A Demonstration of Photo- and Geotaxes in nauplii of Artemia salina

Daniel C. Koblick              Illinois Institute of Technology
                               Life Sciences Building
                               3101 S. Dearborn
                               Chicago IL 60616
                               312-567-3480 (Biology office)
                                   567-3490 (DCK office)
                                   567-3600 (IIT switchboard)

Objectives:

     This presentation was designed for High School students, but can be 
modified for use in Middle School.  Students are presented with an easily
observed orientation behavior of small crustaceans with respect to light and
gravity, given some description of the habitat and feeding behavior of the
organisms and encouraged to relate the phenomena observed to the needs of the
organisms in terms of adaptation. 

Materials Needed:

     1 plastic shoebox with lid.
     1 or 2 liters of 4% NaCl.
     A vial of brine shrimp (Artemia salina) eggs.
     1 siphon, consisting of a two foot length of india rubber tubing with six 
         inch segments of glass tubing inserted in each end.
     2 wire pinch clamps
     1 one liter flask (a clean milk carton will do).
     1 100 ml. graduate cylinder.
     1 approx. one cm internal diameter glass tube, about eight inches long.
     2 black rubber stoppers to fit above tube (Size 00).

Procedure:

Hatching of brine shrimp eggs.
     Brine shrimp eggs are available from tropical fish dealers.  Purchased in 
quantity (1/2 to 1 lb.) from a biological supply house such as Carolina Biological 
Supply or Ward's Natural Science Establishment greatly reduces the unit price.  
Each egg is as small as a grain of fine sand.  A teaspoonful contains many 
thousands of eggs.  The eggs will remain viable for several years if kept cool 
and dry.  If placed in a 2% to 4% salt solution the eggs will hatch into tiny 
larvae (known as nauplii---singular = nauplius).  Do not use iodized salt, nor 
reagent grade salt; best results are obtained using sea salt, which is usually 
available in the gourmet section of your supermarket.  A 4% salt solution is 
easily made up by making a saturated solution (add about 8 ounces of salt to two 
cups of water in a quart jar, agitate vigorously, and allow excess salt to 
settle), and diluting 100 ml of the saturated supernatant solution to one quart 
with water.  (If you use tap water, allow it to stand overnight before use to 
get rid of the chlorine it has been treated with.)  The best way of obtaining 
nauplii free of unhatched eggs and dead larvae (large numbers of eggs do not 
hatch, and many more die within minutes of emergence from the egg), is to fill a 
transparent plastic shoe box with 4% salt solution to a height of 1 to 11/2 
inches, raise one end of the box about 1/2 inch by placing a wedge under it, so 
that the depth varies as it does in a swimming pool, and sprinkle about 1/2 
teaspoonful of dry eggs over the surface.  If this is done gently, the eggs will 
float on the surface.  Penetration of the salt solution into the eggs initiates 
development.  Oxygen is required, and this is obtained directly from the air by 
the floating eggs.  At room temperature, nauplii will emerge about 36 hours 
after wetting; at 30 degrees centigrade, emergence time is about 24 hours.  If 
the boxes are not disturbed during this period, unhatched eggs remain on the 
surface and the rapidly swimming nauplii are found in the solution.  These can 
be separated by siphoning the bulk of the solution into a beaker or other 
container.  After a short time, the nauplii will sink to the bottom of the 
vessel (this behavior is called a positive geotaxis) and a concentrated 
suspension of them can be made by transferring them to a test-tube by means of a 
dropper pipette. 

Demonstration of Phototaxis:

      Stopper one end of the glass tube and, holding the tube vertically, 
transfer the concentrated suspension of nauplii to the tube.  Fill to the top 
with salt solution and stopper the upper end in such a way as to exclude all air 
bubbles.  This can be done by holding a fine pin in the open end of the glass 
tube while introducing the stopper.  The pin distorts the stopper and provides a 
pathway for excess solution and air to escape when the stopper is pushed into 
the tube; if the pin is then carefully pulled out (holding the stopper in 
place), no air bubbles will be trapped in the tube.  Invert the tube a few times 
to spread the nauplii evenly and place it on a horizontal surface. Direct the 
light from a penlite flashlight on one end of the glass tube and cover the 
remainder of the tube with a paper towel.  After a few minutes, almost all of 
the nauplii will be found at the lit end of the tube.  If the opposite end of 
the tube is then illuminated, the nauplii will again move to the illuminated 
end.  (Because of reflections of light in the glass tube, not all the nauplii 
move to the point of greatest illumination; some will remain in the body of the 
tube.  If a round bottomed test-tube had been used for this demonstration, a 
large number of nauplii would be found at the center of curvature of the semi-
spheroidal part of the tube.  This is because the bottom acts as a crude lens, 
concentrating the light at its focal point.) 

Questions For General Discussion:

     Beside the light gradient, what other gradients might be present in the 
tube?  (Gravitational gradient, chemical gradients of oxygen, carbon dioxide, 
excretory products of the nauplii themselves.)  In what ways did the experiment 
try to minimize the effects of these other gradients?  (Equal distribution of 
artemia at the beginning of the experiment, horizontal light gradient compared 
to vertical gravitational gradient, exclusion of air bubble.)  Of what use is 
this built-in response to the nauplii?  Why does the unpurified seasalt produce 
better yields of nauplii than purified table salt or iodized salt? 

Background:

     The brine shrimp lives in salt swamps such as those often found inland of 
the dunes at the seashore, in man-made evaporation ponds used to obtain salt 
from the ocean, and in salt lakes such as those found in the intermountain 
desert region of the western United States.  (Multicultural note: large salt 
water lakes are found in many parts of the world; One of the larger of these is 
the Caspian Sea, into which the famous Volga River flows.)  Few other organisms 
can tolerate such a high concentration of salt as is found in these locales.  
Sea water varies from about 2.9% to about 3.5% salt, depending on the latitude 
and the time of year.  The Great Salt Lake, in northern Utah, undergoes long 
term variations in salt content, but since records have been kept it has varied 
erratically between 25 and 35%.  Adult brine shrimp can tolerate a salt content 
of as much as 50%.  Since few organisms can grow at such high concentrations as 
that found in the Great Salt Lake, brine shrimp from this locale do not have 
much selection in the way food.  They live almost entirely on the photosynthetic 
green alga (singular of algae) Dunaliella.  Like many other primitive aquatic 
plants this organism is attracted to light, rising to the surface in the 
daytime, and sinking at night.  The positive phototaxis of Artemia keeps it at 
the same depth as its prey.