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Color change in Dolabrifera dolabrifera (sea hare) in response to substrate change

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Title:
Color change in Dolabrifera dolabrifera (sea hare) in response to substrate change
Translated Title:
Cambio de color en Dolabrifera dolabrifera (liebre de mar) en respuesta al cambio del sustrato
Creator:
Argiris, Jennay
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Text in English

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Subjects / Keywords:
Marine animals ( lcsh )
Animales marinos ( lcsh )
Sea slugs ( lcsh )
Babosas de mar ( lcsh )
EAP Fall 2017
EAP Otoño 2017
Costa Rica--Guanacaste--Cuajiniquil
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Reports

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Abstract:
Dolabrifera dolabrifera is an Opisthobranch (sea slug) known for its cryptic coloration. This coloration is an important defense mechanism, but D. dolabrifera have never been studied to see if they change colors to increase their cryptic nature. After photographing 12 D. dolabrifera on different substrates, the color of the slugs and their substrate were determined. These colors were then depicted as hue values. Each D. dolabrifera was photographed three times, in different tide pools and over time. Every D. dolabrifera was graphed with the hue value found for the slug, substrate and reference for the three photographs taken. After analyzing the graphs, I found a correlation between the slug and substrate hue in eight out of the twelve trials. D. dolabrifera changes its color based on its substrate. ( ,, )
Abstract:
Dolabrifera dolabrifera es una Opisthobranch (babosa del mar) conocido por su coloración críptica. Esta coloración es un mecanismo de defensa importante, pero nunca se ha estudiado para ver si los D. dolabrifera cambian de color para aumentar su naturaleza crítptica. Después de fotografiar 12 D. dolabrifera en diferentes charcas de mareas a través del tiempo, se determine el color de las babosas y su sustrato. Estos colores fueron luego representados como valores de tono. Cada D. dolabrifera fue fotografiada tres veces, en diferentes charcos de mareas y con el tiempo. Cada D. dolabrifera fue graficado con el valor de tono encontrado para la babosa, sustrato y referencia para las tres fotografías tomadas. Después de analizar los gráficos, encontré una correlación entre los matizes de la babosa y del sustrato en ocho de los doce ensayos. D. dolabrifera cambia su color en función de su sustrato.
Biographical:
Student affiliation: Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara.

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Monteverde Institute
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Monteverde Institute
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This item is licensed with the Creative Commons Attribution Non-Commercial No Derivative License. This license allows others to download this work and share them with others as long as they mention the author and link back to the author, but they can’t change them in any way or use them commercially.
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M39-00634 ( USFLDC DOI )
m39.634 ( USFLDC Handle )

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Tropical Ecology Collection [Monteverde Institute]

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Argiris 1 Color c hange in Dolabrifera dolabrifera (sea hare) in response to substrate c hange Jennay Argiris Department of Molecular, Cellular and Developmental Biology University of California, Santa Barbara EAP Tropical Biology and Conservation Program, Fall 2017 15 December 2017 ABSTRACT Dolabrifera dolabrifera is an Opisthobranch (sea slug) known for its cryptic coloration. This coloration is an important defense mechanism, but D. dolabrifera have never been studied to see if they change colors to increase the ir cryptic nature. After photographing 12 D. dolabrifera on different substrates the color of the slugs and their substrate w ere determined These colors were then depicted as hue values. Each D. dolabrifera was photographed three times in different tide pools and over time. Every D. dolabrifera was graph ed with the hue value found for the slug, substrate and reference for the three photographs taken After analyzing the graphs, I found a correlation between the slug and substrat e hue in eight out of the twelve trials. D. dolabrifera changes its color based on its substrate. R ESUMEN Dolabrifera dolabrifera es una Opisthobranch (babosa de l mar) conocido por su coloraci—n cr’ptica. Esta coloraci—n es un mecanismo de defensa importante, pero nunca se ha e studiado para ver si los D. dolabrifera cambian de color para aumentar su naturaleza cr’ptica. DespuŽs de fotografiar 12 D. dolabrifera en diferentes charcas de mareas a travŽs del tiempo, se determine el color de las babosas y su sustrato. Estos colores f ueron luego representados como valores de tono. Cada D. dolabrifera fue fotografiada tres veces, en diferentes charcos de mareas y con el tiempo. Cada D. dolabrifera fue graficado con el valor de tono encontrado para la babosa, sustrato y referencia para l as tres fotograf’as tomadas. DespuŽs de analizar los gr‡ficos, encontrŽ una correlaci—n entre los matizes de la babosa y del sustrato en ocho de los doce ensayos. D. dolabrifera cambia su color en funci—n de su sustrato. Cryptic coloration is an importa nt mechanism in nature that utilizes color to disguise an organism. It is a prime example of natural selection that can b e found throughout aquatic and terr estrial environments from octopuses to katydids. Most of these organisms match their background while some use disruptive coloration to hide the outline of the ir body (Stevens et al. 2009) A zebra exhibits the latter form of cryptic coloration. It obviously does not blend in to the African Savannah, but when zebras are i n a group, their stripes mak e it very difficult to distinguish individuals. On the other hand many organisms like stick bugs, have colorat ion that allows them to match their surroundings. Dolabrifera dolabrifera is an Opisthobranch found in the Pacific Ocean off the coast of Costa Rica that possesses cryptic coloration by background matching They grow to an average of 10 centimeters and are gene rally shades of green and brown, to match their algae and rock filled substrate ( Rudman, 1999). A substrate is a surface where organisms l ive, eat or traverse. They also have two oral tentacles and two rolled rhinophores on top of their head that sense

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Color c hange in Dolabrifera dolabrifera Argiris 2 chemical cues (Rudman, 1999). D. dolabrifera have fused parapodia everywhere but two flaps for respiration and can be found as deep as 16 me ters (Rudman, 2003). D. dolabrifera belong to the Anaspidea order commonly known as the sea hares. Anaspidea do not possess a hard shell, and unlike their close relative s the nudibranch s do not have chemical defenses associated with nematocyst uptake to discourage predators (Greenwood, 2004) With no known physical defenses, sea hares usually release an ink and opaline mixture. This deters predators by being unpalatable or a phagomimic a compound that mimics food so the sea hare can sneak away (Love Chezem et al. 2013). However, D. dolabrifera posses the opaline gland, but not the ink gland to ward off these predators (Prince, 2007) Because of this, D. dolabrifera's main defense against predation appears to be its background matching In many organis ms, this type of matching is static and broadly matches many substrates While this could be D. dolabrifera's only defense I observed that indiv iduals' color seemed to faintly change over time D o D. dolabrifera change their color to better match their substrate ? MATERIALS AND METHODS D ata was collected at the tide pools of La Islita in Cuajiniquil, Costa Rica between 8:30 AM and 10:30 AM during mid to low tide. Once I found a slug, I took picture with a piece of cardboard present in the photograph as a reference. If the reference moved in the same pattern as the slug and substrate, the correlation between the slugs and substrate would likely be due to the entire image taki ng on a different hue because on camera o r lighting issues. The camera used was a Fujifilm FinePix XP90. Every photo displayed the D. dolabrifera and the reference either fully shaded or fully lit with the reference and the surrounding substrate similarly lit. This D. dolabrifera was then careful ly moved into another tide pool to see if it would change color The new tide pool was chosen at random so as not to bias the results. Within a minute of being placed in the new pool, the slug was photographed again. I took a final photo a fter the slugs ha d grown accustomed to their new substrate for 15 minutes I repeated this with 27 D. dolabrifera Slugs 5, 6, and 18 hid before the final picture could be taken, reducing the sample size to 24. I analyzed photographs using the Digital Color Meter, located in the Utilities fo lder in Apple computers (Klein, 2015 ) This application calculates the amount of red, blue and green (RGB) present in the photo. I had the applications meter set to the largest aperture size and the display was set to sRGB so it would calculate red, green and blue. Data was taken at three points along the posterior, median plane and two more on the posterior, lateral sides of the D. dolabrifera. Multiple data points had to be taken because the aperture of the digital color meter was not large enough to incl ude the entire slug. Each point provided a n RGB value The multiple data points from the slug were averaged to find the red, green and blue value that best represented the overall color of the slug The substrate value was found by getting RGB values for t he substrate directly around the D. Figure 1 : The white boxes are the apertures and measure the red, gre en and blue within the squares. More values were taken, than these three, but this exhibits a sample from the slug, reference and substrate

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Color c hange in Dolabrifera dolabrifera Argiris 3 dolabrifera Values were taken around the entire slug, roughly 0.25 to 0.5 centimeters and the red, green and blue values were averaged Two more values were taken for the referen ce and these were also averaged. An example of this analysis for the slug, substrate and reference is illustrated in Figure 1 The end values for red, green and blue were then converted into HSB hue, saturation and brightness values using a color converter at www.colorizer.org Hue, saturation and brightness are best represented in a three dimensional c onical shape as illustrated in Figure 2 (Jewett, 1997). This conversion was used to account for discrepancies in brightness and because hue is a bet ter indicator of "color" (Karcher, 2003). Hue depicts color using one number while RGB has three different numbers that do not properly represent the color unless the three values are analyzed together. Hue is represented in 360 o with red at 0 o /360 o blu e at 240 o and green at 120 o illustrated in Figure 3 (Vandevenne, 2004) All samples that had values over 180 o were not included because the graphs did not correctly illustrate the close relationship between the highest and lowest values. For example, 0 o and 359 o look very far apart when graphed even though they are actually only 1 o away from each other. This required me to discount nine D. dolabrifera (slugs 4, 13, 17, 18, 19, 20, 21, 26 and 27). RESULTS In 66.67% of the graphs, there was a correlation between the subs trate and the slug coloration (T able 1). On the other hand, the correlations between the reference and slug and the reference and substrate were both 16 .67%. A graph was made for each of the 24 D. dolabrifera that were found after 15 minutes (refer to appendix). The 1 on the x axis refers to the slug in its Figure 2 : Hue is the value around the edge of the cone, strictly representing the color. The brightness takes darkness into account while the s aturation is in charge of lighter shades of a hue. F igure 3 : Hue represented in 360 with no saturation (light) or brightness (dark) included

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Color c hange in Dolabrifera dolabrifera Argiris 4 original substrate, 2 is immed iately after the slug was relocated, and 3 is the slug 15 minutes after it was relocated. I examined the graphs to determine if there were any relationships. These graphs were then divided into a correlation between the slug and the substrate, the slug and the reference, and the substrate and the reference. An example of a strong slug and substrate r elationship was present with slug 23 (Figure 4) It is easy to see how close the slug and substrate T able 1 : This table summarizes the graphical analysis of the slugs, substrates and references by their correlation s. The specific graphs that follow each relationship are in Dolabrifera graphs. These graphs are then changed into percentages and number of graphs out of 12 graphs Figure 4 : This graph exhibits a strong correlation between substrate and slug hue and no correlation to the reference "! #! $! %! &!! &"! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+"' 089: 09;<=>?=@ /@A@>@BC@

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Color c hange in Dolabrifera dolabrifera Argiris 5 follow each other in terms of directionality and hu e Some graphs proved to be inconclusive with neither the slug, substrate or reference showing a relationship that I could easily discern (Figure 5 ) This was due to the lines all showing different directionalities and no pattern among the three lines at the three different point s. I took slugs 1, 3, 7 and 24 out The substrate matched the referen ce for slugs 14 and 15 (Figure 6 ). Slugs 12 and 25 showed a correlation between the Figure 6 : The slug and reference follow the same pattern F igure 5 : The slug, substrate and reference all follow different patterns

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Color c hange in Dolabrifera dolabrifera Argiris 6 slug and the reference (Figure 7 ). Both of these relationships were only found in 16.67% of the graphs. The complete breakdown of which gr aphs expressed which correlations are shown in Table 1. Matches between categories were most likely to occur between D dolabrifera and the substrates (X 2 test =6 .00, df=2, p<0.01). D ISCUSSION From the data presented, I found evidence that D. dolabrifera change their color to match their substrate 66.67% of the graphs exhibit ed a correlation between the slugs and their substrate. After using a chi squared test, this value did prove to be statistically significant. The reference is not following the same pattern, so the correlation is not due to general lighting and shading diffe rences. The 16.67% of cases that indicated a relationship between the slug and the reference show when the slug did not change color in response to its environment. When the substrate paralleled the reference, the substrates happened to be similar in color to each other and the D. dolabrifera did not change accordingly. Color change has never previously been studied in D. dolabrifera, although their cryptic coloration is well known. It is possible that being able to change color makes them better defended against predators. Previous studies on defense mechanisms of D. dolabrifera by Ghazali have seen no release of mucus or ink and no physical acts such as rearing' or fl exing'. Rearing' is when an Opisthobranch lifts anteriorly and flexing' is when it t wists while simultaneously lengthening its body. This indicates that D. dolabrifera employ no physica l defense when interacting with predators. Although, the paper did note their ability to release a "milky substance" when confronted with a possible predator These possible predators included hermit crabs and blacktail snappers. They are referred to as possible predators because there is no F igure 7 : The substrate and the reference are following the same pattern

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Color c hange in Dolabrifera dolabrifera Argiris 7 proof that they actually prey on sea slugs, but they are a potential threat, as they are carnivorous and reside in the same habitat as the D. dolabrifera ( 2006) Upon further investigation the "milky substance was discovered to be released by an opaline gland. According to Johnson, t here are usually two g lands present in Opisthobranchia used for chemical defenses the opaline gland and the ink gland The ink produced, which contains the enzyme escapin, and the opaline, which contains L lysine and L arginine, are then co released to discourage predators (2009) The opaline and ink mimic feeding behavior, distractin g their predator long enough for the sea hare to escape (Prince, 2007) Because the D. dolabrifera does not release ink, the predator can see i t try to escape and is less effective Because of their weak chemical and physical defense s more evolutionary we ight is placed on a specific D. dolabrifera's coloration and ability to match its surroundings to evade predators. A D. dolabrifera that can change color to better match its substrate should be more difficult for predators to find. As a result, being able to change color to match one's substrate would increase a D. dolabrifera's fitness. Over the course of the study, glare from the sun in some of the photographs made analysis difficult. When possible, sections of the photograph that exhibited a glare wer e avoided. If the glare was over a large area of the slug, areas with glare were used but data points affected by glare were also taken along the substrate and on the reference. Slight reflections on the water as observed in the photographs could also aff ect values taken. This was tested by taking a few samples while using an umbrella to decrease the reflections. These sample values were compared to samples that had not been under an umbrella. The comparison did not reveal any discernable d ifferences betwe en the pictures or the associated hue values. Further research should be conducted on possible sea hare predators. Because no Opisthobranches possess a hard skeleton, it is impossible to know what preys on them based on organism's stomach contents. This has led to a lack of knowledge on sea hare predator s The few acknowledged predators are only known because there was a first hand witness to see the sea slug be ing eaten. Even the study done by Johnson did not utilize recognized predators (2009) In regar ds to D. dolabrifera, a n important first question to ask is if their predators even possess color vision. Based on the coloration of D. dolabrifera, it can be assumed that they did have predators that chose for cryptic coloration. If there is more known ab out sea hare predators, the evolution of D. dolabrifera color change can be better understood. ACKNOWLEDGMENTS I would like to thank Frank Joyce for a LOT of help throughout this project. I would also like to thank Xinia, AndrÂŽs, Mauricio and Johnny for opening their home to me. A big thanks to John for joining me to the tide pools and listening to my ever changing project ideas. My paper would not have made it past the first draft without the aid of Miguel Ochoa and Federico Chinchilla. And finally, a big thank you to everyone in the program for the support and great memories! LITERATURE CITED "Colorizer Color Picker and Converter (RGB HSL HSB/HSV CMYK HEX LAB)." Colorizer Color Picker and Converter, colorizer.org/.

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Color c hange in Dolabrifera dolabrifera Argiris 8 Ghazali S. R. (2006). Displays of defense: behavioral differences in antagonist avoidance in four opisthobranch mollusks. Water Resources Center Archives Greenwood, P. G., Garry, K., Hunter, A., & Jennings, M. (2004). Adaptable defense: a nudibranch mucus inh ibits nematocyst discharge and changes with prey type. The Biological Bulletin 206 (2), 113 120. Jewett, Tom. "HSB: Hue, Saturation and Brightness." Color Tutorial, Department of Computer Engineering and Computer Science, Emeritus, 1997, www.tomjewett.com/colors/hsb.html Johnson, P. M., Kicklighter, C. E., Schmidt, M., Kamio, M., Yang, H., Elkin, D., ... & Derby, C. D. (2006). Packaging of chemicals in the defensive secretory glands of the sea hare Aplysia californica. Journal of Experimental Biology 209 (1), 78 88. Love Chezem, T., Aggio, J. F., & Derby, C. D. (2013). Defense through sensory inactivation: sea hare ink reduces sensory and motor responses of spiny lobsters to food od ors. Journal of Experimental Biology 216 (8), 1364 1372. Prince, J. S. (2007). Opaline gland ultrastructure in Aplysia californica (Gastropoda: Anaspidea). Journal of Molluscan Studies 73 (2), 199 204. Prince, J. S., & Johnson, P. M. (2006). Ultrastructural comparison of Aplysia and Dolabrifera ink glands suggests cellular sites of anti predator protein production and algal pigment processing. Journal of Molluscan Studies 72 (4), 349 357. Stevens, M., & Merilaita, S. (2009). Defining disrupti ve coloration and distinguishing its functions. Philosophical Transactions of the Royal Society of London B: Biological Sciences 364 (1516), 481 488. Rudman, W.B. "Dolabrifera Dolabrifera (Rang, 1828)." Dolabrifera Dolabrifera The Sea Slug Forum, 26 Apr. 2003, www.seaslugforum.net/find/doladola Vandevenne, L. (2004). Lode's computer graphics tutorial. Texture Generation using Random Noise

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Color c hange in Dolabrifera dolabrifera Argiris 9 APPENDIX "! #! $! %! & ()*+,-*./**01 0*2)*34*+563-+0)607/57* 089:+& 089: 09;<=>?=@ /@A@>@BC@ "! #! $! %! &!! & ()*+,-*./**01 0)607/57*+53-+0*2)*34* 089:+" 089: 09;<=>?=@ /@A@>@BC@ "! #! $! %! &!! &"! &#! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+' 089: 09;<=>?=@ /@A@>@BC@ D! &!! &D! "!! "D! '!! 'D! #!! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+# 089: 09;<=>?=@ /@A@>@BC@ "! #! $! %! &!! &"! &#! &$! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+E 089: 09;<=>?=@ /@A@>@BC@ "! #! $! %! &!! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+% 089: 09;<=>?=@ /@A@>@BC@

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Color c hange in Dolabrifera dolabrifera Argiris 10 "! #! $! %! &!! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+F 089: 09;<=>?=@ /@A@>@BC@ "! #! $! %! &!! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+&! 089: 09;<=>?=@ /@A@>@BC@ &! "! '! #! D! $! E! %! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+&& 089: 09;<=>?=@ /@A@>@BC@ "! #! $! %! &!! &"! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+&" 089: 09;<=>?=@ /@A@>@BC@ D! &!! &D! "!! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+&' 089: 09;<=>?=@ /@A@>@BC@ "! #! $! %! &!! &"! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+&# 089: 09;<=>?=@ /@A@>@BC@

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Color c hange in Dolabrifera dolabrifera Argiris 11 "! #! $! %! &!! &"! &#! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+&D 089: 09;<=>?=@ /@A@>@BC@ "! #! $! %! &!! &"! &#! &$! &&E" &&E' &&E% ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+&$ 089: 09;<=>?=@ /@A@>@BC@ D! &!! &D! "!! "D! '!! 'D! #!! &&%$ &&%E &"!' ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+&E 089: 09;<=>?=@ /@A@>@BC@ D! &!! &D! "!! "D! '!! 'D! #!! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+&F 089: 09;<=>?=@ /@A@>@BC@ D! &!! &D! "!! "D! '!! 'D! & ()*+,-*./**01 0)607/57*+53-+0*2)*34* 089:+"! 089: 09;<=>?=@ /@A@>@BC@ D! &!! &D! "!! "D! '!! 'D! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+"& 089: 09;<=>?=@ /@A@>@BC@

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Color c hange in Dolabrifera dolabrifera Argiris 12 Appendix 1 : Graphs made for slugs 1 27. Slugs 5, 6, and 18 are not present because their data collection was incomplete. "! #! $! %! &!! &"! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+"" 089: 09;<=>?=@ /@A@>@BC@ "! #! $! %! &!! &"! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+"' 089: 09;<=>?=@ /@A@>@BC@ D! &!! &D! "!! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+"# 089: 09;<=>?=@ /@A@>@BC@ D! &!! &D! "!! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+"D 089: 09;<=>?=@ /@A@>@BC@ D! &!! &D! "!! "D! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+"$ 089: 09;<=>?=@ /@A@>@BC@ D! &!! &D! "!! "D! '!! 'D! #!! & ()*+,-*./**01 0*2)*34*+53-+0)607/57* 089:+"E 089: 09;<=>?=@ /@A@>@BC@