Food preference of the green shore crab, Hemigrapsus oregonensis, before and after exposure to
food treatments
Chris Blackhurst
Saint Martin’s University
Dr. Hartman and Dr. Olney
Senior Seminar
April 23, 2007
Table of Contents
Abstract
1
Introduction
2
Materials and Methods
4
Results
8
Discussion
11
Literature Cited
14
Acknowledgements
15
Abstract
The green shore crab, Hemigrapsus oregonensis, is an abundant crab in much of the
Pacific Northwest of the United States. They are found typically under protective objects, like
rocks or sand, and scavenge the bottom of the intertidal areas for food. As most crabs, they use
odor to detect their food. This study has found that H. oregonensis did not prefer to feed on beef
or shrimp initially. After being fed beef or shrimp H. oregonensis choose to eat foods that they
were familiar with over other foods or not feeding. An aquarium vacation feeder-fed control
group was shown to prefer feeding over not feeding. H. oregonensis was tested in a modified Ymaze in three intervals of food treatment exposure; no exposure, one week exposure, two weeks
exposure.
1
Introduction
Oceanic intertidal zones are rich in species diversity and composition. Rocky intertidal
zones particularly support a wide range of adaptations among species. Interactions among
species are a delicate thing and small disturbances can have a big impact in a system with so
many factors (Addessi, 1994). Understanding how different stimuli affect organisms and
ecosystems as a whole is important to understand the impact humans have on such a system. The
use of sensory input is the most basic form of interaction an organism has with its environment.
One such sense is that of smell and chemosensitivity. The use of chemical stimuli is common
among intertidal organisms. Chemosensitivity typically manifests in response to odor detection.
While it is not intertidal the shrimp, Palaemonetes pugio, has displayed an attraction to
specific chemoattractants in various food sources in a study by Carr and Derby. Using various
natural and artificial mixtures of foods and their componenets Carr and Derby were able to show
that some foods had specific chemoattractans which the shrimp preferred over others.
Observation of P. pugio showed that the shrimp moved toward certain mixtures of
chemoattractants more aggressively as well as displayed other increases in activity (Carr and
Derby, 1986).
In a similar study by Derby and Atema the Walking Legs Lobster Homarus americanus
was excided and attracted to specific mixtures of amino acids and other organic compounds. 35
compounds were exposed to H. americanus as well as a number of extracts of prey. H
americanus was observed to prefer the odor of prey items over chemical compounds
individually. Some mixtures were also used that produced a response less that that of prey items
but more than individual chemicals in H. americanus (Derby and Atema, 1982). The studys done
2
by both Carr and Derby, and Derby and Atema show that shrimp and lobster respond to chemical
odor of prey respectively.
Zimmer-Faust et al. was able to show a direct correlation between the presence of food
odor and the navigation of intertidal crabs. Using fluorescent enhanced odor plumes of
mimicking food sources it was seen that various species of intertidal crabs would be attracted
towards the odor plumes. Crabs were less focused as the odor plume diffused in the water further
away from the source however as the crabs approached the source their paths became more
focused. This shows that some intertidal crabs use chemicals to navigate toward prey and food
(Zimmer-Faust et al. 1995).
Ristvey and Rebach have show that the crab Cancer irroratus actually responds to
familiar foods more than unfamiliar foods. C. irroratus was fed one of two mussel types for a
period of 28 days. After the 28 days the crabs were exposed to effluents made from each of the
mussel types as well as a third. Observations were made concerning the level of activity the
crabs displayed when exposed to these effluents. Ristvey and Rebach observed that crabs
responded more actively to the effluent of the mussel which they had been feeding on. Such
reactions may have been due to simple chemical memory which dictates preference or an actual
choice for foods familiar to the crab (Ristvey and Rebach, 1999).
Considering the widespread use of chemosensitivity shown among crustaceans it is
expected that other crustaceans use chemicals sensing in similar manners. While C. irroratus is
not a intertidal organism, similar utilization of odor response would be expected among intertidal
species. Among intertidal crabs in Washington State, perhaps the most abundant is the green
shore crab, Hemigrapsus oregonensis (Oliver and Schmelter, 1997). In this study, the food
preference of intertidal shore crab, H. oregonensis, was tested when presented foods before and
3
after familiarization test the hypothesis that H. oregonensis will select food with which it is
familiar more than other foods.
Materials and Methods
This study was conducted from February 13, 2007 to April 9, 2007. Hemigrapsus
oregonensis were collected at low tide from Budd Inlet in Olympia Washington. The collection
site was a rocky hill on the south side of the inlet near the Port of Olympia. The hill was
artificially created during dredging of the inlet and inlaid with rocks for stability. Crabs were
found under rocks on-site during low tides. Criteria for collection were crabs of carapace width
greater than 2 cm. Sex of each crab was not a factor for collection. Crabs were kept temporarily
in an acrylic container 18 cm x 32 cm x 13 cm with seawater between 5 cm and 7 cm covering
them. Salinity of collection area was measured with a refractometer at 20 ppt and the temperature
measured with a digital thermometer at 9o C.
H. oregonensis are small crabs approximately 3cm to 5cm in carapace width and dark
green in color. The legs usually have hair on them. H. oregonensis are typically found under
rocks and other protecting structures in intertidal areas (Niesen, 1997).
Housing and conditions
Crabs selected for study were housed in a 208.2 L artificial seawater tank. Water
temperature was 10o ± 3oC and salinity was kept at 22 ppt ± 3 ppt. These artificial conditions are
modeled after conditions observed at collection site. Water was filtered mechanically through
cotton and carbon mesh filters. Water flow was further enhanced through the use of a second
pump to force more water though the filters.
Tank acclimation and environment
4
Three populations of twenty crabs were kept in acrylic tanks measuring 8 cm x 32 cm x
13 cm. 7 – 12 cm of rock was placed along the bottom of the tubs to provide a substrate for the
crabs. Each population had 15 males and 15 females. Acrylic tubs were submerged in the 55
gallon tanks to allow water to be circulated to maintain the temperature and to filter any waste.
Crabs were allowed to acclimate to lab conditions for 24 hours before their first Y-maze test.
During acclimation crabs were not fed to encourage a food choice response in the crabs during
the initial interval.
Modified Y-Maze
The modified Y-maze used in this experiment consisted of an open box 28 cm x 13cm x
12cm of acrylic (Figure 1). Ramps coming from the middle of the box were constructed
approximately 8 cm long. Each ramp was placed face to face forming an enclosed test area. A
central pit was created by the ramps. This pit was filled with aquarium rock forming a surface
which the crabs could navigate. Each ramp was built 3 cm short of the back wall, the opening
formed trapped H. oregonensis in the end containing the food they chose during the trial.
5
Figure 1. This modified Y-maze allowed the crabs to assort to either side or stay in the center. Food was present in
each end of the maze allowing crabs to peruse either food but not both, as crabs fall into the food chamber to access
the food.
Preparation of food
Food was aged for two days at room temperature, approximately 18o C to 20o C to
increase the potency of the food odor. After two days the food was frozen to keep the level of
aging uniform for all food during the duration of the experiment. Before introduction into the
experiment, the food was thawed and cut into pieces weighing 10 g. Uniform amounts of food
were used to ensure that consumption of food among the groups could be watched
comparatively. The foods used were fresh beef and fresh raw shrimp (Wight et al., 1990).
Initial response to food
After being allowed to acclimate to the artificial salt water environment the each crab
population of 30 crabs were placed in the modified Y-Maze represented in Figure 1 and allowed
to move freely in the maze for 2 hours. Observations were recorded on where the crabs were at
6
the end of the 2 hours. Each end of the maze contained a food treatment; one end of the maze
had 10g of beef and while the other end had 10g of shrimp. Each food was rubbed across the
surface of the respective back wall to maximize odor.
Familiarization of food and feeding.
In each trial populations of 30 crabs were maintained for two weeks in the tank. At the
initial, one, and two week intervals each population was run through the Y-maze. This process
was replicated three times. In each trial one population of 30 crabs was fed beef, another
population was fed shrimp and another was fed a composite vacation feeder. The vacation feeder
as well as the aforementioned aged shrimp and beef were added one of three respective
populations. At the end of the first and second week all food was removed from the tanks and
food withheld for one full day. After 1 day without food the crabs were tested in the Y-maze.
After testing 10 g of food was then added to each respective population.
Modified Y-Maze tests
Observations were made as to how many crabs sought the food with which they were
familiar as compared to the other food provided. Procedures concerning food testing methods
were adapted from those used on large rock crabs by Ristvey and Rebach (1999).Food was
placed in each end of the Y-maze, at one end was beef and at the other was shrimp. Each type of
food was smeared across the back wall of one side of the maze respectively to maximize odor
exposure. Vacation feeder food was omitted from testing due to its lack of odor and use as a
neutral control.
Data Analysis.
The number of crabs that assorted themselves over two hours to food they had been
conditioned with was compared to the number who assorted themselves to the food in which
7
they were not conditioned, as well as those that did not sort to any food during testing. Data were
compared using a two-way analysis of variance test (ANOVA) to determine if there was
significant difference in each feeding treatment of the number of crabs who sort to different
foods and the time interval of testing. If the ANOVA showed no significant interaction (p>0.05)
among the factors then the individual factors were examined. If there was interaction, one-way
ANOVA tests were run for the treatment comparing the number of crabs to food choice for each
individual time interval. If one-way ANOVA tests showed significance, a multiple comparisons
were examined with Tukey tests. All statistical was conducted using Minitab statistical software
(Minitab 2006).
Results
The number of Hemigrapsus oregonensis that ran through the modified Y-maze was
recorded in terms of where the organisms were at the end of a two hour trial. Three replicates for
all three feeding treatments were produced. Crabs were counted as either having sorted to beef or
shrimp. Crabs that did not sort to either food were recorded as not being sorted.
Two-way analysis of variance (ANOVA) tests were run for all replicates of each of the
three treatments. The two-way ANOVA compared time since exposure to food to the type of
food used. Two-way ANOVA testing for the beef-fed H. oregonensis showed a significant
interaction among the factors of time and feeding treatment (F = 16.02; df = 4; p = 0.0005). Beef
fed crabs showed a preference for the beef end of the maze over no preference or the shrimp end
(F = 77.72; df = 2; p = 0.0005). There was no significant difference how crabs sorted and
duration of exposure to each feeding treatment (F = 0.07; df = 2; p = .932).
Two-way ANOVA results for H. oregonensis in the shrimp treatment had signifigant
interaction among the factors of time and feeding treatment (F = 14.54; df = 4; p = 0.0005).
8
Crabs the shrimp-fed group chose the shrimp end of the maze significantly more often than no
choice or the beef end of the maze(F = 80.49; df = 2; p = 0.0005). Shrimp-fed H. oregonensis
also showed no significant difference in terms of length of exposure to food and how the crabs
sorted (F = .001; df = 2; p = 0.991).
A two-way ANOVA showed no significant interaction among feeding treatments,
duration of exposure, and how the crabs sorted in the vacation fed group (F=0.263; df = 4; P =
0.069) so both feeding treatments and duration of exposure were examined independently. At the
initial interval before H. oregonensis had been exposed to any food in captivity, there was no
significant difference as to where the crabs sorted (F = 3.43; df = 2; p = 0.102). After being
exposed to vacation feeders at the one week interval, H. oregonensis showed significant
differences among food choices (F=19.50; df =2; p = 0.002). A Tukey test on the data showed
that choices for shrimp were significantly greater than choosing to not pursue food (97.80%
confidence level). At the two week interval vacation-fed crabs showed a significant difference in
where they assorted themselves (F = 9.38; df = 2; p = 0.014). A subsequent Tukey test showed
that H. oregonensis chose feeding significantly more than not pursuing food but did not prefer
either food (97.80% confidence level).
H. oregonensis tended to choose food it had been fed previously in both the beef
treatments (Figure 2) and shrimp treatment (Figure 3). This trend was evident after one week of
feeding and reinforced by similar repetitions after the second week of feeding. While Vacationfed H. oregonensis did not tend toward either beef or shrimp, they did tend to choose food, either
beef or shrimp, over not feeding (Figure 4).
9
Beef-Fed
25
Number of Crabs
20
Beef
15
none
Crabs
10
shrimp
5
0
initial
week 1
week 2
Time
Figure 2. Mean number of beef-fed crabs of three replicates that sorted to different treatments at the initial
pretreatment interval, after one week of treatment and two weeks of treatment. After one week and two weeks of
being fed beef, more H. oregonensis chose beef than either shrimp or no food. Error bars are represented as one
standard error about the mean.
Vacation-Fed
15
10
Beef
Shrimp
none
Crabs
20
Number of Crabs
25
5
0
initial
week 1
Week 2
Time
Figure 3. Mean number of vacation-fed crabs of three replicates that sorted to different treatments at the
initial pretreatment interval, after one week of treatment and after two weeks of treatment. It can be seen that H.
oregonensis chose food options over not feeding. Error bars are represented as one standard error about the mean.
10
Shrimp-Fed
25
Beef
15
Shrimp
none
Crabs
Number of Crabs
20
10
5
0
initial
week 1
Week 2
Time
Figure 4. Mean number of shrimp-fed crabs in three replicates that sorted to different treatments at the
initial pretreatment interval, after one week of treatment and after two weeks of treatment. It can be seen that after
one week and two weeks of being fed shrimp more H. oregonensis chose shrimp over either beef or not feeding.
Error bars are represented as one the standard error about the mean.
Discussion
Several studies have shown that crustaceans use the olfactory sense to detect and pursue
prey. In the field the blue crab, Callinectes sapidus, has been shown to forage using odor through
their reaction to introduced odor chemicals. Odors of popular prey items were introduced into an
area of the typical foraging ground of C. sapidus. In the treated C. sapidus was seen to forage
more aggressively than untreated areas (Finelli et al., 2000). While other crustaceans such as the
hermit crab, Cilibanarius antillensis, has been shown to associate physical locations to chemical
cues. Sea grass odor was used to attract C. antillensis. The selected sea grass is the natural haven
of C. antillensis and the observed attraction to the odor shows that the hermit crab uses the odor
as a means of finding the seagrass. Chemosensitivity of C. antillensis is fine enough that such
cues are detectable from most other odors. These distinctions are specifically diferentiat location
from other used odors such as the odors of prey (Chiussi et al,. 2001). However among the
11
studies examining the importance of odor, few had been done for any species of crab in terms of
odor memory.
Data obtained through this study directly supports my hypothesis that H. oregonensis
would choose food they had been previously exposed to over unfamiliar food. This study
compared feeding treatments of beef, shrimp, and vacation feeder and time exposed to such
treatments. H. oregonensis was found to statistically be more prone to seek feeding treatments
they had been exposed to for food over other treatments. As seen in Figure 2. beef-fed crabs
would later seek out beef over shrimp. This is complimented by Figure 4 in which those crabs
fed shrimp can be seen perusing shrimp food over beef after feeding experience.
Figure 3 shows no significant difference between choosing beef and those choosing
shrimp in crabs fed vacation feeder treatment. This suggests that food selection in other
treatments was not based on caloric benefit but rather that crabs chose food based on previous
experience with that food.
Food preference in Cancer irroratus was shown to be a function of odor learning as these
crabs show preference to prey they are familiar with (Ristvey and Rebach 1999). My results
similarly show that food selection in H. oregonensis is influenced by previous experience. These
results, in conjunction with those of Ristvey and Rebach, suggest that crabs in general may use
this chemical learning in prey selection. The specific design of this study did not differentiate
between a true choice and a chemically dictated choice similar to the study by Ristvey and
Rebach. In turn it remains unclear as to why these crabs choose familiar foods over others, which
may indeed be higher in nutrients and calories.
If such prey preference is a learned response it may be that crabs learn other things. It is
possible that while crabs remember prey they have eaten recently, they may also remember how
12
they accessed such prey. A crab opening a clam may remember how it opened the clam and be
able to apply that experience to future clams.
This study has shown that H. oregonensis preferentially chose food based on experience.
It does not show why familiar food is chosen over others. Future research should include a
comparative analysis of food sources. To show these results coupled with showing that one food
was significantly more valuable in terms of caloric value and other nutrients would suggest that
odor preference is a function of simple chemical memory, a genetic development that allows
crabs to survive. If crabs were to show a preference for the more valuable food it would show
true learning, choosing the most beneficial food because it is the most beneficial.
13
Literature Cited
Addessi, L. 1994. Human disturbance and long-term changes on a rocky intertidal community.
Ecological Applications 4(4):786-797.
Carr. W. E. S., Derby. C. D. 1986 Behavioral chemoattractants for the shrimp, Palaemonetes
pugio: identification of active components in food extracts and evidence of synergistic
mixture interactions. Chemical Senses. 11:49-56.
Chussi, R., Diaz, H., Rittschof, D., Forward Jr., R.B. 2001. Orentation of the hermit crab
Cilibanarius antillensis:effects of visual and chemicals cues. J. of Crustacean Biology.
21(3):593-605.
Derby, C. D., and J. Atema. 1982. Chemosensitivity of walking legs of the lobster Homarus
americanus: neurophysiological response spectrum and thresholds. Journal of
Experimental Biology. 98:303-315.
Finelli, C.M., Pentcheff, N.D., Zimmer, R.K., Wethey, D.S. 2000. Ohysical constraints on
ecological process: a field test of odor mediated foraging. Ecology. 81(3):784-797.
Minitab Inc. 2006. Minitab Release 15.1.0.0 Statistical Software. Minitab Inc., Quality Plaza,
Pennsylvania State College.
Niesen, T.M. 1997. Marine life of the Pacific Northwest. Gulf Publishing, Huston, Texas. 66-69
Oliver, J., Schmelter, A. Life history of the native shore crabs Hemigrapsus oregonensis and
Hemigrapsus nudus and their distribution, relative abundance and size frequency
distribution at four sites in Yaquina Bay, Oregon [Internet]. Corvallis (OR): Oregon State
University; 1997 [cited 2007 Mar 15]. Available from http://oregonstate.edu/~yamadas
/crab/ch5.htm
Ristvey, A., Rebach, S. 1999. Enhancement of the response of rock crabs, Cancer irroratus, to
prey odors following feeding experience. Biological Bulletin. 197(3):361-367.
Wight, K., Francis, L., Eldridge, D. 1990. Food aversion learning by the Hermit Crab Pagurus
granosimanus. Biological Bulletin, 178-3:205-209.
Zimmer-Faust, R.K., C.M. Finelli, N.D. Pentcheff, and D.S. Wethey. 1995. Odor plumes and
animal navigation in turbulent water flow: A field study. Biological Bulletin 188: 511516.
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Acknowledgements
I would like to thank Dr. Mary Jo Hartman for helping me design this experiment and understand
the statistical analyses, both Dr. Hartman and Dr. Margaret Olney for repeated efforts to refine
my presentation of this study, Cheryl Guglielmo for helping me set up this experiment again and
again and giving me the freedom to fiddle with things until they were just right. I would also like
to give special acknowledgement to Marie Payne for helping me in every step of this process and
for keeping me sane through repeated setbacks and breakdowns.