Comparison of Feeding Behavior in Eyestalk-Ablated and Eyestalk-Intact Uca Pugilator Fiddler

Crabs

Anonymous
AP Biology Period 2
December 20, 2013

Abstract:
The main objective of this experiment was to examine the how the presence of eyestalks
in Uca pugilator fiddler crabs affects the feeding behavior of these crabs. A minor objective was
to speculate about a possible mechanism for how FIF works. The hypothesis developed proposed
the following: if the presence of eyestalks affects the feeding behavior of fiddler crabs, then the
crabs with intact eyestalks will eat less than the crabs with ablated eyestalks, as measured by the
twice upward movement of their claws to their mouths, when placed in a feeding assay
containing glucose. There were two main groups tested in this experiment, which were a group
of eight eyestalk-intact male fiddler crabs and a group of four eyestalk-ablated female crabs. The
group of eight eyestalk-intact male fiddler crabs was split into two smaller groups of four each so
that it would be easier to observe the feeding behavior of the crabs. The method for conducting
the experiment involved the use of a feeding assay that contained the following concentrations of
glucose: 0 M, which is just seawater, 0.125 M, 0.25 M, 0.5 M, and 1 M. The original hypothesis
created was rejected by the data because the results indicated that the eyestalk-intact fiddler crabs
and the eyestalk-ablated fiddler crabs exhibited fairly similar feeding behavior patterns, even
though it was expected that the feeding behavior of the eyestalk-ablated group would have
plateaued earlier than the feeding behavior of the eyestalk-intact group. It is possible that factors
such as social interaction among the groups of fiddler crabs had a profound impact on the data
obtained from the experiment.
Purpose:
The purpose of this experiment is to test how the presence of eyestalks in fiddler crabs
affects their feeding behavior. It was hypothesized that if the presence of eyestalks affects the
feeding behavior of fiddler crabs, then the crabs with intact eyestalks will eat less than the crabs
with ablated eyestalks, as measured by the twice upward movement of their claws to their
mouths, when placed in a feeding assay containing glucose.

Introduction:
Sand fiddler crabs of the species Uca pugilator typically are found in burrows that are in
the intertidal to surpratidal zones of salt marshes, sandflats, and mangrove swamps in tropical
and temperate parts of the world, and they forage for food on the sediments in the low intertidal
zone based on chemical cues (2). It has been found that the most important determining factors
for where fiddler crabs forage are the sediment organic content and the sediment water content
(2). Also, the sediment water content seemed to have a greater influence on the patchiness of the
foraging environment of the fiddler crabs than the food content itself (2). These crabs feed by
bringing pieces of the sediment to the buccal, or mouth, region with their chelae and then process
through the material, determining which substance is to be swallowed and which substance is to
be removed (1). Females can use either of their claws whereas males can only use the minor claw
they possess. Larger sediment particles are removed out of the buccal area and then collected
into a small ball. The twice upward movement of its claws to its mouth can determine whether or
not the crab is feeding as opposed to testing the material for food content. The optimal foraging
strategy of the fiddler crabs is affected by the physical characteristics of the sediment: total
organic content of at least 1.0%, a mixture of sediment grains dominated by 0.125 to 0.250 mm
sizes, and complete saturation of water (1). Differences in these factors would thereby affect the
effectiveness of the foraging strategy of the crabs.
Fiddler crabs feed when the chemoreceptors of the end segment, or dactyls, of their
walking legs are stimulated, which can occur by several types of stimulatory chemicals, such as
amino acids, small peptides, and sugars. It has been found that glucose is a type of sugar that is
stimulatory to some types of fiddler crab species, such as Uca pugilator. Glucose is a 6-carbon
ring sugar with the formula C6H12O6. Glucose can be found in the environment in the intertidal
zone when the tide comes in. The same can be said for other types of sugars as well, although
they may not be as likely to be encountered, since glucose tends to be more prevalent in sandflat
environments. If the fiddler crabs were sensitive to more than one type of sugar, then they would
have an advantage because the chemoreceptors on their legs would sense these other types of
sugar and the crabs would be able to feed. They would not be inhibited by the availability of only
a certain type of sugar, such as glucose. Moreover, it is reasonable to say that if crabs are less
sensitive to one type of sugar than to another type of sugar, then they are probably more likely to
encounter the type of sugar that they are more sensitive to, which would explain the difference in
sensitivity as they may have evolutionary evolved over time to have chemoreceptors that are
sensitive to only a certain type of sugar. Additionally, if the types of sugars vary seasonally, then
the sensitivity of the crab to the sugar could change as well, especially if there were a particular
hormone or chemical affecting the sensitivity.
In this lab, the question being tested involved testing how the presence of eyestalks
affects the feeding behavior of fiddler crabs. This topic was chosen because the reactions of the
fiddler crabs to the feeding stimuli are regulated by a hormone-like factor known as the feeding
inhibitory factor, or FIF, which is produced in the sinus gland of the eyestalk. FIF is released
when the hemolymph glucose level of the crabs is elevated above a threshold; the factor reduces
the intensity of the crabs’ responses to the sugar (3). Crabs that contain low levels of FIF in their
hemolymph are hypersensitive to sugars. Therefore, it was hypothesized that if the presence of
eyestalks affects the feeding behavior of fiddler crabs, then the crabs with intact eyestalks will
eat less than the crabs with ablated eyestalks, as measured by the twice upward movement of
their claws to their mouths, when placed in a feeding assay containing glucose. This result is
expected because the fiddler crabs without eyestalks would lack sinus glands and would not
produce FIF; therefore, eyestalk-ablated fiddler crabs would experience a hypersensitivity to
sugars that would cause them to feed more than eyestalk-intact fiddler crabs. In order for this
hypothesis to be proven true, the experimental results would have to show that eyestalk-ablated
crabs exhibited greater levels of feeding activity than the eyestalk-intact crabs. However, if the
opposite results were obtained, meaning that the eyestalk-intact crabs exhibited greater feeding
activity than the eyestalk-ablated crabs, the hypothesis would be rejected. Also, if the two groups
of fiddler crabs, both the eyestalk-ablated group and the eyestalk-intact group, demonstrated
similar feeding patterns, then the hypothesis would be rejected as well because this would
suggest that the presence of the eyestalk, and the sinus gland that produces FIF, has no effect on
the feeding behavior of Uca pugilator, even though other research has shown that the presence of
the eyestalk will affect the feeding behavior.
Methods:

Four black bowls were obtained and then filled with seawater each, approximately enough to
cover the bottom surface of the bowl so that there remained an even level of seawater. One
member of the group obtained four eyestalk-intact male fiddler crabs, two members obtained
another group of four eyestalk-intact male fiddler crabs, and another member obtained a group of
four eyestalk-ablated female fiddler crabs. All of the fiddler crabs had been starved for 24 hours
previous to the experiment in order to ensure that they would be sensitive to the feeding cues.
One person recorded the data for each of the trials conducted and also served as the timer. Each
person handling the fiddler crabs was wearing plastic gloves and transferred the crabs to the
appropriate location by picking them up from behind and placing them in the desired place. The
method used for this experiment involved a feeding assay, in which the following concentrations
of glucose were used: 0 M, 0.125 M, 0.25 M, 0.5 M, and 1 M (3). Samples of each these
concentrations were obtained and then poured into each of the five lids respectively. Just enough
was poured so that there remained an even layer of the fluid covering the bottom of the lid. This
amounted to approximately 25 mL of each solution. All the crabs were tested in increasing order
of the sugar concentrations. Each group was first placed in the 0 M glucose solution, which is
just seawater and then allowed to acclimate for 15 seconds. Another lid was placed on top of the
lid filled with the glucose solution to ensure that the crabs would not crawl out. Next the crabs
were observed for one minute to see if the feeding behavior was exhibited. A movement with the
small claw in the male eyestalk-intact group and a movement with either claw in the female
eyestalk-ablated group at least twice during the observation period were counted as responses.
After the one-minute period of observation, the crabs were then transferred to the seawater rest
bowl and given one-minute rest. The lid was placed back on top of the resting bowl but inverted
with a little gap between the edge of the bowl and the edge of the lid so that the crabs would
receive air. Then, the crabs were transferred to the next bowl of glucose concentration for the
next observation. They were placed in the rest bowls for one minute every time before being
placed in the next lid filled with the varied concentration of glucose. This same procedure was
conducted for each of the three groups of crabs in an identical manner, with the observer(s)
counting the number of responses. Once a fiddler crab exhibited the appropriate response, it was
not necessary to continue observing that particular crab in that particular glucose concentration
because it already exhibited the desired behavior. One crab was switched in the male eyestalk-
intact group 2 after being placed in the rest bowl between the tests for 0.25 M glucose
concentration and 0.50 glucose concentration because it had a missing leg, which affected its
response in the feeding assay. This procedure constituted trial one. Once trial one was
completed, the crabs were allowed to rest in the seawater rest bowls for ten minutes. After this
rest period, trial two was completed in the same manner as trial one; however, two of the crabs in
the male eyestalk-intact group 1 were changed for two different male eyestalk-intact crabs
because they seemed to not be responding at all, which is not the typical behavior for fiddler
crabs. The rest of the procedure remained the same and the person who was the designated
recorder and timer collected the data for trials 1 and 2.

Results:
The experiment was conducted on December 5, 2013 between the time period of 11:43
a.m. and 1:17 p.m.

Data Table 1: Data for Trial 1 Using Feeding Assay

0M 0.125M 0.25M 0.5M 1M

Male Eyestalk-intact 0/4 3/4 2/4 4/4 4/4

Male Eyestalk-intact 0/3 3/3 3/3 4/4 4/4

Female Eyestalk-ablated 0/4 2/4 3/4 4/4 4/4

Data Table 2: Data for Trial 2 Using Feeding Assay

0M 0.125M 0.25M 0.5M 1M

Male Eyestalk-intact 0/4 2/4 2/4 3/4 3/4

Male Eyestalk-intact 0/4 0/4 3/4 4/4 4/4

Female Eyestalk-ablated 0/4 2/4 2/4 4/4 4/4

Data Table 3: Data for Male Eyestalk-intact Crabs Across All Trials
Group Number Total Fed: Total Percentage
Crabs (at end of Trial)
1 13:20 65.0%
Trial 1
2 14:17 82.4%

1 10:20 50.0%
Trial 2
2 11:20 55.0%

1 and 2 48:77 62.3%

Data Table 4: Data for Female Eyestalk-ablated Crabs Across All Trials
Group Number Total Fed: Total Percentage
Crabs (at end of Trial)
1 13:20 65.0%
Trial 1
1 12:20 60.0%
Trial 2
1 and 2 25:40 62.5%
Combined

Figure 1:
 Number of Crabs that Fed at Varied Glucose Concenrations For Trial 1
1
Fraction of Crabs That Fed

3/4

Male Eyestalk-intact 1
2/4
Male Eyestalk-intact 2
Female Eyestalk-ablated
1/4

0
0M 0.125M 0.25M 0.5M 1M
Glucose Concentration (in mol/L)

Figure 2:

Number of Crabs that Fed at Varied Glucose Concentrations for Trial 2

3/4

Male Eyestalk-intact 1
2/4
Male Eyestalk-intact 2
Female Eyestalk-ablated
1/4

0
0M 0.125M 0.25M 0.5M 1M
Glucose Concentration (in mol/L)

Discussion/Conclusion:
Based on the data collected from trials 1 and 2, the hypothesis initially proposed has been
rejected because the data is contrary to what was expected for the outcome of the experiment and
suggests that the feeding behavior of eyestalk-intact fiddler crabs and eyestalk-ablated fiddler
crabs are similar. Graph 1 for the first trial shows how the feeding response for two groups, male
eyestalk-intact crabs group 1 and female eyestalk-ablated crabs, seemed to plateau at the same
place: between 0.5 M glucose concentration and 1 M glucose concentration. For the male
eyestalk-intact group 2, the feeding response plateaued earlier, around 0.125 M glucose
concentration. This contradicts the expected outcome, which is that the feeding responses for the
female eyestalk-ablated crab group would plateau earlier, meaning at a lower concentration, than
the other two male eyestalk-intact crab groups. When the graph plateaus, this suggests that
feeding behavior has reached a maximum limit and will continue at that level for the rest of the
concentrations tested. Therefore, since female eyestalk-ablated crabs do not have eyestalks and
therefore do not have sinus glands that would produce the FIF, it is expected that they would eat
more in their hypersensitive state. Since the feeding behavior recorded did not show this but
rather depicted the opposite, this would suggest that the presence of the eyestalk does not affect
the feeding behavior of fiddler crabs, even though other research has shown that the presence of
eyestalks does affect the feeding behavior (3).
Graph 2 suggests a similar conclusion, that the feeding behavior of the eyestalk-intact
fiddler crabs and eyestalk-ablated fiddler crabs is the same; furthermore, what is interesting is
that male eyestalk-intact group 1 followed a similar trend as the female eyestalk ablated group.
This is surprising because it is expected that the feeding for the female eyestalk-ablated group
would have plateaued earlier than that of the male eyestalk-intact group 1. In addition, each
group demonstrated two plateaus of feeding behavior as opposed to just one. For the male
eyestalk-intact group 1, this occurred between the 0.125 M and 0.25 M glucose solutions as well
at the 0.5 M and 1 M glucose solutions. For the male eyestalk-intact group 2 this occurred
between the 0 M and 0.125 M glucose solutions as well as between the 0.5 M and 1 M glucose
solutions. For the female eyestalk-ablated group this occurred between the 0 M and 0.125 M
glucose solutions as well as between the 0.5 M and 1 M glucose solutions. Referring back to
Data Table 1, Data Table 2, Data Table 3, and Data Table 4, it can see that the conclusion made
based on the data is justifiable. In particular, the last row for Data Table 3 and Data Table 4
suggest that overall the feeding behavior of the eyestalk-intact male fiddler crab groups and the
eyestalk-ablated female fiddler group is not affected the presence or lack thereof of eyestalks,
since for the eyestalk-intact male groups overall the feeding percentage across all trials was
62.3% and for the eyestalk-ablated female group it was 62.5%. Even though there is a slight
increase of 0.2% in the feeding behavior of the eyestalk-ablated female group when compared to
the eyestalk-intact male group, it cannot be concluded that there was a significant difference
between the feeding behavior of the eyestalk-intact crab groups and the eyestalk-ablated crab
groups.
 Overall the design of the experiment served the purpose of answering the experimental
question about how the presence of eyestalks in Uca pugilator fiddler crabs affects their overall
feeding behavior, even though the hypothesis was falsified. However, there were many factors
and confounding variables that could have and may have affected the results of the experiment.
One such factor is that all of the eyestalk-intact crabs were male crabs and all of the eyestalk-
ablated crabs were female crabs. This posed an issue because the male crabs for the most part
seemed to be generally more active than the female crabs, which suggests that perhaps this active
nature of theirs caused them to eat more than usual than compared to eyestalk-ablated male
crabs. Perhaps this is one reason why the eyestalk-intact male crabs exhibited similar feeding as
the eyestalk-ablated female crabs. Another factor is that the ablation procedure may have injured
the female crabs, thereby causing them to feel uncomfortable and not feed as much as they
normally would have even with intact eyestalks. So, if they were to feed less with intact
eyestalks because of an injury, then they would not be expected to eat even more with ablated
eyestalks, which could be another reason why the data collected for the feeding behavior for the
three different groups is so similar.
Another aspect to examine is the aggression of the males toward each other. It seemed as
though the male crabs spent more time waving their claws at each other and fighting than sifting
through the water for potential sources of food. This sort of aggression was absent in the female
group. Also, the male crabs continually attempted to crawl out from the lid containing the
glucose solution and therefore did not spend much of the one minute time period in the layer of
the solution. Instead, they remained closer to the outer surface of the lid where there were only a
few drops of the solution present. These actions also caused acts of aggression among the males
but not among the females, who remained in the liquid and did not move much. Perhaps this is
because in many species of organisms males tend to be more aggressive than females; however,
it is also important to remember that the female crabs could not see each other because they did
not have any eyestalks, so it possible that they were unaware of their surroundings and the crabs
around them. The male crabs on the other hand could view the other males around them with
their intact eyestalks. This poses a question about the social behavior of fiddler crabs, and
specifically Uca pugilator crabs, toward each other among groups of males only, groups of
females only, and groups of males and females combined. The social interaction within these
three different groups could affect the feeding behavior of the crabs and create competition
between individuals.
 One other change that may have affected the feeding behavior of the fiddler crabs is that
some of the crabs were changed in the male eyestalk-intact crab groups, either because they had
a missing leg or because they were not responsive at all to the stimuli of glucose and did not
exhibit the normal behavior expected of a fiddler crab. This in turn could have affected the social
interaction among the crabs, for they did not have much time to acclimate to the new crab(s).
Since the difference in gender could have an effect on the feeding behavior of the fiddler crabs, it
would be more accurate in the future to use crabs of the same gender, so as to avoid introducing
new variables in the experiment that are not being tested or accounted for. Also, a blind was not
placed between the observer and the bowl for the eyestalk-intact crabs, which may have caused
another source of error in the lab design. Therefore, in the future, a blind should be used in order
to minimize any distraction for the fiddler crabs from the observer. Perhaps another reason why
the eyestalk-intact fiddler crabs crawled towards the edge of lid with the glucose solution is
because they could view the observer and were moving towards them. In general, the
experimental design would have worked effectively had it employed controls for the variables
presented by the situation.
Even though the data provided by this lab would not support the function of FIF in fiddler
crabs, other research conducted would (3). Feeding behavior is controlled by a negative feedback
loop that involves the production of FIF in a typical fiddler crab. A possible mechanism for how
FIF works is the following: the fiddler crab senses its food with the chemoreceptors on its legs,
which would respond to certain stimuli, such as glucose particles. The stimulus then generates an
action potential to be transferred by neurons that would allow for the stimulus to be processed by
the brain of the fiddler crab. As a greater amount of the stimuli is received by the chemoreceptors
on the legs of the fiddler crab, the amount of cell signaling will increase as the production of FIF
increases. More specifically, the brain will signal the sinus glands to produce more FIF by
releasing a hormone that would bind a cell receptor of the cells in the sinus glands. Once the
hormone binds to the receptor, it becomes activated and undergoes a conformational change that
results in the activation of a G-protein that is bound to the receptor. The G-protein becomes
activated when it releases GDP in exchange for GTP, which in turn binds to the receptor and is
what causes the conformational change that releases the G-protein from the receptor. Next, the
G-protein works on membrane processes that can lead to the development of cyclic AMP, a
second messenger. This molecule causes an enzymatic cascade in which one protein kinase that
is activated by cyclic AMP can activate other proteins. This leads to signal amplification within
the cell. The response to this transduction of the signal is the production and release of FIF, the
feeding inhibitory factor, which dampens the crab’s response to sugar. FIF causes the
chemoreceptors to become more desensitized to the sugar particles, which eventually causes the
crab to stop eating. In an eyestalk-ablated fiddler crab, there is essentially no production or
minimal production of FIF, which results in hypersensitivity of the crab to sugar. There would
not be any inhibition factor that would halt the crab from continuing to eat, which resembles a
positive feedback loop in some ways, since the continuation to eat in response to the stimulus is
amplified.

It would be interesting to conduct the experiment again using four groups instead of two,
which would include the following: eyestalk-intact male fiddler crabs, eyestalk-ablated male
fiddler crabs, eyestalk-intact female fiddler crabs, and eyestalk-ablated female fiddler crabs.
Using these groups would control for the variable of gender and provide for more in-depth
analysis of the data obtained. Further studies about the aggression in male fiddler crabs could be
conducted as well in order to provide more information. It would also be beneficial to gain more
information about the social interaction of the fiddler crabs, especially among different gendered
fiddler crabs, in order to understand how this factor may affect the experiment overall. Learning
more about the particular feeding behavior in Uca pugilator fiddler crabs could help scientists
apply the knowledge gained to other similar organisms, thus deciphering how the nervous system
works in various organisms and the role of certain substances, such as FIF, in those processes.
Works Cited/ Bibliography
1. Reinsel, Kathleen A., and Dan Rittschof. "Environmental Regulation of Foraging in the
Sand Fiddler Crab Uca Pugilator." Journal of Experimental Marine Biology and
Ecology 187 (1994): 269-87. Web. 17 Dec. 2013.
2. Rittschof, Dan, and C. U. Buswell. "Stimulation of Feeding Behavior in Three Species of
Fiddler Crabs by Hexose Sugars." Chemical Senses 14.1 (1989): 121-30. Web. 18
Dec. 2013.
3. Sears, Margaret A., and Dan Rittschof. "Control of Chemically Stimulated Feeding
Behavior in Sand Fiddler Crabs Uca Pugilator: Evidence for Hemolymph
Feeding Inhibitory Factor." Journal of Chemical Ecology 17.12 (1991): 2337-346.
Web. 2 Dec. 2013.