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Animal Behaviour: Biology 3401

Laboratory 3: The effects of physical and biological factors on various invertebrate organisms

Parts of this lab were taken from or adapted from: Glase, J. C., M. C. Zimmerman, and J. A. Waldvogel. 1992. Investigations in orientation behavior. Pages 1-26, in Tested studies for laboaratory teaching, Volume 6 (C. A. Goldman, S.E. Andrews, P.L. Hauta and R. Ketcham, Editors). Proceedings of the 6th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 161 pages. Questions to Prepare You for this Laboratory

• What are three types of orientation stimuli that can be sensed by some organisms but not humans?

• What characterizes a taxis orientation response? • What characterizes a kinesis orientation response? • Is the manner in which you would locate the source of an odor in a dark room a taxis, a

kinesis, both, or neither? • Is the manner in which you would locate the source of a sound in a dark room a taxis, a

kinesis, both, or neither?

Introduction Humans have been aware of animal orientation, migration, and navigation for

thousands of years. Whole civilizations have thrived or perished based on their understanding of the movements made by principal animal food sources. Yet, it was not until early in the 20th century that a rigorous analysis of orientation mechanisms began, when our knowledge of sensory systems and how other animals detect the world improved considerably.

Orientation refers to the spatial organization of movements. Since movements are elements of behavior, orientation and behavior are intimately associated. For simplicity, we will define behavior as any overt manifestation of life by an animal, especially one that takes the form of movements. A behavior pattern is the fundamental unit of behavior, and is defined as a sequence of movements characterized by a specific configuration in time and space. This underscores the special significance that spatial organization has for behavior. Every behavior is spatially oriented in some way. Whether an animal walks, grooms, catches prey, or interacts with a social partner, "where" and "in which direction" are indispensable features of its behavior pattern. Thus, we can define orientation as the process that animals use to organize their behavior with respect to spatial features.

The specific orientation systems used by an animal correspond to the features of its environment. Many terrestrial organisms are sensitive to humidity levels, and are therefore capable of orienting with respect to moisture gradients. But humidity is an environmental feature that is not relevant in a totally aquatic habitat, and as a result animals that live in water must use physical gradients based on other parameters (e.g., temperature or salinity) to help direct their movements. Some orientation stimuli are available to both terrestrial and aquatic organisms; these include gravity, light and

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magnetism.

This laboratory exercise will allow you to investigate orientation behavior in a variety of animals. It will help you understand the scientific method by giving you experience in conducting and interpreting data involving animal orientation. ������

Orienting Stimuli ������ In orientation studies, one first attempts to identify the nature of the stimuli to

which the animal is orienting. Light, gravity, sound, and mechanical stimuli, as well as temperature, chemical, and moisture gradients are all likely candidates. As with a moth flying into a candle, the nature of the orienting stimulus may be clearly apparent. However, if the animal is orienting to a stimulus for which humans have no receptor organs, identification of that stimulus will be much more difficult. Orientation to ultraviolet and polarized light, magnetism, electrical fields, and some acoustic stimuli are of this sort. Frequently, organisms respond simultaneously to several stimuli while orienting. Thus, one must be cautious in interpreting observations of orientation behavior since the stimulus most obvious to human senses may not be the most important factor determining the animal's behavior. Frequently an orienting stimulus also elicits a behavioral response. For example, in prey-catching and courtship behavior, the animal often first orients toward the prey or mate, then performs the appropriate behavior to capture the prey or attract the mate. The presence of the prey or potential mate in the environment causes the animal to orient appropriately as well as to perform other behaviors. At the same time that an animal is stalking prey or courting, it is also using gravity as a stimulus for body orientation relative to the earth. As a tuna pursues a mackerel in the open ocean, the mackerel elicits and orients the predatory behavior of the tuna, but gravity and light stimuli are also used by the tuna for general body orientation.

In addition to species differences for a given orientation behavior, the nature of the orienting stimulus itself may vary as a function of the animal's age. Many nestling birds, for example, show a gaping response which elicits parental feeding. When the nestlings first hatch they are blind, and the gaping response is released by mechanical or auditory stimuli provided by the parent birds. The nestlings gape vertically, with gravity being the main orienting stimulus. Later, after a nestling can see, the sight of the parent bird not only elicits the gaping response, but also orients it. ���������

Classification of Orientation Responses ������ The ways that animals orient to their environment are diverse, and certain

schemes have been developed to classify these responses in reference to underlying similarities. The classification system presented in this laboratory was first suggested by Fraenkel and Gunn (1961 - The Orientation of Animals). ������ Kinesis ���

One important distinction that Fraenkel and Gunn make depends on whether the animal's body is oriented with respect to the stimulus source. A movement that does not involve orientation with reference to a stimulus source is known as a kinesis where the stimulus produces either a change in the speed of the animal's movement (orthokinesis) or in the animal's turning rate (klinokinesis). These two responses effectively change the

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position of the animal with respect to the stimulus source. Several examples should clarify this point.

Isopods (terrestrial crustaceans) prefer moist habitats. In some species, as the relative humidity of the environment increases, the amount of time the animal is stationary also increases. This response tends to keep an isopod in damper areas. As another example, some insects cannot detect the direction of an odor gradient, but their rate of locomotion varies with the strength of the odor. Thus, if an insect moves rapidly at low concentrations of a chemical and slowly at high concentrations, it should eventually arrive at the source of the odor. The human body louse (Pediculus corporis) finds its host by a kinetic response to a number of stimuli including temperature, humidity, and odor. When in a favorable environment with respect to these stimuli, the louse travels in straight lines. However, if it encounters an unfavorable environment, it turns until a favorable environmental zone is once again encountered. In summary, a kinesis involves quantitative variations in an animal's speed or turning rate with no fixed orientation of the body relative to the stimulus source. ������ Taxis

In a taxis, the animal's body is oriented in some linear manner relative to a stimulus; either directly toward it, directly away from it, or at a fixed angle to it. Locomotion may or may not be involved in a taxis. This kind of response may be shown for light, heat, moisture, gravity, sound, chemicals, or other stimuli.

For the next two weeks you will be examining the effect of various physical and biological stimuli on the behavior of a variety of invertebrates. Materials and Methods A. Physical Factors 1. Phototaxis in Hermit Crabs How do hermit crabs respond to light? Do the crabs exhibit a kinesis or taxes when presented with light and dark environmental choices? If the crabs exhibit phototaxis is it negative or positive phototaxis? What does it mean to be negatively phototactic and why would this be advantageous? What does it mean to be positively phototactic and why would this be advantageous?

• Place 6 hermit crabs in a dissecting tray. Cover half of the tray with a dark piece of

cloth or cardboard and shine a light over the other half of the tray. For each trial, place the crabs in the center of the tray and leave them for 2-3 minutes. After the 2-3 mins count the number of crabs in light and dark areas (you have seen this type of sampling before… it is called instantaneous or scan sampling). To control for any directional bias, remove the cloth/paper from one half of the tray and place it over the other half of the tray. Move the light to the other side of the tray. Wait another 2-3 minutes and record the number of crabs in light and dark areas. Repeat this experiment until you have done 5 trials with the light/dark on

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each side of the tray. Do the hermit crabs prefer light or dark conditions? Record your results in Table 1.

Table 1: Data table for phototaxis experiment using hermit crabs

Section Number in Dark Number in Light Observation

#

1 2 3 4 5 6 7 8 9 10

Mean S.D.

• Some animals exhibit goal-directed orientation. They will determine where a

stimulus originates and head directly towards or away from it. Place a hermit crab in the single tail of a Y-shaped arena. In the starting tail of the arena there should be low light. At one arm of the arena a strong light and in the other arm complete darkness. Is the hermit crab moving? Watch the crab until it has chosen an arm and has stayed there for 3-4 minutes. Repeat this experiment switching which arm has a strong light and which is dark until each condition has been presented five times on either side. Did the hermit crab move into the darkness or head towards the light? If it chose the lit arm, did it head directly towards the light or did it travel around in circles and eventually make its way there?

• Record your results in Table 2.

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Table 2: Goal-oriented movement in a hermit crab

Section Dark Strong Light No movement (Low light)

Observation #

1 2 3 4 5 6 7 8 9 10

Mean S.D.

2. Humidity and Light Preference in sowbugs

These terrestrial crustaceans, sometimes called sowbugs or pillbugs, are common inhabitants of leaf litter and soil. They feed on decaying organic material as well as algae, moss, and bark. Isopods have a pair of compound eyes, two pairs of antennae (although only the second pair is prominent), and seven pairs of legs. When disturbed or desiccated they will roll up into a ball, looking rather pill-like. Experiment one: This experiment will be conducted in an elongated Plexiglas chamber that can be used to collect quantitative data on the response of individuals to light, humidity, or a combination of these stimuli (Fig. 1). At one end there is anhydrous CaCl2 (a desiccant) and a wet paper towel in the other end. This sets up a humidity gradient that we are assuming is changing consistently over the length of the apparatus. For this part of the experiment, observe the animals under red light (most invertebrates have a lower sensitivity to red light). After about 5 minutes, place 10 individuals in the chamber via the central stoppered hole. Every minute count the number of sowbugs in each section of the chamber and enter the data on the sheet on the next page (Table 3). Continue counting for 30 minutes.

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Introduce sowbugs here

 

Desiccant (CaCl2)

Moistened paper towel

Experimental Setup for Sowbug Choice Experiments

   

1 2 3 4 5 6 7 8 9 10 SECTION

 Dry Humid

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Table 3 – Data Table for Sowbug Wet vs. Dry Choice Experiment

Dry Humid Section 1 2 3 4 5 6 7 8 9 10

Observation #

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Mean S.D.

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Experiment 2. This experiment is essentially a repeat of Experiment 1 but testing the response of sowbugs to light vs dark. Use the chamber with one end covered in electrical tape. Introduce 10 sowbugs into the chamber as before and wait one minute before beginning observations. After each minute, count the number of animals in the dark and in the light sections (You have to count the number in the light section and subtract from 10) and enter the data in Table 4. Continue counting for a minimum of 10 minutes and stop when the number of animals in one section is 0 for three successive minutes. The Interaction of Factors After you have gained some insight into the sowbug's response to humidity and illumination independently (and have collected enough data to support your views!), you can examine the interplay between these two stimulus types. Is the response to humidity changed under conditions of high illumination? What if the individuals are offered a brightly illuminated, humid environment versus a dark, dry environment? Experiment 3 In this experiment, you will be looking at the interaction between humidity and light in the response of sowbugs. Set up the experiment as in experiment 1 but put one end of the chamber (either the humid or dry end) into the opaque chamber. Introduce ten sowbugs into the chamber and wait one minute before beginning observations. After each minute, count the number of animals in the dark and in the light sections. As before, continue counting for a minimum of 10 minutes and stop when the number of animals in one section is 0 for three successive minutes. Enter the data into Table 5. Repeat the experiment but this time switch the end the chamber that is in the opaque chamber. Introduce ten fresh (i.e. untested) sowbugs into the chamber and repeat the experiment as above.

Introduce sowbugs here

 

Desiccant or Moistened paper towel

Experimental Setup for Sowbug Combined Choice Experiments

   

1 2 3 4 5 6 7 8 9 10 SECTION

 

Desiccant or Moistened paper towel

Opaque chamber

 

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Table 4 – Data Table for Sowbug Light vs. Dark Choice Experiment

Section Dark Light Observation

#

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Mean S.D.

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Table 5 – Data Table for Sowbug Combination Factors Experiment Observation # Dry -Dark Humid – Light Dry – Light Humid - Dark

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Mean S.D.

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Experiment two: To better understand the orientation response of isopods, examine the response of single individuals within the apparatus (with the humidity gradient in place). Place one sowbug in the apparatus. Observe the movement of the bug in the apparatus for 5 minutes and track its progress by marking its paths (to scale) with a pen and some paper. Use a map measurer to find (1) the total distance travelled in both environments, and (2) the total distances of "direct" travel (i.e., maintaining a straight line). Also, count the number of sharp curves (of more than 90o. ) Repeat this exercise with 3 more sowbugs for a total of 4 trials. Record your data in Table 6. Do your data indicate whether the response shown is a kinesis or a taxis? If a taxis is involved, what would you do to determine the taxis type? Table 6: Data table for sowbug orientation response

Slug # Distance (cm) travelled in dry

Distance (cm) travelled in wet

Direct travel (cm)

# of sharp turns

1 2 3 4

3. Thigmotaxis, chemotaxis and Phototaxis in flatworms (Dugesia) Certain animal species orient themselves in the environment so as to maximize contact with solid surfaces. This type of orientation behavior is called thigmotaxis. Animals showing thigmotaxis tend to aggregate under solid objects and their orientation is sometimes incorrectly assumed to be a response only to light. How do flatworms respond to touch? Based on where they are touched does their response vary? For example, do the flatworms react the same if they are touched on their anterior end as they do if touched posteriorly? Thigmotaxis-Experiment one: Place a flatworm in the center of a petri dish. Wait until the worm begins to move then time how long it takes to reach the outer rim of the dish. Stop the experiment if this does not happen within 5 minutes. Repeat this exercise 2 more times. Record your data in Table 7.

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Table 7: Data table for flatworm thigmotaxis

Flatworm # Time to edge of dish (seconds or minutes if more appropriate)

1 2 3

Phototaxis-Experiment one: Place a flatworm in a petri dish that has duct tape covering one half to provide a dark environment. Wait 3-5 minutes. Has the worm made a clear choice preference of dark or light environments? Repeat this experiment with a second flatworm. Do the flatworms prefer the light or dark environment? Does this mean that the flatworm is positively or negatively phototactic? Chemotaxis- Experiment one: Place a flatworm at one end of a petri dish. Place a pinch of cat food at the other end of the dish. How does the worm respond to the food? Watch the flatworm for 5-10 minutes. Has it moved towards the food? Repeat this experiment but this time use a glass bead as the food item. Is the response to the bead similar to that of the cat food? Choice experiment! Place some cat food at one end of the dish and a glass bead at the other end. Put the flatworm in the center of the dish (try to orient the worm so that it is not facing either food choice). Is the flatworm moving towards the cat food or the bead? Watch the worm for about 5 minutes. Does the worm appear to be interested in the food choice it moved towards (i.e. does it appear to be eating or did it move towards the item and keep on moving past it?) Record your observations in Table 8. Table 8: Observations from flatworm chemotaxis experiment Condition Observations Cat Food

Glass bead

Cat food and glass bead

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4. Geotaxis in snails/slugs Background: Gastropods (including limpets, whelks, slugs and snails) generally move slowly and ‘sluggishly’. For that reason, their locomotion is relatively easy to study. Under certain conditions they move consistently in one direction, and are apparently well-oriented with respect to gravity. Also, the direction of their movements often seems to be influenced by the paths taken by other nearby snails. This portion of the laboratory concerns the study of three aspects of the locomotory behaviour of a terrestrial snail Helix apersa: (1) undisturbed movements, (2) gravity responses, and (3) the tendency to follow trails left by other individuals. Helix aspersa is commonly found in gardens, hedges and woodlots. This snail is normally inactive during daylight and under dry conditions, and moves and feeds especially at night or after rain. It feeds on living vegetable material, from which fragments are abraded by means of a toothed radula. In winter and during prolonged dry periods, Helix seals up the aperture of its shell with a film of mucus hardened with calcium phosphate. Many, if not all snails, will be quiescent at the start of the laboratory session and it is essential to stimulate them and have them moving about actively immediately before use. This is best done by immersing the snails in tepid (lukewarm) water for a few minutes. As soon as a snail shows signs of activity it should be removed from the water, dabbed dry and introduced to the experimental set-up. Handle the snails gently, especially when lifting them up from an attached position. They are not likely to move in a natural, consistent way if you roughly tear them loose from a substrate. PART 1: Movements in relation to gravity (geokineses/geotaxis) are to be studied as follows. If Helix aspera (the land snail) is available use Helix. Otherwise, use the slugs available in lab. • Place a fresh slug on the centre of a clean plexiglass plate that has a grid of squares

drawn on the reverse side. Clamp the plate in a vertical position with one edge on the bench top (angle of inclination=90o). As soon as the slug begins moving, start a stopwatch, and plot its course to scale on a separate sheet of graph paper. After 5 minutes, or when the slug has reached the edge of the glass plate (whichever occurs first), remove it. Determine the angle of orientation of its path with respect to the side of the plate in contact with the bench. Do this by drawing a straight line on the graph paper that represents as accurately as possible the direction of the slug’s path. If a sharp change in direction occurred during the test, two separate angles of orientation should be determined.

• By using a piece of string to estimate the distance travelled by the snail, and the time

between start and end of the test, determine its speed of movement (cm/min). • Repeat the above procedure with two more slugs (thus yielding 3 replicates), being

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sure to wash the glass plate thoroughly after running each animal. • Then change the angle of inclination of the plane to 45o from the horizontal and repeat

above procedure with the same three slugs (or fresh slugs if the supply is large enough).

• Finally, repeat the steps again with the plate at 70 and 25o inclination. 3 slugs each for

a total of 12 separate tests. • • Record all your data in Table 9. • Prepare a graph showing the mean angle of orientation and the mean speed of

movement of your slugs in relation to the angle of inclination of the plate. Each mean value should be based on 3 replicate measurements. In discussing your results, consider the following: What are the sensory structures that enable a slug to detect changes in gravity? Where are they located? What are some environmental variables that could affect the behavior of slugs in this laboratory exercise? What might be the significance of the observed responses to the slugs in nature? Are they taxes or kineses?

Experimental set-up for snail geotaxis experiment

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Table 9: Data table for slug geotaxis experiment Plate angle (degrees)

Slug # Slug path angle (degrees)

Speed (cm/min) Distance (cm)

90 1 90 2 90 3

45 1 45 2 45 3

75 1 75 2 75 3

25 1 25 2 25 3

PART 2: Chemoreception in slugs (trail following) • Purpose: to study trail following by determining the degree to which the mucous trails

of a ‘blazer’ and a ‘follower’ snail coincide.

• The tendency for one animal to follow the recently laid mucous trail of another conspecific has been demonstrated in several gastropods, including terrestrial snails. Examine trail following in slugs as follows.

• Method: Place one active slug (the ‘trail blazer’) at centre of a piece of clean

plexiglass lying flat on the bench top. Plot to scale the path of this slug until it has moved 15-20cm (within 5-8 minutes). Then remove it and place a second slug (the ‘follower’) at the same starting point, and oriented in the same direction as the blazer. Observe and plot the path of the follower on the same graph paper until it has moved at least as far as the blazer did. Remove the follower and determine the coincidence index (C.I.) of the two mucous trails using the following equation:

C.I. = Lc/(Square root of (Lb X Lf)) Where Lc is the length of the follower trail that coincides with the blazer trail, Lb is the length of the blazer trail and Lf is the length of the follower trail. Use string to estimate each of these distances.

• Repeat the above procedure with two more pairs of slugs, making sure that the glass

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plate is washed between tests. Summarize your data in a table (Table 10), showing values of Lc, Lb, Lf and C.I. for each pair of slugs and an overall mean (SD) C.I. Discuss the possible functional significance of trail following in a slug.

Table 10: Data from trail following in slugs

Trial # Lc Lb Lf CI 1 2 3

Mean SD 5. Humidity and mealworms/adult flour beetles: Experiment 1: This experiment is a repeat of the experiment conducted with the sowbugs and will be conducted in an elongated Plexiglas chamber that can be used to collect quantitative data on the response of individuals to light, humidity, or a combination of these stimuli (Fig. 1). At one end there is anhydrous CaCl2 (a desiccant) and a wet paper towel in the other end. This sets up a humidity gradient that we are assuming is changing consistently over the length of the apparatus. For this part of the experiment, observe the animals under red light (most invertebrates have a lower sensitivity to red light). After about 5 minutes, place 10 individuals in the chamber via the central stoppered hole. Every minute count the number of flour beetles in each section of the chamber and enter the data in Table 11. Continue counting for 30 minutes. Experiment 2. This experiment is essentially a repeat of Experiment 1 but testing the response of mealworms/flour beetles to light vs dark. Use the chamber with one end covered in electrical tape. Introduce 10 mealworms/flour beetles into the chamber as before and wait one minute before beginning observations. After each minute, count the number of animals in the dark and in the light sections (You have to count the number in the light section and subtract from 10) and enter the data in Table 12. Continue counting for a minimum of 10 minutes and stop when the number of animals in one section is 0 for three successive minutes. The Interaction of Factors After you have gained some insight into the mealworm/flour beetles's response to humidity and illumination independently (and have collected enough data to support your views!), you can examine the interplay between these two stimulus types. Is the response to humidity changed under conditions of high illumination? What if the individuals are offered a brightly illuminated, humid environment versus a dark, dry environment? Experiment 3 In this experiment, you will be looking at the interaction between humidity and

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light in the response of mealworms/flour beetles. Set up the experiment as in experiment 1 but put one end of the chamber (either the humid or dry end) into the opaque chamber. Introduce ten mealworms/flour beetles into the chamber and wait one minute before beginning observations. After each minute, count the number of animals in the dark and in the light sections. As before, continue counting for a minimum of 10 minutes and stop when the number of animals in one section is 0 for three successive minutes. Enter the data into Table 13. Repeat the experiment but this time switch the end the chamber that is in the opaque chamber. Introduce ten fresh (i.e. untested) mealworms/flour beetles into the chamber and repeat the experiment as above.

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Table 11: Data Table for Mealworm/flour beetle Wet vs. Dry Choice Experiment

Dry Humid Section 1 2 3 4 5 6 7 8 9 10

Observation #

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Mean S.D.

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Table 12 – Data Table for mealworm/flour beetle Light vs. Dark Choice Experiment

Section Dark Light Observation

#

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Mean S.D.

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Table 13 – Data Table for mealworm/flour beetle Combination Factors Experiment Observation # Dry -Dark Humid – Light Dry – Light Humid - Dark

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Mean S.D.

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6. Chemoreception in flour beetles (Tenebrio molitor; effect of pheromones on mate attraction) • See additional hand-out… this is found online.

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Assignment: Every week you will have an assignment to complete. The lab assignments are handed in the following week in lab. Make sure you are doing the right assignment… the assignments for each week depend on which group you are placed in (and therefore, which exercises you completed). This is detailed below. Group work and assignments: Group A Week one: Exercise: Phototaxis in Hermit Crabs and light and humidity preference in sowbugs. Assignment: Write a full manuscript on the effect of humidity and light on sowbugs Week two: Exercise: Light and humidity preference in mealworms and snail/slug geotaxis and trail following Assignment: Write a formal results section for the work with the slugs (the geotaxis work as well as trail following). Week three: Exercise: Chemoreception in flour beetles and exercises with flatworms Assignment: Write a formal discussion for the pheromone work with the flour beetles. Group B Week one: Exercise: Chemoreception in flour beetles and exercises with flatworms Assignment: Write a formal discussion for the pheromone work with the flour beetles. Week two: Exercise: Phototaxis in Hermit Crabs and light and humidity preference in sowbugs. Assignment: Write a full manuscript on the effect of humidity and light on sowbugs Week three: Exercise: Light and humidity preference in mealworms and snail/slug geotaxis and trail following. Assignment: Write a formal results section for the work with the slugs (the geotaxis work as well as trail following).

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Group C Week one: Exercise: Light and humidity preference in mealworms and snail/slug geotaxis and trail following. Assignment: Write a formal results section for the work with the slugs (the geotaxis work as well as trail following). Week two: Exercise: Chemoreception in flour beetles and exercises with flatworms Assignment: Write a formal discussion for the pheromone work with the flour beetles. Week 3: Exercise: Phototaxis in Hermit Crabs and light and humidity preference in sowbugs. Assignment: Write a full manuscript on the effect of humidity and light on sowbugs