Paramecium Cells Research Articles

Quantum Weak Value Enabled Quantitative Phase Imaging

AbstractQuantitative phase imaging (QPI) has been a powerful approach in bioimaging because it can protect specimens from staining and quantify important physical properties of biological specimen, such as local thickness and refractive index. Here, a simple mechanism is proposed to implement QPI based on the quantum weak‐value theory. Two weak values are employed. One is momentum weak value, which inherently gives the phase gradient of the cells and tissues, and the other is polarization weak value leading to the biased imaging. Following the essence of weak value amplification in precision metrology, the weak value can also enhance the contrast in imaging. By combining two converse bias images and a phase gradient intensity image to capture phase gradient data, the original phase of the specimen can be quantitatively reconstructed. A proof‐of‐principle experiment is conducted by imaging the phase targets and unstained Paramecium cells. The ability to capture quantitative phase data is validated through imaging various samples with a phase resolution, demonstrating height deviations of less than 10 nm compared to atomic force microscopy (AFM). This work proposes weak‐value‐based QPI that possess the potential for improving the measurement precision and provides deep insights into physics and applications of weak value.

Anoctamin-like protein 1 regulates repolarization in Paramecium behavioral responses.

Paramecium exhibits responsive behavior to environmental changes, moving either closer to or further away from stimuli. Electrophysiological experiments have revealed that these behavioral responses are controlled by membrane potentials. Anoctamin, a Ca2+-activated Cl- channel, is involved in the regulation of membrane potential in mammals. However, it remains uncertain whether Cl- channels like anoctamin regulate Paramecium behavior. Herein, replacement of external Cl- ions with acetate ion and application of Cl- channel blocker niflumic acid (NFA, 0.1 μM) increased spontaneous avoiding reactions (sARs). Hence, we hypothesized that anoctamin is involved in the stabilization of membrane potential fluctuation. Paramecium cells in which the anoctamin-like protein 1 gene was knocked down displayed frequent sARs in the culture medium without external stimulation. Treatment of anoctamin-like protein 1-knockdown cells with the Ca2+ chelator BAPTA or Ca-channel blocker nicardipine reversed the increase in sARs. Electrophysiological experiments revealed extension of membrane depolarization when positive currents were applied to anoctamin-like protein 1-knockdown cells. We concluded that anoctamin-like protein 1 works as a Cl-channel and stabilizes the membrane potential oscillation, reducing sARs.

EDOT-pyrrole copolymer conductive nanoparticles and electrodes enhance galvanotaxis in Paramecium tetraurelia; a simple biological model for explorative experiments in therapeutic electrical stimulation

The galvanotaxis response of Paramecium tetraurelia was explored as a preliminary model for in vitro electrostimulation of other eukaryotic cells. Interestingly, electrodes coated with 3,4-ethylenedioxythiophene (EDOT) – pyrrole (Py) copolymer induced galvanotaxis in a similar manner and with even greater efficiency than conventional electrodes. Furthermore, in the absence of agar bridges, there was no release of toxic species into the cell medium or any deleterious effects on cell viability after several cycles of electrostimulation for 1 min. The intrinsic cytotoxicity of the copolymer was also investigated in Paramecium tetraurelia and mammalian cell lines. The copolymer did not show any toxic effects at concentrations below 1 mg/ml. Finally, it was discovered that galvanotaxis can be increased by up to 150 % concerning the control (with electrostimulation of 3V for 60s) in Paramecium cells treated with 10 μg/mL copolymer particles for 1 h and that showed endocytosis. Galvanotaxis is voltage-dependent, as it only increases by 100 % at 1.5 V, suggesting a sensitive physiological level effect on the Ca2+ currents and voltage-dependent Ca2+ channels of the cilia.

The phase range extension and accuracy improvement in Fresnel biprism-based digital holography microscopy

We report a highly stable and affordable dual-wavelength digital holographic microscopy system based on common-path geometry. A Fresnel biprism is used to create an off-axis geometry, and two diode laser sources with different wavelengths λ1 = 532nm and λ2 = 650nm generate the dual-wavelength compound hologram. In order to extend the measurement range, the phase distribution is obtained using a synthetic wavelength Λ1 = 2930.5nm. Furthermore, to improve the system's temporal stability and reduce speckle noise, a shorter wavelength (Λ2 = 292.5nm) is used. The feasibility of the proposed configuration is validated by the experimental results obtained with Molybdenum trioxide, Paramecium, and red blood cell specimens.

Bioremediation of heavy metals using a novel species of ciliate Paramecium multimicronucleatum isolated from industrial wastewater

The introduction of heavy metals by industries in the aquatic ecosystem is a threatening alarm for living organisms. Bioremediation is an eco-friendly and inexpensive technique as an alternative to chemical methods for wastewater treatment. Wastewater samples were collected from ponds receiving effluents from the Kot Lakhpat Industrial zone, Lahore (Pakistan). Heavy metal-resistant ciliate, Paramecium multimicronucleatum was isolated and characterized with the help of 18SrRNA biomarker. The ciliate showed optimum growth at 25 °C ± 1 °C and pH 7. Growth patterns of P. multimicronucleatum were observed with and without metal stress in wheat grain medium. The minimum inhibitory concentration of cadmium, copper, zinc, and lead was 60, 70, 110, and 160 μg ml−1, respectively. The maximum uptake by Paramecia for Cd, Cu, and Zn was recorded as 90%, 82%, and 93% respectively after 96 h of exposure in each case. While 90% of lead ions were absorbed by Paramecium cells after 48 h of exposure. The order of uptake ability by Paramecium sp. was Zn2+ > Cd2+ > Cu2+ > Pb2+. This preliminary study of exploring bioremediation ability of this ciliate would be helpful for investigating it further using advanced molecular techniques.

Methods for Paramecium tetraurelia ciliary membrane protein identification and function.

In this chapter we provide some tools to study the ciliary proteins that make it possible for Paramecium cells to swim by beating their cilia. These proteins include many ion channels, accessory proteins, peripheral proteins, structural proteins, rootlets of cilia, and enzymes. Some of these proteins are also found in the soma membrane, but their distinct and critical functions are in the cilia. Paramecium has 4000 or more cilia per cell, giving it an advantage for biochemical studies over cells that have one primarily cilium per cell. Nonetheless, a challenge for studies of many ciliary proteins in Paramecium is their low abundance. We discuss here several strategies to overcome this challenge and other challenges such as working with very large channel proteins. We also include for completeness other techniques that are critical to the study of swimming behavior, such as genetic crosses, recording of swimming patterns, electrical recordings, expression of very large channel proteins, RNA Interference, among others.

The evolutionary conserved proteins CEP90, FOPNL, and OFD1 recruit centriolar distal appendage proteins to initiate their assembly.

In metazoa, cilia assembly is a cellular process that starts with centriole to basal body maturation, migration to the cell surface, and docking to the plasma membrane. Basal body docking involves the interaction of both the distal end of the basal body and the transition fibers/distal appendages, with the plasma membrane. Mutations in numerous genes involved in basal body docking and transition zone assembly are associated with the most severe ciliopathies, highlighting the importance of these events in cilium biogenesis. In this context, the ciliate Paramecium has been widely used as a model system to study basal body and cilia assembly. However, despite the evolutionary conservation of cilia assembly events across phyla, whether the same molecular players are functionally conserved, is not fully known. Here, we demonstrated that CEP90, FOPNL, and OFD1 are evolutionary conserved proteins crucial for ciliogenesis. Using ultrastructure expansion microscopy, we unveiled that these proteins localize at the distal end of both centrioles/basal bodies in Paramecium and mammalian cells. Moreover, we found that these proteins are recruited early during centriole duplication on the external surface of the procentriole. Functional analysis performed both in Paramecium and mammalian cells demonstrate the requirement of these proteins for distal appendage assembly and basal body docking. Finally, we show that mammalian centrioles require another component, Moonraker (MNR), to recruit OFD1, FOPNL, and CEP90, which will then recruit the distal appendage proteins CEP83, CEP89, and CEP164. Altogether, we propose that this OFD1, FOPNL, and CEP90 functional module is required to determine in mammalian cells the future position of distal appendage proteins.

Label‐free single‐shot imaging with on‐axis phase‐shifting holographic reflectance quantitative phase microscopy

Quantitative phase microscopy (QPM) has been emerged as an indispensable diagnostic and characterization tool in biomedical imaging with its characteristic nature of label-free, noninvasive, and real time imaging modality. The integration of holography to the conventional microscopy opens new advancements in QPM featuring high-resolution and quantitative three-dimensional image reconstruction. However, the holography schemes suffer in space-bandwidth and time-bandwidth issues in the off-axis and phase-shifting configuration, respectively. Here, we introduce an on-axis phase-shifting holography based QPM system with single-shot imaging capability. The technique utilizes the Fizeau interferometry scheme in combination with polarization phase-shifting and space-division multiplexing to achieve the single-shot recording of the multiple phase-shifted holograms. Moreover, the high-speed imaging capability with instantaneous recording of spatially phase shifted holograms offers the flexible utilization of the approach in dynamic quantitative phase imaging with robust phase stability. We experimentally demonstrated the validity of the approach by quantitative phase imaging and depth-resolved imaging of paramecium cells. Furthermore, the technique is applied to the phase imaging and quantitative parameter estimation of red blood cells. This integration of a Fizeau-based phase-shifting scheme to the optical microscopy enables a simple and robust tool for the investigations of engineered and biological specimen with real-time quantitative analysis.

Membrane traffic and Ca2+ signals in ciliates

A Paramecium cell has as many types of membrane interactions as mammalian cells, as established with monoclonal antibodies by R. Allen and A. Fok. Since then, we have identified key players, such as SNARE proteins, Ca2+ -regulating proteins, including Ca2+ -channels, Ca2+ -pumps, Ca2+ -binding proteins of different affinity, etc., at the molecular level, probed their function and localized them at the light and electron microscopy level. SNARE proteins, in conjunction with a synaptotagmin-like Ca2+ -sensor protein, mediate membrane fusion. This interaction is additionally regulated by monomeric GTPases whose spectrum in Tetrahymena and Paramecium has been established by A. Turkewitz. As known from mammalian cells, GTPases are activated on membranes in conjunction with lumenal acidification by an H+ -ATPase. For these complex molecules, we found in Paramecium an unsurpassed number of 17 a-subunit paralogs which connect the polymeric head and basis part, V1 and V0. (This multitude may reflect different local functional requirements.) Together with plasmalemmal Ca2+ -influx channels, locally enriched intracellular InsP3 -type (InsP3 R, mainly in osmoregulatory system) and ryanodine receptor-like Ca2+ -release channels (ryanodine receptor-like proteins, RyR-LP), this complexity mediates Ca2+ signals for most flexible local membrane-to-membrane interactions. As we found, the latter channel types miss a substantial portion of the N-terminal part. Caffeine and 4-chloro-meta-cresol (the agent used to probe mutations of RyRs in man during surgery in malignant insomnia patients) initiate trichocyst exocytosis by activating Ca2+ -release channels type CRC-IV in the peripheral part of alveolar sacs. This is superimposed by Ca2+ -influx, that is, a mechanism called "store-operated Ca2+ -entry" (SOCE). For the majority of key players, we have mapped paralogs throughout the Paramecium cell, with features in common or at variance in the different organelles participating in vesicle trafficking. Local values of free Ca2+ -concentration, [Ca2+ ]i , and their change, for example, upon exocytosis stimulation, have been registered by flurochromes and chelator effects. In parallel, we have registered release of Ca2+ from alveolar sacs by quenched-flow analysis combined with cryofixation and X-ray microanalysis.

Thermal adaptation in a holobiont accompanied by phenotypic changes in an endosymbiont.

How and if organisms can adapt to changing temperatures has drastic consequences for the natural world. Thermal adaptation involves finding a match between temperatures permitting growth and the expected temperature distribution of the environment. However, if and how this match is achieved, and how tightly linked species change together, is poorly understood. Paramecium bursaria is a ciliate that has a tight physiological interaction with endosymbiotic green algae (zoochlorellae). We subjected a wild population of P. bursaria to a cold and warm climate (20 and 32℃) for ∼300 generations. We then measured the thermal performance curve (TPC) for intrinsic rate of growth (rmax ) for these evolved lines across temperatures. We also evaluated number and size of the zoochlorellae populations within paramecia cells. TPCs for warm-adapted populations were shallower and broader than TPCs of cold-adapted populations, indicating that the warm populations adapted by moving along a thermal generalist/specialist trade off rather than right-shifting the TPC. Zoochlorellae populations within cold-adapted paramecia had fewer and larger zoochlorellae than hot-adapted paramecia, indicating phenotypic shifts in the endosymbiont accompany thermal adaptation in the host. Our results provide new and novel insight into how species involved in complex interactions will be affected by continuing increasing global temperatures.

In situ remediation efficacy of hybrid aerogel adsorbent in model aquatic culture of Paramecium caudatum exposed to Hg(II)

Silica-gelatin hybrid aerogel of 24 wt% gelatin content is an advanced functional material suitable for the high performance selective adsorption of aqueous Hg(II). The remediation efficacy of this adsorbent was tested under realistic aquatic conditions by exposing cultures of Paramecium caudatum to Hg(II) and monitoring the model cultures by time-lapse video microscopy. The viability of Paramecium was quantified by analyzing the pixel differences of the sequential images caused by the persistent movement (motility) of the cells. The viability of Paramecium displays a clear exposure-response relationship with Hg(II) concentration. Viability decreases with increasing Hg(II) concentration when the latter is higher than 125 μg L−1. In the presence of 0.1 mg mL−1 aerogel adsorbent, the viability of the cells decreases only at Hg(II) concentrations higher than 500 μg L−1, and 220 min survival time was measured even at 1000 μg L−1 Hg(II). The effective toxicity of Hg(II) is lower in the presence of the aerogel, because the equilibrium concentration of aqueous Hg(II) is low due to adsorption, thus Paramecium cells do not uptake as much Hg(II) as in the un-remediated cultures. Video imaging of Paramecium cultures offers a simple, robust and flexible method for providing quantitative information on the effectiveness of advanced materials used in adsorption processes for water treatment.

Autolysis of Chlorella variabilis in Starving Paramecium bursaria Help the Host Cell Survive Against Starvation Stress.

The endosymbiosis between Paramecium bursaria and Chlorella spp. is mutualistic. Symbiotic algae localize beneath the host Paramecium cell cortex compete for their attachment sites with preexisting organelle trichocysts. To examine the relationship between P. bursaria trichocysts and their symbiotic algae, algae-bearing or alga-free P. bursaria were starved for several days and the changes in the number of Chlorella sp. and presence or absence of trichocysts were evaluated. We conducted an indirect immunofluorescence microscopy with an anti-trichocyst monoclonal antibody against P. bursaria cells. Indirect immunofluorescence microscopy demonstrated that under starvation and darkness conditions, the immunofluorescence of trichocysts in alga-free P. bursaria decreased much faster than that in the normal algae-bearing P. bursaria. In the latter case, our observations proposed the possibility that the nutrition obtained from symbiotic algal digestion may promote trichocysts synthesis. This algal digestion mechanism may permit host P. bursaria cells to survive for a longer time under starvation condition. To the best of our knowledge, this may be a new benefit that host P. bursaria gain from harboring symbiotic algae.

Diversity of RNA interference pathways in regulation of endogenous and exogenous sequences expression in ciliates Tetrahymena and Paramecium

RNA interference plays a major role in biology of ciliates. Diverse small RNAs regulate many processes in vegetative cells of ciliates Tetrahymena and Paramecium. Different types of endogenous and exogenous nucleotide sequences induce different RNAi pathways resulting in silencing of the homologous sequences in the macronuclear genome. Likely this way ciliates are able to quickly inactivate heterogeneous sequences and to adapt efficiently to the environmental conditions and external stimuli.

Crucial Role of Legionella pneumophila TolC in the Inhibition of Cellular Trafficking in the Protistan Host Paramecium tetraurelia.

Legionella pneumophila is a facultative intracellular Gram-negative bacterium, which is a major causative agent of Legionnaires’ disease. In the environment, this bacterium survives in free-living protists such as amoebae and Tetrahymena. The association of L. pneumophila and protists leads to the replication and spread of this bacterium. Thus, from a public health perspective, their association can enhance the risk of L. pneumophila infection for humans. Paramecium spp. are candidates of natural hosts of L. pneumophila, but their detailed relationships remain unclear. In the present study, we used an environmental strain, L. pneumophila Ofk308 (Ofk308) and Paramecium tetraurelia st110-1a to reveal the relationship between L. pneumophila and Paramecium spp. Ofk308 was cytotoxic to P. tetraurelia in an infection-dependent manner. We focused on TolC, a component of the type I secretion system, which is a virulence factor of L. pneumophila toward protists and found that cytotoxicity was dependent on TolC but not on other T1SS components. Further, the number of bacteria in P. tetraurelia was not associated with cytotoxicity and TolC was not involved in the mechanism of resistance against the digestion of P. tetraurelia in Ofk308. We used a LysoTracker to evaluate the maturation process of P. tetraurelia phagosomes containing Ofk308. We found that there was no difference between Ofk308 and the tolC-deletion mutant. To assess the phagocytic activity of P. tetraurelia, Texas Red-conjugated dextran-uptake assays were performed. Ofk308 inhibited phagosome formation by P. tetraurelia through a TolC-dependent mechanism. Further, we evaluated the excretion of Legionella-containing vacuoles from P. tetraurelia. We found that P. tetraurelia failed to excrete undigested Ofk308 and that Ofk308 remained within cells through a TolC-dependent mechanism. Our results suggest that TolC is essential for L. pneumophila to remain within Paramecium cells and to show cytotoxicity. Because of the high mobility and high cell division rate of Paramecium spp., living with Paramecium spp. would be beneficial for L. pneumophila to expand its habitat. To control Legionaries’ disease, understanding the ecology of L. pneumophila in the environment is essential.

Complexation of Eu(III), Pb(II), and U(VI) with a Paramecium glycoprotein: Microbial transformation of heavy elements in the aquatic environment

This study investigated the interaction of inorganic aqueous Eu(III), Pb(II), and U(VI) with Paramecium sp., a representative single-celled protozoan that lives in freshwater. Living and prekilled Paramecium cells were tested. The prekilled cells were killed with a fixative. After 24 h exposure of the cells to inorganic aqueous solutions containing Eu(III) or U(VI), analyses by microparticle-induced X-ray emission with a focused beam (<1 μm) did not detect Eu and U in the living cells, whereas Eu and U were detected in the prekilled cells. Size exclusion chromatography coupled with on-line ultraviolet–visible detection and elemental detection by inductively coupled plasma mass spectrometry of the aqueous phases collected after the living cell experiments revealed that a fraction of the Eu, Pb, and U in the aqueous phase bound to a large (ca. 250 kDa) Paramecium biomolecule and formed a metal-organic complex. The characteristics of the biomolecule were consistent with those of the soluble glycoproteins covering the surfaces of Paramecium cells. These results show that Paramecium cells transform inorganic aqueous Eu, Pb, and U to organic complexes. This paper discusses the relation between this novel complexation and the sorption of these heavy elements on Paramecium cells.

Short-pulse neodymium:yttrium–aluminium garnet (Nd:YAG 1064 nm) laser irradiation photobiomodulates mitochondria activity and cellular multiplication of Paramecium primaurelia (Protozoa)

Few studies exist to explore the potential photobiomodulation (PBM) effect of neodymium:yttrium–aluminium garnet (Nd:YAG) laser irradiation using a flat-top handpiece delivery system. In this study, we explored the photobiomodulation effect of that laser, on Paramecium primaurelia. The parameters for the different study groups were: 0.50W, 10Hz, 100msp, 30J/cm2; 0.75W, 10Hz, 100msp, 45J/cm2; 1.00W, 10Hz, 100msp, 60J/cm2; 1.25W, 10Hz, 100msp, 75J/cm2 and 1.50W, 10Hz, 100msp, 90J/cm2. Our results suggest that only the parameter 0.5W, 10Hz, 100msp, 30J/cm2 positively photobiomodulates the Paramecium cells inducing an increment in oxygen consumption, endogenous ATP synthesis and fission rate rhythm. Applying the laser energy with parameters of 1.25W, 10Hz, 100msp, 75J/cm2 and 1.50W, 10Hz, 100msp, 90J/cm2, induce adverse effect on the Paramecium cells, which protect themselves through the increase in Heat Shock Protein-70 (HSP70). The data presented in our work support our assumption that, when using appropriate parameters of irradiation, the 1064nm Nd:YAG laser with flat-top handpiece could be a valuable aid for effective clinical application of PBM.

Early stages of functional diversification in the Rab GTPase gene family revealed by genomic and localization studies in Paramecium species.

New gene functions arise within existing gene families as a result of gene duplication and subsequent diversification. To gain insight into the steps that led to the functional diversification of paralogues, we tracked duplicate retention patterns, expression-level divergence, and subcellular markers of functional diversification in the Rab GTPase gene family in three Paramecium aurelia species. After whole-genome duplication, Rab GTPase duplicates are more highly retained than other genes in the genome but appear to be diverging more rapidly in expression levels, consistent with early steps in functional diversification. However, by localizing specific Rab proteins in Paramecium cells, we found that paralogues from the two most recent whole-genome duplications had virtually identical localization patterns, and that less closely related paralogues showed evidence of both conservation and diversification. The functionally conserved paralogues appear to target to compartments associated with both endocytic and phagocytic recycling functions, confirming evolutionary and functional links between the two pathways in a divergent eukaryotic lineage. Because the functionally diversifying paralogues are still closely related to and derived from a clade of functionally conserved Rab11 genes, we were able to pinpoint three specific amino acid residues that may be driving the change in the localization and thus the function in these proteins.

Photobiomodulation by Infrared Diode Laser: Effects on Intracellular Calcium Concentration and Nitric Oxide Production of Paramecium.

In Paramecium, cilia beating is correlated to intracellular calcium concentration ([Ca2+ ]i) and nitric oxide (NO) synthesis. Recent findings affirm that photobiomodulation (PBM) can transiently increase the [Ca2+ ]i in mammalian cells. In this study, we investigated the effect of both 808 and 980 nm diode laser irradiated with flat-top hand-piece on [Ca2+ ]i and NO production of Paramecium primaurelia, to provide basic information for the development of new therapeutic approaches. In the experiments, the laser power in CW varied (0.1; 0.5; 1; and 1.5 W) to generate the following respective fluences: 6.4; 32; 64; and 96 J cm-2 . The 6.4 J cm-2 did not induce PBM if irradiated by both 808 and 980 nm diode laser. Conversely, the 32 J cm-2 fluence had no effect on Paramecium cells if irradiated by the 808 nm laser, while if irradiated by the 980 nm laser induced increment in swimming speed (suggesting an effect on the [Ca2+ ]i, NO production, similar to the 64 J cm-2 with the 808 nm wavelength). The more evident discordance occurred with the 96 J cm-2 fluence, which had the more efficient effect on PBM among the parameters if irradiated with the 808 nm laser and killed the Paramecium cells if irradiated by the 980 nm laser. Lastly, the 980 nm and 64 or 96 J cm-2 were the only parameters to induce a release of stored calcium.

Assessment of agglomeration, co-sedimentation and trophic transfer of titanium dioxide nanoparticles in a laboratory-scale predator-prey model system

Nano titanium dioxide (nTiO2) is the most abundantly released engineered nanomaterial (ENM) in aquatic environments. Therefore, it is prudent to assess its fate and its effects on lower trophic-level organisms in the aquatic food chain. A predator-and-prey-based laboratory microcosm was established using Paramecium caudatum and Escherichia coli to evaluate the effects of nTiO2. The surface interaction of nTiO2 with E. coli significantly increased after the addition of Paramecium into the microcosm. This interaction favoured the hetero-agglomeration and co-sedimentation of nTiO2. The extent of nTiO2 agglomeration under experimental conditions was as follows: combined E. coli and Paramecium > Paramecium only > E. coli only > without E. coli or Paramecium. An increase in nTiO2 internalisation in Paramecium cells was also observed in the presence or absence of E. coli cells. These interactions and nTiO2 internalisation in Paramecium cells induced statistically significant (p < 0.05) effects on growth and the bacterial ingestion rate at 24 h. These findings provide new insights into the fate of nTiO2 in the presence of bacterial-ciliate interactions in the aquatic environment.

Ciliary metachronal wave propagation on the compliant surface of Paramecium cells

Ciliary movements in protozoa exhibit metachronal wave-like coordination, in which a constant phase difference is maintained between adjacent cilia. It is at present generally thought that metachronal waves require hydrodynamic coupling between adjacent cilia and the extracellular fluid. To test this hypothesis, we aspirated a Paramecium cell using a micropipette which completely sealed the surface of the cell such that no fluid could pass through the micropipette. Thus, the anterior and the posterior regions of the cell were hydrodynamically decoupled. Nevertheless, we still observed that metachronal waves continued to propagate from the anterior to the posterior ends of the cell, suggesting that in addition to hydrodynamic coupling, there are other mechanisms that can also transmit the metachronal waves. Such transmission was also observed in computational modeling where the fluid was fully decoupled between two partitions of a beating ciliary array. We also imposed cyclic stretching on the surface of live Paramecium cells and found that metachronal waves persisted in the presence of cyclic stretching. This demonstrated that, in addition to hydrodynamic coupling, a compliant substrate can also play a critical role in mediating the propagation of metachronal waves.