Lab Activty On Osmoregulation

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A Laboratory Exercise In Osmoregulation

By

Bonnie Leksrisawat, Ann S. Cooper and Robin L. Cooper

PURPOSE

The purpose of this experiment is to observe an organism undergoing a


homeostatic regulation in osmolarity with environments of varying salinity. In addition,
one will examine if an organism can regulate osmolarity and volume after losing a
substantial amount of hemolymph.

PREPARATION

The crayfish

INTRODUCTION

In order for cells to survive they must be able to maintain the proper osmolarity
and balance of ions such as, potassium, sodium, and chloride within their intracellular
and extra cellular environments. The freshwater ancestry of vertebrates is striking
relation to the relative concentration of ions in intra-and extra-cellular environments.
Vertebrates have about one-third the concentration extracellular ions of seawater
(Randall et al. 2002). If the osmolarity and/or concentration of key ions is not regulated
within the tolerated range for animals the weakened metabolism, brain damage, and
even death can occur. The osmolarity and ion concentrations is heavily influenced on
the hydration and water balance of the animal.

The excretory system plays a major role in osmoregulation. In humans rapid


excretion starts in the nephrons of the kidneys. Here blood plasma is filtered and
regulation of the ultrafiltrate is controlled by reabsorbed or secretion of ions and water
back into the body. Humans are osmoregulators as they internally monitor and regulate
the blood osmolarity.

The organism that will be studied in this laboratory exercise, the crayfish, will be
examined for its ability to survive and homeostatic regulate osmolarity with various
insults on its osmoregulatory abilities. The crayfish has an open circulatory system and
hemolymph is the fluid that is pumped through the heart and into the open body cavity.
The hemolymph is in some regards analogous to blood in the vertebrates.

An important adaptation that crayfish have is the ability to osmoregulate from the
time they hatch and during their development (review in Charmantier 1998). The
crayfish has several adaptive mechanisms by which it osmoregulates. One key


 
 

mechanisms is the ability to intake water through the anus. This intake of water is
essential in osmoregulation and also essential for the organism to molt its exoskeleton
(Vogt, 2002; Muramoto, 1981). Another key adaptive mechanisms is the ability to keep
out excess ions from its internal environment because of the low permeability of its
integument. Additionally, through its excretory antenneal glands it produces dilute urine
(Lienemann 1938; Krogh 1939; Gross 1957; Riegel and Kirschner 1960; Shaw 1960;
Lockwood 1962; Potts and Parry 1964; Kerley and Pritchard 1967; Riegel 1970;
Bielawski 1971;Fisher 1972; Mills and Geddes 1980; Dunel-Erb et al. 1982,1997; Henry
and Wheatly 1988; Péqueux 1995; Wheatly and Gannon 1995; Barradas et al. 1999).

Fluid retention is commonly measured by use of body weight in clinical situations


for humans (Fagan et al., 2008; Wada et al., 2008) and animals (Boscan et al., 2010).
Likewise body weight can be used experimentally to determine fluid loss and gain as a
function in an animal’s ability to volume and osmoregulate. Approximately 30% of the
weight for a blue crab or a lobster is hemolymph (Gleeson and Zubkoff, 1977; Guirguis
and Wilkens, 1995). We assume this percentage also holds to be approximately true for
crayfish in these experiments. By extracting a significant amount of the hemolymph and
placing the crayfish back into the water, one can determine the amount of time it takes
the crayfish to replace the lost hemolymph. In addition, we will examine the conductivity
of the various environments in which the crayfish are exposed. If feasible, we will also
test the conductivity of the hemolymph from some of the crayfish.

Conductivity is a measure a material's ability to conduct an electric current and


can be used, for this experiment, as an indirect measure of ionic strength of a solution.
Resistance, which is an electrical measurement expressed in ohms, is the opposite of
conductivity. Therefore conductivity can be expressed as 1/ ohms. The conductivity
measure is related to ionic strength. The measure does not indicate what specific ions
are present. However, in these experiments we are using sea water that is diluted and
we can use conductivity as a relative measure of the amount of dilution. Also, the goal
here is to measure the hemolymph from crayfish after volume defect and exposure to
various osmotic environments. As a side note, a change in osmolarity due to non
charged substances will not be detectable by this measure in conductivity. In the future
we hope to have a vapor pressure osmometer for direct measures of hemolymph
osmolarity.

PROCEDURES:

1. Choose two crayfish from the ice container and dry it off with a paper towel.
2. Take the initial weight of the crayfish.

HEMOLYMPH:

1. Hold the first crayfish behind its claws


2. Position your hand so the tail is spread out. Or have your partner hold the tail
down.


 
 

3. Insert the needle between the thorax (abdomen) and the tail. Be sure not to go
towards the middle because this is where the central nerve cord is located.
4. Draw about 3 mL of hemolymph for a large size crayfish (7-10 grams in body
weight). Then quickly dab the insertion spot with a paper towel so the blood will
clot.
5. Weigh the crayfish.
6. Place the crayfish into the container containing distilled water.
7. Wait 30 minutes
8. Dry the crayfish and take the final weight.

Placement in Different Containers:

1. Place the second crayfish (that was previously weighed) into the seawater.
2. Wait 30 minutes
3. Dry the crayfish and take the final weight.

QUESTIONS TO ADDRESS:

Explain why the kangaroo rat and other desert animals do not have to drink much water.
Where does it gain most of its water from?

Considering surface to volume ratio, would a larger or smaller mammal loss water more
rapidly?

Where is ADH (antidiuretic hormone), also known as vasopressin, produced?

Label the parts of the nephron: Bowman’s Capsule, distal convoluted tubule, proximal
convoluted tubule, efferent arteriole, afferent arteriole, collecting duct, ascending loop of
Henle, descending loop of Henle.

Describe the function of each part of the nephron:


 
 

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