Lab Exercise - Habituation in Mosquitos and Fruit flies

Animal Behavior - Biology 4200/5430

Bowling Green State University, Fall 2009

Lab Exercise: Habituation/Sensitization in house mosquito (Culex pipiens) larvae and adult fruit flies (Drosophila melanogaster)

Objectives:

Conduct experiments on habituation - a simple type of behavioral plasticity - in mosquito larvae (Culex pipiens)
The laboratory is designed to teach you behavioral observations within an aquatic setup
Test whether there is an association between two variables using Spearman's Rank Correlation

Discussion and Pre-Lab Preparation: Aside from <Imprinting>, several types of behavioral plasticity can be distinguished. <Habituation> refers to a gradual decline in response to recurrent stimuli which were not accompanied by adverse effects.. Alternatively, <Sensory Adaptation> refers to decreased signalling of a peripheral sense organ with continued exposure to the stimulus. <Classical Conditioning> or <Associative Learning> occurs when an assocation is formed between a response and a stimulus. <Trial and Error Learning> or <Operant Conditioning> Behavior may change after an action proved rewarding. Insight Learning or Reasoning is the ability to respond correctly the first time to a situation different from any previously encountered.

Mosquito larva (Culex pipiens). © lobsterman

In this lab exercise we explore habituation in larvae of the mosquito (Culex pipiens) a representative of the order Diptera - featuring two wings. The Northern House Mosquito (Culex pipiens) is the most common night-flying mosquito in the northern US and Canada but there are more than 100 species of mosquitos in North America and close to 3000 worldwide. Mosquitos undergo complete metamorphosis, with four stages to their life cycle - egg, larva, pupa, and adult. The female deposits 100-300 eggs in a raft-like mass at the water surface. After hatching from the eggs, larvae feed for 2 weeks head-down suspended from the surface water film. Dipteran larvae come in many forms and shapes. The larval stage is aquatic, feeding on bacteria, algae, and other tiny organisms found in the water. Culex larvae have a breathing tube located at the base of their tail. They must expose the tail to the surface of the water in order to breathe. The body is covered brushy hairs that help it swim and prevent it from sinking. Larvae undergo four growth periods known as instars. They shed their skins and become larger with each molt. Following the fourth instar, larvae rise to the surface of the water and pupate. Now the breathing tube is located in the head region. After 1-3 days, the outer covering of the pupal body splits open and the adult emerges. For additional information see a page on Mosquito biology with videos of Mosquito behavior.

Larvae are generally found at the surface. When they are disturbed they respond by swimming down towards the substrate. We will elicit this flight reaction in various ways and explore whether responses change with repeated presentation. Habituation is a common process that is observed in all animals from anemones, which lack a centralized nervous system, to humans. It is suggested that such plasticity helps an organism deal with a changing and often unpredictable enviroment. The neural basis for habituation has been studied extensively in the defensive gill withdrawal reflex in another invertebrate - the marine snail Aplysia californica.

Exercise 1: Behavioral responses of mosquito larvae to stimuli

Question: Do mosquito larvae flee from various stimuli, (a) a rapid increase in ambient light, (b) a rapid decrease in ambient light, or (c) slight tap on the side of the glass?

Experiment: Count the number of mosquito larvae that have fled from the surface as a result.

Preparation: For every pair of students we need a small tank or glass container, a pipette to transfer the critters, a watch, and a lamp, Fill the container to a depth of 2-3cm with dechlorinated water.

Procedures: Place 10 larvae into your observation container and do not disturb for a 5 minute acclimation period (no changes in light, no tapping on glass). Record the number of larvae at the surface. Set one of the test stimuli: (a) turn on lamp above tank, (b) turn off lamp (after it had been on for the 5 minutes acclimation period), or (c) single tap on glass with pencil. Every10 seconds record the number of larvae at the surface for 3 minutes.

Answer questions: What stimulus proved most disturbing to the larvae?

Exercise 2: Habituation of mosquito larvae

Question: Do mosquito larvae habituate to repeated application of various stimuli, (a) a rapid increase in ambient light, (b) a rapid decrease in ambient light, or (c) slight tap on the side of the glass?

Experiment: Investigate the reactions of a single mosquito larvae.

Preparation: same as exercise 1

Procedures: Place 3 larvae into your observation container and do not disturb for a 5 minute acclimation period (no changes in light, no tapping on glass). Focus on one individual which is currently hanging upside down at the water surface. Set one of the test stimuli: (a) turn on lamp above tank, (b) turn off lamp (after it had been on for the 5 minutes acclimation period), or (c) single tap on glass with pencil. Estimate how deep the larva fled and measure the amount of time the larva stays away from the surface. Stay with the same individual and continue to focus on it. Repeat same stimulus and measurements in 3 minute intervals for 15+ minutes.

Answer questions:

Do you expect an effect of habituation if you mix different stimuli for this experiment? - If you have time left in the lab you might try this experiment right now.
How can you distinguish the effects of fatigue, sensory adaptation and habituation?

Exercise 3: Drosophila and light or dark from above

Question: Do fruit flies change their behavior with repeated application of a rapid increase in ambient light?

Experiment: Investigate the reactions of 10 fruit flies

Preparation: Place 10 fruit flies into the provided container and let the flies acclimate for ~3 minutes.

Procedures: Present a light stimulus from above and leave on for 10 seconds. Count number of individuals at top after 10-second stimulus and knock down to bottom. Repeat light stimulus every 30 seconds for 10 minutes. Repeat this experiiment at ambient light levels. A dark stimulus in now provided by shielding the tank with your hand.

Answer questions:

In what way does repeated application of light from above change the behavior of fruit flies?

Exercise 4: Escape in Drosophila

Question: Do fruit flies show escape when they experience a rapid decrease in ambient light, or a slight tap on the side of the glass?

Experiment: Investigate the reactions of single fruit flies

Preparation: Place 10 fruit flies into the provided container and let the flies acclimate for ~3 minutes. Focus on one flie.

Procedures: Use your hand to create a shadow from above and measure the number of flies that jump immediately following presentation of the stimulus.

Answer questions:

In what way does repeated application of shadow from above change the behavior of fruit flies?

Review of Statistical Concepts

We have previously discussed the differences between independent (X) and dependent (Y) variables. Typically, we have thought about such variables in the context of a simple behavioral experiment, where the experimenter (i.e., you) systematically varies a single variable of interest (X) to see whether it causes a change in some measured behavior (Y). Whether X causes a change in Y is subsequently evaluated by determining the probability of attaining the measured differences in Y due to chance alone. If the probability of attaining these results due to chance is sufficiently small (p<0.05), it can be concluded that X has indeed caused a change in Y. In essence, we have formally rejected our null hypothesis (that there are no differences in the behavior of interest) and accepted our alternative hypothesis (that systematic alterations in X have changed the behavior of interest).

In this laboratory exercise, rather than determining whether one variable (X) causes a change in another variable (Y), we will simply measure whether two variables are associated with each other. In other words, we are not determining whether there is a causal relationship between two variables; instead we are measuring the association between two variables without regard to the existence of a causal relationship.

Correlational analysis is the typical statistical approach used to measure the degree of association between two variables. Instead of dealing with an X and Y variable, we view both variables as Y’s and set out to determine the relationship between them.

We will use a statistical technique called Spearman’s rank correlation to measure habituation in mosquito larvae and fruit flies. One variable (Y1) to be measured will be the time since the initial stimulus (exercise 3) or the order of stimulus presentations (exercise 4). The other variable (Y2) to be measured will be either the number of individuals at the top (exercise 3) or the distance traveled from the top of the container (exercise 4).

In thinking about next week’s lab (that uses regression analysis), it will be important to understand that correlation and regression do not measure the same thing, even though they are mathematically similar.

Review an example of such an analysis using Spearman's Rank Correlation and a Data sheet that is useful for recording the data.

Links of interest

Information on mosquitos is based partially on background and care instruction for mosquitos from Carolina Biological Supply Co.

last modified: 9/2/05