The Effects of Uv Radiation on Adult and Larval Behavior and Chromatophore Size in Octopus Rubescens

THE EFFECTS OF UV RADIATION ON ADULT AND LARVAL BEHAVIOR AND CHROMATOPHORE SIZE IN OCTOPUS RUBESCENS

Wyatt Brown Abstract Ultraviolet (UV) radiation—especially UV-B radiation—can have detrimental effects on marine larvae and elicit behavioral changes in many adult marine organisms. Although there has been a considerable amount of research on chromatophores in marine organisms, little is known about the relationship between cephalopod chromatophores and exposure to UV-B radiation. This study investigates the effects of 280 nm, 330 nm and 488 nm wavelengths on paralarva Octopus rubescens, as well as the behavior and chromatophore changes of adult Octopus rubescens under non-specific UV-B conditions. Paralarvae chromatophore size significantly increased after exposure to each wavelength but there was no significant difference in average percent change between the respective wavelengths. Adult octopuses exhibited no significant behavioral changes or chromatophore color changes in response to UV-B conditions; however, they tended to spend more time in darker conditions when given the chance.

Keywords: Octopus rubescens, cephalopod, chromatophore, ultraviolet radiation, UV-B, UV-A, PAR, Paralarvae, Artemia

1. Introduction up to 30 m, thus affecting organisms living in shallow-water to intertidal communities Photosynthetically active radiation (Kirk, 1994). Over time, however, organisms (PAR) describes the 400-700 nm range of have adapted to limit the damage caused by light emitted by the sun that photosynthetic UV-B radiation. For instance, the green sea organisms can use for photosynthesis. Besides urchin (Strongylocentrotus droebachiensis) PAR, the sun also emits three types of ultra- utilizes mycosporine-like amino acids in early violet (UV) radiation: UV-A (400-320 nm), development to reduce UV-induced damage UV-B (320-280 nm), and UV-C (280-100 (Adams and Shick, 2001). nm). The ozone layer blocks all UV-C radi- While a great deal of research has ation and most UV-B radiation, but allows looked at the effects of UV-B radiation on UV-A radiation to reach the earth’s surface. marine organisms, no studies have explored Because UV-A radiation has been considered its effects on cephalopods. One explanation relatively harmless, most studies have focused is cephalopods generally live at depths where on the biological destructiveness of UV-B UV-B radiation cannot reach. However, radiation (Speekmann et al., 2000). UV-B the tide pool octopus, Octopus rubescens, radiation can penetrate the water column often resides in the intertidal zone where

UV-B radiation is a natural part of the rubescens targeting another prey species. environment. Furthermore, the pelagic O. Furthermore, O. rubescens could become rubescens juveniles—or paralarvae—form more conspicuous to predators if it uses its large feeding schools in the open ocean chromatophores to block out UV-B radiation where they are subjected to UV-B radiation instead of camouflaging to its environment. (Onthank and Cowles, 2011). Faced with a Consequently, an increase in UV-B similar situation, larval crustaceans evolved penetration as a result of the depleting ozone to utilize chromatophores to protect their layer may negatively impact O. rubescens bodies against UV-B radiation (Morgan, survival (Madronich, et al., 1998). Christy, 1996). Despite extensive research Based on this logic, I hypothesized on UV radiation and larval chromatophores, that when presented with the choice between there are no published studies on cephalopod PAR and UV-B radiation, adult octopuses chromatophores and UV-B exposures. would seek refuge in UV-B free environments. Studies suggest the use of Also, I predicted that O. rubescens would chromatophores and iridophores to exhibit a more dramatic color change in create polarized light could be a “hidden” UV-B radiation environments than darker communication pathway between octopuses environments. Lastly, I hypothesized that (Shashar, et al., 1996), since cephalopod the octopus paralarvae would discriminate photoreceptors are capable of detecting between different wavelengths by expanding linear polarization of incoming light and their chromatophores more in harmful cephalopod iridophores can then transform UV-B wavelengths than UV-A and PAR this light into polarized reflective patterns wavelengths. (Mathger, et al., 2009). The iridophores are so fine-tuned that they can reflect specific 2. Materials and Methods wavelengths depending on the space and 2.1 Paralarvae collection and maintenance thickness between them (Land, 1972). This Octopus rubescens embryos were is accomplished by the chromatophores collected from Tomales Bay in Marshall, expanding and retracting in different California. During low tide, rocks were groupings to effectively control the amount of carefully lifted up until a substantial nest light that reaches the iridophores (Mathger was found and approximately 1/16 of the and Hanlon, 2007). Since chromatophore egg strands were clipped and placed in a 17 expansion or contraction controls iridophore by 28 by 17 cm holding tank with seawater. reflection of specific wavelengths, it is realistic The paralarvae hatched on the way back to to consider that they could also function to the Bodega Marine Laboratory and were block UV-B radiation. transferred into large mason jars. Circular UV-B radiation can be an important holes were cut out of the plastic lids and then factor in not only determining the behavioral covered with a fine filter to allow minimal characteristics of Octopus rubescens—such water flow into the jars. Water was changed as which environment it prefers—but also daily and any dead paralarvae were removed in helping better explain the function of its from the jars. chromatophores. If O. rubescens is affected by UV-B radiation, this could cause the 2.2 Artemia culturing and feeding species to shift further down the sea floor Artemia were hatched using standard to avoid penetrating UV-B radiation. This methods to feed the paralarvae (Seixas, et habitat shift due to UV-B radiation may al., 2008). 12 liters of 0.45 micron filtered have ecological implications such as O. seawater were placed into the Artemia tank

UC Davis | EXPLORATIONS: THE UNDERGRADUATE RESEARCH JOURNAL Vol. 17 (2015) W. Brown p.2 3 and two teaspoons of cysts were placed into wavelengths were controlled using Felix the seawater. Air was bubbled up from the software. Paralarvae images were captured bottom of the tank and the Artemia were before and after exposure to 280, 330 and allowed to hatch for 24h. Artemia were 488 nm wavelengths by taking photos collected by removing the air tube from the using a Photometrics CoolSnap camera water and directing a light towards the middle and NIS-Elements software. 11 paralarvae of the tank to concentrate the Artemia. One were used for each wavelength for a total cup of water was drained from the bottom of 33 paralarvae. Images were analyzed of the tank to remove the cysts and then the using Image J by measuring the same water was filtered through a 105 µm mesh net chromatophore before and after exposure to a to collect the Artemia until 5 cm of top water specific wavelength. remained in the tank. Concentrated Artemia was then transferred to a 250 ml beaker of 2.5 Adult octopuses filtered seawater and fed to the paralarvae. One adult Octopus rubescens was Artemia consumption by the captured on the mudflats of Bodega Harbor paralarvae was monitored using fluorescence during low tide and the other was borrowed microscopy. This method involved using from the Bodega Marine Laboratory Aquatic Calcein-AM, which enters the cells of Resource Group (ARG). Between experiments organisms where it is cleaved by esterase animals were housed in tanks with constant and becomes trapped inside of cells in its water flow and fed a single purple shoreline fluorescent form, Calcein. Artemia were crab (Hemigrapsus nudus) daily that was bathed in Calcein-AM for four hours before collected from the rocks along the mudflats of being fed to the octopus larvae. Paralarvae Bodega Harbor. were then examined under a fluorescent ARG helped create the 29 x 36 cm microscope using excitation/emission plastic panels used to conduct the experiment. wavelength of 488/519 nm. One panel allowed UV radiation to pass through (UV-transparent or UV-T) and the 2.3 Agar Plates other blocked UV radiation (UV-free or UV- Agar plates were made in order to F). The third panel was a black, opaque plastic keep the paralarvae confined when capturing divider, which did not allow light to pass images under the stereo or compound through the tank and is subsequently referred microscopes. 0.5 g of agar was dissolved at to as “Dark.” The panels were used to create 36 0C in 50 ml of seawater, and then poured three different treatments: UV-T/UV-F, onto microscope slides. After cooling in UV-T/Dark, and UV-F/Dark. Each treatment the refrigerator, 3 mm diameter holes were was conducted separately. punched into the agar to create wells to house Experiments were carried out in a 33 the paralarvae during image capturing. x 152 x 33 cm flow through water tank built by ARG. Drop-down dividers were placed in 2.4 Measuring the chromatophores of the the tank 40 cm apart to control the size of the paralarvae environment, allowing the octopus to better Paralarvae were pipetted into an detect differences created by the panels. To agar well and imaged using a Nikon AZ100 prevent UV rays from crossing the middle and Microzoom (Melville, NY) stereomicroscope. affecting the UV-blocked side of the tank, an The fiber optic cable was removed from a opaque divider was placed between the two PTI Fluorometer (Edison, NJ) and placed 4 plastic panels to a depth of 10 cm from the cm away from the agar slides. The different top of the tank, allowing the octopus to move

UC Davis | EXPLORATIONS: THE UNDERGRADUATE RESEARCH JOURNAL Vol. 17 (2015) W. Brown p.3 4 from one side to the other. Two 122 cm, 1900 average chromatophore size for paralarvae lumen UV-B bulbs were placed 37 cm above significantly increased from 668.5 μm² to the tank and were only turned on when the 1656.2 μm² (p=0.002), 801.6 μm² to 2473.8 experiment began. The treatment (UV-T/UV- μm² (p=0.00294), and 1080.1 μm² to 2047.5 F, UV-T/Dark, UV-F/Dark) as well as which μm² (p=0.00109) respectively. panel the octopus started under was chosen at Figure 3 depicts the average random and then the proper lids were placed percent change in chromatophore size for on the tank accordingly. paralarvae exposed to 280, 330 and 488 nm To begin each experiment, a single wavelengths. A one-way ANOVA test in JMP adult octopus was placed in a lidless 17 by 28 software revealed that for 280, 330 and 488 by 17 cm holding container and transferred nm wavelengths there was no significant into the experimental tank. The octopus was difference between the average percent allowed to leave the holding container on change in chromatophore area of 303.5%, its own to minimize stress. The panel the 382.8% and 287.3% respectively (p=0.435). octopus was under as well as the color of the However, the different wavelengths octopus was recorded at one-minute intervals appeared to result in differential location of for twenty minutes. Color was determined chromatophore expansion and behavioral on an intensity scale: 1=light peach, 2=solid response, with the 488 nm wavelength tan/ light brown, 3=spotted red, brown and causing peripheral chromatophores to white, 4=light red, and 5=dark red. Lights expand and the 280 nm wavelength resulting were turned off for twenty minutes between in contortion of the paralarvae’s body and trials to reset the initial conditions. During tentacles. the last day of the experiment, the UV-B bulbs were changed out with fluorescent bulbs to 3.2 Adult octopuses simulate PAR and the same experiments as Figure 4 shows the percent time O. described above were conducted to see if non rubescens spent under each panel. Analysis UV-B radiation also had significant effects on for O. rubescens environment preference was octopus behavior and chromatophore color. based on the assumption that O. rubescens would choose either side of the tank equally 3. Results (ratio 1:1). I used chi-squared tests to compare 3.1 Paralarvae whether octopuses spent more than half of Paralarvae that were fed Artemia the time under a given filter. The UV-T/Dark bathed in Calcein-AM (CAM) were visible and UV-F/Dark treatments rejected this using a bright-field/fluorescent microscope null hypothesis, indicating that O. rubescens using the 488 nm excitation/519 nm emission preferred the dark side of the tank compared wavelengths. Figure 1a/b shows before to the UV-Transparent (X^2=18.41, p< feeding (left) and after feeding (right) photos, 0.0005) and the UV-blocked (X^2=10.24, confirming that the Artemia took up CAM and p=0.0025) side of the tank in each that only paralarvae that consumed Artemia experiment. For the UV-T/UV-F treatment, were visible under 488 nm excitation. however, the chi-squared test supported the Figure 2 shows the results from the null hypothesis of a 1:1 ratio (X^2 = 0.46, p > Image J analysis of paralarvae chromatophore 0.25). areas before and after exposure to 280, Figure 5 shows that when given the 330 and 488 nm wavelengths. A one- option, O. rubescens always moved to the way ANOVA test in JMP software and darker side of the tank. O. rubescens never follow-up Tukey’s t-tests showed that the moved from the dark side to the UV-T or

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PAR sides of the tank and only moved from observation coincides with studies that have the dark side to the UV-F side of the tank shown cephalopod vision to be most sensitive one time. Furthermore, O. rubescens showed to the blue (450-495 nm) portion of the no difference in the number of switches per spectrum (Mäthger, et al., 2006). Future opportunity when presented with the choice studies should address if this response is due to go from either the UV-T or UV-F side of the to blue wavelengths traveling furthest into tank to the other. the water column and consequently being Figure 6 shows the average color utilized for either camouflage or the “hidden” for adult octopuses under different light communication pathway via polarized light conditions. Octopuses exhibited five different mentioned earlier (Mathger, et al., 2009). displays and were thus graded on an intensity Due to time constraint and scale from 1-5 (Fig. 7a-e). A one-way ANOVA paralarvae mortality, I was not able to utilize test revealed a significant difference in different options to capture images of their octopus color under the tested panels (p< chromatophores. Figure 8a shows the best 0.0001) and further TUKEY analysis showed image I was able to capture throughout my that the octopuses were a different color entire experiment and highlights the difficulty under dark panels than octopuses in UV- in measuring selected chromatophores due to blocked and UV-transparent environments. paralarvae movement and poor image quality. A color camera in another laboratory was 4. Discussion much better at capturing all of a paralarva’s 4.1 Paralarvae chromatophore changes—especially those The significant increase in paralarvae located in the head and abdomen regions— chromatophore area after exposure to 280, and those images would have been easier to 330 and 480 nm wavelengths suggests that analyze and could have yielded more accurate O. rubescens can detect ultraviolet as well results (Figure 8b). However, the fluorometer as visible radiation. While this evidence fiber optic system was not transportable supports my first hypothesis that they can and thus, these experiments could not be sense UV-B radiation, the data from analyzing conducted. the percent change in chromatophore area Since research has yet to provide a does not support my second hypothesis that successful standardized method for rearing O. rubescens would discriminate and respond octopus paralarvae, it was imperative that differently to the tested wavelengths. I ensured my paralarvae were kept healthy My observations, however, provided throughout the experiment (Iglesias, et al., responses that were difficult to quantify. 2007). Consequently, a significant quantity The paralarvae appeared to be most of my time was spent caring for the newly affected by 280 nm wavelength (UV-B) hatched octopus by determining the best as they contorted their body in abnormal manner to house them, as well as their food shapes and exhibited more movement than preference. The CAM labeling technique the paralarvae exposed to the other two confirmed results from other studies showing wavelengths—possibly due to a compensatory moderate success in feeding Artemia to escape response. Furthermore, I noticed that paralarvae (Villanueva, et al., 2002). the location of chromatophore expansion seemed wavelength dependent. Only 4.2 Adult Octopuses certain wavelengths consistently elicited Since they are aware of their surroundings the peripheral chromatophores to expand— when placed in a new environment, specifically the 488 nm wavelength. This octopuses often alter their behavior leading

UC Davis | EXPLORATIONS: THE UNDERGRADUATE RESEARCH JOURNAL Vol. 17 (2015) W. Brown p.5 6 to inaccurate experimental results (Sinn, et under the dark panel compared to the UV-T al., 2001). For example, when I first handled and UV-B panels. There was not a sufficient the octopuses they continued to change their quantity of data to explain how octopus color chromatophore pattern from what I classified changes under PAR conditions so more trials as an unstressed temperament or a “1” (Fig. need to be performed in the future. 7a) to what I classified as a “3” (Fig. 7c) when Future studies should also conduct I approached the tank. Over time, however, longer time trials to better gauge how O. they displayed a “3” less as I worked around rubescens would spend its time in natural them. This skin pattern reappeared when the sunlight, which is characterized by more octopus were introduced to a new individual intense ultraviolet radiation. This would or presented with stressful stimuli (cleaning allow the evaluation of O. rubescens behavior the tank with a net). Consequently, “3” was in a more natural setting. In the end, a determined to be more characteristic of a great deal of time and patience is needed to stressed octopus. A “4” (Fig. 7d) and a “5” overcome the complexity of working with an (Fig. 7e) were typical patterns displayed when invertebrate that is exceptionally intelligent. the octopuses was transported past a bright environment such as close to a window. Acknowledgements Therefore, chromatophore expansion to a “4” I would like to thank my advisor Gary or “5” could be a mechanism to block UV-B Cherr, and my other professors, Ernie Chang, radiation, but more research is needed to Steven Morgan and Jim Clegg for teaching explore this possibility. Overall it took time the spring 2014 class and for their constant to determine how to acclimate the octopuses guidance throughout the quarter. I would also to the experimental tank so they would not like to thank Carol Vines for her assistance in flash this defensive chromatophore pattern lab, Molly Engelbrecht for providing library and remain in one corner. I was finally able to resources, the ARG staff for helping build the obtain data once I provided the octopus more equipment for my experiment, and my fellow time to acclimate. spring 2014 class for making my time at The environment preference Bodega truly memorable. Lastly, I would like experiment and subsequent analysis did to thank Sukkrit Nimitkul and Sarah Gravem not support my hypothesis that adult O. for being the most helpful and dedicated TAs rubescens would shift habitats to find refuge I have encountered at Davis. from UV-B radiation. It instead suggested that O. rubescens does not detect differences created by the UV-T or UV-F panels and the slight variance in time spent under each panel during the UV-T/UV-F treatment was due to chance. Moreover, the results from the chi squared analysis of the UV-T/Dark and UV-F/Dark treatments in combination with changes per opportunity strongly supports previous studies where O. rubescens preferred darker habitats (Mather, O’Dor, 1991). The analysis of mean color change under each panel suggests that adult O. rubescens only respond to differences in light intensity since octopus had significantly lower color intensity

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Figure 1a: Before ingestion of the Artemia bathed in Calcein-AM

Figure 2

Figure 1b: After ingestion of the Artemia bathed in Calcein-AM

Figure 1a/b: The success in feeding paralarvae the Artemia bathed Calcein-AM. Im- ages on the right are after exposure to 488 nm excitation.

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Figure 2: The area of the paralarvaes’ chromatophores before and after exposure to 280, 330, and 488 nm wavelengths. In each case, chromatophore size significantly increased.

Figure 3: The average percent change of paralarvae chromatophore size after exposure to 280, 330 and 488 nm wavelengths. There is no significant difference between the three wavelengths.

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Figure 4: The percent time O. rubescens spent under each panel in the different treatments.

Figure 5: The number of switches/opportunity for all the possible choices.

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Figure 6: The average color of adult O. rubescens under different panels. Octopuses were graded on a 1-5 intensity scale as follows: light peach (1), solid tan/light brown (2), spotted red, brown and white (3), light red (4), and dark red (5). Similar letters above the bars rep- resent results that are significantly different.

Figure 7a: 1= Light Peach

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Figure 7b: 2 = Solid Tan/Light Brown Figure 7c: 3 = Solid Tan/Light Brown

Figure 7d: 4 = Light Red Figure 7e: 5 = Dark Red

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Figure 8a: Paralarvae Figure 8b: Paralarvae

Figure 8a/b: The figure on the left is an image of O. rubescens paralarvae captured with a Nikon AZ100 Microzoom stereomicroscope while the right shows an image of O. rubescens paralarvae captured with a colored camera.

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