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-A subclass of pheromones is necromones, which contain oleic acid as a primary component. -Some pheromones trigger aggregation between organisms, others can trigger separation. -Necromones trigger necrophoric (organisms remove their dead) or necrophobic (organisms avoid their dead) behavior. -Arthropods use pheromones, this includes sowbugs and cockroaches. Chemical signals are picked up by antennae.
When given a choice between two identical shelters, sowbugs will display necrophobic behavior by aggregating in the shelter that does not contain oleic acid.
III. Biological Rationale:
-C.D. Rollo study showed that cockroaches display necrophobic behavior around oleic acid (dead conspecifics). -Semi-social behavior and necrophobic activity are linked, while social behavior and necrophoric activity are linked. -Cockroaches and Sowbugs are both arthropods, and they both display semi-social behavior. -Evidence shows necromones being old method of risk avoidance (420 Ma). All arthropods shared common ancestor at some point.
I – Introduction:
Some arthropods use a class of chemicals called pheromones for intraspecific communication (2.1). A subclass of these pheromones is called necromones (2.3), which signal the death or injury of conspecifics (2.4). Studies have shown that the primary component of some necromones is oleic acid, a type of unsaturated fatty acid (2.4). Organisms can react in two different ways to these chemical signals: Social species, which need to live in colonies in order to survive, undergo necophoric behavior, where organisms actively remove their dead from the nest or colony. Semi-social species, which can operate both individually and in a group, undergo necrophobic behavior, where the organisms actively avoid their dead, injured, or anything excreting oleic acid (2.4).
These avoidance and removal responses are adaptive in the sense that when the organism senses the acid, it signals that there may be danger; disease or a predator, which should be avoided. By avoiding areas containing oleic acid, or removing the dangerous individual, the organisms are effectively avoiding a potentially hazardous situation. One species, cockroaches, exhibit necrophobic behavior, and are also semi-social (2.4). The experimentally tested species, sowbugs, are also semi-social organisms, and use pheromones for intraspecific communication (1.3).
This raises the question “do sowbugs exhibit necrophobic behavior?” Therefore, we hypothesized that when given a choice between two identical shelters, sowbugs will display necrophobic behavior by aggregating in the shelter that does not contain oleic acid. A null hypothesis would state that when given a choice between two identical shelters, sowbugs will not exhibit a preference between aggregating in a shelter that contains oleic acid and one that does not.
We think this hypothesis is rational due to several factors. An experiment by C.D. Rollo investigating cockroaches showed that 100% of cockroaches displayed necrophobic behavior when exposed to dead conspecifics (2.4). Evidence also shows that semi-social behavior and necrophobia are linked, as displayed by the cockroaches.
Sowbugs, as mentioned earlier, are also semi-social because of individual feeding and group aggregation. It would then be expected that they would also exhibit necrophobic behavior. Evidence also shows that necromones are a very old risk avoidance mechanism (2.4), if sowbugs display necrophobic behavior, it would show that necrophobia as an adaptive trait developed before crustacea diverged into its own subphylum.
-Dependent Variables: How many sowbugs are found in each shelter after 20 minutes. -Independent Variables: One shelter contains oleic acid and one does not. B. All the constant variables concern the shelters, surrounding environment, and sowbugs. These should not interfere with the independent and dependent variables. The independent variable (the shelter containing oleic acid) will affect the dependent variable (number of sowbugs in each shelter) due to their hypothetical aversion to oleic acid.
II – Experimental Design and Methods:
1. Obtain 3 large tin bowls, 1 spoon, 1 pair scissors, 6 ketchup containers, 1 pipet, 3mL oleic acid, 6mL distilled water, 6 filter papers, and 30 sowbugs. 2. Place 10 randomly selected sowbugs in each of the 3 empty tin bowls for 3 minutes to allow them to acclimate to the environment. 3. During the 3 minute period, soak each filter paper with 1mL of distilled water, because sowbugs have been shown to prefer moist environments.
4. Add 1mL of oleic acid to three of the wet filter papers, this creates the independent variable. One will go in each tin. 5. Using the scissors, cut two small doors in each ketchup container on opposite sides, so that when the container is placed upside down, the sowbugs may enter it. Having 2 doors in each increases the chance of sowbugs finding an entrance.
6. After 3 minutes, quickly place the wet filter papers on opposite sides of the tin (noting which one contains oleic acid) and cover them with the ketchup containers. The doors should face away from the center, towards the edges of the tin. The placement of the oleic acid-containing paper should be random, no preference for either side of the tin. 7. Wait 20 minutes, observing the sowbug’s behavior. This 20 minute period gives them time to find each shelter and determine a preference. Do not disturb them during this time. Keep track of how many sowbugs are inside of each shelter, and record these numbers after 20 minutes. Do not count the bugs that have not picked a shelter after this period.
B. Controlled Variables:
Moisture, light, temperature, time, number of sowbugs, surrounding environment, number of doorways to shelters, size of shelters, treatment of sowbugs before and during experiment, origin of sowbugs, all environments are set up exactly the same way.
-The sowbugs will choose independently choose their preferred shelter. -The sowbugs all come from the same environment, and will act similarly to sowbugs in their natural environment. -The materials for the experiment are not contaminated.
-The sexes and ages of the sowbugs will not affect their preference for oleic acid vs. non-oleic acid. -20 minutes is enough time for the sowbugs to decide which shelter they prefer.
D. Opportunities for Bias:
Group members could affect the outcome of the experiment by altering the environment or encouraging the sowbugs to favor one shelter over the other. To prevent this, 2 repetitions of the experiment could be done. E. List of Materials:
3 large tin bowls, 1 spoon, 1 pair scissors, 6 ketchup containers, 1 pipet, 6mL oleic acid, 12mL distilled water, 6 filter papers, and 30 sowbugs.
A. Data Analysis:
We will do a chi-squared goodness of fit test, and calculate the p value (with a significance level of 0.05). B. Results:
If our hypothesis is supported, the p value will be less than or equal to 0.05 with 1 degree of freedom. This will reject the null hypothesis. If we accept the null hypothesis, the p values will be greater than 0.05 with 1 degree of freedom. If the chi-squared value is extremely large, it will indicate that the data strongly supports the hypothesis.
III – Results:
The sowbugs initially explored both shelters, but did not remain long in either. The majority remained outside the shelters, circling the edge of the tin. After 10 minutes more begin to aggregate in each shelter for a longer period of time, with some preference towards the non-oleic shelters.
Table 1. Number of sowbugs in each shelter, one containing oleic acid. N = total number of sowbugs in each tin, only numbers of sowbugs found in shelters were recorded. | # Sowbugs in Oleic Shelter| # Sowbugs in Non-Oleic Shelter| N| Tin A| 0| 5| 5|
Tin B| 0| 9| 9|
Tin C| 0| 4| 4|
Total| 0| 18| 18|
The results of our experiment shown in Table 1 demonstrate that 18 sowbugs preferred the non-oleic shelter, and 0 preferred the oleic shelter (x2 = 18, d.f. = 1, N = 18, p < 0.001).
For this experiment, we hypothesized that the sowbugs, because of an adaptive aversion to oleic acid, when given a choice between two moist, dark shelters (one containing oleic acid), would choose the shelter that did not contain oleic acid. Our results, as can be seen in Table 1, support this hypothesis, and reject the null hypothesis that they would have no preference. Because sowbugs are semi-social, similar to cockroaches, they would be expected to exhibit an aversion to the presence of oleic acid, just as cockroaches do as seen in the C.D. Rollo study.
When we presented the sowbugs with a choice between a shelter containing oleic acid, and a shelter that did not contain oleic acid, the sowbugs showed an overwhelming preference to the shelter that did not contain the acid. 18 sowbugs were found in the clean shelter, while none were counted in the shelter containing the necromones. Overall, the design of the lab was well executed, but there were several factors that inhibited greater accuracy. First, the distribution of light is an important factor for sowbug aggregation. Within each of our tins, the light entered at a slightly different angle, which would be expected to, in turn, affect sowbug behavior.
The placement of the shelters was also source of uncertainty because three different individuals placed and set them up, preventing the start of the experiment to be perfectly simultaneous and the tins to be completely identical. Finally, the distribution of the oleic acid was a source of error, as it did not absorb evenly into the paper, but rather remained on top, in droplets. This may have affected the sowbug’s perception of each shelter to some extent.
Since one important idea behind this experiment is that the aversion to oleic acid may have developed within athropods before the divergence of crustacean and hexapods a long time ago, testing multiple species within crustacea with this same experiment might present evidence to further support the hypothesis. Because the sowbugs within our experiment exhibited strongly necrophobic behavior when exposed to oleic acid, the hypothesis is supported, as is the idea that the aversion to necromones is a trait that evolved in arthropoda before the divergence of crustacea and hexapoda.
E. Lab Conclusion:
The primary question within this lab concerned whether or not sowbugs would exhibit an aversion to necromones (containing oleic acid) because of their semi-social behavior and evolutionary history. By designing an experiment oriented entirely around the sowbug aversion to oleic acid, we were forced to think critically about the relationships between the organisms and necromones, and develop a better overall understanding. Because of previous knowledge, we were able to design an experiment that presented the sowbugs with two environmental factors they favor: moisture and darkness.
By placing two moist, dark shelters in a tin enclosure, we gave the sowbugs a place to aggregate, but by placing oleic acid in one shelter, we could better examine their responses to the necromone. From the start of the experiment, it was quite clear that they had an aversion, any which entered the oleic shelter immediately exited. This helped us understand what exactly necrophobic behavior in a real life situation looks like. We also learned about the importance of being as random as possible, for example, random selection of sowbugs, tins, shelters, etc. These are all sources for potential bias, which would lead to inaccurate results.