Veils in the Water: “Schools” of Protozoa

swarm-205622
“Veil” in week-old sample from Washington Park Arboretum

Sample some pond water, bring it home and let it sit quietly for a week under a light, and you may experience a fascinating sight: ghostly veils and arcs hovering in the water.

After sitting quietly under a desk lamp for a week, a little thrift store aquarium full of water, pond plants, and algae from the Washington Park Arboretum developed these eerie white arcs hovering above a clump of algae (See arrow). They are very difficult to record -it is frustrating to persuade the autofocus on a camera to lock onto them – but with multiple exposures and much processing, it was possible to create a credible image.

Carefully inserting a pipette, I sucked up the area, capturing a few milliliters of what appeared to be clear, completely unremarkable water – but again the microscope showed another world. Spread across the field were a swarm of tiny green, peanut-shaped organisms, each swimming with a peculiar curliqued movement: several rounds in a circular spiral, then a rest, then another circle, then a pause, and so on:

Still images revealed small, peanut-shaped flagellates, each with two curled flagella:

swarm-213326

swarm-flagellates-213758

These are tentatively identified as Cryptomonad algae, possibly Chilomonas (thanks to Wim von Egmond and Vincente Meneu, Amateur Microscopy Facebook group).

I have noted two other occurrences of protozoan schooling.  In one case, delicate but very distinct cloudy veils formed in a mini-aquarium of pond water after it had been set up for several weeks; unfortunately, I was not able to take a sample.  However, in my very first observation of protozoan veils, a 3 cm, cloudy band appeared one morning against the glass of my original mini-aquarium.  A 4X microscope objective on my Samsung cell phone camera showed a school of tiny organisms:

20151211_005258-ce-sharpened
Protozoan school in aquarium, 8-10X

Under the microscope, the tiny dots resolved into a swarm of dinoflagellates:

 Only one other microscopist has commented on this phenomenon on the web:

“In 2006 I noticed a discretely-shaped “veil” about 2 – 3 millimeters in extent, moving through the water of a mini-aquarium where I was also looking for hydras. The shape formed was like a section of an auger bit, or the twisted form of a DNA molecule (but only about 1/2 to 3/4 of a “twist”), and sometimes like a “saddle shape.” It also turned out to comprise something that looked most like Chlamydomonas, but I did not have good enough lighting (mirror understage only) to see bacteria.

I have often wondered since then, how many people are aware of protozoans swimming in organized schools, and what mechanism they use to communicate their positions to each other. The shapes they formed had VERY definite edges (L. Hizer, personal communication, Facebook comment, 2016/09/24)”.

As noted by Mr. Hizer, this phenomenon raises fascinating questions, .  How are these swarms created? Is there a mechanism by which the cohesion of these protozoan “schools” is maintained?  If so, on what basis does it function?  Chemotaxis, light sensitivity, or some unknown mechanism by which protozoa sense each other, rather like flocking behavior in  birds?

The slide of the Cryptomonads also showed many slender bacteria – could this swarm be a collection of organisms clustering around a food source, rather like killer whales around a school of herring, or a response to some bacterial metabolic product?

swarm-bacteria-213655
Many bacteria in a Cryptomonad swarm.  What is their significance?

The most likely answer comes from research on something called “microscale nutrient patches“.  Early modeling studies on movements of plankton, especially unicellular organisms and marine bacteria, considered lake or sea water as a homogeneous medium, but modern thinking suggests that nothing could be further from the truth.  Consider that our environment – the air around us – is anything but homogeneous.  If you ride a motorcycle outside the enclosed environment of a car, you will experience dozens of micro-environments in the course of an hour’s ride:  hot and fragrant near a newly-mown hayfield, damp and cool in a gully, smelly behind the exhaust of a bus, windy and salty near the ocean. Not to mention the aroma wafting downwind from a roadside rib joint…

Now transpose this image to the Bering Sea or a pond basking in the sun.  Not only is the chemical environment different at different depths, but a large body of recent research suggests that every plant, invertebrate, or single-celled organism releases a cloud of chemicals in its immediate environment (see Blackburn). Many of these are vacuumed up by motile bacteria  as food, while others serve as attractants for other unicellular creatures.  Observing the motile bacterium P. haloplanktis, Vahora noted chemotaxtic attraction occurring within seconds  when a single cell of the alga Thalassiosira was introduced into the culture.

On consideration, this is not surprising; the world is full of gradients of chemical attractants and repulsants.  Lemon sharks can detect tuna oil several hundreds of meters away at a concentration of one part per 25 million.  Feeding releases blood and body fluids that attract more sharks, etc.  Anyone who has observed a family hovering outside the kitchen while the Thanksgiving turkey cooks should immediately grasp the concepts of chemotaxis and nutrient patches.  Coco Chanel has made millions from the principle of chemical attraction.

This phenomenon has been extensively studied, and has been documented in many species. The unicellular flagellate algae Chlamydomonas and Dunaliella tertiolecta have been shown to be chemotactic to several substances, including ammonia. The ciliate Paramecium has become the model organism for these studies on protozoa, with development of commercial vat cultures and creation of genetic mutants lacking various chemoreceptors (see the excellent review by Houton and Preston).  Such studies can be of enormous importance, as they may shed light on how cells in general (including our own body cells) sense their environment and react.  Chemotaxis plays a critical role in wound healing and embryonic development, and defective chemotaxis is thought to have significant involvement in asthma, atherosclerosis, and chronic inflammatory diseases (see Kamp et al.)

So how does chemotaxis occur, and how do the dinoflagellates and algae in our specimens congregate to form swarms?  Mechanisms seem to be different between bacteria and protozoa.  Bacteria execute what is called in statistics a “random walk“, where locomotion consists of a “run” and a “tumble.” With E. coli, “runs”, or movement in a straight line, occur when the flagella rotate counter-clockwise, and “tumbles”, or directional changes with no forward movement, occur when the flagella rotate clockwise. “Run” length, speed, and frequency of “tumbles” are all variable.  When a cloud of bacteria finds itself in a concentration gradient of nutrients, these variables change in response to the composition of the immediate environment.  Movement up the nutrient gradient is favored, leading to a “biased random walk”  with the cloud drifting toward the food source:

grad_2d
Bacterial “random walk” up nutrient concentration gradient
find_food
Bacterial random walk toward food source (courtesy Schmidt and Kardan)

Bacteria respond to external chemical stimuli through transmembrane “methyl-accepting chemotaxis proteins” or MCPs.  After binding to specific chemicals in the surrounding fluid, MCPs transmit the message across the membrane into the cytoplasm, where Che proteins are activated.  One Che protein type interacts with the “Flagellar Switch Protein”, changing flagellar rotation from counter-clockwise to clockwise and causing a tumble.  Changing tumble frequency biases the random walk toward the higher end of the gradient, and the whole population drifts in this direction.  This is discussed in Wikipedia’s article on chemotaxis.

525px-chtxasprec
Bacterial Transmenbrane Methyl-accepting Chemotactic Protein (MCP)

This is how a cloud of bacteria move.  So what happens in an animal or plant, non-bacterial cell, when chemotaxis occurs?  You can thank all those little creepy organisms  in the soil and water billions of years ago for doing the basic R&D. The mechanisms they developed seem to have worked, because we are still using the same chemicals, whether you are a slime mold, an annelid worm, or a Member of Parliament.  We all use “G Protein Coupled Receptors” or GPCRs, a family of proteins that transmit information across the cell membrane.  GPCRs have a charged N-terminal end on the outside of the membrane, seven fatty helices that span the lipid-rich center of the membrane, and a charged, water-soluble C-terminal end inside the cell.

The N-terminus on the membrane surface (cell surface “receptor”) is configured to bind to a specific chemical or hormone; binding causes a subtle structural shift (conformational change) on the outside segment of the GPCR protein.  This shift in the complex, tangled yarn of the protein echoes across the membrane to the segment inside the cell, where GTP, a signaling chemical or “messenger“, is released, unleashing an enormously complex chemical cascade:

ijms-17-00090-g003
Mechanism by which binding of a small chemoattractant to a membrane surface GCPR releases messengers GTP and GDP, with subsequent activation of actin and myosin fibers via complex chemical pathways.  See Kamp et al. for nomenclature and discussion.

In slime molds and human neutrophils, activation of this mechanism causes the whole cell to rumble forward, “…creating actin-rich pseudopods at the front and retracting the back of the cell using myosin filaments…”  This mechanism is detailed in Kamp et al’s just-published and very complex, but excellent, review article.  In pond critters, this mechanism controls the direction and speed with which they swim toward a food source.  The intracelluar architecture of this process is enormously complicated, and our understanding is in its infancy.

Research on chemotaxis and directed locomotion raises more questions than answers once we get above the level of bacteria.  Paramecia seem to display elements of the both the biased random walk and directed swimming. However, the mechanisms by which higher single-celled organisms interact with their environment are multifaceted, and much more complex than those used by bacteria. Protozoa are large enough that a chemical gradient outside the cell may be reflected in a chemical messenger gradient inside the cytoplasm from front to back; the recently-discovered complex phospholipid PIP3 may be involved in this process.  In addition to releasing internal messenger chemicals, binding of chemoattractants to surface GCPRs causes depolarization and changes in the transmembrane voltage that control swimming (Valentine et al.). Receptors are probably distributed in specific areas over the cell membrane, and there is evidence that they can be synthesized quickly in response to stimuli.

Cilia themselves are covered by specialized extrusions of the membrane, act as sensory organs, and have complex internal functions that involve molecular transport between the tip and base:  “…cilia and flagella are micromachines and they act as cybernetic devices to receive, process and communicate information… (see Linck).  Voltage changes in the ciliary membrane open calcium channels that affect the strength of the ciliary wave, altering direction and speed.  Control of ciliary and flagellar beating, and hence direction and speed of swimming, appears to involve a complex and poorly-understood relationship between ciliary membrane voltage, calcium and magnesium ion movement, and intracellular released cyclicic nucleotides (cAMP and cGMP) (see Andrivon).  Cilia may also be in part coordinated by a system of intracellular fibers and tubules.

These mechanisms have implications for more than the wanderings of protozoa.  A whole spectrum of genetically-coded diseases are now thought to be “ciliopathies“, or inherited defects in ciliary-associated functions.  The body of research in all of these areas is vast and fascinating.

f1-medium
Complexity of ciliary architecture and transport mechanisms (see Emmett et al.)

Amazing that we can couple a little shadowy swarm in a cloudy bottle of water to such profound mechanisms at the root of our own existence!

So what does all this tell us about our little watery veils of algae and dinoflagellates?  The occurrence of these miniature swarms of protozoa is probably associated with a tiny, invisible patch of food.  Chilomonas is a cryptomonad flagellate that has no pigment in its chloroplast (leucoplast) and must survive on soluble compounds.  Its favorite carbon source is acetate, but it will survive equally well on succinate, and also gains nutrition from straight-chain fatty acids and alcohols (see Nisbet, p. 166).  Both succinate and acetate are, however, produced under anaerobic (low oxygen) conditions, and would more likely to be found in sediments and ooze. Another attractant is more likely to be responsible.

It is, however, interesting that the Chilomonas swarm was associated with a proliferation of bacteria.  Were the Chilomonas slurping up waste products of the bacteria, or was it the other way around?  With the other swarm, Dinoflagellates have enormously broad feeding behavior, ranging from photosynthetic species to predatory varieties, so understanding their swarming responses is more uncertain.  Clearly, they were attracted by something in that small area, even though we could not see it.

Protozoan schooling may be much more common than previously recognized.  Even when present, protozoan clouds are very difficult to see with the naked eye.  Like ribbons of smoke rising in the air, these delicate aquatic veils are apparent only with appropriate lighting, ideally from the side.  They would be virtually invisible through the water’s surface in ordinary sunlight.  Furthermore, they would form only in very still waters and be dispersed by even a gentle current.  You are encouraged to look for these delicate structures next time you leave a bottle of pond water on the window sill.

THE VALUE OF AMATEUR MICROSCOPY:

This is the beauty of natural science:  a simple observation of  phenomena in a bottle of muddy water opens a path to principles critical to the function of life itself.

Discovering this delicate and fleeting phenomenon is a testament to the potential value of work done by dedicated amateur microscopists with careful observational skills and a desire to communicate their findings.  Even modest equipment can allow worthwhile observations like these (done with a 45-year old Nikon S-Kt, a cell phone camera, and an international group of helpful fellow enthusiasts).  Professional scientists are too often torn between teaching, grant writing, and gaining tenure, not to mention their own research (often farmed out to graduate students), to have time for interesting natural science.  Research topics are often strongly influenced by what will be funded, and observing pond water is not often the best path up the academic ladder.

The situation is similar in astronomy.  A night of observing time on the Keck Telescope costs $53,700.  Obviously, this is one good reason why new asteroids and comets are often detected by amateurs with basic equipment, time to spend, and no research committees to satisfy.  As an example of the value of amateurs to astronomy, NASA is presently promoting a new program (part of the ORISIS asteroid sampling mission) to engage amateur astronomers in documenting new asteroids, an avenue of research that can now be done with a good 8″-10″ telescope and CCD camera.  “Citizen Science” is well organized within NASA, and there are many established avenues for competent amateurs to make scientific contributions.

Although scientific opportunities outside astronomy or established institutions are miserably infrequent (see Kathlyn Mills article in Wired-UK, and Bruce Bigelow’s still-relevant 1996 article in The Scientist), some formal opportunities exist for citizen scientists. Some of these may provide opportunities for microscopists.  Citizenscience.gov is a federal web site sponsored by the General Services Administration and the Wilson Center; as of October 2016, it listed 303  federally-sponsored natural science projects needing citizen scientist participation.  Europe has the European Citizen Science Association, and Australia has the Australian Citizen Science Association.

There is less public attention and less funding for the world of the very small as compared to NASA’s world of the very large, but amateurs are making definite contributions.  Popular Mechanics article, “Inside Amateur Science: The Best in Out-of-Lab Research“includes the work of Ely Silk, who makes fluorescence LED illuminators for microscopists. The Diatom Herbarium of Drexel University and much of the database of New Zealand diatomology were largely built from the collections of amateur diatomologists.  Loren Bahls‘ recent paper details the important role played by “…citizen volunteers…(and)…discusses the process of engaging citizen volunteers in diatom collection and the value of citizen collections in building diatom herbaria, in cataloging diatom biodiversity, and in expanding our knowledge of diatom biogeography, especially in remote regions with difficult access…”

More than anything, there is an enormous need for microscopists who will popularize this essentially unknown avocation.  Microscopy is a better-known pastime in Britain, which has a long tradition in this area.  In the U.S. and Canada, I am met with either fascination or blank looks.  At best, I am viewed as a harmless eccentric; I worst, I am still waiting to be picked up by the police during my midnight marsh wanderings.  There is a great need for people who will set up a chair and a portable microscope under a tree at a lakeside beach and wait for curious children and parents to congregate.  Try it!

REFERENCES:

American Museum of Natural History.  “Myth 5:  Sharks can Detect a Single Drop of Blood in the Ocean.”  http://www.amnh.org/learn/pd/sharks_rays/rfl_myth/myth_page5.html

Andrivon C.  “Membrane Control of Ciliary Movement in Ciliates.”  Biol Cell. 63:133-42 (1988).  https://www.ncbi.nlm.nih.gov/pubmed/2849495

Blackburn, N., Fenchel T, Mitchell JG.  “Microscale nutrient patches in plankton habitats shown by chemotactic bacteria.” Science 282: 2254-2256

Clark, R.  “Combined Amateur Telescopes for Asteroid Detection.” Polymath Blog, April 12, 2016. http://exoscientist.blogspot.com/2016/04/combined-amateur-telescopes-for.html

Emmer, B.T., et al.  “Molecular Mechanisms of Protein and Lipid Targeting to Ciliary Membranes.”  J. Cell Sci. 123: 529-536 (2010). http://jcs.biologists.org/content/123/4/529

Govorunova, E.G. & Sineshchekov, O.A. “Chemotaxis in the Green Flagellate Alga Chlamydomonas.”  Biochemistry (Moscow) (2005) 70: 717

Haake, A.  “Inside Amateur Science:  The Best in Out-of-Lab Research.” Popular Mechanics Online, June 10,2009.  http://www.popularmechanics.com/science/a4343/4321192/

Houten, J.V. and Preston, R.R.  “Eukaryotic Unicells:  How Useful in Studying Chemoreception?”  https://www.uvm.edu/~biology/faculty/documents/EucaryoticUnicells-HowUsefulinStudyingChemoreception.pdf

Kamp, M.E. et al.  “Function and Regulation of Heterotrimeric G Proteins during Chemotaxis”.  Int J Mol Sci. 2016 Jan; 17(1): 90.  Published online 2016 Jan 14.  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4730333/

Linck, R.W.  “Cilia and Flagella.” In: eLS, Citable Reviews in the Life Sciences, July 2015. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001258.pub3

Nisbet, B. Nutrition and Feeding Strategies in Protozoa. Croom Helm Ltd, London, p. 166 (1984).  https://books.google.com/books?id=tj6SBgAAQBAJ&pg=PA166&lpg=PA166&dq=nutrition+of+chilomonas&source=bl&ots=nTn9X3R8V9&sig=NDCdeH70_MQIAkMOwtOt0aZQ-mo&hl=en&sa=X&ved=0ahUKEwiHwuq9qszPAhUQ9GMKHTbRDvEQ6AEIPzAH#v=onepage&q=nutrition%20of%20chilomonas&f=false

Schmidt, A. and Kardan, M. “Chemotaxis:  How a Small Organism finds a Food Source.”  http://www.mit.edu/~kardar/teaching/projects/chemotaxis(AndreaSchmidt)/

Vahora, N.  “Micro-scale interactions between chemotactic bacteria and algae”.  Thesis, M. Eng., Massachusetts Institute of Technology, Dept. of Civil and Environmental Engineering.  http://hdl.handle.net/1721.1/60787

Valentine, M. et al.  “Chemosensory Transduction in Paramecium.” Jpn. J. Protozool. 41:1-7 (2008).  https://www.uvm.edu/~biology/faculty/documents/ChemosensoryTransductioninParamecium2008.pdf

Wikipedia.  “Chemotaxis.”  https://en.wikipedia.org/wiki/Chemotaxis

Wikipedia.  “Lemon Shark”   https://en.wikipedia.org/wiki/Lemon_shark

Advertisements
Veils in the Water: “Schools” of Protozoa

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s