Argiris 1

Color change in dolabrifera (sea ) in response to substrate change 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 ) 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 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.

RESUMEN

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 críptica. 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.

Cryptic coloration is an important mechanism in nature that utilizes color to disguise an organism. It is a prime example of natural selection that can be found throughout aquatic and terrestrial environments from octopuses to katydids. Most of these organisms match their background while some use disruptive coloration to hide the outline of their 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 in a group, their stripes make it very difficult to distinguish individuals. On the other hand, many organisms, like stick bugs, have coloration 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 generally shades of green and brown, to match their algae and rock filled substrate (Rudman, 1999). A substrate is a surface where organisms live, eat or traverse. They also have two oral and two rolled on top of their head that sense Color change 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 meters (Rudman, 2003). D. dolabrifera belong to the order- commonly known as the sea . Anaspidea do not possess a hard shell, and unlike their close relatives the nudibranchs, 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 organisms, this type of matching is static and broadly matches many substrates. While this could be D. dolabrifera’s only defense, I observed that individuals’ color seemed to faintly change over time. Do D. dolabrifera change their color to better match their substrate?

MATERIALS AND METHODS

Data 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 taking on a different hue because on camera or 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 carefully 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 after the slugs had 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 folder 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 include the entire slug. Each point provided an RGB value. The multiple data points from the slug were averaged to Figure 1: The white boxes are the apertures find the red, green and blue value that best and measure the red, green and blue within represented the overall color of the slug. The the squares. More values were taken, than substrate value was found by getting RGB values these three, but this exhibits a sample from for the substrate directly around the D. the slug, reference and substrate Color change 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 reference 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 conical shape as illustrated in Figure 2 (Jewett, 1997). This conversion was used to account for discrepancies in brightness and because hue is a better 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 360o with red at 0o/360o, blue at 240o, and green at 120o illustrated in Figure 3 (Vandevenne, 2004). All samples that had values over 180o were not included Figure 2: Hue is the value around the because the graphs did not correctly illustrate the close edge of the cone, strictly representing relationship between the highest and lowest values. the color. The brightness takes For example, 0o and 359o look very far apart when darkness into account while the graphed even though they are actually only 1o away saturation is in charge of lighter from each other. This required me to discount nine D. shades of a hue. 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 substrate and the slug coloration (Table 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 3: Hue represented in 360 with no saturation (light) or brightness (dark) included Color change in Dolabrifera dolabrifera Argiris 4

Table 1: This table summarizes the graphical analysis of the slugs, substrates and references by their correlations. 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

original substrate, 2 is immediately 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 relationship was present with slug 23 (Figure 4). It is easy to see how close the slug and substrate

Slug 23

120

100

80

60

HUE (DEGREES) 40

20

0 1 2 3 SEQUENCE AND SUBSTRATE

Slug Substrate Reference

Figure 4: This graph exhibits a strong correlation between substrate and slug hue and no correlation to the reference Color change in Dolabrifera dolabrifera Argiris 5 follow each other in terms of directionality and hue. 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

Figure 5: The slug, substrate and reference all follow different patterns the three lines at the three different points. I took slugs 1, 3, 7 and 24 out. The substrate matched the reference 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 Color change in Dolabrifera dolabrifera Argiris 6 slug and the reference (Figure 7). Both of these relationships were only found in 16.67% of the

Figure 7: The substrate and the reference are following the same pattern graphs. The complete breakdown of which graphs expressed which correlations are shown in Table 1. Matches between categories were most likely to occur between D. dolabrifera and the 2 substrates (X test=6.00, df=2, p<0.01).

DISCUSSION

From the data presented, I found evidence that D. dolabrifera change their color to match their substrate. 66.67% of the graphs exhibited 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 differences. 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 ‘flexing’. ‘Rearing’ is when an Opisthobranch lifts anteriorly and ‘flexing’ is when it twists while simultaneously lengthening its body. This indicates that D. dolabrifera employ no physical 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 Color change 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, there are usually two glands present in 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, distracting their predator long enough for the sea hare to escape (Prince, 2007). Because the D. dolabrifera does not release ink, the predator can see it try to escape and is less effective. Because of their weak chemical and physical defenses, more evolutionary weight 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 were 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 affect 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 differences between 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 predators. The few acknowledged predators are only known because there was a first hand witness to see the being eaten. Even the study done by Johnson did not utilize recognized predators (2009). In regards to D. dolabrifera, an 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 about 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, Andrés, 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/. Color change 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 mucus inhibits 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 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 odors. Journal of Experimental Biology, 216(8), 1364-1372. Prince, J. S. (2007). Opaline gland ultrastructure in Aplysia californica (: 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 disruptive 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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APPENDIX

Slug 1 Slug 2

80 100 60 80 60 40 40 20 HUE (DEGREES) HUE (DEGREES) 20 0 0 1 2 3 1 2 3 SEQUENCE ABND SUBSTRATE SUBSTRATE AND SEQUENCE

Slug Substrate Reference Slug Substrate Reference

Slug 3 Slug 4

140 400 120 350 100 300 250 80 200 60 150 40

HUE (DEGREES) HUE (DEGREES) 100 20 50 0 0 1 2 3 1 2 3 SEQUENCE AND SUBSTRATE SEQUENCE AND SUBSTRATE

Slug Substrate Reference Slug Substrate Reference

Slug 7 Slug 8

160 100 140 120 80 100 60 80 60 40

HUE (DEGREES) 40 HUE (DEGREES) 20 20 0 0 1 2 3 1 2 3 SEQUENCE AND SUBSTRATE SEQUENCE AND SUBSTRATE

Slug Substrate Reference Slug Substrate Reference

Color change in Dolabrifera dolabrifera Argiris 10

Slug 9 Slug 10

100 100

80 80

60 60

40 40 HUE (DEGREES) 20 HUE (DEGREES) 20

0 0 1 2 3 1 2 3 SEQUENCE AND SUBSTRATE SEQUENCE AND SUBSTRATE

Slug Substrate Reference Slug Substrate Reference

Slug 11 Slug 12

80 120 70 100 60 50 80 40 60 30 40 20 HUE (DEGREES) HUE (DEGREES) 10 20 0 0 1 2 3 1 2 3 SEQUENCE AND SUBSTRATE SEQUENCE AND SUBSTRATE

Slug Substrate Reference Slug Substrate Reference

Slug 13 Slug 14

200 120 100 150 80 100 60 40

HUE (DEGREES) 50 HUE (DEGREES) 20 0 0 1 2 3 1 2 3 SEQUENCE AND SUBSTRATE SEQUENCE AND SUBSTRATE

Slug Substrate Reference Slug Substrate Reference

Color change in Dolabrifera dolabrifera Argiris 11

Slug 15 Slug 16

140 160 120 140 100 120 80 100 80 60 60 40

HUE (DEGREES) 40 HUE (DEGREES) 20 20 0 0 1 2 3 1172 1173 1178 SEQUENCE AND SUBSTRATE SEQUENCE AND SUBSTRATE

Slug Substrate Reference Slug Substrate Reference

Slug 17 Slug 19

400 400 350 350 300 300 250 250 200 200 150 150

100 HUE (DEGREES) 100 HUE (DEGREES) 50 50 0 0 1186 1187 1203 1 2 3 SEQUENCE AND SUBSTRATE SEQUENCE AND SUBSTRATE

Slug Substrate Reference Slug Substrate Reference

Slug 20 Slug 21

350 350 300 300 250 250 200 200 150 150 100 100 HUE (DEGREES) HUE (DEGREES) 50 50 0 0 1 2 3 1 2 3 SUBSTRATE AND SEQUENCE SEQUENCE AND SUBSTRATE

Slug Substrate Reference Slug Substrate Reference

Color change in Dolabrifera dolabrifera Argiris 12

Slug 22 Slug 23

120 120 100 100 80 80 60 60 40 40 HUE (DEGREES) 20 HUE (DEGREES) 20 0 0 1 2 3 1 2 3 SEQUENCE AND SUBSTRATE SEQUENCE AND SUBSTRATE

Slug Substrate Reference Slug Substrate Reference

Slug 24 Slug 25

200 200

150 150

100 100

50 50 HUE (DEGREES) HUE (DEGREES)

0 0 1 2 3 1 2 3 SEQUENCE AND SUBSTRATE SEQUENCE AND SUBSTRATE

Slug Substrate Reference Slug Substrate Reference

Slug 26 Slug 27

250 400 350 200 300 150 250 200 100 150

HUE (DEGREES) HUE (DEGREES) 100 50 50 0 0 1 2 3 1 2 3 SEQUENCE AND SUBSTRATE SEQUENCE AND SUBSTRATE

Slug Substrate Reference Slug Substrate Reference

Appendix 1: Graphs made for slugs 1-27. Slugs 5, 6, and 18 are not present because their data collection was incomplete.