wee et al., 2024). Yet, our understanding of these diseases is still not fully understood (Hyland & Brody, 2021).
Researchers have identified 1,999 unique ciliary genes, accounting for approximately 10% of all known human genes (Van Sciver & Caspary, 2024). Despite their importance, however, many cilia-associated proteins remain poorly characterized. Rhophilin associated tail protein 1–like (ROPN1L) and testis-expressed protein 9 (TEX9) have been identified through proteomic and transcriptomic studies as candidate components of cilia or flagella in humans, mice, and flies, with some evidence suggesting roles in flagellar or sperm tail structure. However, direct functional analysis of these proteins is limited. To address this gap, we used RNA interference to examine the functions of Smed-ropn1l and Smed-tex9, planarian homologs of the human genes, in Schmidtea mediterranea, a model well suited for studying motile cilia, and evaluated their roles in ciliary structure and function.
ROPN1L is a scaffold protein in the ROPN1/ROPN1L–Dependent 2 (R2D2) family that anchors protein kinase A (PKA) signaling complexes to cilia and flagella (Bontems et al., 2014). In human bronchial epithelial cells, ROPN1L is identified as one of 37 key cilia-associated genes upregulated during mucociliary differentiation, based on transcriptomic analysis (Ross et al., 2007). Proteomic and immunolocalization data from the same study further demonstrates that ROPN1L localizes to the ciliary axoneme, providing direct evidence of its association with cilia (Ross et al., 2007). Functional evidence for the role of ROPN1L in microtubule-based motility comes from mouse models, where knockout of Ropn1l or its paralog rhophilin associated tail protein 1 (Ropn1) results in moderately reduced sperm motility, while deletion of both genes causes complete sperm immotility, structural disruption of the fibrous sheath, and loss of A kinase anchoring protein 3 (AKAP3) in sperm flagella (Fiedler et al., 2013). Consistent with these findings, qPCR and RNA-seq analyses of germ cells from men with cryptorchidism, a congenital condition in which one or both testes fail to descend into the scrotum, reveals ROPN1L as the most downregulated gene among several fertility-associated targets, including AKAP4, IZUMO1, and SPAG6, further supporting its role in human fertility (Sun et al., 2023).
Functional and comparative genomic analysis of TEX9 across eukaryotes suggests a role for TEX9 in cilia biology. Phylogenetic profiling across 100 eukaryotic genomes identifies TEX9 as a candidate ciliary protein, and cross-species comparisons of flagellated and non-flagellated organisms places TEX9 within a highly conserved subset of six proteins associated with cilia-related centriolar satellites (Wu et al., 2025). Consistent with these predictions, CilioGenics—an integrative platform combining single-cell RNA sequencing, protein–protein interactions, comparative genomics, transcription factor network analysis, and text mining—identifies TEX9 among 256 candidate ciliary genes, including 89 previously annotated by CiliaCarta and 28 with experimental validation (Pir et al., 2024). TEX9 is detected in human testes via proteomic analysis, suggesting an association with sperm development and cilia or flagella formation (Vandenbrouck et al., 2020). Direct evidence for ciliary association is provided by immunofluorescence studies demonstrating localization of TEX9 to ciliary basal bodies in cultured human kidney proximal tubule epithelial (HK2) cells (Nevers et al., 2017). Functional support for a role in cilia and flagella comes from loss-of-function studies, where RNAi knockdown of TEX9 in Drosophila causes sensory cilia defects and impaired sperm flagellar motility, and C. elegans mutants exhibit dye-fill defects indicative of structural or trafficking abnormalities in sensory cilia (Dobbelaere et al., 2023).
Widely studied for its regenerative abilities, S. mediterranea (freshwater planaria) has a fully sequenced genome and robust molecular tools, including RNAi for gene function analysis, making it an ideal model for studying ciliary genes. The planarian is a low-cost, easily maintained laboratory model and is one of few multicellular organisms with an external multiciliated epithelium. Cilia are used in planarian locomotion and are external and visible in live specimens under the microscope (Rabiasz & Ziętkiewicz, 2023; Rompolas et al., 2009; Rompolas et al., 2010). Ventral epithelial cells bear multiple cilia that beat against a secreted mucus layer, together with muscle contraction, to drive locomotion, similar to mucus transport in the human airway, reproductive tract, and brain ventricles. Defects in ciliary function cause visibly impaired locomotion (Rompolas et al, 2010). Additionally, protonephridia depend on ciliary movement for waste filtration and osmoregulation, and ciliary disruption can impair this function, resulting in fluid retention and edema. (Reddien et al., 2005; Rink et al., 2009; Rink et al., 2011; Thi-Kim Vu et al., 2015).
RNAi-mediated knockdown of ropn1l and tex9 in S. mediterranea resulted in clear phenotypic changes in swim rate when compared to neg.cont.(RNAi) (Fig. 1A; Extended data video 1 – neg.cont.(RNAi) 20 sec., Extended data video 2 – ropn1l(RNAi) 20 sec., Extended data video 3 – tex9(RNAi) 20 sec.). One week post-RNAi treatment, gliding locomotion was assessed using 20 second video segments, and distance traveled over time was determined using z-projection in Fiji/Image J. Quantitative analysis (n = 30) demonstrated that control worms (neg.cont.(RNAi)) had a mean swim rate ± SEM of 1.30 ± 0.05 mm/s, whereas ropn1l(RNAi) and tex9(RNAi) worms had significantly reduced swim speeds. ropn1l(RNAi) mean swim rate was 0.96 ± 0.17 mm/s and tex9(RNAi) mean swim rate was 0.87 ± 0.09 mm/s, representing decreases of 26.4% and 33.2%, respectively. (Fig 1C).
Differential interference contrast microscopy images of cilia located in the lateral region of planarian heads were taken, and these indicated reduced cilia length for ropn1l(RNAi) and tex9(RNAi) treated worms (Fig 1B). Quantitative analysis demonstrated significant reductions in cilia length in ropn1l(RNAi) and tex9(RNAi) worms, corresponding to decreases of 37.1% and 38.7%, respectively (mean ± SEM: 6.94 ± 0.40 µm and 6.77 ± 0.30 µm), relative to negative control (neg.cont.(RNAi)) worms (11.04 ± 0.19 µm) (Fig 1D). These data strongly suggest that functionally, these genes are important for ciliary integrity. In addition, edema in the trunk region of a small number of the ropn1l(RNAi) and tex9(RNAi) worms was observed, indicating potential defects in fluid regulation by the flame cells of the protonephridia (Reddien et al.,2005; Rink et al., 2009; Rink et al., 2011; Thi-Kim Vu et al., 2015). This edema was not observed in any of our neg.cont.(RNAi) worms (Extended data image 1 – edema observations).
Single-cell transcriptomic analysis of ropn1l and tex9 obtained from Planaria Single Cell Database (https://shiny.mdc-berlin.de/psca/) reveals that these genes exhibit strongest expression in specific neuronal subtypes, epidermal populations, protonephridia, and a distinct pharyngeal cell type described as epidermal-related (Fig. 1E, F) (Plass et al., 2018). In addition, single-cell transcriptomic data from Planarian Digiworm (https://digiworm.wi.mit.edu/) indicates that some of the highest-expressing cells for both genes are found in the protonephridial cluster (cluster 29), further supporting a role in protonephridial ciliary function (Fincher et al., 2018). This observation provides a link to the edema phenotype observed in a subset of RNAi knockdown animals. The lineage diagrams (Fig 1E, F) also depict inferred differentiation trajectories, in which increased gene expression toward the distal tips reflects enrichment in terminally differentiated cell types. Because the formation of motile cilia is expected to occur late in differentiation, the localization of ropn1l and tex9 expression toward terminal branches is consistent with their proposed roles in motile ciliogenesis. Across major cell categories including secretory, muscle, gut, and parenchymal populations both genes show comparatively low expression, whereas enrichment is observed in specific neurons, epidermis, protonephridia, and pharyngeal lineages, all of which include tissues known to harbor abundant motile cilia (Fincher et al., 2018).
Our results show that Schmidtea mediterranea is a valuable in vivo model for studying conserved ciliary genes. Since defects in motile cilia are linked to human disease, analyzing ROPN1L and TEX9 helps clarify shared molecular mechanisms across tissues and species. Although ROPN1L and TEX9 function through distinct molecular pathways, both genes are strongly associated with spermatogenesis and sperm flagellar structure, suggesting a shared role in motile microtubule-based systems (Sharma et al., 2014). We therefore presented these genes together in an exploration of their potential involvement in ciliary structure and function. In summary, our findings demonstrate that ROPN1L and TEX9 are required for the maintenance of normal ciliary length and motile function in multiciliated cells in planarians. Integration of single-cell expression analyses with RNAi-mediated knockdown phenotypes—including impaired gliding motility, shortened cilia, and posterior abdominal edema—provides compelling functional evidence that these genes are conserved components essential for proper motile cilia assembly and performance. These results extend prior proteomic and transcriptomic associations by establishing direct roles for ROPN1L and TEX9 in ciliary biology.
","references":[{"reference":"Bontems F, Fish RJ, Borlat I, Lembo Fdr, Chocu S, Chalmel Fdr, et al., Lane. 2014. C2orf62 and TTC17 Are Involved in Actin Organization and Ciliogenesis in Zebrafish and Human. PLoS ONE 9: e86476.
","pubmedId":"","doi":"10.1371/journal.pone.0086476"},{"reference":"Cebrià F, Newmark PA. 2005. Planarian homologs ofnetrinandnetrin receptorare required for proper regeneration of the central nervous system and the maintenance of nervous system architecture. Development 132: 3691-3703.
","pubmedId":"","doi":"10.1242/dev.01941"},{"reference":"Dobbelaere J, Su TY, Erdi B, Schleiffer A, Dammermann A. 2023. A phylogenetic profiling approach identifies novel ciliogenesis genes in\n Drosophila\n and\n C. elegans. The EMBO Journal 42: 10.15252/embj.2023113616.
","pubmedId":"","doi":"10.15252/embj.2023113616"},{"reference":"Fiedler SE, Dudiki T, Vijayaraghavan S, Carr DW. 2013. Loss of R2D2 Proteins ROPN1 and ROPN1L Causes Defects in Murine Sperm Motility, Phosphorylation, and Fibrous Sheath Integrity1. Biology of Reproduction 88: 10.1095/biolreprod.112.105262.
","pubmedId":"","doi":"10.1095/biolreprod.112.105262"},{"reference":"Fincher CT, Wurtzel O, de Hoog T, Kravarik KM, Reddien PW. 2018. Cell type transcriptome atlas for the planarian\n Schmidtea mediterranea. Science 360: 10.1126/science.aaq1736.
","pubmedId":"","doi":"10.1126/science.aaq1736"},{"reference":"Gogoi C, Pitt R, Mazur K, Naraharisetti R, Johnson K. 2026. undefined. microPublication Biology. 2026","pubmedId":"","doi":"10.17912/MICROPUB.BIOLOGY.001887"},{"reference":"Horani A, Ferkol TW. 2021. Understanding Primary Ciliary Dyskinesia and Other Ciliopathies. The Journal of Pediatrics 230: 15-22.e1.
","pubmedId":"","doi":"10.1016/j.jpeds.2020.11.040"},{"reference":"Hyland RM, Brody SL. 2021. Impact of Motile Ciliopathies on Human Development and Clinical Consequences in the Newborn. Cells 11: 125.
","pubmedId":"","doi":"10.3390/cells11010125"},{"reference":"Nevers Y, Prasad MK, Poidevin L, Chennen K, Allot A, Kress A, et al., Lecompte. 2017. Insights into Ciliary Genes and Evolution from Multi-Level Phylogenetic Profiling. Molecular Biology and Evolution 34: 2016-2034.
","pubmedId":"","doi":"10.1093/molbev/msx146"},{"reference":"Newmark PA, Reddien PW, Cebrià F, Alvarado ASn. 2003. Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians. Proceedings of the National Academy of Sciences 100: 11861-11865.
","pubmedId":"","doi":"10.1073/pnas.1834205100"},{"reference":"Pir MS, Begar E, Yenisert F, Demirci HC, Korkmaz ME, Karaman A, et al., Kaplan. 2024. CilioGenics: an integrated method and database for predicting novel ciliary genes. Nucleic Acids Research 52: 8127-8145.
","pubmedId":"","doi":"10.1093/nar/gkae554"},{"reference":"Plass M, Solana J, Wolf FA, Ayoub S, Misios A, Glažar P, et al., Rajewsky. 2018. Cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics. Science 360: 10.1126/science.aaq1723.
","pubmedId":"","doi":"10.1126/science.aaq1723"},{"reference":"Rabiasz A, Ziętkiewicz E. 2023. Schmidtea mediterranea as a Model Organism to Study the Molecular Background of Human Motile Ciliopathies. International Journal of Molecular Sciences 24: 4472.
","pubmedId":"","doi":"10.3390/ijms24054472"},{"reference":"Reddien PW, Bermange AL, Murfitt KJ, Jennings JR, Sánchez Alvarado A. 2005. Identification of Genes Needed for Regeneration, Stem Cell Function, and Tissue Homeostasis by Systematic Gene Perturbation in Planaria. Developmental Cell 8: 635-649.
","pubmedId":"","doi":"10.1016/j.devcel.2005.02.014"},{"reference":"Rink JC, Gurley KA, Elliott SA, Sánchez Alvarado A. 2009. Planarian Hh Signaling Regulates Regeneration Polarity and Links Hh Pathway Evolution to Cilia. Science 326: 1406-1410.
","pubmedId":"","doi":"10.1126/science.1178712"},{"reference":"Rink JC, Vu HTK, Alvarado ASn. 2011. The maintenance and regeneration of the planarian excretory system are regulated by EGFR signaling. Development 138: 3769-3780.
","pubmedId":"","doi":"10.1242/dev.066852"},{"reference":"Rompolas P, Azimzadeh J, Marshall WF, King SM. 2013. Analysis of Ciliary Assembly and Function in Planaria. Methods in Enzymology,Cilia, Part B : 245-264.
","pubmedId":"","doi":"10.1016/B978-0-12-397944-5.00012-2"},{"reference":"Rompolas P, Patel-King RS, King SM. 2009. Schmidtea mediterranea: a model system for analysis of motile cilia. Methods Cell Biol 93: 81-98.
","pubmedId":"","doi":"https://doi.org/10.1016/S0091-679X(08)93004-1"},{"reference":"Rompolas, P., Patel-King, R. S., & King, S. M. (2010). An outer arm Dynein conformational switch is required for metachronal synchrony of motile cilia in planaria. Molecular biology of the cell, 21(21), 3669–3679. https://doi.org/10.1091/mbc.E10-04-0373
","pubmedId":"","doi":""},{"reference":"Ross AJ, Dailey LA, Brighton LE, Devlin RB. 2007. Transcriptional Profiling of Mucociliary Differentiation in Human Airway Epithelial Cells. American Journal of Respiratory Cell and Molecular Biology 37: 169-185.
","pubmedId":"","doi":"10.1165/rcmb.2006-0466OC"},{"reference":"Sharma E, Künstner A, Fraser BA, Zipprich G, Kottler VA, Henz SR, Weigel D, Dreyer C. 2014. Transcriptome assemblies for studying sex-biased gene expression in the guppy, Poecilia reticulata. BMC Genomics 15: 10.1186/1471-2164-15-400.
","pubmedId":"","doi":"doi.org/10.1186/1471-2164-15-400"},{"reference":"Sun W, Zhang X, Wang L, Ren G, Piao S, Yang C, Liu Z. 2023. RNA sequencing profiles reveals progressively reduced spermatogenesis with progression in adult cryptorchidism. Frontiers in Endocrinology 14: 10.3389/fendo.2023.1271724.
","pubmedId":"","doi":"10.3389/fendo.2023.1271724"},{"reference":"Van Sciver RE, Caspary T. 2024. A prioritization tool for cilia-associated genes and their in vivo resources unveils new avenues for ciliopathy research. Disease Models & Mechanisms 17: 10.1242/dmm.052000.
","pubmedId":"","doi":"10.1242/dmm.052000"},{"reference":"Vandenbrouck Y, Pineau C, Lane L. 2020. The Functionally Unannotated Proteome of Human Male Tissues: A Shared Resource to Uncover New Protein Functions Associated with Reproductive Biology. Journal of Proteome Research 19: 4782-4794.
","pubmedId":"","doi":"10.1021/acs.jproteome.0c00516"},{"reference":"Vu HTK, Rink JC, McKinney SA, McClain M, Lakshmanaperumal N, Alexander R, Sánchez Alvarado A. 2015. Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ. eLife 4: e07405.
","pubmedId":"","doi":""},{"reference":"Wagner DE, Ho JJ, Reddien PW. 2012. Genetic Regulators of a Pluripotent Adult Stem Cell System in Planarians Identified by RNAi and Clonal Analysis. Cell Stem Cell 10: 299-311.
","pubmedId":"","doi":"10.1016/j.stem.2012.01.016"},{"reference":"Wee WB, Kinghorn B, Davis SD, Ferkol TW, Shapiro AJ. 2024. Primary Ciliary Dyskinesia. Pediatrics 153: 10.1542/peds.2023-063064.
","pubmedId":"","doi":"10.1542/peds.2023-063064"},{"reference":"Wu B, Long C, Liu J, Huang X, Ma S, Ma Y, et al., Li. 2025. A subset of evolutionarily conserved centriolar satellite core components is crucial for sperm flagellum biogenesis. Theranostics 15: 7025-7044.
","pubmedId":"","doi":"10.7150/thno.117118"}],"title":"Insights into Human Ciliopathies: Gene Silencing of ropn1l and tex9 in Schmidtea mediterranea Indicate Association with Ciliary Structure and Motility
","reviews":[],"curatorReviews":[]},{"id":"80835f1e-e1c0-4899-a9a3-c4181f510436","decision":"publish","abstract":"Cilia are microtubule-based organelles essential for motility, sensory signaling and development. In humans, motile cilia facilitate fluid movement, and their dysfunction causes ciliopathies, including infertility. We used RNAi-mediated knockdown of two human spermatid flagellar genes in Schmidtea mediterranea to assess effects on ciliary function and locomotion. Knockdown of Smed-ropn1l and Smed-tex9 significantly reduced planarian swim rate by 26.4% and 33.2% respectively and shortened cilia by 37.1% and 38.7% respectively. These findings highlight the critical roles of ROPN1L and TEX9 in cilia function and the use of planarians as a valuable model for studying ciliopathies.
","acknowledgements":"
We thank Kaleigh Powers and Dr. Jason Pellettieri (Keene State College) for their valuable guidance, Dr. Mark Scimone (University of New Hampshire) for assistance with DIC microscopy. We are also grateful to Dr. Megan Thompson (Oyster River High School) and Dr. A. Sanchez Alvarado (Stowers Institute) for generously providing the planaria used in this study.
","authors":[{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["dataCuration","formalAnalysis","investigation","validation","writing_originalDraft","visualization","writing_reviewEditing"],"email":"Rachel.Pitt@unh.edu","firstName":"Rachel ","lastName":"Pitt","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["dataCuration","formalAnalysis","investigation","validation","writing_originalDraft","visualization","writing_reviewEditing"],"email":"chayanika.gogoi@unh.edu","firstName":"Chayanika","lastName":"Gogoi","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["investigation"],"email":"Angelina.Calnan@unh.edu","firstName":"Angelina","lastName":"Calnan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["investigation"],"email":"Lata.Patnaik@unh.edu","firstName":"Lata","lastName":"Patnaik","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of New Hampshire at Manchester, Manchester, New Hampshire, United States"],"departments":["Department of Life Sciences"],"credit":["conceptualization","formalAnalysis","fundingAcquisition","investigation","methodology","project","resources","supervision","validation","writing_reviewEditing"],"email":"kristen.johnson@unh.edu","firstName":"Kristen C","lastName":"Johnson","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":" https://orcid.org/0000-0002-1825-0265"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"20 sec swim video of neg.cont (RNAi) worms","doi":"10.22002/z5vja-gfv92","resourceType":"Audiovisual","name":"Extended data video 1.mp4","url":"https://portal.micropublication.org/uploads/1dcb1441dd3d5a4e47d137ea62d079de.mp4"},{"description":"20 sec swim video of ropn1l(RNAi) worms","doi":"10.22002/5m8tf-2aq36","resourceType":"Audiovisual","name":"Extended data video 2.mp4","url":"https://portal.micropublication.org/uploads/81718a714afb503b091eb832cfdf7b83.mp4"},{"description":"20 sec swim video of tex9(RNAi) worms","doi":"10.22002/f41a4-hgf75","resourceType":"Audiovisual","name":"Extended data video 3.mp4","url":"https://portal.micropublication.org/uploads/7f9e500c2994653f6c35b04a9f56bac3.mp4"},{"description":"edema observations","doi":"10.22002/s8625-b8x92","resourceType":"Image","name":"Extended data image 1 - edema observations.jpg","url":"https://portal.micropublication.org/uploads/c462d033a0bdc6863d41653ffc1d9cd4.jpg"}],"funding":"Student research participation and publication support were funded by the Cell Biology Education Consortium (CBEC): Path to Publication (Award ID #2316122) through the National Science Foundation (NSF) and NH-INBRE (NIH Award: P20GM103506).
","image":{"url":"https://portal.micropublication.org/uploads/0ca3ef7d3ff1a8a06f822292e1bc7a8a.jpg"},"imageCaption":"A) Pathway tracing of RNAi-mediated knockdown worms shows reduced gliding motility. Overlays of sequential frames from 20 second video segments, illustrating the distance travelled by individual flatworms in control (neg.ctrl.(RNAi)), ropn1l(RNAi), and tex9(RNAi) worms. Z-projection within FIJI/Image J software was used to perform the path tracing analysis. Scale bar = 10mm B) Differential Interference Contrast Microscopy images at 63X magnification show planarian cilia length. Gene knockdown of ropn1l and tex9 resulted in visibly shorter cilia. Scale bar = 10µm. C) RNAi-mediated knockdown impairs planarian swim speed. Mean swim speed (± SEM) for neg.ctrl.(RNAi) worms was 1.3 ± 0.05 mm/s. ropn1l(RNAi) and tex9(RNAi) worms displayed 0.96 ± 0.17 mm/s and 0.87 ± 0.09 mm/s swim speeds, respectively (****p<0.0001, one-way ANOVA, n=30). D) RNAi-mediated knockdown reduces cilia length in planarians. Mean cilia length (± SEM) in neg.ctrl.(RNAi) worms was 11.04 ± 0.19 µm. ropn1l(RNAi) and tex9(RNAi) worms exhibited 6.94 ± 0.40 µm and 6.77 ± 0.30 µm cilia lengths, respectively (****p<0.0001, one-way ANOVA, n=5). E) Single-cell RNA-Seq expression of ropn1l (dd_Smed_v6_4888_0) in planarian cell clusters. Expression of ropn1l is strongly enriched in pharyngeal, epidermal, and specific neuronal cells with lower expression in other lineages (lineage diagram created using Planaria Single Cell Database; https://shiny.mdc-berlin.de/psca/). F) Single-cell RNA-Seq expression of tex9 (dd_Smed_v6_7007_0) in planarian cell clusters. Expression of tex9 is strongly enriched in pharyngeal and neuronal cells with lower expression in other lineages (lineage diagram created using Planaria Single Cell Database; https://shiny.mdc-berlin.de/psca/).
","imageTitle":"Knockdown of Smed-ropn1l and Smed-tex9 reduces planarian gliding locomotion and cilia length
","methods":"Planarian maintenance and feeding protocol
Planarians (S. mediterranea asexual strain ClW4) were maintained in a dark environment at room temperature in vessels containing water prepared as follows: 1.6mM NaCl, 1.0mM CaCl2, 1.0mM MgSO4, 0.1mM KCl, 1.2mM NaHCO3 (Cebrià & Newmark, 2005). Worms were fed once weekly with organic beef liver paste. After two hours of feeding, any remaining food was removed using plastic transfer pipettes, and a small volume of fresh planarian water was added to top off the vessel. A complete water change was performed every two days to remove accumulated excretory waste and maintain optimal water quality (Rompolas et al., 2013).
RNAi plasmid cloning and feeding
Following the methods established in our previous study (Gogoi et al., 2026), RNAi constructs were generated and administered in Schmidtea mediterranea with gene-specific modifications as described below. FASTA transcript sequences for ropn1l (dd_Smed_v6_4888_0_1) and tex9 (dd_Smed_v6_7007_0_1) were obtained from the PlanMine database, and Benchling was used to design primers generating 200–800 bp fragments with plasmid-homologous overhangs compatible with the pPR-T4P vector. A 543 bp fragment of ropn1l and a 615 bp fragment of tex9 were amplified from cDNA using gene-specific primers with homology arms indicated in capital letters and gene-specific regions in lowercase (primer sequences available under Reagents). Total RNA was isolated from homogenized animals, reverse transcribed to cDNA, and target fragments were amplified using either Q5 or Taq polymerase, verified by gel electrophoresis, purified, and quantified to calculate a 2:1 insert-to-backbone ratio. Inserts were cloned into pPR-T4P using Gibson Assembly for ropn1l and InFusion assembly for tex9. Assembled constructs were transformed into E. coli DH5α, screened by M13 PCR, purified, and sequence-verified by nanopore sequencing prior to RNAi food preparation. The C. elegans gene unc-22 with no known homolog in the planarian genome was used as a negative control neg.cont.(RNAi) (Wagner, 2012). Verified plasmids were subsequently transformed into E. coli HT115 for dsRNA production, and induced cultures were mixed with homogenized liver for feeding. Following a one-week starvation period, animals were fed six times over two weeks (every 2-3 days). For each RNAi condition, three experimental dishes containing ten animals each were analyzed (n=30).
Seven days after the final RNAi feeding, phenotypic assays were performed as follows:
Cilia Phenotype Analysis:
Planarians were immobilized by incubation in a relaxant solution (1% HNO3, 0.8325% formaldehyde, and 50 mM MgSO4) for 5 minutes prior to mounting on glass slides. Specimens were imaged using differential interference contrast (DIC) optics on a Zeiss LSM 510 Meta confocal microscope with a tungsten halogen lamp using a Plan Apochromat 63x/1.4 oil immersion objective lens with pixel size of 0.07μm/px. Images were captured using a Teledyne Lumenera Infinity 3 camera with Infinity Analyze software. For each condition, five worms were analyzed. Images were captured from both the left and right sides of the head region, near the eyes, where cilia are more densely distributed. From each image, the lengths of 10 individual cilia were measured, resulting in 10 data points per image. Measurements were performed using Fiji/ImageJ software to calculate the mean cilia length for both control and RNAi-treated planarians.
Gliding Assays:
Planarian locomotion was recorded over a 20-second period using an Andonstar AD246S-M HDMI Digital Microscope. Videos were analyzed in Fiji ImageJ, where z-projection was applied to generate tracks of individual worms. The lengths of these tracks were measured to calculate average swimming speed (mm/s).
Statistical Analysis:
Gliding velocity and cilia length measurements were quantified using FIJI/ImageJ software. For gliding velocity analysis, 30 individual worms were analyzed per condition. For cilia length analysis, 5 worms were used per condition, and for each worm, images were acquired from both left and right sides of the animal. Ten cilia were measured per side, yielding a total of 100 measurements per condition, which were averaged to obtain a single mean value per worm for statistical analysis.
The normality of replicates was confirmed using the Shapiro–Wilk test. Both RNAi groups (ropn1l(RNAi) and tex9(RNAi)) were compared with the same negative control neg.cont.(RNAi) animals. Analyses were conducted using GraphPad Prism version 10.6.1. One-way ANOVA revealed significant differences among groups for both cilia length (df=4, p < 0.0001) and gliding velocity (df = 29, p < 0.0001), and post hoc analysis using Dunnett’s multiple comparisons test further confirmed that both ropn1l(RNAi) and tex9(RNAi) worms differed significantly from negative control animals (neg.cont.(RNAi)) in both gliding speed and length of cilia (p < 0.0001 for all comparisons).
","reagents":"Category | Reagents | Use/Notes | Source of Reagent |
Plasmids & Vectors | pPRt4p plasmid | Expression vector backbone for cloning & RNAi experiments | Gifted by Jason Pellettieri |
Bacterial Strains | HT115 E. coli | RNAi feeding strain (RNase III deficient, used in dsRNA expression) | Gifted by Jason Pellettieri |
| NEB 5-alpha E. coli | High efficiency cloning strain for plasmid propagation | New England Biolabs |
Primers | ropn1l-fwd | CATTACCATCCCGaagctgccattcgtacccagcc | Invitrogen |
| ropn1l-rev | CCAATTCTACCCGtgcttggttgaacgaggccacc | Invitrogen |
| tex9-fwd | CATTACCATCCCGtccagcaaattccaacacgttcca | Invitrogen |
| tex9-rev | CCAATTCTACCCGgctttctgacgctctagccttctgc | Invitrogen |
Cilia are small hair-like structures that are highly evolutionarily conserved and found on the surface of most eukaryotic cells (Horani & Ferkol, 2021). There are two main types of cilia, primary (non-motile) and motile which include flagella and nodal cilia (Hyland & Brody, 2021). Motile cilia and related microtubule-based structures, such as flagella, are essential for a wide range of cellular and developmental processes across eukaryotes. Ciliopathies are a set of human diseases arising from genetic mutations that affect cilia structure or function. Many ciliopathies currently lack curative treatments, although ongoing research is exploring gene therapy approaches that aim to address underlying causes rather than only managing symptoms (Wee et al., 2024). Yet, our understanding of these diseases is still not fully understood (Hyland & Brody, 2021).
Researchers have identified 1,999 unique ciliary genes, accounting for approximately 10% of all known human genes (Van Sciver & Caspary, 2024). Despite their importance, however, many cilia-associated proteins remain poorly characterized. Rhophilin associated tail protein 1–like (ROPN1L) and testis-expressed protein 9 (TEX9) have been identified through proteomic and transcriptomic studies as candidate components of cilia or flagella in humans, mice, and flies, with some evidence suggesting roles in flagellar or sperm tail structure. However, direct functional analysis of these proteins is limited. To address this gap, we used RNA interference to examine the functions of Smed-ropn1l and Smed-tex9, planarian homologs of the human genes, in Schmidtea mediterranea, a model well suited for studying motile cilia, and evaluated their roles in ciliary structure and function.
ROPN1L is a scaffold protein in the ROPN1/ROPN1L–Dependent 2 (R2D2) family that anchors protein kinase A (PKA) signaling complexes to cilia and flagella (Bontems et al., 2014). In human bronchial epithelial cells, ROPN1L is identified as one of 37 key cilia-associated genes upregulated during mucociliary differentiation, based on transcriptomic analysis (Ross et al., 2007). Proteomic and immunolocalization data from the same study further demonstrates that ROPN1L localizes to the ciliary axoneme, providing direct evidence of its association with cilia (Ross et al., 2007). Functional evidence for the role of ROPN1L in microtubule-based motility comes from mouse models, where knockout of Ropn1l or its paralog rhophilin associated tail protein 1 (Ropn1) results in moderately reduced sperm motility, while deletion of both genes causes complete sperm immotility, structural disruption of the fibrous sheath, and loss of A kinase anchoring protein 3 (AKAP3) in sperm flagella (Fiedler et al., 2013). Consistent with these findings, qPCR and RNA-seq analyses of germ cells from men with cryptorchidism, a congenital condition in which one or both testes fail to descend into the scrotum, reveals ROPN1L as the most downregulated gene among several fertility-associated targets, including AKAP4, IZUMO1, and SPAG6, further supporting its role in human fertility (Sun et al., 2023).
Functional and comparative genomic analysis of TEX9 across eukaryotes suggests a role for TEX9 in cilia biology. Phylogenetic profiling across 100 eukaryotic genomes identifies TEX9 as a candidate ciliary protein, and cross-species comparisons of flagellated and non-flagellated organisms places TEX9 within a highly conserved subset of six proteins associated with cilia-related centriolar satellites (Wu et al., 2025). Consistent with these predictions, CilioGenics—an integrative platform combining single-cell RNA sequencing, protein–protein interactions, comparative genomics, transcription factor network analysis, and text mining—identifies TEX9 among 256 candidate ciliary genes, including 89 previously annotated by CiliaCarta and 28 with experimental validation (Pir et al., 2024). TEX9 is detected in human testes via proteomic analysis, suggesting an association with sperm development and cilia or flagella formation (Vandenbrouck et al., 2020). Direct evidence for ciliary association is provided by immunofluorescence studies demonstrating localization of TEX9 to ciliary basal bodies in cultured human kidney proximal tubule epithelial (HK2) cells (Nevers et al., 2017). Functional support for a role in cilia and flagella comes from loss-of-function studies, where RNAi knockdown of TEX9 in Drosophila causes sensory cilia defects and impaired sperm flagellar motility, and C. elegans mutants exhibit dye-fill defects indicative of structural or trafficking abnormalities in sensory cilia (Dobbelaere et al., 2023).
Widely studied for its regenerative abilities, S. mediterranea (freshwater planaria) has a fully sequenced genome and robust molecular tools, including RNAi for gene function analysis, making it an ideal model for studying ciliary genes. The planarian is a low-cost, easily maintained laboratory model and is one of few multicellular organisms with an external multiciliated epithelium. Cilia are used in planarian locomotion and are external and visible in live specimens under the microscope (Rabiasz & Ziętkiewicz, 2023; Rompolas et al., 2009; Rompolas et al., 2010). Ventral epithelial cells bear multiple cilia that beat against a secreted mucus layer, together with muscle contraction, to drive locomotion, similar to mucus transport in the human airway, reproductive tract, and brain ventricles. Defects in ciliary function cause visibly impaired locomotion (Rompolas et al, 2010). Additionally, protonephridia depend on ciliary movement for waste filtration and osmoregulation, and ciliary disruption can impair this function, resulting in fluid retention and edema. (Reddien et al., 2005; Rink et al., 2009; Rink et al., 2011; Thi-Kim Vu et al., 2015).
RNAi-mediated knockdown of ropn1l and tex9 in S. mediterranea resulted in clear phenotypic changes in swim rate when compared to neg.cont.(RNAi) (Fig. 1A; Extended data video 1 – neg.cont.(RNAi) 20 sec., Extended data video 2 – ropn1l(RNAi) 20 sec., Extended data video 3 – tex9(RNAi) 20 sec.). One week post-RNAi treatment, gliding locomotion was assessed using 20 second video segments, and distance traveled over time was determined using z-projection in Fiji/Image J. Quantitative analysis (n = 30) demonstrated that control worms (neg.cont.(RNAi)) had a mean swim rate ± SEM of 1.30 ± 0.05 mm/s, whereas ropn1l(RNAi) and tex9(RNAi) worms had significantly reduced swim speeds. ropn1l(RNAi) mean swim rate was 0.96 ± 0.17 mm/s and tex9(RNAi) mean swim rate was 0.87 ± 0.09 mm/s, representing decreases of 26.4% and 33.2%, respectively. (Fig 1C).
Differential interference contrast microscopy images of cilia located in the lateral region of planarian heads were taken, and these indicated reduced cilia length for ropn1l(RNAi) and tex9(RNAi) treated worms (Fig 1B). Quantitative analysis demonstrated significant reductions in cilia length in ropn1l(RNAi) and tex9(RNAi) worms, corresponding to decreases of 37.1% and 38.7%, respectively (mean ± SEM: 6.94 ± 0.40 µm and 6.77 ± 0.30 µm), relative to negative control (neg.cont.(RNAi)) worms (11.04 ± 0.19 µm) (Fig 1D). These data strongly suggest that functionally, these genes are important for ciliary integrity. In addition, edema in the trunk region of a small number of the ropn1l(RNAi) and tex9(RNAi) worms was observed, indicating potential defects in fluid regulation by the flame cells of the protonephridia (Reddien et al.,2005; Rink et al., 2009; Rink et al., 2011; Thi-Kim Vu et al., 2015). This edema was not observed in any of our neg.cont.(RNAi) worms (Extended data image 1 – edema observations).
Single-cell transcriptomic analysis of ropn1l and tex9 obtained from Planaria Single Cell Database (https://shiny.mdc-berlin.de/psca/) reveals that these genes exhibit strongest expression in specific neuronal subtypes, epidermal populations, protonephridia, and a distinct pharyngeal cell type described as epidermal-related (Fig. 1E, F) (Plass et al., 2018). In addition, single-cell transcriptomic data from Planarian Digiworm (https://digiworm.wi.mit.edu/) indicates that some of the highest-expressing cells for both genes are found in the protonephridial cluster (cluster 29), further supporting a role in protonephridial ciliary function (Fincher et al., 2018). This observation provides a link to the edema phenotype observed in a subset of RNAi knockdown animals. The lineage diagrams (Fig 1E, F) also depict inferred differentiation trajectories, in which increased gene expression toward the distal tips reflects enrichment in terminally differentiated cell types. Because the formation of motile cilia is expected to occur late in differentiation, the localization of ropn1l and tex9 expression toward terminal branches is consistent with their proposed roles in motile ciliogenesis. Across major cell categories including secretory, muscle, gut, and parenchymal populations both genes show comparatively low expression, whereas enrichment is observed in specific neurons, epidermis, protonephridia, and pharyngeal lineages, all of which include tissues known to harbor abundant motile cilia (Fincher et al., 2018).
Our results show that Schmidtea mediterranea is a valuable in vivo model for studying conserved ciliary genes. Since defects in motile cilia are linked to human disease, analyzing ROPN1L and TEX9 helps clarify shared molecular mechanisms across tissues and species. Although ROPN1L and TEX9 function through distinct molecular pathways, both genes are strongly associated with spermatogenesis and sperm flagellar structure, suggesting a shared role in motile microtubule-based systems (Sharma et al., 2014). We therefore presented these genes together in an exploration of their potential involvement in ciliary structure and function. In summary, our findings demonstrate that ROPN1L and TEX9 are required for the maintenance of normal ciliary length and motile function in multiciliated cells in planarians. Integration of single-cell expression analyses with RNAi-mediated knockdown phenotypes—including impaired gliding motility, shortened cilia, and posterior abdominal edema—provides compelling functional evidence that these genes are conserved components essential for proper motile cilia assembly and performance. These results extend prior proteomic and transcriptomic associations by establishing direct roles for ROPN1L and TEX9 in ciliary biology.
","references":[{"reference":"Bontems F, Fish RJ, Borlat I, Lembo Fdr, Chocu S, Chalmel Fdr, et al., Lane. 2014. C2orf62 and TTC17 Are Involved in Actin Organization and Ciliogenesis in Zebrafish and Human. PLoS ONE 9: e86476.
","pubmedId":"","doi":"10.1371/journal.pone.0086476"},{"reference":"Cebrià F, Newmark PA. 2005. Planarian homologs ofnetrinandnetrin receptorare required for proper regeneration of the central nervous system and the maintenance of nervous system architecture. Development 132: 3691-3703.
","pubmedId":"","doi":"10.1242/dev.01941"},{"reference":"Dobbelaere J, Su TY, Erdi B, Schleiffer A, Dammermann A. 2023. A phylogenetic profiling approach identifies novel ciliogenesis genes in\n Drosophila\n and\n C. elegans. The EMBO Journal 42: 10.15252/embj.2023113616.
","pubmedId":"","doi":"10.15252/embj.2023113616"},{"reference":"Fiedler SE, Dudiki T, Vijayaraghavan S, Carr DW. 2013. Loss of R2D2 Proteins ROPN1 and ROPN1L Causes Defects in Murine Sperm Motility, Phosphorylation, and Fibrous Sheath Integrity1. Biology of Reproduction 88: 10.1095/biolreprod.112.105262.
","pubmedId":"","doi":"10.1095/biolreprod.112.105262"},{"reference":"Fincher CT, Wurtzel O, de Hoog T, Kravarik KM, Reddien PW. 2018. Cell type transcriptome atlas for the planarian\n Schmidtea mediterranea. Science 360: 10.1126/science.aaq1736.
","pubmedId":"","doi":"10.1126/science.aaq1736"},{"reference":"Gogoi C, Pitt R, Mazur K, Naraharisetti R, Johnson K. 2026. undefined. microPublication Biology. 2026","pubmedId":"","doi":"10.17912/MICROPUB.BIOLOGY.001887"},{"reference":"Horani A, Ferkol TW. 2021. Understanding Primary Ciliary Dyskinesia and Other Ciliopathies. The Journal of Pediatrics 230: 15-22.e1.
","pubmedId":"","doi":"10.1016/j.jpeds.2020.11.040"},{"reference":"Hyland RM, Brody SL. 2021. Impact of Motile Ciliopathies on Human Development and Clinical Consequences in the Newborn. Cells 11: 125.
","pubmedId":"","doi":"10.3390/cells11010125"},{"reference":"Nevers Y, Prasad MK, Poidevin L, Chennen K, Allot A, Kress A, et al., Lecompte. 2017. Insights into Ciliary Genes and Evolution from Multi-Level Phylogenetic Profiling. Molecular Biology and Evolution 34: 2016-2034.
","pubmedId":"","doi":"10.1093/molbev/msx146"},{"reference":"Newmark PA, Reddien PW, Cebrià F, Alvarado ASn. 2003. Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians. Proceedings of the National Academy of Sciences 100: 11861-11865.
","pubmedId":"","doi":"10.1073/pnas.1834205100"},{"reference":"Pir MS, Begar E, Yenisert F, Demirci HC, Korkmaz ME, Karaman A, et al., Kaplan. 2024. CilioGenics: an integrated method and database for predicting novel ciliary genes. Nucleic Acids Research 52: 8127-8145.
","pubmedId":"","doi":"10.1093/nar/gkae554"},{"reference":"Plass M, Solana J, Wolf FA, Ayoub S, Misios A, Glažar P, et al., Rajewsky. 2018. Cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics. Science 360: 10.1126/science.aaq1723.
","pubmedId":"","doi":"10.1126/science.aaq1723"},{"reference":"Rabiasz A, Ziętkiewicz E. 2023. Schmidtea mediterranea as a Model Organism to Study the Molecular Background of Human Motile Ciliopathies. International Journal of Molecular Sciences 24: 4472.
","pubmedId":"","doi":"10.3390/ijms24054472"},{"reference":"Reddien PW, Bermange AL, Murfitt KJ, Jennings JR, Sánchez Alvarado A. 2005. Identification of Genes Needed for Regeneration, Stem Cell Function, and Tissue Homeostasis by Systematic Gene Perturbation in Planaria. Developmental Cell 8: 635-649.
","pubmedId":"","doi":"10.1016/j.devcel.2005.02.014"},{"reference":"Rink JC, Gurley KA, Elliott SA, Sánchez Alvarado A. 2009. Planarian Hh Signaling Regulates Regeneration Polarity and Links Hh Pathway Evolution to Cilia. Science 326: 1406-1410.
","pubmedId":"","doi":"10.1126/science.1178712"},{"reference":"Rink JC, Vu HTK, Alvarado ASn. 2011. The maintenance and regeneration of the planarian excretory system are regulated by EGFR signaling. Development 138: 3769-3780.
","pubmedId":"","doi":"10.1242/dev.066852"},{"reference":"Rompolas P, Azimzadeh J, Marshall WF, King SM. 2013. Analysis of Ciliary Assembly and Function in Planaria. Methods in Enzymology,Cilia, Part B : 245-264.
","pubmedId":"","doi":"10.1016/B978-0-12-397944-5.00012-2"},{"reference":"Rompolas P, Patel-King RS, King SM. 2009. Schmidtea mediterranea: a model system for analysis of motile cilia. Methods Cell Biol 93: 81-98.
","pubmedId":"","doi":"https://doi.org/10.1016/S0091-679X(08)93004-1"},{"reference":"Rompolas, P., Patel-King, R. S., & King, S. M. (2010). An outer arm Dynein conformational switch is required for metachronal synchrony of motile cilia in planaria. Molecular biology of the cell, 21(21), 3669–3679. https://doi.org/10.1091/mbc.E10-04-0373
","pubmedId":"","doi":""},{"reference":"Ross AJ, Dailey LA, Brighton LE, Devlin RB. 2007. Transcriptional Profiling of Mucociliary Differentiation in Human Airway Epithelial Cells. American Journal of Respiratory Cell and Molecular Biology 37: 169-185.
","pubmedId":"","doi":"10.1165/rcmb.2006-0466OC"},{"reference":"Sharma E, Künstner A, Fraser BA, Zipprich G, Kottler VA, Henz SR, Weigel D, Dreyer C. 2014. Transcriptome assemblies for studying sex-biased gene expression in the guppy, Poecilia reticulata. BMC Genomics 15: 10.1186/1471-2164-15-400.
","pubmedId":"","doi":"doi.org/10.1186/1471-2164-15-400"},{"reference":"Sun W, Zhang X, Wang L, Ren G, Piao S, Yang C, Liu Z. 2023. RNA sequencing profiles reveals progressively reduced spermatogenesis with progression in adult cryptorchidism. Frontiers in Endocrinology 14: 10.3389/fendo.2023.1271724.
","pubmedId":"","doi":"10.3389/fendo.2023.1271724"},{"reference":"Van Sciver RE, Caspary T. 2024. A prioritization tool for cilia-associated genes and their in vivo resources unveils new avenues for ciliopathy research. Disease Models & Mechanisms 17: 10.1242/dmm.052000.
","pubmedId":"","doi":"10.1242/dmm.052000"},{"reference":"Vandenbrouck Y, Pineau C, Lane L. 2020. The Functionally Unannotated Proteome of Human Male Tissues: A Shared Resource to Uncover New Protein Functions Associated with Reproductive Biology. Journal of Proteome Research 19: 4782-4794.
","pubmedId":"","doi":"10.1021/acs.jproteome.0c00516"},{"reference":"Vu HTK, Rink JC, McKinney SA, McClain M, Lakshmanaperumal N, Alexander R, Sánchez Alvarado A. 2015. Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ. eLife 4: e07405.
","pubmedId":"","doi":""},{"reference":"Wagner DE, Ho JJ, Reddien PW. 2012. Genetic Regulators of a Pluripotent Adult Stem Cell System in Planarians Identified by RNAi and Clonal Analysis. Cell Stem Cell 10: 299-311.
","pubmedId":"","doi":"10.1016/j.stem.2012.01.016"},{"reference":"Wee WB, Kinghorn B, Davis SD, Ferkol TW, Shapiro AJ. 2024. Primary Ciliary Dyskinesia. Pediatrics 153: 10.1542/peds.2023-063064.
","pubmedId":"","doi":"10.1542/peds.2023-063064"},{"reference":"Wu B, Long C, Liu J, Huang X, Ma S, Ma Y, et al., Li. 2025. A subset of evolutionarily conserved centriolar satellite core components is crucial for sperm flagellum biogenesis. Theranostics 15: 7025-7044.
","pubmedId":"","doi":"10.7150/thno.117118"}],"title":"Insights into Human Ciliopathies: Gene Silencing of ropn1l and tex9 in Schmidtea mediterranea Indicate Association with Ciliary Structure and Motility
","reviews":[],"curatorReviews":[]}]}},"species":{"species":[{"value":"acer saccharum","label":"Acer saccharum","imageSrc":"","imageAlt":"","mod":"TreeGenes","modLink":"https://treegenesdb.org","linkVariable":""},{"value":"achillea millefolium","label":"Achillea millefolium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"acinetobacter baylyi","label":"Acinetobacter baylyi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"actinobacteria bacterium","label":"Actinobacteria bacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adelges tsugae","label":"Adelges tsugae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adenocaulon chilense","label":"Adenocaulon chilense","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aedes japonicus","label":"Aedes japonicus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aegorhinus vitulus","label":"Aegorhinus vitulus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alaimidae","label":"Alaimidae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"allobates femoralis","label":"Allobates femoralis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alnus glutinosa","label":"Alnus glutinosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alosa aestivalis","label":"Alosa aestivalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alosa pseudoharengus","label":"Alosa pseudoharengus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alternaria alternata","label":"Alternaria alternata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"amynthas agrestis","label":"Amynthas Agrestis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ancylostoma caninum","label":"Ancylostoma caninum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ancylostoma ceylanicum","label":"Ancylostoma ceylanicum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anemone multifida","label":"Anemone multifida","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anguilla rostrata","label":"Anguilla rostrata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anisakis simplex","label":"Anisakis simplex","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anomala albopilosa","label":"Anomala albopilosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anthomyiidae sp","label":"Anthomyiidae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anthomyiidae sp","label":"Anthomyiidae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"arabidopsis","label":"Arabidopsis","imageSrc":"arabidopsis.png","imageAlt":"Arabidopsis graphic by Zoe Zorn CC BY 4.0","mod":"TAIR","modLink":"https://arabidopsis.org","linkVariable":""},{"value":"architeuthis dux","label":"Architeuthis dux","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"arion vulgaris","label":"Arion vulgaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"armeria","label":"Armeria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"artemia","label":"Artemia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"arthrobacter sp.","label":"Arthrobacter sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ascaridia","label":"Ascaridia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ascaridia galli","label":"Ascaridia galli","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"asparagopsis taxiformis","label":"Asparagopsis taxiformis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"astatotilapia burtoni","label":"Astatotilapia burtoni","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"avena sativa","label":"Avena sativa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aves","label":"Aves","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus","label":"Bacillus (firmicutes)","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus cereus","label":"Bacillus cereus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus mycoides","label":"Bacillus mycoides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus subtilis","label":"Bacillus subtilis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus thuringiensis","label":"Bacillus thuringiensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus toyonensis","label":"Bacillus toyonensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus wiedmannii","label":"Bacillus wiedmannii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacteria","label":"Bacteria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacteriophage","label":"Bacteriophage","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bactrocera","label":"Bactrocera sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"batrachospermum gelatinosum","label":"Batrachospermum gelatinosum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"betula lenta","label":"Betula lenta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"betula nigra","label":"Betula nigra","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bombus dahlbohmii","label":"Bombus dahlbohmii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bombus terrestris","label":"Bombus terrestris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bombyx mori","label":"Bombyx mori","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bos taurus","label":"Bos Taurus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brachygobius doriae","label":"Brachygobius doriae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brassica oleracea","label":"Brassica oleracea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brassica rapa","label":"Brassica rapa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brugia malayi","label":"Brugia malayi","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"burkholderia thailandensis","label":"Burkholderia thailandensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"buttiauxella","label":"Buttiauxella","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caenorhabditis brenneri","label":"Caenorhabditis brenneri","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis briggsae","label":"Caenorhabditis briggsae","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"c. elegans","label":"Caenorhabditis elegans","imageSrc":"c-elegans.jpg","imageAlt":"C. elegans graphic by Zoe Zorn CC BY 4.0","mod":"WormBase","modLink":"https://wormbase.org","linkVariable":""},{"value":"caenorhabditis inopinata","label":"Caenorhabditis inopinata","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis japonica","label":"Caenorhabditis japonica","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis nigoni","label":"Caenorhabditis nigoni","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caenorhabditis remanei","label":"Caenorhabditis remanei","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis tropicalis","label":"Caenorhabditis tropicalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"calidifontibacillus","label":"Calidifontibacillus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"calidifontibacillus erzuremensis","label":"Calidifontibacillus erzuremensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"calliphora sp","label":"Calliphora sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caltha sagittata","label":"Caltha sagittata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cambarus latimanus","label":"Cambarus latimanus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"candida albicans","label":"Candida albicans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"canis familiaris","label":"Canis familiaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cannabis sativa","label":"Cannabis sativa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caretta caretta","label":"Caretta caretta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cassiopea xamachana","label":"Cassiopea xamachana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caulobacter vibrioides","label":"Caulobacter vibrioides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cephalopods","label":"Cephalopoda","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cerastium arvense","label":"Cerastium arvense","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ceriodaphnia","label":"Ceriodaphnia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ceroglossus suturalis","label":"Ceroglossus suturalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chaetoceros","label":"Chaetoceros","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chamaecrista fasciculata","label":"Chamaecrista fasciculata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chilicola chalcidiformis","label":"Chilicola chalcidiformis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chitinimonas","label":"Chitinimonas","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chlamydomonas reinhardtii","label":"Chlamydomonas reinhardtii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chromobacterium","label":"Chromobacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chrysemys picta","label":"Chrysemys picta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chrysoperla rufilabris","label":"Chrysoperla rufilabris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"citrus","label":"Citrus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"clavibacter sp.","label":"Clavibacter sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"colinus virginianus","label":"Colinus virginianus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"crassostrea virginica","label":"Crassostrea virginica","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"crithidia fasciculata","label":"Crithidia fasciculata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cutibacterium acnes","label":"Cutibacterium acnes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cyanobacteria","label":"Cyanobacteria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"daphnia","label":"Daphnia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"daphnia pulex","label":"Daphnia pulex","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"diabrotica virgifera","label":"Diabrotica virgifera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"diabrotica virgifera virgifera virus 1","label":"Diabrotica virgifera virgifera virus 1","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"d. discoideum","label":"Dictyostelium discoideum","imageSrc":"dicty.png","imageAlt":"D. discoideum","mod":"dictyBase","modLink":"http://dictybase.org","linkVariable":""},{"value":"diptera","label":"Diptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dotocryptus bellicosus","label":"Dotocryptus bellicosus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"drechmeria coniospora","label":"Drechmeria coniospora","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"drosophila","label":"Drosophila","imageSrc":"drosophila.png","imageAlt":"Drosophila graphic by Zoe Zorn CC BY 4.0","mod":"FlyBase","modLink":"https://flybase.org/doi/","linkVariable":"doi"},{"value":"dryopteris campyloptera","label":"Dryopteris campyloptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dryopteris expansa","label":"Dryopteris expansa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dryopteris intermedia","label":"Dryopteris intermedia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dugesia dorotocephala","label":"Dugesia dorotocephala","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"elasmobranchii","label":"Elasmobranchii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"embryophyta","label":"Embryophyta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"enoploteuthis chunii","label":"Enoploteuthis chunii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"enterobacter aerogenes","label":"Enterobacter aerogenes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"enterococcus raffinosus","label":"Enterococcus raffinosus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"epichloë coenophiala","label":"Epichloë coenophiala","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"equus caballus","label":"Equus caballus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"erigeron sp","label":"Erigeron sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"eristalis","label":"Eristalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"eruca vesicaria","label":"Eruca vesicaria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"erwinia carotovora","label":"Erwinia carotovora","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"erythronium americanum","label":"Erythronium americanum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"escherichia coli","label":"Escherichia coli","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"eukaryota","label":"Eukaryotes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"felis catus","label":"Felis catus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"francisella novicida","label":"Francisella novicida","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"francisella tularensis","label":"Francisella tularensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"fraxinus americana","label":"Fraxinus americana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"fucus distichus","label":"Fucus distichus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"fungi","label":"Fungi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"gasteropelecus sp.","label":"Gasteropelecus sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"geranium sp","label":"Geranium sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"girardia","label":"Girardia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"glaucomys volans","label":"Glaucomys volans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"glycine max","label":"Glycine max","imageSrc":"","imageAlt":"","mod":"Soybase","modLink":"https://soybase.org","linkVariable":""},{"value":"glyptemys insculpta","label":"Glyptemys insculpta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"gossypium hirsutum","label":"Gossypium hirsutum","imageSrc":"","imageAlt":"","mod":"CottonGen","modLink":"https://www.cottongen.org/","linkVariable":""},{"value":"gromphadorhina portentosa","label":"Gromphadorhina portentosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"gryllodes sigillatus","label":"Gryllodes sigillatus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"haliotis rufescens","label":"Haliotis rufescens","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hepacivirus hominis","label":"Hepatitis C Virus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"herpes simplex virus type 1","label":"Herpes simplex virus type 1","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"human","label":"Human","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"human coronavirus oc43","label":"Human coronavirus OC43","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hydra vulgaris","label":"Hydra vulgaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hydropsyche sp","label":"Hydropsyche sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hymenoptera","label":"Hymenoptera","imageSrc":"","imageAlt":"","mod":"Hymenoptera Genome Database","modLink":"https://hymenoptera.elsiklab.missouri.edu/","linkVariable":""},{"value":"hypochaeris radicata","label":"Hypochaeris radicata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hypodynerus vespiformis","label":"Hypodynerus vespiformis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"iflaviridae","label":"Iflaviridae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"iflavuris","label":"Iflavirus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ipomoea hederacea","label":"Ipomoea hederacea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ischnomera","label":"Ischnomera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ischnomera ruficollis","label":"Ischnomera ruficollis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"julidochromis marlieri","label":"Julidochromis marlieri","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"juniperus virginiana","label":"Juniperus virginiana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"kluyveromyces marxianus","label":"Kluyveromyces marxianus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"l. casei","label":"L. casei","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lacticaseibacillus casei","label":"Lacticaseibacillus casei","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"larentiinae sp","label":"Larentiinae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"laurus nobilis","label":"Laurus nobilis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lepidoptera","label":"Lepidoptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"leucanthemum vulgare","label":"Leucanthemum vulgare","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"linepithema humile","label":"Linepithema humile","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"liometopum occidentale","label":"Liometopum occidentale","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lolium arundinaceum","label":"Lolium arundinaceum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lumbriculus variegatus","label":"Lumbriculus variegatus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lumbricus terrestris","label":"Lumbricus terrestris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lupinus polyphyllus","label":"Lupinus polyphyllus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lycorma delicatula","label":"Lycorma delicatula","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lynx rufus","label":"Lynx rufus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"magnaporthe oryzae","label":"Magnaporthe oryzae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mammalia","label":"Mammalia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"manihot esculenta","label":"Manihot esculenta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"medicago lupulina","label":"Medicago lupulina","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"meloidogyne","label":"Meloidogyne","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mimus polyglottos","label":"Mimus polyglottos","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bryophyta","label":"Mosses","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mouse","label":"Mouse","imageSrc":"","imageAlt":"","mod":"MGI","modLink":"https://informatics.jax.org","linkVariable":""},{"value":"m. minutoides","label":"Mus minutoides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mycobacterium smegmatis","label":"Mycobacterium smegmatis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"nakaseomyces glabratus","label":"Nakaseomyces glabratus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"nauphoeta cinerea","label":"Nauphoeta cinerea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"neurospora","label":"Neurospora","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"n. benthamiana","label":"Nicotiana benthamiana","imageSrc":"","imageAlt":"","mod":"Solgenomics Network","modLink":"https://solgenomics.net/organism/Nicotiana_benthamiana/genome","linkVariable":""},{"value":"nicotiana tabacum","label":"Nicotiana tabacum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"noctuidae","label":"Noctuidae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"noctuidae sp","label":"Noctuidae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"nothobranchius furzeri","label":"Nothobranchius furzeri","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"onchocerca volvulus","label":"Onchocerca volvulus","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"orconectes virilis","label":"Orconectes virilis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ormia ochracea","label":"Ormia ochracea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"o. sativa","label":"Oryza sativa","imageSrc":"","imageAlt":"","mod":"Gramene","modLink":"https://www.gramene.org/","linkVariable":""},{"value":"other","label":"Other","imageSrc":"","imageAlt":"","mod":null,"modLink":null,"linkVariable":null},{"value":"oxalis enneaphylla","label":"Oxalis enneaphylla","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"paenarthrobacter nicotinovorans","label":"Paenarthrobacter nicotinovorans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"paenarthrobacter nicotinovorans","label":"Paenarthrobacter nicotinovorans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pantoea","label":"Pantoea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pantoea agglomerans","label":"Pantoea agglomerans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"papaver sp","label":"Papaver sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"paramecium bursaria","label":"Paramecium bursaria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"partitiviridae","label":"Partitiviridae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pelodiscus sinensis","label":"Pelodiscus sinensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"perezia recurvata","label":"Perezia recurvata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"petromyzon marinus","label":"Petromyzon marinus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"photinus pyralis","label":"Photinus pyralis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"photinus pyralis associated partiti-like virus","label":"Photinus pyralis associated partiti-like virus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"photinus pyralis iflavirus 1","label":"Photinus pyralis iflavirus 1","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"physcomitrium patens","label":"Physcomitrium patens","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pinus strobus","label":"Pinus strobus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pinus taeda","label":"Pinus taeda","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"platycheirus","label":"Platycheirus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"plectus sambesii","label":"Plectus sambesii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pogonomyrmex occidentalis","label":"Pogonomyrmex occidentalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"poncirus trifoliata","label":"Poncirus trifoliata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"populus deltoides","label":"Populus deltoides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"potato virus y","label":"Potato virus Y","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"primula magellanica","label":"Primula magellanica","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pristionchus pacificus","label":"Pristionchus pacificus","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"prunus persica","label":"Prunus persica","imageSrc":"","imageAlt":"","mod":"Genome Database for Rosaceae","modLink":"https://www.rosaceae.org/","linkVariable":""},{"value":"psalmopoeus iriminia","label":"Psalmopoeus iriminia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudanabaena sp.","label":"Pseudanabaena sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas","label":"Pseudomonas","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas aeruginosa","label":"Pseudomonas aeruginosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas glycinae","label":"Pseudomonas glycinae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas putida","label":"Pseudomonas putida","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas syringae","label":"Pseudomonas syringae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pterophyllum scalare","label":"Pterophyllum scalare","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"python regius","label":"Python regius","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"quercus macrocarpa","label":"Quercus macrocarpa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ralstonia solanacearum","label":"Ralstonia solanacearum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ranitomeya imitator","label":"Ranitomeya imitator","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ranunculus peduncularis","label":"Ranunculus peduncularis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"rat","label":"Rat","imageSrc":"","imageAlt":"","mod":"RGD","modLink":"https://rgd.mcw.edu","linkVariable":""},{"value":"rheinheimera","label":"Rheinheimera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ribes rubrum","label":"Ribes rubrum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"sars-cov-2","label":"SARS-CoV-2","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. cerevisiae","label":"Saccharomyces cerevisiae","imageSrc":"yeast.png","imageAlt":"Yeast graphic by Zoe Zorn CC BY 4.0","mod":"SGD","modLink":"https://yeastgenome.org","linkVariable":""},{"value":"saccharomyces paradoxus","label":"Saccharomyces paradoxus ","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. uvarum","label":"Saccharomyces uvarum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"schistosoma","label":"Schistosoma","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"schizosaccharomyces japonicus","label":"Schizosaccharomyces japonicus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. pombe","label":"Schizosaccharomyces pombe","imageSrc":"pombe.png","imageAlt":"Pombe graphic by Zoe Zorn © Caltech","mod":"PomBase","modLink":"https://www.pombase.org/reference/PMID:","linkVariable":"pmId"},{"value":"schmidtea mediterranea","label":"Schmidtea mediterranea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"senecio sp","label":"Senecio sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"simocephalus","label":"Simocephalus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"siraitia grosvenorii","label":"Siraitia grosvenorii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"solanum lycopersicum","label":"Solanum lycopersicum","imageSrc":"","imageAlt":"","mod":"Solgenomics Network","modLink":"https://solgenomics.net/organism/1/view/","linkVariable":""},{"value":"sorghum","label":"Sorghum","imageSrc":"","imageAlt":"","mod":"SorghumBase","modLink":"https://www.sorghumbase.org","linkVariable":""},{"value":"spiroplasma eriocheiris","label":"Spiroplasma eriocheiris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"staphylococcus aureus","label":"Staphylococcus aureus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"staphylococcus epidermidis","label":"Staphylococcus epidermidis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"steinernema carpocapsae","label":"Steinernema carpocapsae","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"https://wormbase.org","linkVariable":""},{"value":"steinernema hermaphroditum","label":"Steinernema hermaphroditum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"stenotrophomonas geniculata","label":"Stenotrophomonas geniculata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"streptococcus gordonii ","label":"Streptococcus gordonii ","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"streptococcus mutans","label":"Streptococcus mutans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":" streptococcus pneumoniae","label":"Streptococcus pneumoniae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. purpuratus","label":"Strongylocentrotus purpuratus","imageSrc":"","imageAlt":"","mod":"Echinobase","modLink":"https://www.echinobase.org","linkVariable":""},{"value":"strongyloides ratti","label":"Strongyloides ratti","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"sulfolobus","label":"Sulfolobus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"symphoricarpos albus","label":"Symphoricarpos albus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"syncirsodes","label":"Syncirsodes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"synechococcus elongatus","label":"Synechococcus elongatus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"syrphidae","label":"Syrphidae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tarantobelus jeffdanielsi","label":"Tarantobelus jeffdanielsi","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"taraxacum officinale","label":"Taraxacum officinale","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tatochila theodice","label":"Tatochila theodice","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tetrahymena","label":"Tetrahymena","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tetramorium immigrans","label":"Tetramorium immigrans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tomato brown rugose fruit virus","label":"ToBRFV","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trachemys scripta","label":"Trachemys scripta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tribolium castaneum","label":"Tribolium castaneum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trichoptera","label":"Trichoptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trichuris muris","label":"Trichuris muris","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"trifolium repens","label":"Trifolium repens","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trypoxylus dichotomus","label":"Trypoxylus dichotomus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tsuga canadensis","label":"Tsuga canadensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ulva expansa","label":"Ulva expansa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"universal","label":"Universal","imageSrc":"","imageAlt":"","mod":null,"modLink":null,"linkVariable":null},{"value":"vargula hilgendorfii","label":"Vargula hilgendorfii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"vespula vulgaris","label":"Vespula vulgaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"virus","label":"Virus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"watasenia scintillans","label":"Watasenia scintillans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"wolbachia pipientis","label":"Wolbachia pipientis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"xenopus","label":"Xenopus","imageSrc":"xenopus.png","imageAlt":"Xenopus graphic by Zoe Zorn CC BY 4.0","mod":"XenBase","modLink":"https://xenbase.org","linkVariable":""},{"value":"xenorhabdus griffiniae","label":"Xenorhabdus griffiniae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"yramea cytheris","label":"Yramea cytheris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"zaprionus indianus","label":"Zaprionus indianus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"zea mays","label":"Zea mays","imageSrc":"","imageAlt":"","mod":"MaizeGDB","modLink":"https://www.maizegdb.org","linkVariable":""},{"value":"zebrafish","label":"Zebrafish","imageSrc":"zebrafish.png","imageAlt":"Zebrafish graphic by Zoe Zorn CC BY 4.0","mod":"ZFIN","modLink":"https://zfin.org","linkVariable":""}]}},"pageContext":{"id":"4942ceba-576d-4e56-bb67-3ffd2b78569c","citedBy":[],"parsedCsv":{"csvHeader":[],"csvData":[]}}}, "staticQueryHashes": ["2114697108"]}