Abstract Over thirty years ago anecdotal accounts of the undescribed Larger Pacific Striped Octopus suggested behaviors previously unknown for octopuses. Beak-to-beak mating, dens shared by mating pairs, inking during mating and extended spawning were mentioned in publications, and enticed generations of cephalopod biologists. In 2012–2014 we were able to obtain several live specimens of this species, which remains without a formal description. All of the unique behaviors listed above were observed for animals in aquaria and are discussed here. We describe the behavior, body color patterns, and postures of 24 adults maintained in captivity. Chromatophore patterns of hatchlings are also shown.

Citation: Caldwell RL, Ross R, Rodaniche A, Huffard CL (2015) Behavior and Body Patterns of the Larger Pacific Striped Octopus. PLoS ONE 10(8): e0134152. https://doi.org/10.1371/journal.pone.0134152 Editor: Claude Wicker-Thomas, CNRS, FRANCE Received: April 30, 2015; Accepted: July 6, 2015; Published: August 12, 2015 Copyright: © 2015 Caldwell et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: Funding was provided to RLC by the University of California Museum of Paleontology. This funding group had no influence on the authors' study design, interpretation, or communication. Competing interests: The authors have declared that no competing interests exist.

Introduction The ‘Larger Pacific Striped Octopus’ (LPSO; Fig 1) first appeared in peer-reviewed literature in 1977, as an illustration of a juvenile without further discussion [1]. Along with Octopus chierchiae Jatta, 1889 and Octopus zonatus Voss, 1968 from the Western Atlantic and Caribbean [2], LPSO is one of the ‘Harlequin’ octopuses identified by their semi-permanent stripes and spots [3]. The first indications of its unique behavior came in 1982, in a broader discussion of ritualized (body color) patterns in cephalopods [4]. Although mentioned only briefly, early accounts of this octopus based on field collections and groups housed in large tanks listed intriguing behaviors previously unknown for octopuses. These accounts included beak-to-beak mating, dens shared by mating pairs, inking during mating, and extended spawning [4–6]. LPSO was reported to form colonies of 30–40 individuals living at high density, including co-occupancy of dens by pairs presumed to be mates [4,5]. Despite the potential interest of these unusual behaviors to cephalopod biologists, 25 years ago a full ethological description was rejected and the original observations never resubmitted for publication. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Artwork depicting Larger Pacific Striped Octopus. by AR. https://doi.org/10.1371/journal.pone.0134152.g001 In 2012, additional live specimens of LPSO became available through the aquarium trade. Based on observations of these individuals maintained in the laboratory from 2012–2014, we provide photographic and video evidence and behavioral descriptions that confirm many observations reported in previous accounts about this species [4,5], including beak-to-beak mating, co-occupancy of a den by a mating male and female pair, extended spawning, and unique prey-capture methods. This behavioral catalog describes prominent behaviors related to feeding, den construction, mating, aggression, egg-laying, and senescence observable in the laboratory. Body color patterns and postures of adults maintained in captivity, and chromatophore patterns of hatchlings are also shown. Octopus body color patterns and textures are determined by the presence, location, and pigment composition of fixed anatomical structures and muscles in the skin [7]. While each of these individual components varies in visual intensity from moment to moment, together they ultimately determine the range of body color patterns and postures an octopus can employ, and are thought to offer a species-specific means for identification [8,9]. Although a species description of LPSO is forthcoming by other authors, we provide detailed photographs and descriptions of live individuals that should allow for easy identification, and provide a guide for future ethological studies.

Materials and Methods Unless otherwise stated, observations reported here are based on live animals kept in captivity from 2012–2014 at the University of California, Berkeley and California Academy of Sciences, USA. Habitat information from the collector is also provided. We also include limited references to observations from the 1970’s, which are drawn from observations and collections performed by A. Rodaniche at the Naos Island Marine Laboratory of the Smithsonian Tropical Research Institute in the Republic of Panama. At the time, limited observations were made in the field, but most descriptions were based on captive individuals held in the laboratory in March 1975 and May 1977. All observations from the 1970’s are explicitly noted where included or cited. Between April 2012 and August 2014 a total of 24 individuals (13 males and 11 females) were hand-collected on SCUBA and obtained from commercial aquarium wholesalers [Quality Marine (18), Live Aquaria (1), Russo’s Reef (4) and Sea Logic (1)]. All specimens originated from the same collector from one location in Nicaragua. They were collected for commercial sale in the aquarium trade by Livan, Jansen & Cia. Ltda., Managua, Nicaragua, a firm that holds permits to collect and export octopus. It appeared to the locally-experienced collectors using SCUBA that there was only one persistent aggregation of octopus at the site of collection. Octopuses were shipped to the wholesalers within four days of capture, and then shipped to the authors within four days of arriving in the United States. At delivery they ranged in size from approximately 15–25 mm mantle length (ML). In the laboratory they were initially housed in plastic containers with removable plastic mesh lids (2–4 L). These containers were placed within larger aquaria (227–757 L) with a closed seawater filtration system. This system allowed physical separation of animals in case of aggression because some octopuses can be cannibalistic in captivity [10] and in the wild [11,12]. This housing system also maintained the possibility for visual and chemical communication between individuals. Water temperature was 20–23°C. Octopuses were fed every other day a variety of crustaceans and molluscs (see Hunting and feeding section). Because animals were active in the day, and did not appear to be active during the night (as verified by haphazardly timed checks), ambient light was sufficient for observations. After several successful matings in controlled environments that resulted in no cannibalism or damage beyond sucker marks on the mantles of males, four male and female pairs were allowed to share a tank. In each of these cases the female was initially kept in her den in a plastic container with the top removed, and the male was allowed access to the entire aquarium. After several weeks of that arrangement, the female’s den was removed from the container and placed in the main tank space. After the last shipment of LPSO was received, a female/female/male trio was housed together. A female/female paring was attempted but was quickly abandoned when the larger female started to eat the eggs she had already laid. A male/male pairing was never attempted. These groups were housed in tanks ranging from 113–454 L with sand substrates and small objects for shelter (see Distribution, habitat and denning information below). Photographs were taken of animals in aquaria with a variety of camera gear. Still images were taken with Nikon D7000, Nikon D800, AF-S MicroNIKKOR 105mm, and Nikon AF-S DX NIKKOR 18–200mm with a Nikon SB 600 strobe (RR), and a Nikon D800 with Nikon 910 strobes (RLC). Videos were taken with a Sony HDR-CX165 (RR), a Sony Handycam HDR-XR260-V (RLC) and an iPhone 4 (RR). We used conventional terminology of gross morphology [13]. Terms used to describe skin morphology follow those defined and illustrated previously [8,9,14–21]. Behavioral terminology follows descriptions in the literature for other species, or were defined here if no established term was available. Modes of locomotion follow those outlined previously [22]. Because we aimed to observe as many behaviors as possible, we were not able to control for observation effort of each animal. Therefore any references to the frequency of observations must be interpreted with caution. This non-invasive study was carried out in accordance with the policies of care outlined by the Association of Zoos and Aquariums, and the Steinhart Aquarium’s Animal Research Committee and Animal Care Committee under the guidance of the Steinhart Aquarium’s Animal Health Department. This study did not require approval of protocols by The University of California, Berkeley Animal Care and Use Committee (our IACUC), which only requires approved protocols for vertebrate animals (Animal Care and Use Program Policy http://www.acuc.berkeley.edu/policies/acu_program.pdf). At this time, and during the period that the research was conducted, no Federal, State or Local regulations have been applied to the study of cephalopod mollusks. We therefore did not seek institutional approval. All animals were provided with an appropriate den in which to reside, and fed live shrimp, crabs, snails and clams. No animals were euthanized. They all died a natural senescent death typical of octopus after reproducing. When males and females were introduced to observe mating and other reproductive behavior, the animals were closely monitored and at any sign of aggression the animals were immediately separated. No damaging injures occurred. Animals were offered food the day after arrival, but were allowed to acclimate to new conditions for several days to a week before a mating opportunity was presented. Because of the close proximity of tanks, many of the octopuses could see each other during this acclimation period, but were not able to physically interact.

Discussion To our knowledge LPSO exhibits several behaviors not previously described for other octopuses. Mating and egg laying in this species appear to be unique. The beak-to-beak mating posture is similar to the head-to-head mating posture typical in decapod cephalopods (squids and cuttlefishes; [32,33]), especially those that mate directly beak-to-beak (e.g. Dosidicus gigas [34]). However, with the exception of LPSO, this mating position appears to be absent among octopuses. All other known octopuses mate using ‘distance’ or ‘mount’ postures, or intermediate positions between the two [32,33,35]. When decapods mate head-to-head the male’s funnel is positioned near the female’s buccal region, where spermatophores are often placed during copulation [33,36,37]. Male squids and cuttlefishes mating in the head-to-head posture can flush the female’s buccal region, clearing the area for placement of his sperm, or potentially removing the existing sperm of rival males [38,39]. Beak-to-beak mating by LPSO would not confer this advantage in sperm-competition to males. Rather than depositing spermatophores for external sperm storage as in decapods, male octopuses place spermatophores internally in the oviducts of the female, which is located inside the mantle and not within reach of male flushing via jetting [33,40]. Unlike with distance and mounting postures observed in other types of octopus, beak-to-beak mating in LPSO frequently involves grappling, and allows rapid full envelopment of the male into the female’s oral web, behaviors that are typically associated with aggressive contests in octopuses [14,27]. We have no direct evidence that the beak-to-beak mating and feeding postures by LPSO confer specific advantages, although we speculate on three non-mutually exclusive hypotheses: allowing for females to maintain their brooding-typical posture while mating and/or eating, allowing for simultaneous feeding by a mating male and female pair, and limitation of mating access to females. The resting posture of brooding LPSO involves the oral surface facing outward while the dorsal mantle faces the eggs. The beak-to-beak posture might facilitate mating with females that are brooding, but still likely to lay more eggs. As with mating in the ‘distance’ position in other octopuses [41], female LPSO can also feed while mating beak-to-beak. This mating position might allow for sucker alignment and simultaneous beak-to-beak feeding associated with mating and/or pairing behavior, including during brooding. The cases of beak-to-beak feeding behavior were observed by a pair that, at other times, mated repeatedly and co-occupied den space. Previously, pair bonding has been noted to occur in this species [5]. However, thus far no observations have been designed to verify this behavioral aspect of LPSO. We do not know if these behaviors are associated with long-term pairings in the wild, as can occur for up to two weeks in group-living Abdopus aculeatus [41]. Finally, beak-to-beak mating might allow males to monopolize mating access to females. In octopuses, females can mate with two males simultaneously (one per oviduct), and in some cases more males try to mate with the female than she has oviducts available (Octopus cyanea [42]; Vulcanoctopus hydrothermalis [43]; Octopus kaurna [21], Octopus bimaculoides [32]). Beak-to-beak mating might limit access to a mating female to one male at a time because she has one buccal area, a situation that might confer competitive mating advantages in this group-living species. Egg laying in LPSO is especially prolonged compared to that exhibited by other octopuses. In shallow-water octopuses, female senescence typically begins shortly after egg laying, and death coincides approximately with hatching [31]. This generalization does not apply to LPSO, whose females lay eggs for up to six months and brood for up to eight months. Females continue to feed, lay at least hundreds of additional eggs, and accept additional copulations long after the first eggs have hatched. Although possibly common in deep-sea cirrate octopodids (e.g. Opistoteuthis [44,45]), few shallow-water octopuses are known to exhibit extended spawning. Iteroparity, a form of extended spawning in which multiple batches of eggs are laid, is thought to increase lifetime fecundity of very small cephalopods (as in Octopus chierchiae [6]), which are limited by ovary space in the mantle cavity [46]. While this tactic would increase long-term fecundity of LPSO as well, this species is of average size rather than small, reaching a mantle length of at least 70 mm while laying eggs. It appears that numerous selective pressures or evolutionary constraints may have led to the rare but phylogenetically widespread expression of this trait. Although the striking high contrast dark brown and white body color pattern of LPSO is unique among octopuses, similarities in body color pattern and texture can be found. Octopus chierchiae exhibits dark bars and stripes, similar to that of LPSO, but over a predominantly pale rather than dark background [6]. Octopus zonatus, another Harlequin octopus, bears body patterning similar to O. chierchiae [2]. The deimatic display of Abdopus spp. often involves pale spots over a dark background [47], but resting patterns and skin textures are more complex than those of LPSO [22,47]. Finally Thaumoctopus mimicus and Wunderpus photogenicus exhibit body patterns that incorporate high-contrast dark brown and pale spots and bars visible at rest [48]. However, in T. mimicus the elements of these patterns are less clearly delineated [49], and in W. photogenicus [50] they are reversed, with bars on the arms and pale spots on the mantle rather than vice versa in LPSO. Skin texture of LPSO is not unique. The granular skin, including on the dorso-lateral surfaces of the arms, is similar to that found in Amphioctopus spp. [17,51]. LPSO also takes on a resting posture common in Amphioctopus (Fig 3C; Figure 5A in [51]; Figure 7 in [52]). However LPSO lacks the flap-like primary papillae in a diamond configuration on the mantle, and dark arm stripes along the dorsolateral edge of arms typically found in Amphioctopus. Instead, LPSO bears low conical mantle papillae. Along with W. photogenicus, Hapalochlaena lunulata [53] and O. chierchiae, LPSO bears individually unique and consistently distinguishable body color patterns. Behavioral and biological similarities with Octopus chierchiae are numerous [6] and could possibly be attributed to close phylogenetic relationships (Wright, Caldwell, Ross unpublished data). We have no evidence for speculating on similarities between T. mimicus, W. photogenicus, Amphioctopus and LPSO other than these octopuses all occupy a sandy bottom habitat [21,51]. On multiple occasions SCUBA divers have found groups of LPSO living in very close proximity to one another ([4,5], collections in 2012–2014), a situation that might have strong implications for behavioral interactions. Many octopuses are known to have an ecologically clumped distribution according to habitat features or resources (e.g. Abdopus aculeatus, Octopus insularis, Octopus vulgaris [22,54,55]). Those populations with grouped dens and/or especially high local densities appear also to exhibit more frequent intraspecific interactions, aggression, and/or more complex mating associations than exhibited by more solitary octopuses [27,41,56–59]. Where suitable habitat is limited to small islands of hard substrate surrounded by a soft substrate, Octopus tetricus constructs dens in high densities, which are maintained successively by different individuals [56]. We do not know whether this situation also applies to LPSO. The SCUBA divers who collected LPSO between 2012 and 2014 reported targeting a single aggregation that persisted in the same area for over two years. This observation suggests group site fidelity, which might extend beyond the life span of a single individual. Likewise, we do not know whether LPSO living in groups visually recognize conspecifics, which is associated with reduced physical aggression and increased localized avoidance with familiar individuals in Octopus vulgaris [60]. If so, then it is possible that their individually unique body color patterns might facilitate this form of recognition, and allow for the unique suite of interspecific interactions observed here in LPSO, such as repeated mating, and food and den sharing.

Acknowledgments We thank Lukas Jansen of Livan, Jansen & Cia. Ltda., Managua, Nicaragua, who first alerted us of the occurrence of LPSO off the Pacific coast of Nicaragua and provided all of the LPSO collected 2012–2014. We thank Elizabeth Palomeque for her patronage and support of this project, as well as Jean Auplay, Marisa Avila and Kalin Ross for their help in the care of the animals during this study. This manuscript was improved by constructive comments from Stephanie Bush, Roger Hanlon and two anonymous reviewers.

Author Contributions Conceived and designed the experiments: RLC RR AR CH. Performed the experiments: RLC RR AR. Analyzed the data: RLC RR CH. Contributed reagents/materials/analysis tools: RLC RR. Wrote the paper: RLC RR AR CH.