1) The study examined the effects of varying light wavelengths (red vs blue) on the physiology and behaviors of hermit crabs (Coenobita clypeatus). 2) Hermit crabs exposed to red light showed decreased feeding, and increased aggressive and exploratory behaviors compared to those exposed to blue light. 3) Hermit crabs exposed to red light also had higher numbers of serotonin-positive cells in the eyestalk compared to those exposed to blue light. The number of serotonin-positive cells correlated with the duration of behaviors observed.

1. A
INTRODUCTION
METHOD
RESULTS
REFERENCES
DISCUSSION
Rachel S. Clein, Ingrid K. Tulloch, Larry D. Fort, Shanice L. Hastings & Sarthak Shah
Stevenson University, Stevenson, Maryland.
For more information contact rclein@Stevenson.edu
HYPOTHESES
ACKNOWLEDGEMENTS
Varying Wavelengths of Light Alters Serotonin Positive
Cells and Associated Behaviors in Coenobita clypeatus
Light pollution can have profound impacts on the physiology and behavior of many
organisms. Coenobita clypeatus is a crustacean species typically known as the purple
pincer hermit crab. These crabs are of ecological and commercial importance on the
eastern shore of Maryland. However, human activity can and has altered environmental light
in the habitat of this organism. We therefore sought to examine the physiological and
behavioral effects of varying environmental light conditions in C. clypeatus. In our previous
studies, we demonstrated that short wavelength light (blue) exposure resulted in increased
feeding behaviors and decreases in all other activities (Fort & Tulloch, 2016). We also
observed decreases in feeding and increases in aggression and exploratory behaviors when
exposed to longer wavelengths, such as red light (Fort & Tulloch, 2016).
We aimed to replicate our previous behavioral studies and pilot-test a neurochemical
response thought to be involved in feeding and aggressive behaviors (Briffa & Elwood,
2007; Frazer & Hensler,1999). The neurochemistry examined was the number of serotonin
(5-HT) positive cells in the hermit crab eyestalk after chronic exposure to red or blue light.
We hypothesized that 5-HT positive cells in the eyestalk would be greater in crabs exposed
to red light compared to crabs exposed to blue light, and that there would be a relationship
between the duration of behaviors and the number of 5-HT positive cells.
Figure 1. Varying Light Conditions. Tank 1 as it appeared under red light conditions 620-750 nm (left), under blue light conditions at
450-495 nm (middle), and under natural light (right). Data presented in previous work showed blue and red light behaviors differed
from behaviors under natural light (Fort and Tulloch, 2016)
Day 1 – 6 Habituation
to Terrarium and
Natural Light
Day 7-9
Red or Blue Light
Exposure
Day 9
Euthanization and
Dissection of
Eyestalks
After Dissection
Serotonin
Immunohistochemistry
Hermit crabs of both sexes (n = 6) were weight matched and given food and water ad libitum under constant
temperature and humidity.
Video recordings were taken between 6:30 and 10:30 pm for nine days and the following behaviors coded
and quantified.
 Feeding Behaviors: entering food dish and touching food with pincers, eating food, drinking water
 Aggressive Behaviors: mounting other crabs’ shells, fighting other crabs
 Exploratory Behaviors: climbing around shelter, touching other crabs’ shells, touching objects in the
shelter
5-HT positive cells were labeled by immunohistochemistry using Rabbit anti-serotonin antibody and visualized
using Goat Anti-Rabbit secondary antibody with DAB Peroxidase. They were quantified with NIH ImageJ
software using cell counter add-on.
Figure 5. Representative image of immunohisotochemical staining of eyestalk
tissue from a crab exposed to red light (a) and a crab exposed to blue light (b).
0
5
10
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20
25
30
35
40
45
50
0 10 20 30 40 50 60 70 80
DurationofFeedingBehavior
(Minutes)
Number of Positive 5-HT Cells
Figure 2. Mean duration of feeding behavior per light condition (a), and scatterplot of its correlation
with 5-HT- positive cells (b). Duration of feeding behavior decreased under red light [t(4) = -8.103,
p = 0.001.] and was negatively correlated with number of positive 5-HT cells (r = -.930, p =
0.007).
Figure 3. Mean duration of aggressive behaviors per light condition (a), and scatterplot of its
correlation with the number of positive 5-HT cells (b). Duration of aggressive behavior increased
under red light. [t(4) = 5.358, p = 0.006] and was positively correlated with the number of 5-HT
positive cells ( r = .875, p = 0.022).
Figure 4. Mean duration of exploratory behaviors per light condition (a) and scatterplot of its
correlation with the number of 5-HT-positive cells (b). Duration of exploratory behaviors [t(4) =
3.313, p = 0.030] increased under red light and was positively correlated with 5-HT-positive cells
(r = .843, p = 0.035).
 Significant decreases in feeding and increases in aggression and exploration under
red light conditions support our previous findings that varying wavelength of light
alters behaviors in C. clypeatus.
 Duration of these behaviors correlate with serotonin positive cells in the eyestalk,
suggesting a role for serotonin the behavioral response to varying wavelength of light.
 The findings provide a novel method for examining the effects of light pollution on
physiology and behavior in an invertebrate model.
 Future studies will replicate the experiment with a larger number of organisms and a
between-groups design that includes baseline light.
 Direct manipulation of serotonin under varying light conditions might also provide
mechanistic explanation for these findings.
Briffa, M., & Elwood, R.W. (2007). Monamines and decision making during contests in the
hermit crab Pagurus bernhardus. Animal Behavior, 73, 605-612.
Fort, L & Tulloch I.K. (2016) Behaviors of a captive Coenobita clypeatus in the presence of
varying light stimuli. Modern Psychological Studies 21(2): 23-32
Frazer, A., & Hensler, J.G. (1999). Serotonin. In Siegel, G.J., Agranoff, B.W., Albers, R.W.
(Eds.), Basic Neurochemistry: Molecular, Cellular and Medical Aspects. (6th ed.).
Philadelphia, PA: Lippincott-Raven.
This research is made possible through funding from Stevenson University School of
Humanities and Social Sciences and Department of Psychology. Research infrastructure
access provided by Stevenson University School of Sciences.
Feeding Behaviors and 5-HT Cells
Aggressive Behaviors and 5-HT Cells
Exploratory Behaviors and 5-HT Cells
2a 2b
3a 3b
*
*
4a 4b*
5-HT-Positive Cells in Eyestalk Tissue
5a 5b
Figure 6. Mean number of positive 5-HT cells per light
condition. Greater 5-HT cells were in the eyestalks of crabs
exposed to red light than crabs exposed to blue light t(4) =
12.102, p = .000.
5-HT-Positive Cells per Light Condition
*
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DurationofExploratory
Behaviors(MInutes)
Number of Positive 5-HT Cells
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0 10 20 30 40 50 60 70
DurationofAggressiveBehaviors
(Minutes)
Number of Positive 5-HT Cells