Project
Summary:
Our research has revealed promising results
on further understanding the feeding mechanisms of Daphnia. Using electrochemistry
and a Schlieren optical video system we have developed a new approach
that in real time observes changes in the feeding pattern of Daphnia.
Presently,
different species of Daphnia are utilized by various
government agencies, including the EPA, as test organisms
in toxic research. Our
findings can be applied as a new biological
test method for determining acute lethality of effluents
to different zooplankton species that is
accurate, quick and cost effective.
Methods
Electrochemistry
Fig. 1 The
micropipette (1) is used to deliver
the tracer which is picked up by the
laminar inflow current of the Daphnia and
continues to travels through the feeding
mechanism. The microelectrode (2) placed
in the outflow current, records fluxes
in concentration of the tracer solution
created by the dynamic movements of
the feeding appendages. [White bar = 0.2 mm] |
The
application of microelectrodes (Fig.1) has
been successfully transferred to research in
aquatic
environments. They have been utilized to map chemical
landscapes, trace chemical stimuli, study antennal
morphology of honey bees, and to further our understanding
of the physical constraints of chemoreception in
aquatic environments. When a tracer chemical comes
into contact with a microelectrode’s surface
a chemical reaction occurs that relays a change
in current that can be recorded utilizing a computer
controlled potentiostat. Dopamine can be used as
a tracer because it has been successfully utilized
in aquatic systems, has a diffusion coefficient
of 2 X 10-9 m2 s-1 at 20°C in water, and minute
concentrations of it can be easily detected by
microelectrodes. Therefore by directly monitoring
the movement and temporal changes of dopamine concentrations
we can observe how the Daphnia reacts to different
chemicals and other environmental factors.
Optics
Visualization
of the tracer solution (Fig. 2) as it traveled
through the feeding apparatus of
D. pulex is accomplished by using a Schlieren optical
system. The video system component is used as a
guide when placing recording devices near the organism
and to assess animal condition. The tracer and
test solutions contained 2 mM dopamine, dextran
for the visualization of the flow, and 0.2 mM ascorbic
acid (as an antioxidant).
Fig.
2 Schlieren system images under the two different
conditions with their respective 3D Phase Space
Diagrams. The difference can be seen visually between:
(A) The more regular beat pattern of Daphnia when
no food present; and (B) the more dynamic beat
pattern when food is present. Dopamine concentration
distribution as visualized using phase space diagrams
over 60-s (n=5000) when no food present (C), and
when food present (D). Bar = 0.25 mm.
Results
Our
data suggests that D. pulex has a dynamic feeding
mechanism with a nonlinear periodicity.
When
feeding, a Daphnia’s feeding apparatus
repeats movements, yet they don’t repeat
at exact intervals. This was actually noticed
by Cannon in 1928 when studying filter feeding
and our system supports his observations. PSDs
of lag 3 were compared across treatments and
animal. All exhibited the same type of structure
for each type of treatment, making it possible
to compare changes in the feeding behavior as
a whole. The PSD of the feeding behavior of D.
pulex when no food is present is more concentrated
and predictable, while a PSD when food is present
has a larger spread across 3D space and becomes
more complex (Fig. 2 c & d). This type of
representation suggests that Daphnia adjust their
feeding beat frequencies under different conditions.
They have a shorter delay between similar events
when no food is present, and exhibit a larger
delay and array of strokes when food is present.
Our findings are in agreement with other research
conducted on Daphnia and copepod feeding behavior
under changing food conditions.
Future Work
Evaluation
of the sensory mechanism of various other microorganisms
is possible with
our system.
Calculating both the temporal and intensity of
the response under different conditions should
assist in our understanding of their sensory
ecology. As this new technique is further perfected
we hope
to utilize other organisms as biosensors; creating
situations and visualizing their response to
varying conditions. A new electrochemistry system
has been
designed that works at a higher temporal level
(200 Hz) and allows for a greater amount of data
to be collected at once.
Endless Possibilities
It is noteworthy to understand that with this system
we have exceptional control over the environment
occupied by the organism, and thus can alter
how and when doses of different chemicals or
toxins are delivered to the organisms. We can,
for example, program our system to randomly deliver
single particles from different sources, observe
response of animal to particles en mass; we can
create oil droplets and administer them individually
as well as deliver coated and uncoated particles.
The table on the right is just an example of
treatments we can administer and the types of
questions we can answer by monitoring the trace
of dopamine, which is in fact a direct representation
of the animal’s response.
| Possible Treatments |
Questions Answered |
| Oil Droplets |
Did the animal reject particle? Ingest particle?
Was the temporal frequency altered? Was the
animal aware of the oil? How soon did it become
aware of it? |
Coated vs. Uncoated Particles of Oil or DDT
or any known toxin
|
Was
there a preference for one when coated or
were both rejected? What if particles are
coated with algae extract – is the animal
fooled? How does the animal react to fish kairmone
covered particles?
|
Young growing algae vs. old dying algae
|
Does the organism prefer young algae that
is dividing and probably of better nutritional
value? Does it prefer one species of algae
over another? How does its behavior change
when presented with both at random intervals?
Repetitive? Can it learn to avoid certain particles?
|
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