BIOLOGY LAB REPORT TITLE PREPARED BY I/C NUMBER STUDENT ID GROUP LECTURERS NAME PRACTICAL DATE SUBMISSION DATE

: HABITUATION : : : : : : :

Abstract In this experiment, habituation in snail is being investigated. Each student is needed to find one snail and 10 stimulations are being given to each snail. The time taken for the snail to retreat and re-emerge is recorded and the graph is plotted. From this experiment, I found out that generally the time taken for the snail is decreasing with increasing no. of stimulation. After a few stimuli had been given, the snail became habituated. Besides, I also found that different snails (from other student) need different no. of stimulation to become habituated.

Introduction 1. Snail(1) Snails can be found in gardens, in ponds and even in the sea. They belong to a group of animals with a soft body called molluscs which are related to oysters, clams, and other shellfish. Characteristically they have soft, unsegmented bodies. Normally, their soft bodies are protected by a hard shell. The scientific name for the garden snail is Helix aspersa. The body of the snail is long, moist and slimy. It has a shell to protect its soft body. When the snail is disturbed, it simply withdraws or pulls itself back into its shell. The snail also retreats into its shell and seals the entrance in dry weather to protect its body from drying up. A snail is most active at night and on cloudy days. It does not like the sunshine very much. During very cold weather or winter, it hibernates in the ground. Garden snails are best for this activity. If possible, snails should be collected locally, during warm, wet conditions. Students can find for snails early in the morning or evening hours, and check on leafy plants and under overturned pots. Snails can be housed in any closed container that has air holes. For bedding, moist paper towels work well. The activities in this lesson will not harm snails. Snails eat various leafy foods; young green leaves works well if the snails will be kept up to several weeks. Snails go dormant if they have not been fed for several days, so if it will be a few weeks before they are studied, food should be withheld and their enclosure kept drier; they can survive for several months in a dormant state. The snails can be awakened usually in minutes by being rinsed and fed.

Figure 1: Snail and its life cycle(2)

2. Learning Learning ability is one of many factors which will determine the survival of an animal. Learning is the modification of behavior resulting from specific experiences. There are two main types of behavior: innate and learned. In this biology practical, one form of learned behavior which is habituation is explained with the help of garden snails.

i. Habituation(3)(4) Habituation is learning process exhibited by several different species. Although its quite simple, its been suggested that habituation may be the basis of all other forms of learning. The mechanisms of habituation are usually closely regarded as akin to the mechanisms of learning. An example of habituation is wearing a watch, if you were to put your watch on your other arm it would feel strange and out of place, as it would if you took it off the arm its on now. This is becau se your body has got used to it being on one arm and not the other. If you were to leave the watch on the other arm for a few days then you would forget about it. This process is known as Habituation and can be summed up as when stimuli are neither harmful nor helpful to any creature it will eventually be ignored. A quick way of testing this is to gently touch a snails tentacle, when touched it will withdraw sharply. If it is touched repeatedly eventually the snail will see that this is doing it no harm and not bother withdrawing its tentacle as much. Habituation is a phenomenon that is widespread across the animal kingdom; it helps them survive by balancing responses to either harmful stimuli or stimuli with uncertain significance. An effect of habituation is usually that a response that a habituated response will soon reappear if stimuli are withheld for a long time, the guppy will start retreating again if no shadows are shown for about a day. However, habituation to the shadow the next day will occur quicker because the animal has previously been habituated. This process is known as dishabitation. No animal can afford to ignore potentially dangerous stimuli; the process of habituation is a compromise between saving energy and the animal saving itself. In relation, the snail will not be afraid after a while of no harmful stimuli but when the stimulus is first presented the snail should cower in an attempt to preserve its own life. Humphrey (1933) studied the process of habituation, and he was one of the first. He placed a number of snails on a glass plate; he would then give the plate a sharp jerk that would cause the snails to react and to withdraw reflexively into their shells. After each jerk, the number of snails that had withdrawn was counted, it was found that after several trials the number showing withdrawal reflexes decreased and less and less would withdraw into their shells. Since Humphreys study a variety of techniques have been used to study habituation. They also found repeated stimulation o f the aplysias siphon causes a reduction in the magnitude of gill withdrawal .

Figure 2: Habituation in Aplysia (5) One of the famous experiments investigating habituation is the experiment involving the giant sea slug Aplysia. The giant sea slug called Aplysia californica is often chosen for the studying of memory. Its brain has about 20000 neurons, some of which are large enough to be visible to the naked eye. Aplysia can learn and most importantly it is found that the mechanisms and principles involved in its formation of short- and long-term memories are conserved throughout the animal kingdom, including in humans. Aplysia exhibits a behavior of protective reflex in which the sea slug withdraws its gill into the safety of the mantel cavity in response to a mild touch stimulus to another part of the body called the siphon. If the stimulus is repeated a number of times, the gill withdrawal reflex becomes weaker until finally the animal ignores the touch stimulus. The waning of sensitivity to repeated stimulation is known as habituation and is a very simple form of learning found in all animals, including humans.

At the macromolecule level, it is known that the neurotransmitter involved in the processes is the serotonin. A puff of serotonin alone can substitute for the siphon shock. It is shown further that the serotonin triggers the release of the second chemical messenger called cyclic-AMP. It activates an important type of enzyme called a kinase, which modifies the properties of particular target proteins by adding a phosphate molecule to them; the term for this is protein phosphorylation. The target for this modification in the sensory neuron is a potassium channel protein which is important in the downward phase of the action potential. The net result of phosphorylation is a prolongation of the action potential in the sensory neuron and so more neurotransmitter is released by the sensory neuron. Thus the sensory neuron's synapse with the gill motor neuron is strengthened. In short-term memory, special enzymes quickly remove phosphates from the proteins and return them to their original state, restoring the synaptic strength to its lower pre-sensitized level. However, following repeated serotonin delivery, the level of cAMP-activated kinase is much higher and this allows the crucial step in the formation of long-term memory to occur. This crucial step is the transport from the synapse to the cell body of kinase molecules that have been activated by c-AMP. Once in the cell body the activated kinases enter the nucleus and start to regulate the expression of particular genes. In Aplysia, proteins that result from this process of gene activation are transported back to the synapse where they are used to maintain the strength of synapses already affected by local effects of c-AMP and to grow new synaptic connections. So in Aplysia the conversion of a short-term into a long-term memory involves the reinforcement of the short-term changes in synaptic strength and the growth of new synapses, both of which require the synthesis of new proteins.

Objective To investigate whether snails will become habituated due to the repeated stimulation. Problem Statement Can snail become habituated to a repeated stimulus? Hypothesis When the snail is being given stimulation (touch) repeatedly, it will eventually become habituated. Thus, the snail would not retreat when another touch is given. As number of stimulations between the eyes stalks of the snails increases, the time for the eye stalk withdrawal will decreases , thus showing negative correlation. Null hypothesis There is no significant correlation between the number of pokes on the snails and the time for the eyestalk to be withdrawn. Experiment design Independent measures will be used for this experiment, as they are separate, different snails being used.

Apparatus Stopwatch Materials Garden snails, dampened cotton wool bud, clean and firm surface for the snails and water

Variables : Types of Variables Manipulated Variable: Number of pokes on snails eye stalk (no.) Damp cotton bud used to poke eye stalk of snail ranges from one to ten pokes. Ways to control the variables

Responding Variables: Time for eyestalk withdrawal (s) Using stopwatch, time taken for the eyestalk to rer-emerge calculated

Control Variables: Same size and species snails Assuming same age between samples. Different species or age snails react differently. Each snails trauma control to be minimal, avoiding ethical problems.

Same volume and force of stimulation

Procedure : 1. A garden snail is being captured and placed on a clean, firm surface. The snail is being left for a few minutes so that it has gotten used to the new surrounding and fully emerged from its shell. 2. A cotton wool bud is being dampened with water. 3. The snail is firmly touched between the eye stalks by using the dampened cotton wool bud and the stopwatch is immediately started. 4. The time taken for the snail to retreat and fully emerged is recorded. 5. Procedure in step 3 and step 4 are repeated for a total of 10 touches with the timing for the snails to re-emerge each time. 6. The data is tabulated in a suitable table and other snails data were tabulated as well. 7. A graph of time against no. of stimulation is then plotted.

Risk Assessment Safety precaution In order to avoid any accident or injury during the experiment in laboratory, the precautionary steps should be taken and applied. Wearing lab coat and a pair of suitable shoes are compulsory when conducting an experiment in the lab at all times to protect the skin and clothing from spillage of any chemical substance. For the snail, make sure to be careful, not to make it stressful that it already has due to different surrounding. The surrounding needs to be constantly damp, mimicking the snails real habitat.

Ethical issue There are no ethical problems when dealing with invertebrates. The snails should suffer no psychological harm and will be released straight back to the wild afterwards. In this case the knowledge gained justifies the procedure, as the procedure should cause no harm whatsoever. The experiment will not harm or stress the snails, they arent social animals so they wont mind being caged for a short period as long as the exper iment takes and common garden snails are not an endangered species, they are actually an incredibly common species. Snails are a great model for this experiment because they can be easily handled and manipulated. Snails are advantageous when it comes to experiments because they are easy to keep and maintain, also abundant in surrounding, easy to study plus they are inexpensive. But, humans cant be compared to snail due to their difference in the genetic make-up. In this experiment, the snails are not tested until they died. Only 10 stimulations are being given and after that, they are released back to the environment. In addition, the use of cotton wool buds instead of sharp object can reduce the risk of injury to the snails. Although the snails are simple organisms that may not suffer in the same way as higher animal, they still deserve respect. These snails are released back to their original habitat and reduce the disturbance on the food chain.

Bias The only bias possible in an experiment testing reflexes is experimenter bias, there could be some ambiguity as to whether or not a snail did actually reflexively withdraw or not.

 Reliability This experiment is relatively simple, and as a result there is no reason why it shouldnt work. The results should show correlation with whats expected to happen and it will be repeated a few times to make sure that it works properly. Validity This experiment has been done before by Humphrey (1933) so the procedure will just be following his that was already proved valid, yet it was quite an old experiment so it cant be trusted completely. Also, the presence of a human at the time of the experiment may affect the snails and may make them react more. However, eventually the experiment should still have the same effect.

Results No. of stimulation 1 2 3 4 5 6 7 8 9 10 Snail 1 22.2 21.8 20.3 19.0 14.1 12.8 8.9 4.2 0.0 0.0 Time taken / s Snail 2 27.9 17.5 16.0 12.4 10.9 7.7 5.2 0.0 0.0 0.0 Snail 3 13.4 7.3 5.4 5.2 4.6 3.6 2.3 0.0 0.0 0.0

Table 1: The time taken against no. of stimulation for the three snails is plotted.

Graph 1: Number of pokes against time taken for eye stalk to re-emerge
30

Time taken for the eye stalk to re-emerge/s

25

20

15

Snail 1 Snail 2

10

Snail 3

0 1 2 3 4 5 6 7 8 9 10

Number of pokes

Spearmans rank, rs correlation coefficient. Null hypothesis, H0: No significant correlation between the number of stimulations and the time for eye stalk withdrawal. Alternative hypothesis, H1: Negative correlation between the number of stimulations and the time for eye stalk withdrawal.

Level of significance for a one-tailed test, = 0.05 Reject H0 if test statistics -0.564

Number of pokes 1 2 3 4 5 6 7 8 9 10

Rank stimulation 1 2 3 4 5 6 7 8 9 10

Mean time / seconds 21.17 15.53 13.90 12.20 9.87 8.03 5.47 1.40 0.00 0.00

Rank time

Difference/D

D2

10 9 8 7 6 5 4 3 2 1

-9 -7 -5 -3 -1 1 3 5 7 9

81 49 25 9 1 1 9 25 49 81

Table 2 :Spearmans rank, rs correlation coefficient data.

Spearmans rank, rs correlation coefficient,

Where = rank stimulation rank time = 10

Based on table of critical value and by using significance value of 5% = 0.05 the critical value was found out to be -0.5636.

Rejection region

-0.5636

Reject H0 as there is sufficient evidence to say that there is negative correlation between the variables. Therefore, snails can become habituated to a touch stimulus

DISCUSSION Data Analysis From the graph, we can see that all three snails showed the same pattern for the habituation experiment. All three snails showed negative correlation between the time taken and no. of stimulus which means as the no. of stimulus increases, the time taken for eye stalk withdrawal decreases. Therefore, in general, we can conclude that the time taken for the snails to retreat and re-emerge decreases as the no. of stimulus increases, and at certain point became fully habituated. Comparisons can also be made between all three snails although the trends are almost the same generally. This is due to different individuals of snail used and each snail is only tested once. Firstly, we can see that snail 2 took the longest time to retreat and re-emerge after the 1st stimulation is given with 27.9s, followed by snail 1 and snail 3. Other than that, it is clear that snail 2 and snail 3 had become habituated after the 8th stimulation, while snail 1 needs extra stimulation before being fully habituated (at 9th stimulation). Therefore, we can also conclude that different snails need different amount of stimulation before being habituated. Evaluation Habituation is a decrease in response to a stimulus after repeated stimulus. Nervous system is that the calcium channel in the presynaptic membrane becomes less responsive with repeated stimulations. With fewer calcium channels open, less calcium ions cross into the presynaptic knob. As a result, fewer vesicles move towards presynaptic membrane, fuse, and discharge their neurotransmitter. Less neurotransmitter available to bind to the post-synaptic membrane so the post-synaptic excitatory potential is not high enough (under threshold level) to trigger an action potential. . Thus, the action potential generated is not high enough which cause no response. According to the graph drawn, there is negative correlation between the number of stimulus and the time taken for the eyestalk to fully emerge. This happen due to habituation has takes place sine same stimulus is given and identified as not important or not danger. Thus, the snails doesnt respond after several seconds.

Limitations There are several limitations that have been identified throughout this experiment. Not able to find the average time taken for each snail. This is because, once the snail become habituated due to the given stimulus. It would not react to the nest stimulus. A long period of time needed for the snail to restore normal activity and this will lead to time wastage if the experiment is going to be carried out again. Without extra sets of reading, the average value cannot be calculated thus affecting the validity of the data Difference in snails original habitat and experiment site (the lightning and the environment are too artificial during experiment and captive period). The snail may be unable to adapt to the surrounding, feeling stressful (anthropomorphism), thus affecting the time taken for the eye stalk to re-emerge. The experiment should be done outdoors in the field or the habitat of the snails instead of laboratory or class to give more pleasant and comfortable feeling to the snails toward its environment. The frequency of stimulus is not consistent. Time for the eye stalk to re-emerge is taken and recorded by each and every student individually, thus time taken to record the data and carry out the next stimulus is different per stimulus. Or if touched too frequently, the tentacle may remain withdrawn throughout the experiment. Snails can remember their immediate previous experiences, if a snail is handled roughly it may react to touch by completely withdrawing, or if the snail is just waking up from being dormant it may not respond as quickly. Snails also have individual differences that may contribute to how they react to this experiment. Thus, each and every snail response is differently.

Sources of errors Several sources of error in this experiment were identified and steps were taken to minimize these errors to make the result more accurate. Difference in snails original habitat and experiment site is too pronounced due to different light intensity and humidity. This is reduce by making the experiment site damp to make the snail comfortable. The strength of stimulus may differ according to the students nature. Students that are scared of snail may give small and light stimulus and the others vice versa. Thus, the stimulus given is very much controlled by the individual.

Conclusion From this experiment it is true that the time taken for the snails to retreat and re-emerge decreases with increasing no. of stimulation. Over a no. of stimulation, the snails had become habituated. Other than that, we can also conclude that different snails need different no. of stimulus before being habituated. Thus, the hypothesis is accepted.

References 1. www.animalbehavioronline.com/habituation.html. Accessed on 16th January 2013. 2. http://www.snail-world.com/Snail-Anatomy.html. Accessed on 16th January 2013. 3. C J Clegg, 2008. Edexcel Biology for AS, 209 p . London : Hodder Education. 4. Habituation in snail, Matthew Ferguson 5. http://visual.merriam-webster.com/animal-kingdom/mollusks/snail/Aplysia. Accessed on 16th January 2013