© 2021. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2021) 224, jeb237529. doi:10.1242/jeb.237529

RESEARCH ARTICLE Feel the light: sight-independent negative phototactic response in octopus arms Itamar Katz, Tal Shomrat*,‡ and Nir Nesher*

ABSTRACT manner. This could allow the octopus to retract and protract its arms Controlling the octopus’s flexible hyper-redundant body is a when exposed to danger, even when it is minimally or not at all challenging task. It is assumed that the octopus has poor aware of their position and posture. Extraocular photoreception is a proprioception which has driven the development of unique well-documented phenomenon and has been observed in many and mechanisms for efficient body control. Here we report on such a diverse species (Cronin and Johnsen, 2016). Florey (1966) was the mechanism: a phototactic response of extraocular photoreception. first to report that increased illumination on the squid skin evokes Extraocular photoreception has been observed in many and diverse chromatophore expansion. Later, Packard and Brancato (1993) species. Previous research on cephalopods revealed that increased reported the same observation in octopus skin, and recently the illumination on their skin evokes chromatophore expansion. Recently, mechanism was investigated in Octopus bimaculoides skin by ‘ the mechanism was investigated and has been termed ‘light- Ramirez and Oakley (2015). They termed this behavior light- ’ activated chromatophore expansion’ (LACE). In this work we show activated chromatophore expansion (LACE). Their work on that in response to illumination, the arm tip reacts in a reflex-like manner, O. bimaculoides skin indicates that LACE is induced through folding in and moving away from the light beam. We performed a set of similar opsins, G-protein coupled receptors (GPCRs), to those behavioral experiments and surgical manipulations to elucidate and found in the octopus eye. It has been suggested that the function of characterize this phototactic response. We found that in contrast to the LACE is peripheral, local fine tuning of the camouflage to local activation and control of LACE, the phototactic response is environmental luminance and/or that the skin photoreceptors mediated by the brain, although it is expressed in a reflex-like pattern. serve as supplementary sensory organs to the eyes, sending the Our research results and observations led us to propose that the brain information about the surrounding environment (Buresch phototaxis is a means for protecting the arms in an instinctive manner et al., 2015; Kingston et al., 2015). We have found an additional from potential daytime predators such as fish and crabs, that could body response to light in Octopus vulgaris, which is sight identify the worm-like tips as food. Indeed, observations of the independent and might reveal mechanosensation properties. In octopuses revealed that their arm tips are folded in during the this work, we performed a set of behavioral experiments and daytime, whereas at night they are extended. Thus, the phototactic surgical manipulations to elucidate and characterize the strong response might compensate for the octopus’s poor proprioception by phototactic response of the octopus arm tip to illumination. keeping their arms folded in illuminated areas, without the need to be aware of their state. MATERIALS AND METHODS Animal care and handling KEY WORDS: Octopus vulgaris, Cephalopod, Phototactic, Octopus vulgaris Cuvier 1797 specimens (female and male, 300– Extraocular photoreception 1000 g) were collected by fishermen from the Israeli coast of the Mediterranean Sea. In the lab, each octopus was maintained in an INTRODUCTION individual 130 l tank connected to a semi-open running sea water The octopus has eight long arms that are void of rigid skeletal (SW) system with controlled temperature of 20±2°C and 12 h:12 h elements. The control of motion in such a long and flexible light:dark cycle (illuminated by T5 fluorescent lamps). The ambient appendage is extremely complex, as it involves control of organs light in the aquarium area (indoor system) measured with a HOBO with an extremely large degree of freedom. Not surprisingly, the MX2202 light sensor, was around ∼30 lx (equivalent to octopus possesses poor proprioceptive sensation of its arms motion 0.1 μmol m−2 s−1). The animals were fed raw defrosted fish meat and position in space (Wells, 1978; Zullo et al., 2009), since such a three times a week. All animal handling and experimental system would require a huge computational load. These features procedures were in accordance with the ethics and regulations have forced special evolutionary innovations of exceptional and applicable in the Israel academy and nature conservation authorities. sophisticated mechanisms that allow the octopus efficient control of its body (Levy et al., 2016; Nesher et al., 2014; Sumbre et al., 2005, Minimal light condition 2006). Here, we report on a phototactic response in the octopus arm, In order to test the effect of prolonged change in environmental which is mediated by the brain in an extraocular photoreception illumination conditions on the phototactic response, the aquarium was covered with a black opaque plastic sheet (Fig. 1), the ambient light in the aquarium was below the sensitivity of the light meter Faculty of Marine Sciences, Ruppin Academic Center, Michmoret 40297, Israel. *These authors contributed equally to this work ∼0 lx. Apart from the illumination conditions, all other biotic and abiotic variables were unchanged. The octopuses were held under ‡ Author for correspondence ([email protected]) dark conditions, except for very short periods of 1–2 min, every T.S., 0000-0002-4696-5962 other day, during feeding. The measurements of the phototactic response took about 30 min and were performed under regular

Received 11 September 2020; Accepted 23 January 2021 illumination. Journal of Experimental Biology

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AB

Fig. 1. Experimental setup for the octopus sight-independent response test. (A) Sketch of the experimental apparatus; the aquarium was covered with black opaque plastic. At the top of the aquarium, an open tube allows the octopus to extend an arm out of the aquarium in order to feed. (B) Illumination of the foraging arm, shielded by the lid from the octopus’ sight.

Arm illumination procedures Arm amputation for the examination of LACE and phototactic The influence of direct light on octopus arms was tested by response in an isolated arm illuminating the arms with a LED flashlight (LED 5000 lm, Arm amputations were performed as described by Nesher et al. XML-T6). The flashlight was manually attached perpendicular (2014). In brief, the distal third of the arm was amputated by a (±10 deg) to the aquarium glass and the arm was directly single surgical cut, leaving the proximal portion undamaged. illuminated when it was adjacent to the glass (Movie 1). The Then, the isolated arm section was rinsed in fresh SW and the octopuses were tested throughout the day and no correlation was octopus was returned to the aquarium for recovery. All the animals found between the time of examination and the characteristics of recuperated within a few minutes after awakening from anesthesia the response. and exhibited normal behavior. Arm amputation is followed by For the sensitivity examination, the light intensity was measured regeneration of a functioning arm within several weeks (Fossati and adjusted before each experiment using a HOBO MX2202 light et al., 2013). sensor; the logger was placed in the aquarium adjusted to the glass and was illuminated through the glass of the aquarium, in a similar Transection of the arm nerve cord and peripheral incision manner to the arm illumination. The light measurement was logged The octopuses were anesthetized during the entire surgical at 1 s intervals and simultaneously transmitted through the procedure. Both procedures were done under a dissecting Bluetooth of a handheld cellular phone, for online measurements. microscope, with fine surgical scissors (Fig. 2). The incision site The measured units (in lux) were converted to PPFD was designated at the point of a distal third of the arm. We severed (photosynthetic photon flux density), according to a conversion the arm’s main nerve cord with minimal damage to the surrounding curve performed with a MQ-200 quantum meter and HOBO muscle tissue. A sudden white appearance of the skin distal to the MX2202 light sensor. The adjustment of light intensity was cut indicated a successful transection of the cord (Fig. 2B). achieved by incorporating a light dimmer into the flashlight Peripheral incision was achieved by cutting around the electrical circuits. circumference of the arm while leaving the nerve cord intact (Fig. 2C). Following these procedures, the octopus recovered within Sight-independent test a few minutes after being removed from the anesthetic solution and To test whether the phototaxis is sight independent, the aquarium returned to the aquarium. The octopus’s treated arm was then was covered with black opaque plastic sheet on top and an open tube illuminated and tested for a phototactic response. Following this on the lid which allowed the octopus to extend an arm out of the test, the section distal to the cut was removed in order to promote aquarium and reach for the food outside the tank (Fig. 1A). The arm regeneration. The arms healed in less than a day, and initiation of was illuminated from a distance of ∼20 cm, while foraging arm on regeneration was clearly observed after approximately 2 weeks. the covered lid was out of the octopus’ eyesight (Fig. 1B). In a further experiment, the food on the lid was illuminated and arm Removal of the supraoesophageal brain response was examined when it entered the illuminated area In order to minimize the number of octopuses killed, examination of (Movie 2). the involvement of the supraoesophageal brain mass and the optic lobes in LACE and the phototaxis mechanism were done on six Surgical procedures octopuses that were dedicated for a different study on the All surgical procedures were performed under deep anesthetization neurophysiology of learning and memory (which is dependent on according to a protocol developed by Shomrat, et al. (2008). the supraoesophageal brain mass). Under the dissecting Octopuses were immersed in 2 l seawater (SW) supplemented with microscope, the optic lobes were disconnected and the −1 55 mmol l MgCl2 and 1% v/v ethanol (96%) for 30–45 min. supraoesophageal brain mass was carefully removed by cutting Octopuses were considered deeply anesthetized and ready for the brain tissue on the sides of the esophagus (for more details, see surgical procedures when they exhibited the following: pale skin, no Shomrat et al., 2008, 2011). The octopuses, with only the voluntary movements, and limited or inability of the suckers to suboesophageal brain remaining, were returned to the aquarium attach. and recovered within a few minutes after being removed from the Journal of Experimental Biology

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A PNS CNS

B

Surgical site C

Surgical site

Nerve cord

Ganglion

Fig. 2. Illustration of Octopus vulgaris nerve system anatomy and surgical procedures. (A) Octopus neural anatomy. Illustration of the octopus central nervous system (CNS, in blue) and main peripheral nervous system (PNS, in yellow). The surgical procedures were performed on separate arms in the same animal. (B) Transection of the arm’s nerve cord: a small incision was made on the dorsal side of the arm. The nerve cord was disconnected while most of the muscle and skin surrounding the cutting site remained intact. (C) Peripheral incision: A circular cut through the muscle mass of the arm creates transverse separation of the muscle, leaving the nerve cord intact. anesthetic solution. At the end of the short examination, the ELI50 results were summarized, normal distribution was octopuses were euthanized under deep anesthesia. evaluated using the Shapiro–Wilk test, and difference among datasets were verified by two-tailed paired t-test. Behavior and statistical analysis Behavioral experiments were captured by video camera, and the movies RESULTS were analyzed off-line by a ‘blind observer’ for both motoric reaction The phototactic response and arm behavior. Statistical analysis was performed using JMP Pro, While investigating the effect of light on octopus behavior, we version 15 (SAS Institute Inc.). Sight-independent tests, determining the noticed a curious phenomenon. Illuminating the arm tips of freely effective luminance intensity 50 (ELI50) were examined in a binary behaving octopuses elicited a negative phototactic response, manner (response versus no response). Two-tailed Fisher’sexacttest meaning the arms withdrew from the illuminated area (Movie 1). was used as goodness-of-fit test. Differences among datasets for First, we tested whether this behavior is sight dependent. Four phototactic response latency were verified by repeated measure octopuses were trained to obtain food by extending an arm outside ANOVA, interquartile range method was implemented for deduction the tank through an open tube in the cover of the aquarium (Fig. 1). of outliers, and post hoc Tukey HSD multiple comparison test was The octopuses were ready for the experiment after 2 days of training. implemented when d.f. was higher than one. The top of the aquarium was covered with an opaque plastic sheet to ELI50 was determined with certain adjacent according to the prevent the octopus from seeing the light projected on top of the method of Reed and Muench (1938). Four octopuses were subjected cover while their arm was searching for food (Fig. 1A). When to 3–7 sessions of arm tip illuminations with an increasing gradient the foraging arm reached outside of the aquarium it was of light intensity (see Fig. S2). Every session was composed of ∼10 directly illuminated by ultra-bright LED flashlight (mean±s.e.m.: illuminations per octopus and the percentage of positive response 439.27±4.36 μmol m−2 s−1) on its tip, once per time the arm reached was documented. Then the accumulated percentage of response to out (Fig. 1B). Negative phototactic response occurred in 84% certain light intensity, was calculated for each octopus and the (n=74) of the tip illuminations. In this experiment no food was ELI50 was calculated using the following equation: actually offered, and the octopus’s foraging behavior was a result of the anticipation of food according to the training. ð% . % %Þ ð ð% . %Þ ð ÞÞ 50 50 ¼ I 50 I x ; Next, we tested whether sensing the strong illumination forced ð% . 50% % , 50%Þ ðIð% . 50%ÞIð% , 50%ÞÞ the octopus’s arm to avoid the food. We placed a piece of fish on the ’ ð1Þ covered lid out of the octopuses sight (Movie 2). The food was continuously illuminated. Except for the change in illumination, no where % is the accumulative percentage of positive response, I(%) change in temperature or other abiotic condition at the food area represents the illumination intensity corresponding with the specific were detected. We tested whether the foraging arm would sense the

% of response and I(x) is the ELI50 value. change in illumination around the feeding area and avoid the food. Journal of Experimental Biology

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Indeed, when octopuses approached with their arms, they avoided the procedure described above). The response latency was measured the illuminated food in 88% of the trials (n=26). after 1 week of darkness (n=114, median=0.68 s) and again after an In a further experiment we examined the sensitivity to light additional week in darkness (n=153, median=0.68 s). After these 2 illumination in different sites along the arm (n=6 octopuses). Tip weeks, three of the octopuses were examined again 1 month after illumination resulted in a negative phototactic response in 92.3% of they were returned to regular conditions of 12 h:12 h light:dark cases (n=26). This was significantly different to the 62.3% response cycle (one of the four octopuses died). As depicted in Fig. 3A, the (n=26) following illumination of the middle section of the arms results reveal a significant decrease in the latency of the phototactic (d.f.=1, P=0.039, N=52; two-tailed Fisher’s exact test). Then, we response after the darkness period (d.f.=3, F=23.647, P<0.0001, monitored the latency of the response as an assay for the phototactic N=644; repeated-measure ANOVA). Measurements of latency a response reactivity. The latency was defined as the time from the month after returning to regular light conditions revealed a return to initial moment of illumination until the beginning of the illuminated the initial value range (n=101, median=0.74 s; Fig. 3A). In an arm movement. The response latency was significantly shorter at the additional complementary experiment, we focused on the response tip of the arm (n=13, median=0.77 s) compared with the middle sensitivity. We determined and compared the white light intensity (n=13, median=1.57 s), (d.f.=1, F=14.086, P=0.001, N=26; that triggered a phototactic response in 50% of the illuminations, i.e. repeated-measure ANOVA). No phototactic response was observed the ELI50 (see Materials and Methods for more details) in four at the base of the arms. octopuses before and after 1 week in darkness. The experiments were recorded and analyzed off-line by a trained blind observer Alterations due to changes in environment illumination familiar with octopus behavior. The results revealed a significant Two sets of experiments were done in order to test the adaptability increase in the ELI50 after darkness suggest a decrease in octopus of the responses to changes in environmental illumination and sensitivity after the dark period (d.f.=3, P=0.016, N=4; paired t-test, examine the long-term effect of maintaining the octopus under two-tailed; Fig. 3B). minimal illumination. In both experiments the octopuses were maintained in darkness by completely covering the aquariums with Surgical manipulations opaque nylon sheeting. In the first experiment we initially ruled out For preliminary characterization of the neural pathway and control short term modifications in the response reactivity, by comparing of the phototactic response and to reveal the central or peripheral the latency of the arms response to white light projection in three nervous system involvement, we carried out top-down examinations octopuses before (n=47, median=0.74 s) and after they were kept in through surgical and behavioral procedures (Fig. 2). We examined darkness for 1 h (n=50, median=0.7 s). Then, the octopuses were the findings in comparison to the LACE response. LACE and the maintained under darkness for 1 week, except for very short periods arm phototactic response can be induced in octopuses by direct light of 1–2 min, every other day, during feeding. At the end of this week projection. In an anesthetized octopus that exhibited pale skin and the latency of the arm response was tested again (n=54, lack of voluntary movement, no phototactic response and only median=0.57 s; Fig. S1). Overall, the results showed a significant negligible LACE could be induced, while mild aversive stimulation change in the latency of response (d.f.=2, F=14.443, P<0.0001, of the arm induced local movement and color change in the skin. N=151; repeated-measure ANOVA) while no significant Complete removal of the supraoesophageal section of the brain differences were detected after 1 h. In the second examination we along with disconnection of the optic lobe (suggested to be a pivotal tested the reversibility of the mechanism. First, the response latency motor control for chromatophore patterns; Liu and Chiao, 2017; of four additional octopuses was measured (n=276, median=0.81 s). Messenger, 2001) prevents the phototactic response, but has no Then, the octopuses were maintained under dark conditions (using effect on LACE which can even be activated in isolated skin tissue.

A B n.s. P=0.016 P<0.0001 P 2.0 <0.0001 ) –1

s 10 1.5 –2 mol m

1.0 µ

Latency (s) 5

0.5 ELI50 (

t=0 1 week 2 weeks 6 weeks 0 // 0 12L/12D24D24D 12L/12D 12L/12D 24D

Fig. 3. Adaptation of octopus arm phototactic response to prolonged change in environmental luminance. (A) The phototactic response latency to white flashlight projection, following maintenance of the octopuses in regular illumination conditions [12 h light:12 h dark (12L/12D); 4 octopuses, n=276)]; first week in darkness (24D; 4 octopuses, n=114); second week in darkness (4 octopuses, n=153); 1 month after the octopus was returned to regular illumination conditions (12L/12D; 3 octopuses, n=101). The whisker box plot exhibits significant changes in latency of response before, during and 1 month after 2 weeks of prolonged darkness (as presented in the timeline bar). (d.f.=3, F=23.647, P<0.0001, N=644; repeated-measure ANOVA), post hoc Tukey HSD multiple comparison test P- value presented in the plot (n.s., not significant). All experimental steps were performed sequentially with the same octopuses. Box indicates 25th and 75th percentiles, with whiskers showing the range and bar the median. (B) Prolonged darkness affects phototactic response sensitivity. ELI50 (in μmol m−2 s−1)was determined from four octopuses that were maintained under regular illumination conditions (12 L/12D; mean=6.228±0.698) and after 1 week in dark conditions (24D; mean=10.1±1.149). Results presented as means±s.e.m. Note, the response sensitivity significantly decreased after 1 week of dark conditions (d.f.=3,

P=0.016, N=4; paired t-test, two-tailed). A detailed presentation of the ELI50 for each octopus is shown in Fig. S2. Journal of Experimental Biology

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We preceded these experiments with a set of surgical procedures at and Tar, 1968; Zhukov et al., 2006) and also some vertebrate studies the arm level (Fig. 2). After transection of the arm’s nerve cord (Vistamehr and Tian, 2004) on degradation of the retina (Fig. 2B) the arm become pale and both phototactic response and components, including photoreceptors, owing to prolonged LACE distal to the surgery site are functionally disabled. darkness conditions. The increase in response reactivity may be Interestingly, although transection of the nerve cord prevented the result of modifications at a higher level of the neural network LACE in octopus, following amputation the same arm again showed processing. Therefore, more light will be needed in order to reach LACE but not the phototactic response. Peripheral incision of the the threshold for activation of the response, but once the threshold is arm (Fig. 2C), i.e. around the axon tract while leaving the axon tract achieved, the latency, which is carried out by the nervous system, is intact, eliminates the phototactic response while preserving the shorter. Overall, this set of results revealed that the response LACE. The portion distal to the peripheral incision showed quite behavior may adapt to environmental light conditions, thus normal motor behavior relative to the rest of the animal’s intact arms suggesting an eco-physiological role that may be important for (e.g. typical crawling movement and stretching). In order to test the octopus’s ability to cope with dynamic environmental light whether the peripheral incision prevents the phototactic response as conditions. a result of the traumatic procedure, we did a sham control with a peripheral incision of the arm circumference skin tissue, while Surgical manipulations leaving the muscle mass intact. We found that there was no effect on In order to reveal any nervous system involvement, we carried out either the LACE or phototactic response in this sham control. top-down examinations of the phototactic versus LACE responses, through surgical and behavioral procedures (Fig. 2). The results DISCUSSION (summarized in Table 1) point toward a reflex arc-like control Here, we describe a phenomenon that has not been previously configuration that involves the brain for the phototactic response, in reported, of an extraocular photoreception mechanism in the contrast to the LACE local activation. The surgical manipulations octopus arm. We found that illuminating the tip of the arm causes revealed two interesting findings. The first one is the whitening of a negative phototaxis response (Movie 1). Using behavioral the distal part of the arm after transection of the arm’s nerve cord experiments and surgical manipulations, we characterized this (Fig. 2B), whereas amputating the arm re-demonstrated the newly disclosed behavior and examined the findings in comparison pigmentation and LACE response. The second interesting finding to the LACE response, as both are extraocular photoreception was the ablation of the phototactic responses following a peripheral phenomena. incision (leaving the arm nerve cord intact; Fig. 2C). Both these observations might imply that there is an essential neuronal or Behavioral experiments myogenic conduction pathway (backward or forward) passing The first set of behavioral experiments examined the initialization of through the muscle mass of the arm. Such a conduction pathway has the phototactic response while the arm was out of the octopuses’ not been described in octopus to date. However, the presence of sight. The results of these experiments clearly confirmed that the musculature gap junctions has previously been described in several phototactic response of the arms is sight independent, and that it is works, as in the cuttlefish stomach and between chromatophore sufficient to modify behavior such as food gathering. In a further set muscle cells (Bone et al., 1995). Notably, a simple arm withdrawal of behavioral experiments, we aimed to examine the basic properties reflex can be activated in an isolated arm (Hague et al., 2013); of this mechanism. By illuminating different locations along the arm therefore, the lack of phototactic response is not due to impaired while counting the successful elicited response and their latency, we motor capabilities in an isolated arm. showed that the tip of the arm is much more sensitive to illumination In summary, our results revealed a CNS-mediated motoric compared with the middle of the arm. These results could be due to response in the arm tip that is triggered by photo-sensation. This differences in photoreceptor density. Another explanation could be reflex-like behavior adapts to environmental light conditions and that the phototaxis mechanism is restricted to the tip of the arm and appears to play a functional role for the octopus. Yet, the question is the responses counted after illuminating the middle part of the arm what could be the role of this light sensitive response in a natural were due to scattered light that stimulated the tip. Importantly, these setting? results are in contrast to LACE, which can be initiated at different locations along the arm. Suggested role for the arm phototaxis mechanism Next, we tested the adaptability of the phototactic response to Full awareness of eight long and exceptionally flexible arms prolonged change in the environmental illumination. Maintaining requires a huge and impractical workload on the nervous system. It the octopuses in minimal light condition for 1–2 weeks resulted in is accepted that octopuses possess limited proprioceptive sensation decrease of the response latency (Fig. 3A). The decrease in response latency did not occur after 1 h in darkness (Fig. S1), which ruled out Table 1. Summary of the differences and similarities between LACE and short term modifications. In addition, measurements of latency 1 phototactic response revealed by a series of surgical manipulations month after returning to regular light conditions revealed reversibility of the mechanism as it returned to the initial value Phototactic Phenomenon LACE response (Fig. 3A). In contrast to the decrease of the response latency (greater reactivity) due to prolonged darkness, there is a decrease in Intact octopus + + − sensitivity (the light intensity that elicited response) as reflected by Anesthetized intact octopus Weak Removal of supraoesophageal lobes and + − the ELI50 test (Fig. 3B). These dichotomous effects of a decrease in disconnecting optic tract sensitivity along with an increase in reactivity, suggest a separate Transection of the arm’s nerve cord − − source for each mechanism. The decrease in sensitivity may occur Peripheral incision through the skin only + + due to alterations at the receptor level. For instance, there are several Peripheral incision through the skin and muscle + − reports mainly from studies on invertebrates (Bloom and Atwood, Arm amputation (isolated arm) + −

1981; Eguchi and Waterman, 1979; Meyer-Rochow, 2001; Röhlich +, response; −, no response. Journal of Experimental Biology

5 RESEARCH ARTICLE Journal of Experimental Biology (2021) 224, jeb237529. doi:10.1242/jeb.237529 for their arms (Wells, 1978; Zullo et al., 2009). Consequently, the N.N.; Writing - original draft: I.K., T.S., N.N.; Writing - review & editing: I.K., T.S., octopus is poorly or not at all aware of its arms’ location and posture N.N.; Supervision: T.S., N.N.; Project administration: T.S., N.N.; Funding acquisition: T.S., N.N. when they are out of sight (Gutnick et al., 2011) Although, recently, Gutnick et al. (2020) broadened this view. Previous works have Funding revealed unique and efficient strategies evolved in the octopus that This work was supported by the Israel Science Foundation (ISF) No. 1767/17. allow handling of the arms, independently or with minimum involvement of the CNS (Hochner, 2012; Levy et al., 2016). For Supplementary information example, goal-directed movement (Sumbre et al., 2005, 2001), Supplementary information available online at https://jeb.biologists.org/lookup/doi/10.1242/jeb.237529.supplemental withdrawal response (Hague et al., 2013) and decision making-like behavior (Altman, 1971; Nesher et al., 2014). References During observations of octopus in our laboratory we noticed a Altman, J. S. (1971). Control of accept and reject reflexes in the octopus. Nature recurring behavior that points to the physiological relevancy of arm 229, 204-206. doi:10.1038/229204a0 tip phototaxis mechanism. During the daytime, octopuses kept Bleckmann, H., Budelmann, B. U. and Bullock, T. H. (1991). Peripheral and central nervous responses evoked by small water movements in a cephalopod. theirs arms folded (Movie 3) while at night their arms are spread and J. Comp. Physiol. [A] 168, 247-257. doi:10.1007/BF00218417 fully extended (Movie 3, at 50 s). Several public domain videos (e.g. Bloom, J. W. and Atwood, H. L. (1981). Reversible ultrastructural changes in the YouTube) of octopuses in the wild that were captured during rhabdom of the locust eye are induced by long term light deprivation. J. Comp. daytime or night, also demonstrated this same alternating behavior. Physiol. 144, 357-365. doi:10.1007/BF00612567 Bone, Q., Brown, E. R. and Usher, M. (1995). The structure and physiology of We cannot completely discount the possibility that the phototactic cephalopod muscle fibres. In Cephalopod Neurobiology (ed. N. J. Abbott, R. response is a side effect of strong LACE that causes an aversive Williamson and L. Maddock), pp. 301-329. London: Oxford University Press. sensation due to drastic chromatophore expansion. However, the Buresch, K. C., Ulmer, K. M., Akkaynak, D., Allen, J. J., Mäthger, L. M., results and observations presented here, led us to hypothesize that this Nakamura, M. and Hanlon, R. T. (2015). Cuttlefish adjust body pattern intensity reflex-like behavior is actually a functional mechanism that might with respect to substrate intensity to aid camouflage, but do not camouflage in extremely low light. J. Exp. Mar. Biol. Ecol. 462, 121-126. doi:10.1016/j.jembe. prevent the tip of the arms from being exposed to daytime predators 2014.10.017 such as fish and crabs that could identify the worm-like tips of the Cronin, T. W. and Johnsen, S. (2016). Extraocular, non-visual, and simple arms as food (such as the worm-like lure of the tasseled anglerfish). photoreceptors: an introduction to the symposium. Integr. Comp. Biol. 56, The fact that in contrast to LACE, the phototactic response is 758-763. doi:10.1093/icb/icw106 Eguchi, E. and Waterman, T. H. (1979). Longterm dark induced fine structural controlled by the CNS, allows the octopus to over-ride the instinctive changes in crayfish photoreceptor membrane. J. Comp. Physiol. 131, 191-203. response and to use its arms tips when needed in a goal-directed doi:10.1007/BF00610428 movement. Indeed, our observations revealed that when the octopus Florey, E. (1966). Nervous control and spontaneous activity of the chromatophores identified food by sight, in some cases (depending on the strength of of a cephalopod, Loligo opalescens. Comp. Biochem. Physiol. 18, 305-324. doi:10.1016/0010-406X(66)90189-7 illumination and the state of the octopus) it will extend its arm tip and Fossati, S. M., Carella, F., De Vico, G., Benfenati, F. and Zullo, L. (2013). Octopus reach for the food although it is being illuminated. arm regeneration: role of acetylcholinesterase during morphological modification. Interestingly, Ramirez and Oakley (2015) identified the LACE- J. Exp. Mar. Biol. Ecol. 447, 93-99. doi:10.1016/j.jembe.2013.02.015 activated opsin GPCRs of the octopus, on ciliated sensory cells that Gutnick, T., Byrne, R. A., Hochner, B. and Kuba, M. (2011). Octopus vulgaris uses visual information to determine the location of its arm. Curr. Biol. 21, have been suggested to be mechanoreceptors, homologous to those 460-462. doi:10.1016/j.cub.2011.01.052 in the fish lateral line system (Bleckmann et al., 1991). It is worth Gutnick, T., Zullo, L., Hochner, B. and Kuba, M. J. (2020). Use of peripheral mentioning that recently, non-visual and light-independent sensory information for central nervous control of arm movement by Octopus functions such as temperature sensation (Leung and Montell, vulgaris. Curr. Biol. 30, 4322-4327.e3. doi:10.1016/j.cub.2020.08.037 2017), mechanosensation for hearing (Senthilan et al., 2012) and Hague, T., Florini, M. and Andrews, P. L. R. (2013). Preliminary in vitro functional evidence for reflex responses to noxious stimuli in the arms of Octopus vulgaris. ciliated proprioceptors (Zanini et al., 2018) have been identified for J. Exp. Mar. Biol. Ecol. 447, 100-105. doi:10.1016/j.jembe.2013.02.016 opsin in the fruit fly Drosophila melanogaster. Therefore, it is not Hochner, B. (2012). An embodied view of octopus neurobiology. Curr. Biol. 22, inconceivable that the phototactic response is initiated by opsin R887-R892. doi:10.1016/j.cub.2012.09.001 receptors stimulated by light to elicit a neural circuit that ends with a Kingston, A. C., Kuzirian, A. M., Hanlon, R. T. and Cronin, T. W. (2015). Visual phototransduction components in cephalopod chromatophores suggest dermal motoric response. photoreception. J. Exp. Biol. 218, 1596-1602. doi:10.1242/jeb.117945 In conclusion, this work revealed another extraocular photoreception Leung, N. Y. and Montell, C. (2017). Unconventional roles of opsins. Annu. Rev. mechanism that may provide a further piece in the puzzle of how the Cell Dev. Biol. 33, 241-264. doi:10.1146/annurev-cellbio-100616-060432 octopus controls its arms using a cost-effective computational load and Levy, G., Nesher, N., Zullo, L. and Hochner, B. (2016). Motor control in soft bodied animals-the octopus. prevents its exposure to risk given its limited proprioceptive abilities. Liu, T.-H. and Chiao, C.-C. (2017). Mosaic organization of body pattern control in the optic lobe of squids. J. Neurosci. 37, 768-780. doi:10.1523/JNEUROSCI. Acknowledgements 0768-16.2016 We thank Tal Eyal, Eden Goldfarb, Ivgeni Tsigalnitski and Roi Siegelman for their Messenger, J. (2001). Cephalopod chromatophores: neurobiology and natural preliminary contribution in the framework of annual project during their BSc studies. history. Biol. Rev. Camb. Philos. Soc. 76, 473-528. doi:10.1017/ We thank Michael Apfelbaum for the drawings in the paper in Figs 1 and 2. We thank S1464793101005772 Mai Sadeh and Maria Roubanov for help with movie analysis. We would also like to Meyer-Rochow, V. B. (2001). The crustacean eye: dark/light adaptation, thank Rafi Yavetz and Arik Weinberger from the Maritime Aquaculture Department at polarization sensitivity, flicker fusion frequency, and photoreceptor damage. Ramot-Yam High School, for their help with octopus maintenance and the Zoolog. Sci. 18, 1175-1197. doi:10.2108/zsj.18.1175 experimental apparatus design and setup. We thank Roxanne Halper for editorial Nesher, N., Levy, G., Grasso, F. W. and Hochner, B. (2014). Self-recognition assistance. Lastly, we would like to thank Benny Hochner for fruitful discussions. mechanism between skin and suckers prevents octopus arms from interfering with each other. Curr. Biol. 24, 1271-1275. doi:10.1016/j.cub.2014.04.024 Competing interests Packard, A. and Brancato, D. (1993). Some responses of Octopus The authors declare no competing or financial interests. chromatophores to light. J. Physiol. 459, 429. Ramirez, M. D. and Oakley, T. H. (2015). Eye-independent, light-activated Author contributions chromatophore expansion (LACE) and expression of phototransduction genes Conceptualization: I.K., T.S., N.N.; Methodology: I.K., T.S., N.N.; Software: I.K.; in the skin of Octopus bimaculoides. J. Exp. Biol. 218, 1513-1520. doi:10.1242/

Formal analysis: I.K., T.S., N.N.; Investigation: I.K., T.S., N.N.; Resources: T.S., jeb.110908 Journal of Experimental Biology

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