EXERCISE 4

Osmoregulation

Valerie Sol T. Bengzon

University of the Philippines Visayas Tacloban College

Division of Natural Science and Mathematics

INTRODUCTION

Living cells have developed the ability to persist in the face of a fundamental

contradiction. On the other hand, they preserve an internal composition that is an

optimal milieu for the processes that are essential to the maintenance of the living

state and maintain the ability to regulate that composition as defense against external

perturbations (Evans 2008). Osmoregulation is the maintenance of a constant or

nearly constant osmotic pressure in body fluids regardless of the osmotic pressure in

the external environment(Hill et al. 2012). It is the homeostatic mechanism where

organisms actively regulate the level of water and mineral salts in their bodies or organ

systems.

The earthworm, Lumbricus terrestris, of the phylum Annelida normally lives in

moist soil but during rainy seasons they are exposed to an aquatic environment (Dietz

and Alvarado 1970). Terrestrial oligochaetes such as earthworms, although preferring

moist soil, live indefinitely in freshwater (Roots 1956). They have a cuticle relatively

thinner than the muscle layers, presumably because oxygen absorption is cutaneous.

In the subsoil habitat, oxygen levels may be quite variable, but it is commonly observed

that earthworms surface periodically (Evans 2008). They move to the surface after

heavy rains, which is a phenomenon driven by the low oxygen levels in their flooded

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burrows. It is also possible that osmotic stress of a sudden deluge of freshwater may

also be a factor. Earthworms are able to survive several months in freshwater of the

appropriate ionic composition (Roots 1956). Soil salinity is also an important factor

that determines the survival of earworms because they are highly sensitive to salt

stress. Salinity may reduce their growth at low salt concentrations or cause mortality

at high salt concentrations due to their inability to tolerate high ionic strength. High salt

concentrations destroy their sensitive skin, and the earthworms cannot have control

over the osmotic regulation. In addition, the neurosecretory cells in earthworms play a

vital role in water balance as well as ionic and osmotic regulation.

In this exercise, the osmoregulation of Lumbricus terrestris was investigated.

Specifically, the study aimed to explain responses of Lumbricus sp. to varying moisture

conditions and solute concentrations; and to describe the ion concentration and

volume controlling mechanisms occurring in both osmoregulating and

osmoconforming organisms.

MATERIALS AND METHODS

Twenty-five earthworms were collected within the perimeter of the grounds of

the college. The earthworms were placed on dry paper towels and gently rolled to

remove adhering debris. They were soaked in tap water overnight for at least 8 hours.

The worms were weighed in groups of five and placed in separate petri dishes

containing enough water to immerse the worms. The weights of each set-up was

weighed after 15 minutes and 30 minutes. Each group was transferred to different

beakers containing 100 ml of saline solutions ranging from 0 M (tap water), 0.05 M,

0.1 M, 0.25 M and 0.3 M. Each set-up was weighed at 20 minute intervals for 2 hours.

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The earthworms submerged in tap water solution (0 M NaCl) was removed from the

solution and transferred to a box containing extremely dry soil. The weights of the

worms were determined at 20 minute intervals for 2 hours. The results were recorded

and plotted into a line graph where the weight changes against time. The percentage

gained and lost in weight against time and against osmotic value of saline solution was

also plotted.

RESULTS AND DISCUSSION

Organisms that constitute the phylum Annelida are remarkably diverse and

occupy habitats from open ocean, estuaries, freshwater and soil in terrestrial

environments. A suite of biological adaptations is necessary for survival in each of

these specific niches, but one of the most important adaptations is certainly the

capacity to regulate internal osmotic pressure and the composition of cellular and

tissue osmolytes.

Terrestrial species such as the Lumbricus terrestris, face transient freshwater

challenges and desiccation stress. They face the challenge of conserving wayer

unless they dry out and die. In most cases, they maintain the volume of water and salt

concentration in the body fluid. During growth, they tend to take in more water to

increase its volume. Osmoregulation is observed which is the regulation of body fluids.

An animal can either be an osmoregulators or osmoconformers. Osmoregulators,

such as freshwater and terrestrial animals, maintain a more or less stable internal

osmolarity. They exist in quite stable habitats where they do not face the added burden

of coping with changes in humidity or salinity. On the other hand, osmoconformers,

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such as marine animals, have body fluids isomolar to their environment as they simply

tolerate changes that occur in their bodies.

Figure 1.1 Earthworms submerged in tap water for 30 minutes

The earthworm, Lumbricus terrestris, is capable of volume regulation in dilute

media wherein their total body content remains constant over a wide range of

salinities, but a greater percentage of body water is extracellular in more dilute media

(Carley et al. 1983). In dilute media, the earthworm’s integumental water exchange is

lower which reduces osmotic influx of water.

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WEIGHT (g)

0
0 15 30
TIME (min)

1 2 3 4 5

Figure 1.2 Weight of earthworms submerged in tap water for 30 minutes

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 0
RELATIVE PERCENTAGE (%) -1
-2
-3
-4
-5
-6
-7
-8
-9
-10
15 30
TIME (min)

Series1 Series2 Series3 Series4 Series5

Figure 1.3 Relative percentage of weight gained/lost of earthworm set-ups in tap

water for 30 minutes

After 30 minutes of submersion in tap water, the 5 groups of earthworms lost

water as they decreased in body weight. The body weight decreased in 30 minutes

wherein water moved out of the earthworm’s body. This indicates that the tap water is

hyperosmotic in relation to the earthworm’s internal fluid. Ideally, the earthworms

should gain water and body weight. In tap water, a hypotonic medium, the earthworms

are hyperosmotic. Water would diffuse into the earthworm, resulting to weight gain.

Figure 2.1 Earthworms in different saline concentrations for 2 hours

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 Lumbricus terrestris are soil processor or detritus feeders. The typical osmotic

pressure of lumbricoids is about 150 mOsm. Earthworms are exposed to atmospheres

of varying moisture content and soils with different ionic concentrations. Due to their

wide range of varying conditions, they have developed adaptive mechanisms

important for their survival. With regard to the osmotic environment, earthworms

minimize drastic osmotic water loss via dessication or gain during flooding by rain

water and take advantage of microenvironments in the soil that moderate osmotic

stress.

4.5

3.5

3
WEIGHT (g)

0M
2.5 0.05 M

2 0.1 M
0.25 M
1.5
0.3 M
1

0.5

0
0 20 40 60 80 100 120
TIME (min)

Figure 2.2 Weight of earthworms in different saline concentrations for 2 hours

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 140
120
RELATIVE PERCENTAGE (%)

100
80
60
40
20
0
-20
-40
20 40 60 80 100 120
TIME (min)

TIME 0M 0.05 M 0.1 M 0.25 M 0.3 M

Figure 2.3 Relative percentage of weight gained/lost of earthworms in different saline

concentrations for 2 hours

In the exercise, earthworms submerged in tap water for the first 60 minutes lost

water, however, gained water in the next 60 minutes. As the body weight increased,

the water moved inside of the earthworm’s body because the medium is hypoosmotic

in relation to the earthworm’s internal fluid. For the saline concentrations at 0.05 M,

0.1 M, 0.25 M and 0.3 M, water loss is observed wherein the earthworm’s body weight

decreased. Water moved out of the earthworm’s body because the saline media are

hyperosmotic in relation to the earthworms internal fluid. Earthworms have a coelomic

fluid osmotic pressure 154 mOsm.

Initially, all set-ups had a 100 mL volume of each saline solution. However, on

the process of the exercise conducted weighing the earthworms on each 20 min

interval for 2 hours would incur an error for the final displaced volume. Transferring

the earthworms to another container and patting them dry before weighing displaces

water. Volume displacement would not be an accurate basis, instead the weight and

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relative percentage of the weight gained or lost could be a sufficient basis if the

earthworms gained or lost water.

Figure 2.4 Route of water loss or gain in Lumbricus terrestris

(Laverack and Kerkut 1963)

Water is lost readily through the skin, and it can be gained by the same pathway

from the external environment with equal facility. Earthworms ligatured at both anterior

and posterior ends prior to immersion in water gains weight, about 7%, and then

maintains a constant weight level (Maloeuf 1940 ). Salts can also be taken in via the

body wall. Chloride ions disappear from dilute salt solutions containing worms

because it is thought actively uptake ions (Maloeuf 1940; Van Brink and Rietsema

1949). Water enters through the skin, a process which goes on very easily, and is

removed via the intestine. The loss in weight sustained by worms when handled was

due mainly to expulsion of fluid from the nephridiopores, (Wolf 1940). Maintenance of

water balance depends on balancing passive osmotic water gain driven by solute

transport (mainly ions) and nephridial loss. Lumbricus nephridia secrete a hypotonic

urine and recover ions and organic solutes. The osmotic pressure relationships in

Lumbricus indicate that hypotonic urine formation begins in the middle tube but is

largely formed in the wide tube. The typical osmotic pressure of Lumbricus coelomic

fluid is about 150 mOsm, and the urine may be 10 to 120% of this value.

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 The terrestrial subsoil environment subjects the earthworms to potential

dessication, floods of rainwater and anoxia. Lumbricus terrestris, a terrestrial

oligochaete, ideally would balance the water permeability properties of the body wall

to resist desiccation but allow water gain from the interstitial moisture of the soil (Evans

2008). Their intestine should be adapted to avid absorption of both salts and organic

molecules. Earthworms have a thin cuticle overlaying a mucus secreting membrane

relatively thinner than the muscle layers, presumably because oxygen absorption is

cutaneous. It is unlikely that the cuticle plus epidermal layer provides a serious barrier

to the influx and efflux of water through the body wall, so that changes in environmental

conditions can lead to considerable changes in the water balance of the animal

(Laverack and Kerkut 1963). In the subsoil habitat, oxygen levels may be quite

variable, but it is commonly observed that earthworms surface periodically. This would

cause exposure to high atmospheric oxygen levels. One of the reasons attributed to

the commonly observed phenomenon earthworms moving to the surface after heavy

rains is that they are driven to the surface by low oxygen levels in their flooded

burrows. In a waterlogged soil, earthworms remain partly stretched above the surface

of the soil in the water layer (Laverack and Kerkut 1963). Long survival of earthworms

under water depends not upon the ability to control the water circulation of the body,

but upon the ability to withstand prolonged starvation (Roots 1956). Earthworms will

leave water- logged soil in preference for a drier site if possible.

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 5
4.5
4
3.5
WEIGHT (g)

3
2.5
2
1.5
1
0.5
0
0 20 40 60 80 100 120
TIME (min)

WEIGHT (g)

Figure 3.1 Weight of Earthworms in an Extremely Dry Soil for 2 hours

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RELATIVE PERCENTAGE (%)

-5

-10

-15

-20
20 40 60 80 100 120
TIME (min)

Figure 3.2 Relative Percentage of Weight Gained/Lost of Earthworms in an

Extremely Dry Soil for 2 hours

As the earthworms previously submerged in tap water were transferred in an

extremely dry soil, they continuously lost weight in the 20 minute interval for 2 hours.

A decrease in their body weight indicates water loss. Subjecting the earthworms in an

extremely dry soil is a setup mimicking a natural phenomenon wherein they undergo

cycles of dehydration stress. Dry environmental conditions and fairly water-permeable

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body wall suggest that potentially significant water loss may occur. In previous

laboratory studies, earthworms may lose as much as 60% of the body water content

for short periods and when rehydrated remain viable. From the initial weight of the

earthworms of 4.32 g, they decreased to 2.79 g after 2 hours in the extremely dry soil.

They lost a relative weight percentage of 153% of their initial body weight. Soil

conditions are rarely stable with regard to moisture content, however, and if they

become dry then earthworms lose moisture from the body. In nature, it is also likely

that behavioral responses trigger the migration of earthworms to soil with moisture to

avoid the most severe conditions which could lead to dehydration and death.

Based on the exercise, the comparison with the rate at which the earthworms

gained weight in water and lost in the dry soil could not be made because the

earthworms in tap water did not constantly gain water for 2 hours. Instead, in the first

60 minutes they lost water and in the remaining 60 minutes water gained was

observed as it increased in weight. For the earthworms in soil, they continuously lost

water as they continuously decreased in body weight. Theoretically, the loss of weight

in soil is a faster process than the gaining of weight in water (Adolph 1943). The minor

weight and accompanying volume changes serve to show that under normal field

conditions the earthworm maintains a water equilibrium with fair efficiency (Laverack

and Kerkut 1963).

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 CONCLUSION

In this experiment, Lumbricus terrestris responded to various moisture and

solute concentrations. Osmoregulation is observed in animals such earthworms

wherein they maintain water and ionic balance in their body. In tap water, the

earthworms gained water while in saline solutions they lost water in their body. In

extremely dry soil, water lose was also observed in earthworms. The rate of water lose

in soil is greater than the gain of water in the earthworms.

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 REFERENCES

Dietz, T.H., Alvarado, R.H. 1970. Osmotic and ionic regulation in Lumbricus terrestris

L. The Biological Bulletin, 138(3), 247-261.

Evans, D. 2008. Osmotic and Ionic Regulation: Cells and Animals. CRC Press.

Hill, R.W., Wyse, G.A., Anderson, M. 2018. Animal physiology. New York: Sinauer

Associates/Oxford University Press.

Laverack, M. S., Kerkut, G.A. 1963. The Physiology of earthworms. Oxford: New York,

Paris.

Maloeuf, N.S.R. 1940. The volume and osmo-regulative functions of the alimentary

tract of the earthworm (Lumbricus terrestris) and on the absorption of chloride

from freshwater by this animal. Z. vergl. Physiol 59, 535-552.

Roots, Β.I. 1956. The water relations of earthworms. 2. Resistance to desiccation,

immersion and behaviour when submerged a n d when allowed a choice of

environment. J. exp. Biol. 33, 29-44.

Van Brink, J.M., Andrietsema, J. 1949. Some experiments on the active uptake o f

chlorine ions b y the earthworm {Lumbricus terrestris L.). Physiol, comp, et oecol.

1, 348-351.

Wolf, Α.V. 1940. Paths of water exchange in the earthworm. Physiol. Zoöl. 1 3 , 294-

308.

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 APPENDIX

Table 1.1 Weight of Earthworm Set-ups after 30 minutes of Submersion in Tap Water

TIME WEIGHT (g)

1 2 3 4 5
INITIAL 1.0103 0.9642 0.9348 2.8157 5.0263
15 min 0.9381 0.9212 0.912 2.7643 4.5952
30 min 0.8886 0.8938 0.8764 2.745 4.3515

Table 1.2 Weight Gained/Lost Percentage of Earthworm Set-ups after 30 minutes of

Submersion in Tap Water

TIME PERCENTAGE OF WEIGHT GAINED/LOST (%)

1 2 3 4 5
15 min -7.146392161 -4.459655673 -2.43902439 -1.825478567 -8.576885582
30 min -4.899534792 -2.84173408 -3.808301241 -0.685442341 -4.848496906

Table 2.1 Weight of Earthworm Set-ups after 2 hours of Submersion in Different Saline

Solutions

TIME WEIGHT (g)

0M 0.05 M 0.1 M 0.25 M 0.3 M
0 4.3515 2.745 0.8764 0.8938 0.8886
20 3.5153 2.6915 0.7746 0.7965 0.6226
40 3.4121 2.6612 0.7455 0.6829 0.5677
60 3.3147 2.6378 0.5924 0.6582 0.5767
80 3.4 2.6492 0.6436 0.6525 0.5669
100 3.4469 2.658 0.6145 0.6307 0.5854
120 3.5239 2.6557 0.6692 0.6657 0.5995

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Table 2.2 Weight Gained/Lost Percentage of Earthworm Set-ups after 2 hours of

Submersion in Different Saline Solutions

TIME RELATIVE PERCENTAGE OF WEIGHT GAINED/LOST (%)

0M 0.05 M 0.1 M 0.25 M 0.3 M
20 -19.21636217 -1.948998179 -11.61570059 -10.88610427 -29.93472879
40 -2.371596001 -1.103825137 -3.320401643 -12.70977847 -6.178257934
60 -2.238308629 -0.852459016 -17.46919215 -2.763481763 1.012829169
80 1.960243594 0.415300546 5.842081241 -0.637726561 -1.102858429
100 1.077789268 0.320582878 -3.320401643 -2.43902439 2.081926626
120 1.769504768 -0.083788707 6.241442264 3.915864847 1.586765699

Table 3.1 Weight of Earthworms in Extremely Dry Soil for 2 hours

TIME (Min) WEIGHT (g)

0 4.32
20 4.49
40 4.11
60 3.75
80 3.1
100 2.99
120 2.79

Table 3.2 Weight of Earthworms in Extremely Dry Soil for 2 hours

TIME (Min) WEIGHT (g)

20 3.935185185
40 -8.796296296
60 -8.333333333
80 -15.0462963
100 -2.546296296
120 -4.62962963

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