Here you will find movies of feeding and swimming volvocalean colonies. Enjoy! If you are interested in the evolution of sex in Volvocales, click here.

The general goal of our work is to deconstruct fitness in volvocalean colonies of increasing size and complexity so as to understand the transition from unicellular to multicellular life in this lineage. The specific goal of the project described here is to understand the viability component of fitness by studying motility and feeding capacities. Some theoretical background for the hydrodynamic analysis is given first to give an idea of how we calculate the swimming speeds and swimming force per motile cell using the videos.

A Hydrodynamics Approach to Motility, Mixing of Local Habitat, and Acquisition of Metabolites: A Crucial Aspect of the Viability Component of Fitness

Thanks to Cristian Solari, John Kessler and Ray Goldstein. Visit Ray Goldstein's site for a lot more on Volvox hydrodynamics.

Theoretical Background

Volvocales can be modeled as moving spheres in the low Reynolds number regime (Re = RVr /h < 1; R=colony radius, V=speed, h =viscosity of water, r =density of water). Re is always < 1 even for a large Volvox swimming fast, so we can use the Stokes drag force (F) acting on a moving sphere (F=6phRV). At a low Re, a sedimenting colony reaches a terminal velocity given by an equilibrium between the Stokes drag force and the effect of gravity. Therefore, the negative buoyant force of a colony equals the drag force as expressed in Eq. 1, where ∆M is the average difference in mass between the colony and the displaced water, g is the acceleration of gravity, and V_sed the sedimentation velocity. When motile, a colony’s upward locomotion is governed by a basic force balance equation: force by all flagella = drag proportional to upward velocity (V_up) plus the downward force of gravity which gives Eq. 2. The flagellar force Xf in Eq. 2 is a function of the number X of flagellated cells (somatic or undifferentiated reproductive cells) and the mean upward swimming force per motile cell (f).

    Eq. 1

  Eq. 2

By measuring both V_up and V_sed, from movies similar to those provided here, and using Eq. 3, we can test these relationships and see how a colony organization affects motility.

  Eq. 3

Colonies are video taped on the optical bench shown here in John Kessler's lab and from these tapes V_up, V_sed, and R are measured as are the number of cells and proportion of cell types. These variables allow for the calculation of the other parameters in Eq. 1-Eq. 3. Examples of the video tapes are given next.

Movies

Movie file of Volvox carteri swimming and sinking for calculation of hydrodynamics model. Notice that mature large colonies starting cleavage (after 12 hours) can't swim upwards. (8MB Windows Media, 15MB Quick Time, 5MB Quick Time)

Movie file of Pleodorina californica (notice increasing flow rate into colony by flagellar beating) and Haematococcus (a bi-flagellated unicellular algae).  (4MB Windows Media, 5MB Quick Time, 2MB Quick Time)

The Effect on Feeding of Enhanced Transport Due to Flagella-driven Flows: A Viability Component of Fitness

Thanks to Cristian Solari, John Kessler and Ray Goldstein.

Theoretical Background

We are using particle image velocimetry (PIV) to study flagella-driven flows surrounding sessile colonies of three species of Volvox. It is usually assumed that flagella exist for motility. While it is certainly true that flagella provide motility, we believe it is also true that they produce crucially important transport and mixing flows that aid in resource uptake and the removal of waste products. Passive nutrient uptake is governed by diffusion, and is limited by the gradient of concentration, established by consumption and diffusion. Vigorous stirring, and flow due to swimming, increase supply of resources and removal of waste products at the organism surface thereby ameliorating the diffusion constraints. We call this process "flagellar feeding." To guide research on the tradeoff between fecundity and motility an estimate of the importance of these transport processes becomes necessary. The competition between diffusion and advection is conventionally estimated by the ratio (Peclet number Pe) of time constants for transport. Pe= t_diff/t_adv = LV/D, where L is distance, V the fluid velocity, and D the diffusion coefficient of the resource in water. If Pe<1, diffusion wins, ruling out strong interaction between motility and fecundity. If Pe>1, advection becomes important for uptake; a simple tradeoff between motility and fecundity is ruled out and a model that considers their interaction must be developed. We predict for the Volvocales that the role of flagella beating, yielding chaotic mixing and nutrient uptake, will increase as colony size increases, and concomitantly, so will the interaction between the fitness components. We are studying the factors: force, mixing and long range advection, by measuring flagellar size, beating rate, associated transport of passive tracers, and flow rate, with a high speed camera and Particle Image Velocimetry (PIV) setup shown here in Ray Goldstein's lab.

Generation and Analysis of Dynamic Graphic Data

Volvox colonies accidentally lodged on transparent substrates are ideal subjects for demonstrating the flow of fluid that surrounds them. That flow is entirely due to the action of the peripheral flagella. The orientation and character of the flow field depends on the orientation, e.g. whether the coenobium is seen from the top or side, of the particular imaged algal colony. The orientation dependence is due to azimuthal variations in beat patterns. Future experiments will utilize free-swimming as well as sessile coenobia. The flow field is visualized by entrained particles. These particles, together with the spherical assembly of cells that causes the motion of the fluid that entrains them, are readily observed in bright field. The 1 um diameter entrained flow-marking particles are fluorescent. They serve as high contrast markers when laser-stimulated, with all extraneous illumination removed. Particle Imaging Velocimetry (PIV) uses these particles, analyzed via a long series of pairs of video frames, to generate the vector field characterizing the velocity of the flow generated by the Volvox colonies. In the following sequences we present bright field videos, fluorescence videos, and analyses of the vector fluid flow fields generated by the flagella. The analyses are averaged over 15 second sequences of video, at 30 frames per second. The motion of flagella, and evanescent mixing whorls are detectable by shorter duration analyses. The averages presented demonstrate the long range transport of particles, and, inferentially, molecules, crucially important for the viability of these algae.

Movies where the action (the motion of the fluorescent markers) can be analyzed using the PIV system

Quick Time movies (about 1.2 MB each) showing flagellar feeding of V. carteri (narrow-band laser illumination, bright field) of V. rousseletii (narrow-band laser illumination, bright field) and of V. tertius (narrow-band laser illumination).

Vector field images of flow around Volvox colonies using the PIV system

Below is an image of the vector flow fields for V. carteri (the image on the right is a magnification of the region shown in the left). Click for the corresponding images for V. tertius (two neighboring colonies) and for V. rousseletii. Note the much higher flow (almost double) created by V. rousseletii than by V. carteri. Special thanks to Luis Cisneros for preparing these images.

Pictures Below. PIV-derived fluid velocities (field arrow lengths) surrounding the colonies of (A) V. carteri top view; inset arrow 50 um/s, and (B) V. rouselletii side view; inset arrow 100 um/s. The flows are driven solely by the somatic cells' flagella at the surfaces of the colonies. Note the differences in velocities by examining the ratio of the lengths of the field arrows to the scale arrow. The flows in (B) are approximately double the flows in (A).

What to look for in the movies and PIV analysis

The three Volvox species studied differ in developmental mode, degree of germ soma differentiation and ratio of somatic to reproductive cells (S/R ratio). V. carteri is completely germ/soma differentiated (G/S), the S/R ratio is 142, and during development there is an asymmetric cell division creating gonidia which first grow large and then divide rapidly. V. tertius  is also G/S, but the S/R ratio is smaller at 99, and there is no asymmetric division. Gonidia start small, grow large and then divide slowly, without further growth, to produce equal sized daughter cells. V. rousseletii is not completely germ soma separated (GS/S); the germ cells first have flagella before differentiating, but the S/R ratio at 332 is the largest of the three species. Developmentally rousseletii is quite different, the gonidia start small and grow in between symmetrical cell divisions.

Notice how the colonies are able to bring nutrients from far away. The V. carteri colony is viewed from the top. In the bright field movie, the fluorescent passive markers are barely visible, the Volvox colony is seen clearly. The V. rousseletii colony is viewed "from the side". In the bright field video, note that the concentration of somatic, flagella-bearing cells is much greater than for V. carteri and that the flow is greater (100 microns/sec for rousseletii versus 50 for carteri).  In the PIV vector analysis, note the "exchange of fluid": influx upper left; outflow in lower right. In the V. tertius video, there are two adjacent colonies. Note that some markers have attached to flagella. Their whirling motion can be followed easily in the movies. This motion not only drives "macro-" flows, but also serves to mix and deflect localized parcels of fluid. In the high resolution PIV vector analysis, note the counter streaming over the middle of the colony.

The magnitudes (cm/s) of the flow fields (and beating rates of flagella which are coming soon) along with the shape of the vector field tell the story of enormously enhanced (compared to diffusion, see Peclet number above) long range acquisition/discharge of metabolites due to flagellar action. These data show also (if you look closely at the movies) that the flagella do local stirring. All of these phenomena greatly enhance transport in and out of the colony.

Future directions using PIV to evaluate algal performance

The PIV system allows us to begin to ask rather sophisticated questions about the biology and fitness of aquatic organisms such as the Volvocales under realistic conditions. One of the most important questions in biology is - how do abiotic features of the environment interact with morphological and physiological characteristics of organisms to influence fitness? For example, how does a change in organism size or complexity (somatic / germ cell ratios, motility and flagellar beating) alter the interactions with the physical environment and processes such as energy acquisition? Our goal is to use the vector fields from the PIV along with details of the hydrodynamic condition surrounding the colonies, coupled to measures of metabolism to understand how physical and biological factors interact to influence organism performance. Specifically, we plant to evaluate the relative importance of diffusional versus advective forces in limiting the supply of substrates to the photosynthetic machinery of these aquatic colonies (see "The effect of enhanced transport…", above).

We will use the coupled PIV and chlorophyll fluorescence system discussed below to evaluate volvocalean species of varying complexity and colony sizes. Presumably, colonies of different size experience different physical conditions of the environment, such as an altered boundary layer surrounding the colony. For each species, we will manipulate factors such as the pCO2 of the surrounding media, while measuring associated changes in vector fields (to evaluate how flagellar stirring changes boundary layer conditions) and photosynthetic fluorescence (to evaluate changes in photosynthetic performance result from changes in transport constraints). This will allow us to consider physiology, flagellar behavior and physical factors under controlled conditions to see how the evolution of organism size has been influenced by constraints on resource transport. This is of keen interest to us, as it links the our two measures of fitness (viability and fecundity) through a single adaptive characteristic, the flagella. We will evaluate how flagellar stirring changes boundary layer conditions in response to environmental perturbation, controlling metabolism; and how instantaneous changes in photosynthetic performance result from changes in transport constraints. This will allow us to carefully consider how life history characteristics of colonies in our model system (such as motility, resource acquisition and reproduction) interact. Additionally, it allows us a means to evaluate how changes in organism size may influence the ability for resource use, reproduction and survival.

The PIV system uses a high-speed camera with a fluorescence detection device that can be tuned to evaluate physiological processes associated with photosynthesis. The irradiance system in the PIV can be used to preferentially excite photosynthetic pigments allowing specific evaluation of different processes. Photosynthesis is measured by fluorescence using the following technique - dark adapted samples are irradiated with short wavelengths (< 680 nm), and fluorescence from excited chlorophyll complexes is measured between 680 and 760 nm. The ratio of fluorescence in the dark adapted state (F_o) is compared to the maximum fluorescence (F_m) following saturating irradiation (with a flux density of ~ 5000 moles m^-2 s^-1). The ratio of F_v (variable fluorescence - F_m-F_o) to F_m is considered an estimate of "photosynthetic efficiency" and is useful for understanding both metabolic performance and generalized stress in these organisms (it varies between 0.8 and 0.3, with low to high efficiencies respectively). Similarly, the quantum yield of the photosynthetic apparatus can be calculated by measuring maximum fluorescence next in a light adapted state. The rate of electron transport through the photosystems can be determined from the quantum yield if irradiance is also known, allowing for stochiometrically determined photosynthetic rates.