Argiris 1 Color change in Dolabrifera dolabrifera (sea hare) 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 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. 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 tentacles and two rolled rhinophores 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 Anaspidea order- commonly known as the sea hares. 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.