Poster Y10 - Microfluidics for Hydrodynamics of Ciliated Larvae

Introduction: Most benthic marine invertebrates possess a larval stage that employs a ciliary band for locomotion and feeding. Beyond marine organisms, cilia have evolved for specialized functions in the human body. Difficulties in obtaining, manipulating, and imaging cilia have prevented studies on cilia physics and fluid dynamics. Using marine invertebrate larvae to understand physical characteristics of ciliary action offers logistical advantages.
The Pacific sand dollar, Dendraster excentricus, is abundant along coastal California. Its larvae sequentially develop pair of arms that support a continuous ciliary band that circumscribes the larva. Sand dollar larvae exhibit phenotypic plasticity in which arm length depends on the amount of available food (unicellular algae). Larvae in low-food conditions grow longer arms to support a longer ciliary band which increases feeding capacity.
Sand dollars’ plasticity makes them a useful model system for studying ciliary dynamics. Such studies could further our understanding of larval plasticity, inspire biomimetic designs, and lead to approaches for maintaining species amidst climate change. These investigations could also shed light on unifying principles of ciliary action that can be applied to human diseases that involve compromises in ciliary function (e.g. CFTR).
We developed a microfluidic-miniaturized particle image velocimetry (mPIV) system to characterize cilia-induced hydrodynamics. mPIV was used to generate path tracking (FlowTrace), velocity, and vorticity measurements with flow and no flow. Velocities were used in dimensionless analysis (Reynolds and Péclet numbers) to elucidate how cilia aid in particle capture. The methods were also extended to sea urchin (S. purpuratus) and sea star (P. helianthoides) larvae.

Materials and

Methods: D. excentricus and S. purpuratus adults were collected from the Los Angeles Harbor in San Pedro, CA (33.7088, -118.2806). Adults were spawned by injection of 0.5 M KCl into the body cavity to induce gamete release. Fertilized eggs were stocked at 1 per mL in 20-L vessels at 16° C (±1° C). Larvae were fed the red unicellular algae, Rhodomonas lens, at 1,000 cells per mL. Throughout development, larvae were selected for microfluidic analysis.
The microfluidic mold, a 4 mm x 5 cm straight channel, was cut from acrylic (1.59 mm thick) using a CO2 laser cutter. Polydimethylsiloxane (PDMS) was cured (1:10 linker to base) and poured onto the mold. A craft knife, tweezers, and 1.5 mm hole-punch were used to remove the PDMS, create an inlet/outlet, and apply it to a glass slide via plasma bonding.
The microfluidic channel was filled with fluorescent beads (1.1 µm diameter, Fluoro-Max) in filtered seawater (20° C) (1:100). Larvae were individually loaded into a primed chip. A syringe pump (KDS Legato) pumped the bead/seawater solution during flow conditions (15 µL/min). Larvae were imaged with a fluorescence microscope (Leica DMI3000) (Figure 1A).
ImageJ was used to measure morphology, convert images to video, adjust contrast, and apply FlowTrace path tracking. PIVlab (MATLAB GUI) quantified the velocity and vorticity of fluorescent beads in cilia-induced flow. Videos displaying bead velocity vectors, velocity magnitude (heat map), and vorticity (heat map) were generated (Figure 1B). Velocities were used to calculate two dimensionless numbers: Reynolds number and Péclet number.

Results, Conclusions, and Discussions: The feasibility of tracking cilia-induced flow was shown by generating FlowTrace videos, velocity vector, velocity magnitude, and vorticity videos for larval (low-fed and high-fed) and juvenile sand dollar forms. The juvenile sand dollar showed reduced vortex patterns and velocities due to retracted arms and cilia loss.
Next, we imaged 21 D. excentricus larvae for 11-20 days post fertilization (DPF), each with flow (15 µL/min) and without flow. FlowTrace, velocity, and vorticity videos were generated for each larva (Figure 1C). The same videos were produced for P. helianthoides and S. purpuratus larvae (61-67 DPF) (Figure 1D & E).
Reynolds and Péclet numbers were calculated for sand dollar larvae, using the maximum average velocities across a line of interest (between the mouth and arm tips). Reynolds numbers were < 0.06 and Péclet numbers (for oxygen diffusion) were < 65.
The dimensionless analysis of sand dollar larvae provided insight into how cilia aid in particle transport (i.e., food capture and waste release). Reynolds number was less than 1, suggesting a laminar, viscous-dominated flow, which makes it difficult for the larva to move particles. Péclet number (>1) however, suggestion that ciliary behaviors help showing that cilia move particles via advection, overcoming the larva’s diffusion-barrier.
To investigate cilia-induced hydrodynamics, we used a flat microfluidic channel, allowing for standard microscope imaging and mPIV. A vertically oriented microfluidic chip would more accurately represent an ocean environment and enable the study of cilia during swimming and sinking. A new device and innovative imaging methods are required to image horizontally.
Beyond flow, the microfluidic-mPIV system could test the effect of other conditions on larval cilia (e.g., pH and temperature). Testing these conditions would not only deepen our understanding of cilia but also provide insight into larval responses to changing ocean conditions.

Acknowledgements (Optional): Work was supported in part by awards from CSUPERB, STEM-NET, CSULB BUILD scholarship to W.Chen (NIH; UL1GM118979; TL4GM118980; RL5GM118978) and CSULB-COAST awards.   Authors express gratitude to members of CSULB Marine lab, Pace Lab, and Ahrar Lab.