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James Duong

Bio 205L Wednesday


1:50-4:50PM

The Effect of Aplysia California Ink and Opaline on Hermit Crabs

Introduction

Hermit crabs can sense chemical deterrents released by other organisms in defense

against predators since chemical stimuli are water soluble which can easily access sensory

organs. (Carr, 1988) Chemicals such as ink and opaline released from sea slugs can confuse and

deter hermit crabs by interfering with their behavioral responses. (Love-Chezem et al., 2013) The

secretion of ink of from the ink gland decreases the function of olfactory receptors of

surrounding organisms, preventing them from orienting toward the sea hare. Opaline is a cloudy-

white liquid secreted from the opaline gland that can polymerize and become viscous upon

contact with water. The chemical opaline is known to decrease other predators appetite while the

ink causes confusion. (Johnson, 1999). Presently, we do not know if hermit crabs avoid sea

hares' ink and opaline, but there are some previous studies showing that opaline is a more

successful repellent than ink on hermit crabs' feeding behavior. (Takagi et al., 2010) To

determine if hermit crabs have an avoidance for ink and opaline, we tested this in a y-maze and

hypothesized that hermit crabs are more likely to move toward the area with ASW and stay away

from the ink and opaline.

Methods

A sample of 48 starved hermit crabs (Pagurus samuelis) along with a sea hare (Aplysia

california) were gathered in Point Fermin, Los Angeles. In lab, the crabs were randomly

handpicked from a 46x47x91cm tank. Each crab ranging from 1.5cm to 1.8cm. The control for

the first experiment was artificial salt water (ASW) running down both arms. A Y-shaped maze

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with an inclined of 40°, a length of 63cm, and 38cm for each arm was used as a runway for the

crab to make a decision between the left or right wing with the appropriate treatments. These

treatments were dripped at the end of the two arms with 2 plastic pipettes at a rate 1mL/5s for

every trial. The ink gland (1g) and opaline gland (0.1g) were extracted from a sea hare (Aplysia

california) and smashed with a mortar and pestle. Each gland was then mixed with 500mL of

ASW to make stock. The stock was kept on ice to prevent the ink from oxidizing and degrading.

The ink was made with 20mL of the stock and diluted with 980mL of ASW. The opaline

treatment was made using the same method above. The opaline+ink treatment was made by

mixing 20mL of the ink with 20mL of opaline then diluted in a 960mL of ASW. A total of 48

trials were tested for this experiment. 1) Opaline vs. ASW, 2) Ink vs. ASW, 3) Ink+Opaline vs.

ASW and 4) Ink vs. Opaline. Halfway through each experiment, there was a treatment switch to

eliminate side biases. After each trial, the maze was cleaned with ethanol and rinsed with

deionized water. The p-value for each experiment was found by entering the number of trials and

the number of success through the website "http://graphpad.com/quickcalcs/binomial1.cfm".

Results

The ink was found to create an avoidance behavior in Pagurus samuelis while the opaline

showed no effect. In the first experiment, the opaline seemed to show no effect on crabs tested.

According to Figure 1, half of the crabs (24/48) went toward ASW and the other half (24/48)

went toward opaline yielding a significance of (p= 1.1146). The second test examined if the

hermit crab showed deterrence toward the ink presented in Figure 2. According to the results, the

majority (34/48 crabs) avoided the ink water and migrated to the other arm showing a

significance of (p=0.0055). Only 14/48 crabs were deterred by the arm containing the opaline. In

Figure 3, (25/48) crabs chose ink+opaline arm while (23/48) avoided it with a significance of

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(p=0.8854). Figure 4 illustrates that the crabs were deterred by the ink more since 29/48 went

toward the opaline arm while 19/48 crabs were deterred by opaline (p=0.1934).

Figure 1: Results of the Opaline and ASW choices of 48 Pagurus samuelis (N=48). 24 out of 48 chose the ASW arm
and 24 chose on the opaline side (p=1.1146). Therefore there was not enough conclusive evidence to show a
deterrence toward opaline.

Figure 2: Results of the second set of 48 hermit crabs (N=48) avoidance of the ink treatment. (34/48) of Pagurus
samuelis moved toward the arm where there is no presence of ink. (p=0.0055)

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Figure 3: The third set of 48 hermit crabs (N=48) tested with a combination of both opaline and ink vs ASW. (25/48)
crabs chose the opaline and ink treatment whereas only (23/48) avoided it. (p=0.8854)

Figure 4: The fourth set (N=48) shows (29/48) hermit crabs show a higher preference for opaline than the ink. 19/48
crabs avoided the opaline (p=0.1934)

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Figure 5: The control shows that the sample of Parugus samuelis show a preference toward the left arm of the maze.
(p=0.0595)

Discussion

Our study shows that Pagurus samuelis reveal a deterrence toward the arm containing the

ink, but had no effect on the arm with opaline. Before the experiment, we predicted that hermit

crabs are more likely to move toward ASW over areas containing ink and opaline. Small

crustaceans especially hermit crabs can be easily repelled by uncomfortable chemical secretion,

(Love-Chezem et al., 2013). Hermit crabs might have a stronger reaction to ink than opaline. The

results did not support our hypothesis, however, Takagi et al., (2010), stated that hermit crabs

have a strong deterrence to opaline. Our first experiment contradicts this hypothesis because the

crabs did not show enough statistical data to confirm a deterrence to opaline. According to Figure

1, 50% of the trials chose to the ASW and 50% chose opaline, thus no conclusion can be made

based on the deterrence of opaline.

The tests supports our hypothesis since hermit crabs were vastly deterred from the ink.

Based on Figure 2, 70% of the tested crabs avoided the ink and fled toward the ASW. Takagi et

al., (2010) showed that Paragus samuelis are repelled by ink which also supports our findings.

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The crabs' choices suggests that they have specific receptor types that signal them to avoid areas

with the presence of ink. This result is consistent with earlier reports stating that ink can disrupt

the behavioral response of the crabs.

The third trial also oppose our hypothesis since there were more crabs were not deterred

by the ink+opaline treatment. Even though the ink was merged with the opaline, the hermit crabs

still chose the side with opaline present than the side with no chemicals at all. As a result, this

trial also contradicts Takagi et al., (2010) prediction that opaline and ink cause a strong

deterrence. One possible reason why the crabs were not fully deterred by the chemicals is

because the ink and opaline were not concentrated enough to activate their receptors.

The fourth trial complicates the study that ink is the ultimate cue that deters predators

away. In figure 4, 29 out of 48 crabs avoided the arm with ink and went into the ASW. This

means that there were not enough data to prove a deterrence effect since p=0.1934. The null

effect on ink opposes (Love-Chezem et al., 2013) findings explaining that ink causes confusion

and can inhibit chemoreceptor in many aquatic organisms. In addition, this result also does not

support Tagaki et al., (2010) hypothesis since the opaline did not elicit a stronger deterrence

effect according to the results in figure 4. During the ink and opaline secretion, the chemicals

both work together by blocking the uptake of scent and decreasing appetite (Johnson, 1999) . A

possible reasoning for this trial is that the ink slightly shows a stronger effect on the crabs at

lower concentration. As a result, Aplysia's ink have a greater effect of Parugus samuelis while

opaline fails to cause a response.

There were indeed several limitations that prevented us to run this experiment at full

precision. One discrepancy on the ink+opaline water trial was that some the solutions were not

concentrated enough to illicit an suitable response. As a result, the receptors on the hermit crabs

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were not fully functional in triggering a physiological deterrence. Additionally, some of the

ethanol used to clean the maze might have interfered in the crabs' decision. The sample of crabs

tested might also have been injured when experimented by previous groups so their antennae

could have been damaged. The three days starvation of the crabs makes them less able to pick up

chemical cues.

A conclusion can be drawn from this experiment illustrating that Pagurus samuelis have

no reaction to the chemical opaline, but deterred by ink secreted by the Aplysia california. A

follow up study should test the effect of opaline vs. the presence of a predator by increasing the

concentration of opaline. Since the number of hermit crabs tested was limited, the experiment

should be performed with more trials (N>50).

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Reference

Carr, W. E. S. (1988). The molecular nature of chemical stimuli in the marine environment. In

Sensory Biology of Aquatic Animals (ed. J. Atema, R. R. Fay, A. N. Popper and W. N.

Tavolga), pp. 3-27. New York: Springer-Verlag.

Johnson, P. M., and A. O. D. Willows. 1999. Defense in sea hares (Gastropoda, Opisthobranchia,

Anaspidea): multiple layers of protection from egg to adult. Mar. Freshw. Behav.

Physiol. 32:147–180.

Love-Chezem, Tiffany, Juan F. Aggio, and Charles D. Derby. "Defense Through Sensory

Inactivation: Sea Hare Ink Reduces Sensory And Motor Responses Of Spiny Lobsters To

Food Odors." Journal Of Experimental Biology 8 (2013): 1364. Academic OneFile. Web.

15 Apr. 2014.

Takagi, KK, NN Ono, and WG Wright. "Interspecific Variation In Palatability Suggests

Cospecialization Of Antipredator Defenses In Sea Hares." Marine Ecology Progress

Series 416.(2010): 137-144. Science Citation Index. Web. 15 Apr. 2014.

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