Analysis of Interspecific and Intraspecific Mediterranean Octopus Behavior Annette Brennan, Stephanie Douglas, Cara Fuller, Samantha Gartner, 2016

Home , Ark clam, Barbatia barbata, Callistoctopus, Callistoctopus macropus, Octopus (genus), Octopus salutii

ABSTRACT Species coexistence is theorized to affect the behaviors and adaptations of individuals within the same system. For this study, we conducted two sets of trials looking at the foraging behavior and prey preference of three species of octopus, Octopus sautii, Octopus vulgaris, and Callistoctopus macropus in the harbor of the research station, STARESO, in Calvi, Corsica of September and October 2016. In the foraging behavior trials we documented ten different behaviors, where six were found to have significant relationships (p<0.05) with the species or individual. For the prey preference trials, we documented the octopus’ first choice when given three different prey items. A comparison of individual octopus and their prey choice exhibited a significant relationship (p<0.05). From the results, it was concluded that individual personalities of an octopus have greater influence on behavior than species-specific traits. These individual behaviors may lessen competition and lead to coexistence between the three species studied.

INTRODUCTION literature documenting how such differences in Coexistence of many species in otherwise like species may be associated with ecosystems has been a perplexing question to coexistence. Coexistence of octopus has already ecologists for well over 100 years. This is been studied between Octopus vulgaris and because highly diverse systems exist despite the Callistoctopus macropus by D.V. Meisel et al. well-established idea of competitive exclusion, They found that these two species of octopus which has at its core the principle that similar may coexist despite sharing overlapping species will compete for limited resources and temporal and geographical ranges. They that one consequence of the competition may be determined that this possible coexistence could local extinction of one or more of the be due to the two species differing behaviorally competitors. Many hypotheses have been put (Meisel et al., 2006). forward to account for high diversity, given the It is clear that morphological differences counter weight of competitive exclusion, one of between octopus species have allowed them to the earliest and more important being niche succeed in their environment; specialized diversification (Grassle & Sanders, 1973). This features such as webbing, arm length, and color hypothesis focuses on maintenance of diversity distinguish different octopus species. However, through evolution to reduce niche overlap these interspecific characteristics may not have among species thereby reducing competition and as great of an influence as individual behavior. allowing coexistence. A goal of our study was to follow on from these Under the niche diversification ideas and the literature to assess if three species hypothesis, phenotypic traits evolve in a way of octopus, differing substantially in that minimizes resource overlap. Resources in morphology, have variable foraging behavior this context include anything that could be in and prey selection that would be consistent with limited supply leading to competition, for niche diversification. example food and refuge. Often this is Patterns of foraging behavior and prey manifested in morphological traits or species- preference may differ on a species or individual specific behaviors and there is considerable level. Within the STARESO harbor in Corsica,

1 France three species of Mediterranean octopus due to their commercial importance in the may coexist which could lead to competition. international seafood trade and their prevalence The octopods living in close proximity, may in shallow waters. This species is found world- have altered their behaviors or prey preference if wide in both tropical and semitropical coastal the competition between species or individuals water from 0 to 200 meters in depth (Wood & became too great. The primary goal of this study Day, 1998). They feed mostly on invertebrates, is to determine if the difference in foraging using their radula to drill into shelled organisms behavior and prey preference between species is they cannot pry open. O. vulgaris are especially greater than the difference between each opportunistic, often holding their living prey individual’s behavior and how it could relate to within their web to keep their arms free in case their coexistence. We predict that three species, another prey item becomes available to catch Octopus vulgaris, Octopus salutii, and (Wood & Day, 1998). This species can be Callistoctopus macropus exhibit different identified by its skin mottling and is often foraging behaviors and preferred prey which spotted in or near its den made of rocks (Fiorito allows them to coexist within the STARESO & Gherardi, 1999). harbor. Callistoctopus macropus are known as the Red Octopus or White Spotted Octopus. The MATERIALS AND METHODS Mediterranean variety of this species is Background exclusive to the Mediterranean Sea and Eastern We conducted our study using the three Atlantic Ocean (Taki, 1964). They can be species of octopods that occur in shallow waters recognized by their long, ovate red to brown of the Mediterranean (Morton & Britton, 2000). mantle with distinct white spots along the mantle Octopus salutii is commonly known as and down the arms. They have a shallow web the Long Armed Octopus or Spider Octopus depth and very muscular arms that are 5 to 8 (Gofas, 2004). O. salutii is often confused with times the mantle length (Taki, 1964). C. the Common Octopus, though upon close macropus are often a shallow species, but can be examination O. salutii has a deep web depth and found between 0 and 200 meters in depth longer arms compared to their mantle length (Rocha & Cheikh, 2015). They feed on small (Gofas, 2004, Quetglas, et al., 2005). This fish and invertebrates, and tend to be generalists species occurs throughout the Mediterranean Sea (Meisel et al., 2006). and the East Atlantic Ocean, including in the We caught one O. salutii, three O. Gulf of Biscay and Cadiz between 25 and 800 vulgaris, and two C. macropus (Table 1) during meters in depth (Quetglas et al., 2005). In the September and October 2016 at Station de Mediterranean, this species primarily inhabits Recherche Océanographiques et sous-marines the lower continental shelf, as well as the upper (STARESO) in Corsica, France (42°34’N, slope. The juvenile and adult diet often consists 08°43’E). Divers caught the individuals in the of crustaceans, fishes and cephalopods (Quetglas harbor of STARESO, which is about 100m et al., 2005). O. salutii are rarely caught and across, at depths ranging from 1.8-3.6m. even more rarely studied due to their usual depth. For this reason, little is known about their juvenile biology, as they are infrequently caught, especially with a mantle length less than 9 cm. Octopus vulgaris, or the Common Octopus, is an extensively researched species

2 These containers were 51mm petri dishes, modified with multiple 4mm diameter holes in order to allow the scent of the prey to travel through the aquarium. We taped two petri dishes together using duct tape to either seal a prey item inside or keep empty as a control. In order to limit the octopus’ ability to visually visually learn, we used an opaque plexiglass sheet before and between trials to ensure that the octopus could not see the food and control being randomly placed on the Table 1: A list of the individuals and their opposite side of the tank. We measured and cut corresponding species. the plexiglass to guarantee a correct fit and

Field Methods prevent the octopus from being able to slip past Opportunistic SCUBA and skin diving the partition while preparing the trials. surveys were conducted during the day and night in STARESO harbor to find an octopus of any Foraging Trials species. If an octopus was found, the divers In order to test if the various species of would catch the octopus in a mesh bag and bring octopus foraged differently, ten behaviors it into the lab. exhibited by the individuals during trials were In order to gather prey for both foraging documented. Beginning at 21:15 CET, we and food preference trials, SCUBA and skin turned off all fluorescent lights, turned on red divers flipped rocks in the STARESO harbor to lights to simulate night light, and inserted the search for Red Abalone (Haliotis tuberculata), opaque partition to divide the aquarium into two Rock Crab (Pachygrapsus marmoratus), and a equal sides. In addition, we prepared the small ark clam (Barbatia barbata). We kept the containers for the night, filling one with an prey in an aquarium with an open circuit, and abalone and leaving one empty as a control. At gathered fresh prey multiple times each week. 22:00 we would return to the lab, having given 45 minutes to acclimate to the new surroundings Materials and put the containers on the side of the tank After capturing an octopus in the harbor opposite the octopus. The containers would then at STARESO, we placed it in a large aquarium be covered with rocks to simulate abalone (250 or 300L) with nonuniform rocks and a hiding. After removing the partition, we gave the cinder block inside, giving it the opportunity to octopus 15 minutes to participate in the trial. create a den. Before beginning trials, we left the During this, we documented the starting location octopus in the aquarium for two days to of the octopus and piles, any behaviors acclimate, turning on and off lights to simulate exhibited, and whether the octopus successfully daytime. During this time the octopus were not preyed on the abalone. fed to guarantee hunger for trials. During the trials, we kept the prey in The behaviors documented are as follows: closed containers that could easily be removed if 1. Moved whole body around tank the octopus began to feed. This prevented the 2. Moved arms through water column prey from moving throughout the tank, and 3. Approached either rock pile minimized the octopus eating in every trial. 4. Approached abalone pile first

3 5. Approached abalone pile straight line parallel to the partition, such that 6. Removed abalone from container at rock each one was approximately the same distance pile away from each other. After placing a small rock 7. Brought container back to den from rock on top of each container to keep it stationary, we pile removed the partition and gave the octopus 15 8. Groping minutes to approach and choose a prey. 9. Rock removal We documented start time and any times 10. Speculative pounce that the octopus chose a prey item. In these The final three behaviors are different ways that trials, we gave the octopus the full 15 minutes, the octopus would remove the prey from the even if they had already eaten, in case they had a rock pile. Groping describes the octopus second or third choice prey item. Additionally, reaching its arms into the rock pile to pull the we defined the prey items as being either container out without pulling any rocks off of difficult or easy to access based on the octopus’ the pile first (Fiorito & Gherardi, 1999). Rock initial location in the aquarium and whether or removal refers to the octopus pulling the rocks not they would have to go out of their way to off the pile to reveal the container. Speculative reach a specific prey item.If the octopus had to pounce describes when the octopus observed the go out of its way to reach a prey item, i.e. it went rock pile from afar, then engulfs it, surrounding across the tank to the prey, the difficulty to the pile with its web and suckers, working to get access this prey was deemed “high.” The prey the prey while it remains underneath its body was easy to access if it was in front or next to (Fiorito & Gherardi, 1999). the octopus’ original position, thus deemed If the octopus successfully attacked the “low” difficulty. These trials were conducted abalone pile, the trial would be cut short and the three times each night for three nights, totalling containers removed to avoid satiation. After the nine trials per octopus. trial, we would reinsert the opaque partition and leave for 15 minutes to allow the octopus to Statistical Analysis acclimate once again before continuing with In order to determine typical foraging another trial. These trials were conducted four behavior, ten behaviors were looked for during times each night for three consecutive nights, trials and each trial resulted with either a yes or totalling twelve trials per octopus. no response. After reviewing the results for the behaviors and inputting the results into the Food Preference Trials statistical analysis program JMP, a chi-square After three nights of foraging trials, we test was run to test the significance of behavior began food preference trials to test whether each against species and against the individual species had a significant preferred prey. At octopuses. 21:15 CET we turned off all fluorescent lights We documented which prey an octopus and turned on red lights in addition to inserting would choose first for each trial. This preference our opaque partition. For these trials, we was inputted into JMP and we ran a chi-square prepared four containers, each with an H. test for significance. tuberculata, P. marmoratus or B. barbata inside, Using a critical alpha value of 0.05, we leaving one container empty as a control. compared this value to the Likelihood Ratio p- We then left the the lab and returned at value from the chi-square tests. The chi-square 22:00 to begin our first trial of the evening. We test resulted in both the Likelihood Ratio and placed all four containers into the aquarium in a Pearson p-values, however we opted to use the

4 Likelihood Ratio as it is more robust, using a range of probability distributions. One C. macropus (Individual 6) we captured was severely injured and had only one arm. The data from this individual was removed from our study as an outlier when it failed to exhibit any behavior except moving around the aquarium.

RESULTS Foraging Behavior Results Figure 2a: A summary of the octopods 12 foraging behavior trials Of the ten behaviors studied, six were showing the yes(blue)/no(red) outcome of an octopus approaching found to have significant results. The octopus either pile when comparing individuals. The outcome of each trial is represented as the total of yes and no results. With a p-value of moving around the tank was found to be 0.0207, the comparison is significant. significant on both the species (p=0.0045, p<0.05) and individual level (p=0.0058, p<0.05; Table 2). This behavior was used as a metric for acclimation. The second behavior, moving their arms through the water column, was significant for species comparison (p=0.0260, p<0.05; Figure 1), and not significant when comparing individuals (p=0.0980, p>0.05; Table 2).

Figure 2b: Comparison of individuals with moving their body around the tank during their 12 foraging behavior trials. The outcome of the trials are represented as yes (blue) and no (red). With a p-value of 0.0058, the relationship is significant.

The behaviors exhibited by each octopus when retrieving the abalone from a rock pile included groping (p=0.0471; Figure 3), rock removal (p=0.0415; Figure 4), and speculative

Figure 1: The comparison of species vs. arm movement through pounce (p=0.0074; Figure 5). All of these the water column. A summary yes/no plot for each of the 12 behaviors had significant values for the foraging behavior trials per octopus. The outcome of each trial is represented as the total of yes (blue) and no (red) results. With a p- individual comparison (p<0.05) and non- value of 0.026, the comparison is significant. significant values for the species comparison

(p=0.085, p=0.214, p=0.1264 respectively,

p>0.05; Table 2).

Table 2: Shows the significance values for the behaviors in the foraging trials. *Indicates a p-value less than the alpha value (0.05). Figure 3: A summary yes/no plot for the octopods 12 foraging behavior trials showing the interspecific relationship with groping. Prey Preference Results The outcome of each trial is represented as the total of yes (blue) and no (red) results. With a p-value of 0.0471, the comparison is The first prey choice of each trial was significant. compared to its subsequent octopus and the

species. When comparing individuals, the results were significant (p=0.0061, p<0.05; Figure 6) while the species results were not (p=0.1117, p>0.05; Table 3).

Figure 4: Comparison of five individual octopods exhibiting rock removal during their 12 foraging behavior trials. The outcome of each trial is represented as the total of yes (blue) and no (red) results. With a p-value of 0.0415, the comparison is significant.

Figure 6: The comparison between individuals and the first prey they chose in each of the 9 food preference trials. Red represents the octopus not going for prey(No), green represents control (Co), blue represents clam (Cl), orange is crab (Cr), and turquoise is abalone (A). The p-value is 0.0061 so the relationship is significant.

The first choice of prey (Figure 7) compared to the difficulty to access that prey was found to have significant results (p<0.0001, p<0.05; Table 3).

Figure 5: Comparison of five individual octopods exhibiting speculative pounce during their 12 foraging behavior trials. The outcome of each trial is represented as the total of yes (blue) and no (red) results. With a p-value of 0.0074, the comparison is significant.

6 anxious will presumably retreat to their den to avoid a potential threat. Since the octopods in our study were willing to leave their dens, swim around the tank and the p-values were significant, this could mean that the individuals were acclimated to their new environment (Table 2). This measurement was used only to suggest that the octopuses were accustomed to their new surroundings instead of making a comparison about their level of acclimation; the Figure 7: The relationship between the available prey items, more a species or individual moved is not control (Co), clam (Cl), crab (Cr), abalone (A), and no choice (No) to the difficulty to access prey based on the octopus’ original assumed to mean that one was more acclimated position. The two difficulty levels are represented in red (high than the other. difficulty, H) and in green (low difficulty, L). With a p-value less than 0.0001, there is a significant relationship. Arm movement through the water column was the only behavior to result in The difficulty to access the first prey significance for the comparison between species, using the octopus’ original position was and not the individuals (Figure 1). This behavior compared against individuals and species. Both was used to detect prey before the octopus comparisons yielded non-significant results would move its body (Courage, 2013). The (p=.0648, p=.3587 respectively, p>0.05; Table results suggest that O. salutii and O. vulgaris 3). may be more hesitant species when compared to C. macropus (Figure 1). However, these results should be further looked upon, as this study only had one individual representing O. salutii and C. macropus. Since octopods have the ability to smell their prey through the suckers on their arms, this result provides evidence that some species, Table 3: Shows the significance values for the prey preference especially O. salutii , smell the water before trials. *Indicates a significant p-value less than alpha (0.05) going for their prey, while the other species tend DISCUSSION to go towards their prey with little hesitation The results of both trials supported that (Courage, 2013). individual octopus personalities have a greater When individuals approached either influence over behaviors than a species-wide pile, it suggested that the octopus was foraging. trend. This suggests that the assumed Figures 2a and 2b show the frequency at which coexistence between octopuses in shallow water an individual approached either pile, and how is based on individual personalities rather than a often they moved around the tank. If these behavior consistent on a species-wide scale. figures are put together, a relationship between moving around the tank and approaching the Foraging Behavior piles is seen. A good example of this is The behavior of an octopus moving displayed by individual 4 (Table 1). This C. around the tank is vital to understanding the macropus didn’t approach the piles often but it acclimation of the octopus. An octopus that is did move around the tank frequently (Figures 2a

7 and 2b). This suggests that this octopus may put one octopus has a preferred prey. This may have more energy into moving from place to place created an environment where the octopuses than actually foraging. were more participatory in the foraging trials. Individuals 2 and 3 show a different trend: this individual, an O. vulgaris, appears to Morphology approach the piles at the same frequency that it In our study five of the six octopods had moves around the tank. This implies that if this at least one arm removed. From the two different octopus decides to move it will do so more experiments we performed, the number of limbs frequently when it knows prey is available an octopus had was compared to all results (Figures 2a and 2b). gathered. No significant relationships were Groping, rock removal, and speculative found (p>0.05). This could mean that pounce all showed significance on the morphology has little to no effect on an octopus’ individual, but not species, level (Figures 3, 4, 5 foraging behavior or prey preference. respectively). This supports the idea that each Additionally, five of the octopus had at individual develops its own foraging technique least one limb missing. This small sample size rather than having a generally accepted created an unequal comparison that potentially technique for each species. skewed the data and gave non-significant values for a potentially significant relationship. Further Prey Preference studies should be conducted on whether a lost All individuals were caught in the limb actually affects the behavior of an octopus. harbor of STARESO and they appear to have After the trials, measurements of each developed individual preferences for prey octopus were taken. Although none of these (Figure 6). This could have resulted from food measurements resulted in significant availability near their dens in the field. Another relationships with an octopus’ foraging explanation could be that each individual behavior, nor prey preference, the mantle length, octopod had developed their own prey distance from the midpoint between the eyes to preference resulting from competition between the mantle tip, of O. salutii merits discussion octopuses. (Boletsky et al., 2001). Individuals with a mantle Table 2 indicates the lack of length of 9cm or less are rarely caught (Quetglas significance when comparing difficulty level et al., 2005). It was hypothesized that this could across species and individuals. This could be because the juveniles go deeper than the suggest that each of the individuals are more sampled depths (25-800m) (Quetglas et al., influenced to leave their den by the type of prey 2005). However, finding our octopus in 3m of rather than the difficulty to access this prey. water with a mantle length of 5.5cm could The difference in the amount of effort provide evidence that the juveniles actually live the octopuses are willing to put forth toward in shallower waters and then go deeper later in accessing specific prey items is seen in Figure 8. life. Overall, an octopus is willing to go out of its This life cycle could be due to the way for their favored prey and will investigate the control if it is close or next to them (Figure resources in the shallow versus deeper waters, 8). It is also very likely that they will not since in shallower waters there are usually more approach any prey at all. resources (Eppley & Peterson, 1979). The Upon further consideration, it would juvenile O. salutii could be taking advantage of have been beneficial to perform the prey the benefits of shallow water and have an preference trials first in order to understand if

8 opposite life cycle than what was previously hypothesized. We found that three of the five octopuses studied were juveniles, meaning they were not of reproductive age (Roper et al., 1984). The ages of our octopods could have resulted in data that is not actually representative of a species in its entirety, but only the species’ juvenile population. To estimate the age of the studied individuals, we measured the length of the mantle. Our O. salutii had a mantle length of 5.5cm; adults of this species typically have a mantle length of 9cm or greater, therefore this octopus was categorized as a juvenile (Quetglas Table 4: A summary of the results for the foraging behavior trials. et al., 2005). Adult O. vulgaris have a mantle “X” designates significance. length of 9.5cm or greater for adult males and Prey preference results indicate that 13.5cm or greater for females (Roper et al., individuals have developed their own favored 1984). Two of our three O. vulgaris had a prey and there are not species-specific prey mantle length of 5.8cm, and our third had a choices. An individual is more likely to develop mantle length of 13.5cm, meaning only one of its own preference rather than a preference based our O vulgaris could be categorized as an adult. on species (Table 5). Our C. macropus had a mantle length of 9.9cm, meaning that this octopus was an adult (Boletsky et al., 2001). It can be difficult to draw meaningful conclusions by comparing adults and juveniles of the same species, and even more difficult when comparing adults of one species to juveniles of another. Table 5: Summary of the results from the food preference trials. “X” designates significance. CONCLUSION The foraging behavior results point to Based on the significant individual the possibility that individual octopus develop differences exhibited in the foraging behavior their own personalities. These personalities and prey preference trials, the octopod appear to have greater influence on an personalities support coexistence. The diversity individual’s behavior than species-wide traits. of individuals favors the niche diversification There appear to be no species-specific behaviors hypothesis which would allow the octopuses except for individual prey detection techniques. within STARESO harbor to coexist. An individual’s ability to detect prey is suggested to be an innate behavior where Future Studies foraging techniques appear to be executed on an During our time in Corsica we had many individual basis (Table 4). more hypotheses that we were unable to study due to time constraints. We noticed while

9 looking for octopus, that there seemed to be less beneficial to study exclusively those species to octopus dens than in previous years. This gain more insight on their intraspecific trends. encouraged us to take GPS locations of dens we Finally, it would be interesting to see if found in the harbor, as well as in the north and the trends we found were exclusive to the south areas beyond the harbor. While we didn’t octopus community within the harbor or if they use this information in our study, we hope that apply to the populations on the north and south future classes can use it for a density study over sides. an extended period of time. A similar study could be conducted with Acknowledgements equal population sizes. While we would have We would like to thank Peter Raimondi preferred to have at least three of each species, and Giacomo Bernardi for helping us throughout limited time to conduct our studies and limited our entire project from start to finish. Thank you lab resources prevented us from achieving this to Gary Longo and Kathryn Beheshti for helping goal. us to develop our project in Corsica, providing In addition, past programs have us valuable advice, and catching one of our exclusively studied behaviors and trends of O. octopuses. Finally, thank you to everyone in the vulgaris with significant results. After seeing BIOE159 class that assisted us, especially on how little information there is on the species C. late night dives for prey and octopods. macropus and O. salutii we believe it would be

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