Verification of Tetrodotoxin Utilization Against Predators in Japanese Blue-lined Octopus Hapalochlaena Cf. Fasciata

Verification of Tetrodotoxin Utilization Against Predators in Japanese Blue-lined Octopus Hapalochlaena Cf. 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Fasciata Yuta Yamate, Tomohiro Takatani, Takeshi Takegaki This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3913047/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Many taxa secrete chemicals to avoid predation. The Japanese blue-lined octopus Hapalochlaena cf. fasciata has high levels of potent lethal tetrodotoxin (TTX) in the muscles and skin; thus, it has been hypothesized that TTX is a defense mechanism. However, this hypothesis is based on the relationship between the location and level of TTX possession, and it has not been verified whether TTX is actually secreted in response to predators. In determining whether the external secretion of chemicals is a predator avoidance behavior, TTX must be verified as targeted to predators. In this study, TTX concentrations in the arms (muscle and skin) of octopus decreased after 3 days of predator (moray eel) presentation. In addition, TTX was only secreted in the mucus on the body surface of the octopus in the presence of a predator. Our findings showed that octopuses secrete TTX in the muscle and skin for defense, indicating that H. cf. fasciata does not necessarily require a physical contact attack by the predator to stimulate TTX secretion and can recognize predators by visual or olfactory stimuli, secreting TTX in response. Toxic octopus chemical defense moray eel antipredator defense defensive secretion Figures Figure 1 Figure 2 INTRODUCTION Predators are a threat to prey animals, making them the driving force behind the evolution of defensive traits (Vermeij 1982 ; Brodie and Brodie 1999 ). The possession of toxic or unpleasant chemical substances is a common defensive measure in many taxa (Speed et al. 2012 ; Sugiura 2020 ; Jared et al. 2021 ). For example, several venomous fish have adapted to a benthic lifestyle and erect venomous spines when threatened. When these dorsal spines pierce a predator’s body, the tip of the spine injects a painful proteinaceous toxin (Ziegman and Alewood 2015 ). Many animal species deter predators by secreting toxins from their body surface. Sea cucumbers (family: Holothuriidae) release sticky net-like organs called Cuvierian tubules in response to predator attacks. These tubules not only restrict predator movement but also contain repellent substances (saponins) that deter predator attack (Van Dyck et al. 2010 ). Some species of Dendrobatidae ingest alkaloids by preying, secreting them through their skin (Dumbacher et al. 2004 ; Saporito et al. 2004 ; Takada et al. 2005 ). Over 200 alkaloids have been detected in the skin of Dendrobates pumilio (Saporito et al. 2007 ). Alkaloids may be highly toxic and extremely bitter depending on their composition, and they may deter predator attacks (Saporito et al. 2007 ). The secretion of toxins or unpalatable substances outside the body may not always be for defensive purposes. For example, saponins secreted by sea cucumbers not only deter predator attacks but also attract the small symbiotic crab Lissocarcinus orbicularis (Caulier et al. 2013 ). The grass pufferfish Takifugu niphobles secretes tetrodotoxin (TTX), a potent neurotoxin, from its body surface. However, TTX is released from the cloaca of mature females to attract mature males (Matsumura 1995 ). If the toxin has functions other than defense, then the association between toxin release and the presence of predators should be examined. The association between toxin release and physical stimuli has been investigated in many taxa, but whether the presence of a predator triggers toxin release remains unclear. For example, some species of TTX-bearing pufferfish ( T. pardalis , T. porphyreus , T. flavipterus , T. niphobles , and T. vermicularis ) release TTX from the epidermis upon electrical stimuli (Kodama et al. 1986 ) or handling stimuli (Saito et al. 1985 ), and this response may be a defense mechanism (Kodama et al. 1986 ). The release of toxin in response to a physical stimulus may stop a predator’s attack, but if the attack can be stopped earlier, then the risk of fatal injury is further reduced. Therefore, clarifying when organisms release toxins in defense is important because failure to do so can lead to death. TTX has a wide range of taxa, and this toxin is often associated with predator defense (Noguchi and Arakawa 2008 ). Japanese newts ( Cynops pyrrhogaster ) are known to release TTX from their skin upon frictional stimulation with gauze (Tsuruda et al. 2002 ). The epidermis of the eastern newt Notophthalmus viridescens contains TTX, making it repellent to fish and crustacean predators, and the higher the TTX concentration, the higher the probability of deterring predators (Marion and Hay 2011 ). The TTX-carrying rough-skinned newt Taricha granulosa is sympatrically distributed with the common garter snake Thamnophis sirtalis , a TTX-resistant predator. Areas with newts possessing low TTX levels tend to have snakes with low TTX resistance, and areas with newts possessing high TTX levels tend to have snakes with high TTX resistance. This is a known example of an arm race in which the toxicity of newts increased to avoid predation by snakes, and the resistance of snakes increased so that they can prey on newts repeatedly competing against each other (Brodie and Brodie 1990 ; Brodie and Brodie 1999 ; Hanifin et al. 2008 ; Williams et al. 2010 ). Wild juveniles of tiger pufferfish ( T. rubripes ; toxic) were reported to have a higher survival rate compared with hatchery juveniles (nontoxic) in the same environment (Shimizu et al. 2007 , 2008 ). Gustatory cells of rainbow trout ( Oncorhynchus mykiss ) and Arctic char ( Salvelinus alpinus ) can detect low levels of TTX, which may result in the avoidance of toxic prey that secretes TTX (Yamamori et al. 1988 ; Yamashita et al. 2006 ; Hara 2011 ). Hapalochlaena is the only cephalopod genus that has TTX (Sheumack et al. 1978 ; Yotsu-Yamashita et al. 2007 ; Williams and Caldwell 2009 ; Williams et al. 2011a , b ; Williams et al. 2012 ; Wu et al. 2014 ; Asakawa et al. 2019 ; Yamate et al. 2021 ; Zhang et al. 2023 ; Kim et al. 2023 ). In the blue-lined octopus ( Hapalochlaena fasciata , including H. cf. fasciata in Japan), the highest TTX concentration is found in the posterior salivary glands, and the highest total amount of TTX is contained in the muscle and skin (Williams and Caldwell 2009 ; Yamate et al. 2021 ). In general, the venom in the posterior salivary glands of cephalopods is used for foraging (Ponte and Modica 2017 ). Furthermore, TTX is highly toxic to small crabs (Yamamori et al. 1992 ). Thus, TTX in the posterior salivary glands of Hapalochlaena spp. was used to paralyze prey organisms (Williams 2010 ). However, considering that TTX is highly lethal to some non-TTX-bearing species of carnivorous fish (Saito et al. 1985 ), TTX in the posterior salivary glands of Hapalochlaena spp. may be injected during predator counterattacks. In addition, TTX present in the muscle and skin may be used for defense, but whether TTX secretion occurs in response to the presence of predators has yet to be investigated. Williams et al. ( 2011b ) tested whether hatchlings of the greater blue-ringed octopus ( H. lunulata ), which possesses TTX inherited from their mother, are preyed upon by various predator species. Most predators avoided the hatchlings, but those predators fed food different from the hatchling that contained the same level of TTX as the hatchlings preyed upon them. Therefore, the mucus contains unknown unpleasant substances or toxins other than TTX, and predation may be averted by the hatchling behavior in the predator’s mouth (Williams et al. 2011b ). TTX distribution in the muscle and skin of adult H. fasciata and H. lunulata has been investigated in detail using fluorescent immunostaining for TTX, but no glandular structures were identified as responsible for secreting TTX outside the body (Williams et al. 2012 ). Whether TTX is secreted from the body surface against actual predators must be confirmed to clarify the predator defense function of TTX present in the muscle and skin. Thus, this study aimed to confirm whether TTX concentrations in the muscle and skin decreased in the presence of a predator and whether TTX was contained in the mucus on the body surface following predator presentation. METHODS AND MATERIALS Sampling. H. cf. fasciata were sampled from an artificial fish reef located at a depth of approximately 10 m in Nagasaki (Nagasaki Prefecture, Japan). Two experiments were performed in this study. Sixteen individuals were used in Experiment 1, including 12 individuals collected by scuba divers on September 27, 2019, and December 9, 2020, two collected by fishermen, and two that were provided by other researchers (both collected in Nagasaki Prefecture). In Experiment 2, six individuals collected from the same artificial fish reef on September 13, 2021, and November 17, 2021, were used. Each octopus was housed individually in a small rearing case (W 175 × D 105 × H 105 mm) and submerged in a 300-L trough filled with artificial seawater (salinity: 33‰, water temperature: 20°C). Experiment 1. We examined whether exposure to predators in an aquarium over a 3-day period reduced TTX concentrations in the arms of H. cf. fasciata. The experiment was conducted in a 90-cm-wide glass aquarium (W 900 mm × D 300 mm × H 360 mm) divided into two sections of 30 and 60 cm each by a clear acrylic plate with small holes. Before the experiment commenced, TTX was orally administered to 16 H. cf. fasciata to sufficiently increase TTX levels in the body of all individuals and to compensate for any individuals with low TTX levels at the time of collection. If the TTX level was too low, then changes under the experimental conditions might be below the level of detection. TTX was administered by injecting crab surimi mixed with TTX into frozen crabs with a carapace length of 1–2 cm, and all octopuses were fed the TTX-injected crabs until they stopped eating. The final amount of TTX administered ranged from 60 to 150 µg, depending on the number of crabs the octopus has eaten. In confirming the level of TTX in the muscle and skin before the experiment, all octopuses were anesthetized 24 h after TTX administration with 1% ethanol seawater (Andrews and Tansey 1981 ; Ikeda et al. 2009 ), and tissue samples from the arm tips (approximately 0.1 g) were collected to measure TTX levels. In previous research, TTX levels in the arm and mantle were not markedly different (Yamate et al. 2021 ). Therefore, TTX levels in the arm may reflect TTX levels in the muscle and skin of the whole body. Once the octopuses had recovered from the anesthesia, they were allocated to the predator exposure group (n = 8) or the control group (n = 8). In the predator exposure group, a moray eel ( Gymnothorax kidako ) was introduced into the 60-cm compartment of the experimental tank, and the octopus was exposed to this predator for 3 days. In the control group, the octopus was kept for 3 days without a predator in the 60-cm compartment. At the end of the experiment, all octopuses were euthanized in 5% ethanol seawater (Andrews and Tansey 1981 ; Ikeda et al. 2009 ), and a portion of the arm tissue (approximately 0.1 g) was collected for TTX extraction. Experiment 2. This experiment aimed to investigate the presence of TTX in the mucus on the body surface after predator exposure. It was conducted in an experimental tank (W 450 mm × D 300 mm × H 300 mm) at a depth of 25 cm. The two experimental tanks of the system described in Experiment 1 were prepared per individual, one for predator exposure and the other for predator nonexposure (control). The same moray eels were used as predators in both experiments. Each octopus was housed in a small case (W 55 mm × D 45 mm × H 90 mm) made of clear acrylic plates with small holes, and the lid was closed with Parafilm to prevent them from escaping. Then, the case was hung by a string and suspended in the nonexposure tank for 10 min as the acclimation time and withdrawn from the tank after an additional 10 min. Next, the case was placed in a bath filled with seawater to a depth of approximately 2 cm. Then, the case was opened, and mucus was collected from the body surface of the octopus, with only the dorsal mantle of the octopus exposed out of the seawater. The mucus was collected using gauze (1 cm × 1 cm) held by tweezers, which was slowly rubbed over 1 cm of the dorsal mantle of the octopus before turning the gauze over and rubbing the mantle again in the same manner. After collecting the mucus, the lid of the case was closed and sealed with Parafilm, and then the case was suspended in the predator exposure tank for 10 min for acclimation. Thereafter, a moray eel was introduced as a predator and exposed to the octopus for 10 min. Then, the case was pulled up again, and mucus was collected as previously described. In this experiment, the octopus may unintentionally secrete TTX because of contact stimulation by mucus collection rather than the presence of a predator. Therefore, after the first predator presentation, the series of steps from the nonexposure condition to the predator exposure condition was repeated. If no TTX was detected in the second nonexposure condition and TTX was detected in the predator presentation condition, then TTX was secreted by the octopus in response to the presence of the predator rather than in response to the contact stimuli associated with mucus collection. TTX Analysis. TTX was extracted by adding 300 µL of 0.1% acetic acid per 0.1 g of arm tissue collected in Experiment 1 and 200 µL of 0.1% acetic acid to each mucus sample collected in Experiment 2. The mixture was homogenized by using an ultrasonic crusher and then heated for 10 min at 100°C in a heating block (CTU-Neo, Taitec, Aichi, Japan). After cooling the sample, it was centrifuged at 13,000 × g for 15 min. Then, the supernatant was filtered through a filter (FILTSTAR Syringe Filter Hydrophilic Nylon, Hawach Scientific, Xi’an City, China), transferred to a vial, and stored at − 30°C until assayed. The amount of TTX in each sample was quantified using liquid chromatography mass spectrometry (LC/MS, Online Resource 1) system. The limit of detection and the limit of quantification were 0.0009 nmol/mL (0.003 nmol/g tissue; signal-to-noise ratio [S/N] = 3) and 0.003 nmol/mL (0.09 nmol/g tissue; S/N = 10), respectively. Statistical Analysis . In Experiment 1, multiple regression analysis was conducted using a generalized linear mixed model (GLMM) with TTX concentration in the arm tissue as the response variable, the presence/absence of predator exposure and pre- and post-experiment as explanatory variables, and individual ID as the random effect. P values were calculated using likelihood ratio tests to determine whether the presence or absence of predator presentation was associated with pre- and post-experiment. In Experiment 2, the TTX concentration in the mucus was assumed to be zero if it was below the limit of detection. Multiple regression analysis was conducted using a GLMM with TTX concentration in mucus as the response variable, the presence or absence of predator exposure and the experimental cycle indicating the first (none, 1; presence, 1) and second (none, 2; presence, 2) presentation conditions as explanatory variables, and individual ID as the random effect. P values were calculated by performing likelihood ratio tests for significant effects of predator presentation and experimental cycle. RESULTS Experiment 1. In the group that was not exposed to the predator, the TTX concentration in the arms increased because of the oral administration of TTX. On the contrary, the TTX concentration in the arms decreased in the group exposed to the predator (Fig. 1 ). A significant association was found between the presence or absence of predator presentation and the experimental phase ( P = 0.019), indicating that more TTX was consumed in the predator-exposed condition than was acquired by oral administration (Table 1 ). Table 1 Results of multiple regression analysis of the effect of predator presentation and experimental stage to explain the TTX concentration in samples of arm tissue (ng/g) Variable Estimate Standard error t value Pr (Chi) Intercept 87.95 36.89 2.384 - Presence of predator 129.16 52.18 2.475 - Experimental stage 37.11 42.36 0.876 - Presence of predator: experimental stage −143.64 59.9 −2.398 0.0190 Experiment 2. In 13 of the 20 cases, TTX was detected in the mucus after exposure to the predator. However, TTX was not detected in the mucus of any cases after exposure to predator-free tanks (Fig. 2 ). The main effect of the presence of predator on TTX concentration in mucus was statistically significant ( P < 0.001), indicating that TTX in the muscle and skin was only secreted when a predator was presented (Table 2 ). Table 2 Results of multiple regression analysis of the effect of predator presentation and experimental cycle to explain the TTX concentration in the mucus samples collected from the body surface (ng/g) Variable Estimate Standard error t value Pr (Chi) Intercept 7.852 1.693 4.637 - Presence of predator −7.205 1.872 −3.849 3.259E-04 Experiment cycle −1.294 1.872 −0.691 0.476 DISCUSSION Although many animals contain toxins in their body surface mucus (Noguchi and Arakawa 2008 ; Jared et al. 2021 ), with the exception of some species that spray toxins from their body surface (Brodie and Smatresk 1990 ; Jared et al. 2011; Mailho-Fontana et al. 2014), their secretion is generally not observable to the naked eye. In addition, studies examining whether their toxins are defense traits against predators are limited (Jared et al. 2021 ). This study experimentally showed that TTX was present in the mucus on the body surface immediately after predator exposure (Fig. 2 ), and TTX concentrations in the arms of H. cf. fasciata decreased after exposure to the predator (Fig. 1 ). These results indicate that TTX is secreted into the mucus from the arm muscles and epidermis in response to the presence of predators and is released (consumed) from the body, strongly suggesting that the TTX present in the arms of H. cf. fasciata is used to induce defense against predators. The results of this study highlight several characteristics regarding the defensive use of TTX by H. cf. fasciata . One of these is that TTX secretion into the body surface mucus of this species does not require physical contact stimulation with a predator. In general, the release of chemicals by poisonous organisms against predators is a passive defense, as it occurs simultaneously with or after an attack (Sugiura and Sato, 2018 ; Sugiura 2020 ; Jared et al. 2021 ). Toxin release in amphibians is passive because the poison glands on the body lack muscles. Considering that H. cf. fasciata and moray eel were separated by a clear acrylic plate with small holes, this species actively secretes TTX from its body surface when it recognizes the presence of a predator by visual or olfactory cues. When the moray eel approached the octopus in this experiment, the characteristic blue ring and black stripes that develop during vigilance (Mäthger et al. 2012 ) indicate that the octopus recognized the predator as a danger. Although TTX secreted into the mucus by H. cf. fasciata could be dissolved into seawater, the predator moray eels did not exhibit neither typical TTX intoxication reactions such as dyspnea or paralysis nor behaviors such as avoidance of the octopus observed in this study experiment, which was conducted in a closed aquarium. Therefore, TTX dissolved in the body surface mucus of this species may discourage attack against spatially distant predators prior to attack, as is known for fire salamander ( Salamandra salamandra ; Brodie and Smatresk 1990 ) and the smooth-sided toad Rhaebo guttatus (Mailho-Fontana et al. 2014). However, whether TTX dissolved in mucus or in seawater helps this species avoid predation remains unknown. The hatchling of the TTX-bearing grater blue-ringed octopus ( H. lunulata ) has been observed to be spit out immediately after being taken into the predator’s mouth (Williams et al. 2011b ). Rainbow trout ( O. mykiss ) and Arctic char ( S. alpinus ) are known to reject food containing TTX because they have gustatory receptors that can detect even trace amounts of TTX (Yamamori et al. 1988 ; Yamaha et al. 2006; Hara 2011 ). The TTX in the mucus of H. cf. fasciata may also play a role in minimizing the cost of injury in case of a predator attack. In fact, many adult H. cf. fasciata collected in the field have lost their arms (unpublished data), indicating that they may be able to avoid predation by only losing part of their arms when attacked by predators, although predator attack is not the only cause. The experimental results of this study also provide insights into the origin and internal transport of TTX possessed in this species. The TTX concentration in the arms of H. cf. fasciata in Experiment 1 increased with the oral administration of TTX (Fig. 1 , control group). Therefore, H. cf. fasciata absorbs TTX from its food and transports it to the muscle and skin via the digestive gland within 3 days. This result indicates that this species has a physiological mechanism for the accumulation of TTX in the body from outside the body, which would be one piece of evidence supporting the exogenous nature of TTX in this species. Similarly, orally administered TTX can accumulate in the body of the tiger puffer T. pardalis within 24 h (Gao et al. 2019 ) and the bivalve Paphies australis within 7–13 days (Biessy et al. 2021 ). By contrast, despite the administration of TTX, TTX levels in the arms of individuals exposed to predators decreased by about 30% in 3 days (Fig. 1 ). Although the effect of the length of exposure period must be considered, the magnitude of this percentage reduction indicates that the magnitude of the effect of predation pressure on the amount of TTX in the muscle and skin is not negligible. Substantial differences in individual TTX levels have been observed in H. cf. fasciata , with TTX concentrations in the muscle and skin ranging from 9.61 to 12,900 ng/g, and several individuals with undetectably low TTX levels have been found (Yamate et al. 2021 ). These individual differences may be partially influenced by predation pressure in the habitat. Declarations This study was reviewed and approved by the Fish Experiment Committee of the Faculty of Fisheries, Nagasaki University, in accordance with the Fish Experiment Guidelines (established in February 2016, approval no.: NF-0036). Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Funding: This research was financially supported by the Sasakawa Scientific Research Grant from The Japan Science Society (no. 2020–4013). Yuta Yamate has received research support from The Japan Science Society. Author Contribution Collection of octopus: Yuta Yamate and Takeshi Takegaki. Conception and design of experiments: Yuta Yamate and Takeshi Takegaki. Guidance on chemical analysis: Tomohiro Takatani. Perform experiments and chemical analysis: Yuta Yamate. Writing manuscripts: Yuta Yamate. All authors read and approved the manuscript. ACKNOWLEDGMENTS We thank the members of the Nomozaki Sanwa Fishery Cooperative Association and Mr. Endo of SNC Corporation for allowing us to survey the reefs for the collection of octopus specimens. This research was financially supported by the Sasakawa Scientific Research Grant from The Japan Science Society. References Andrews PLR, Tansey EM (1981) The effects of some anaesthetic agents in Octopus vulgaris . Comp Biochem Physiol C 70:241–247. https://doi.org/10.1016/0306-4492(81)90057-5 Asakawa M, Matsumoto T, Umezaki K, Kaneko K, Yu X, Gomez-Delan G, Tomano S, Noguchi T, Ohtsuka S (2019) Toxicity and toxin composition of the greater blue-ringed octopus Hapalochlaena lunulata from Ishigaki Island, Okinawa Prefecture, Japan. 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Am Soc Nat 120:701–720. https://doi.org/10.1086/284025 Williams BL, Caldwell RL (2009) Intra-organismal distribution of tetrodotoxin in two species of blue-ringed octopuses ( Hapalochlaena fasciata and H. lunulata ). Toxicon 54:345–353. https://doi.org/10.1016/j.toxicon.2009.05.019 Williams BL (2010) Behavioral and chemical ecology of marine organisms with respect to tetrodotoxin. Mar Drugs 8:381–398. https://doi.org/10.3390/md8030381 Williams BL, Hanifin CT, Brodie ED Jr., Brodie ED III (2010) Tetrodotoxin affects survival probability of rough-skinned newts (Taricha granulosa) faced with TTX-resistant garter snake predators (Thamnophis sirtalis). Chemoecology 20:285–290 Williams BL, Hanifin CT, Brodie ED Jr., Caldwell RL (2011a) Ontogeny of tetrodotoxin levels in blue-ringed octopus: maternal investment and apparent independent production in offspring of Hapalochlaena lunulata . J Chem Ecol 37:10–17. https://doi.org/10.1007/s10886-010-9901-4 Williams BL, Lovenburg V, Huffard CL, Caldwell RL (2011b) Chemical defense in pelagic octopuses paralarvae: tetrodotoxin alone does not protect individual paralarvae of the greater blue-ringed octopus ( Hapalochlaena lunulata) from common reef predators. Chemoecology 21:131–141. https://doi.org/10.1007/s00049-011-0075-5 Williams BL, Stark MR, Caldwell RL (2012) Microdistribution of tetrodotoxin in two species of blue-ringed octopuses ( Hapalochlaena lunulata and Hapalochlaena fasciata ) detected by fluorescent immunolabeling. Toxicon 60:1307–1313. https://doi.org/10.1016/j.toxicon.2012.08.015 Wu YJ, Lin CL, Chen CH, Hsieh CH, Jen HC, Jian SJ, Hwang DF (2014) Toxin and species identification of toxic octopus implicated into food poisoning in Taiwan. Toxicon 91:96–102. https://doi.org/10.1016/j.toxicon.2014.09.009 Yamamori K, Nakamura M, Matsui T, Hara T (1988) Gustatory responses to tetrodotoxin and saxitoxin in fish: a possible mechanism for avoiding marine toxins. Can J Fish Aquat Sci 45:2182–2186. https://doi.org/10.1139/f88-253 Yamamori K, Yamaguchi S, Maehara E, Matsui T (1992) Tolerance of shore crabs to tetrodotoxin and saxitoxin and antagonistic effect of their body fluid against the toxins. Bull Jpn Soc Sci Fish 58:1157–1162. https://doi.org/10.2331/suisan.58.1157 Yamashita S, Yamada T, Hara TJ (2006) Gustatory responses to feeding- and non-feeding-stimulant chemicals, with an emphasis on amino acids, in rainbow trout. J Fish Biol 68:783–800. https://doi.org/10.1111/j.0022-1112.2006.00965.x Yamate Y, Takatani T, Takegaki T (2021) Levels and distribution of tetrodotoxin in the blue-lined octopus Hapalochlaena fasciata in Japan, with special reference to within-body allocation. J Molluscan Stud 87:eyaa042. https://doi.org/10.1093/mollus/eyaa042 Yotsu-Yamashita M, Mebs D, Flachsenberger W (2007) Distribution of tetrodotoxin in the body of the blue-ringed octopus ( Hapalochlaena maculosa ). Toxicon 49:410–412. https://doi.org/10.1016/j.toxicon.2006.10.008 Zhang Y, Yamate Y, Takegaki T, Arakawa O, Takatani T (2023) Tetrodotoxin profiles in xanthid crab Atergatis floridus and blue-lined octopus Hapalochlaena cf. fasciata from the same site in Nagasaki, Japan. Toxins 15:193. https://doi.org/10.3390/toxins15030193 Ziegman R, Alewood P (2015) Bioactive components in fish venoms. Toxins 7:1497–1531. https://doi.org/10.3390/toxins7051497 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3913047","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":271243699,"identity":"81566765-3901-4f01-bd2b-a8a5991df53c","order_by":0,"name":"Yuta Yamate","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYDADfihtwAZlMBPQYMAg2UCyFoMDMBYhYHCAx/DTjZo/csa3mx+/+PHHzpiPgceA4UcNA7s5bi3G0jnHDIzN7hwzs+xtSzZjA2ph7DnGwGzZgFOLgXQOm0HithsJZga8Dcw2bPJvDBh4GxiYYU7FZsvvnH8G9ZtnpH8z/POn3gZsy1/8Wsykc9sMEgwkcowf87AdBjuMGZ8tkgfYyqxz+4wNZ9zIKWOWbTtuzMbAVnBY5pgETr/wHWDefDvnm5w8/4z0zR/f/Kk2nN/AvPHhmxqbZFwhpnD/BTwm2CRgLKCTJJJxxZB8A/sDGJv5A7KMHeFIHQWjYBSMghECAHTtUrJLqt29AAAAAElFTkSuQmCC","orcid":"","institution":"Nagasaki University","correspondingAuthor":true,"prefix":"","firstName":"Yuta","middleName":"","lastName":"Yamate","suffix":""},{"id":271243700,"identity":"300d69ac-a4b8-49d4-8b54-3ec61746b684","order_by":1,"name":"Tomohiro Takatani","email":"","orcid":"","institution":"Nagasaki University","correspondingAuthor":false,"prefix":"","firstName":"Tomohiro","middleName":"","lastName":"Takatani","suffix":""},{"id":271243701,"identity":"997f6fad-b068-4c6d-b71e-efd810d6b581","order_by":2,"name":"Takeshi Takegaki","email":"","orcid":"","institution":"Nagasaki University","correspondingAuthor":false,"prefix":"","firstName":"Takeshi","middleName":"","lastName":"Takegaki","suffix":""}],"badges":[],"createdAt":"2024-01-31 07:59:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3913047/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3913047/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50783886,"identity":"cce29abf-cccf-498f-82f8-3622fa7b9b0b","added_by":"auto","created_at":"2024-02-07 08:53:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":71723,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in TTX concentration in the arm tissue (ng/g) prior to predator exposure 3 days later. Left: predator exposure group (n = 8). Right: the control group (no predator exposure; n = 8).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3913047/v1/d3c3075c3874d286550c0f9d.png"},{"id":50783885,"identity":"48b8e7bb-0762-43bf-b4a1-f27cc397a975","added_by":"auto","created_at":"2024-02-07 08:53:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41266,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in TTX concentration in the mucus (ng/g) after exposure to predator-free tanks (control) and after predator exposure.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3913047/v1/4b3c1dcc49450ede3326e27c.png"},{"id":55848240,"identity":"a39096d9-3c1c-4b21-b500-44f090a741dd","added_by":"auto","created_at":"2024-05-04 14:39:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":555785,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3913047/v1/4c7f4eae-b7ce-4790-90d8-452ddc206e01.pdf"},{"id":50783887,"identity":"78282927-3824-4845-bad5-ed1a436cca98","added_by":"auto","created_at":"2024-02-07 08:53:36","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":28018,"visible":true,"origin":"","legend":"","description":"","filename":"onlineresource1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3913047/v1/9c81c98b0fac520db16f02ed.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eVerification of Tetrodotoxin Utilization Against Predators in Japanese Blue-lined Octopus \u003cem\u003eHapalochlaena Cf. Fasciata\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePredators are a threat to prey animals, making them the driving force behind the evolution of defensive traits (Vermeij \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Brodie and Brodie \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The possession of toxic or unpleasant chemical substances is a common defensive measure in many taxa (Speed et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sugiura \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jared et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For example, several venomous fish have adapted to a benthic lifestyle and erect venomous spines when threatened. When these dorsal spines pierce a predator\u0026rsquo;s body, the tip of the spine injects a painful proteinaceous toxin (Ziegman and Alewood \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Many animal species deter predators by secreting toxins from their body surface. Sea cucumbers (family: Holothuriidae) release sticky net-like organs called Cuvierian tubules in response to predator attacks. These tubules not only restrict predator movement but also contain repellent substances (saponins) that deter predator attack (Van Dyck et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Some species of Dendrobatidae ingest alkaloids by preying, secreting them through their skin (Dumbacher et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Saporito et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Takada et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Over 200 alkaloids have been detected in the skin of \u003cem\u003eDendrobates pumilio\u003c/em\u003e (Saporito et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Alkaloids may be highly toxic and extremely bitter depending on their composition, and they may deter predator attacks (Saporito et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe secretion of toxins or unpalatable substances outside the body may not always be for defensive purposes. For example, saponins secreted by sea cucumbers not only deter predator attacks but also attract the small symbiotic crab \u003cem\u003eLissocarcinus orbicularis\u003c/em\u003e (Caulier et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The grass pufferfish \u003cem\u003eTakifugu niphobles\u003c/em\u003e secretes tetrodotoxin (TTX), a potent neurotoxin, from its body surface. However, TTX is released from the cloaca of mature females to attract mature males (Matsumura \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). If the toxin has functions other than defense, then the association between toxin release and the presence of predators should be examined. The association between toxin release and physical stimuli has been investigated in many taxa, but whether the presence of a predator triggers toxin release remains unclear. For example, some species of TTX-bearing pufferfish (\u003cem\u003eT. pardalis\u003c/em\u003e, \u003cem\u003eT. porphyreus\u003c/em\u003e, \u003cem\u003eT. flavipterus\u003c/em\u003e, \u003cem\u003eT. niphobles\u003c/em\u003e, and \u003cem\u003eT. vermicularis\u003c/em\u003e) release TTX from the epidermis upon electrical stimuli (Kodama et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1986\u003c/span\u003e) or handling stimuli (Saito et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1985\u003c/span\u003e), and this response may be a defense mechanism (Kodama et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). The release of toxin in response to a physical stimulus may stop a predator\u0026rsquo;s attack, but if the attack can be stopped earlier, then the risk of fatal injury is further reduced. Therefore, clarifying when organisms release toxins in defense is important because failure to do so can lead to death.\u003c/p\u003e \u003cp\u003eTTX has a wide range of taxa, and this toxin is often associated with predator defense (Noguchi and Arakawa \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Japanese newts (\u003cem\u003eCynops pyrrhogaster\u003c/em\u003e) are known to release TTX from their skin upon frictional stimulation with gauze (Tsuruda et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The epidermis of the eastern newt \u003cem\u003eNotophthalmus viridescens\u003c/em\u003e contains TTX, making it repellent to fish and crustacean predators, and the higher the TTX concentration, the higher the probability of deterring predators (Marion and Hay \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The TTX-carrying rough-skinned newt \u003cem\u003eTaricha granulosa\u003c/em\u003e is sympatrically distributed with the common garter snake \u003cem\u003eThamnophis sirtalis\u003c/em\u003e, a TTX-resistant predator. Areas with newts possessing low TTX levels tend to have snakes with low TTX resistance, and areas with newts possessing high TTX levels tend to have snakes with high TTX resistance. This is a known example of an arm race in which the toxicity of newts increased to avoid predation by snakes, and the resistance of snakes increased so that they can prey on newts repeatedly competing against each other (Brodie and Brodie \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Brodie and Brodie \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Hanifin et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Williams et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Wild juveniles of tiger pufferfish (\u003cem\u003eT. rubripes\u003c/em\u003e; toxic) were reported to have a higher survival rate compared with hatchery juveniles (nontoxic) in the same environment (Shimizu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Gustatory cells of rainbow trout (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e) and Arctic char (\u003cem\u003eSalvelinus alpinus\u003c/em\u003e) can detect low levels of TTX, which may result in the avoidance of toxic prey that secretes TTX (Yamamori et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Yamashita et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Hara \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eHapalochlaena\u003c/em\u003e is the only cephalopod genus that has TTX (Sheumack et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Yotsu-Yamashita et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Williams and Caldwell \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Williams et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011a\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Williams et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Asakawa et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yamate et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the blue-lined octopus (\u003cem\u003eHapalochlaena fasciata\u003c/em\u003e, including \u003cem\u003eH.\u003c/em\u003e cf. \u003cem\u003efasciata\u003c/em\u003e in Japan), the highest TTX concentration is found in the posterior salivary glands, and the highest total amount of TTX is contained in the muscle and skin (Williams and Caldwell \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Yamate et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In general, the venom in the posterior salivary glands of cephalopods is used for foraging (Ponte and Modica \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, TTX is highly toxic to small crabs (Yamamori et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Thus, TTX in the posterior salivary glands of \u003cem\u003eHapalochlaena\u003c/em\u003e spp. was used to paralyze prey organisms (Williams \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, considering that TTX is highly lethal to some non-TTX-bearing species of carnivorous fish (Saito et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1985\u003c/span\u003e), TTX in the posterior salivary glands of \u003cem\u003eHapalochlaena\u003c/em\u003e spp. may be injected during predator counterattacks. In addition, TTX present in the muscle and skin may be used for defense, but whether TTX secretion occurs in response to the presence of predators has yet to be investigated. Williams et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011b\u003c/span\u003e) tested whether hatchlings of the greater blue-ringed octopus (\u003cem\u003eH. lunulata\u003c/em\u003e), which possesses TTX inherited from their mother, are preyed upon by various predator species. Most predators avoided the hatchlings, but those predators fed food different from the hatchling that contained the same level of TTX as the hatchlings preyed upon them. Therefore, the mucus contains unknown unpleasant substances or toxins other than TTX, and predation may be averted by the hatchling behavior in the predator\u0026rsquo;s mouth (Williams et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011b\u003c/span\u003e). TTX distribution in the muscle and skin of adult \u003cem\u003eH. fasciata\u003c/em\u003e and \u003cem\u003eH. lunulata\u003c/em\u003e has been investigated in detail using fluorescent immunostaining for TTX, but no glandular structures were identified as responsible for secreting TTX outside the body (Williams et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhether TTX is secreted from the body surface against actual predators must be confirmed to clarify the predator defense function of TTX present in the muscle and skin. Thus, this study aimed to confirm whether TTX concentrations in the muscle and skin decreased in the presence of a predator and whether TTX was contained in the mucus on the body surface following predator presentation.\u003c/p\u003e"},{"header":"METHODS AND MATERIALS","content":"\u003cp\u003e \u003cem\u003eSampling. H.\u003c/em\u003e cf. \u003cem\u003efasciata\u003c/em\u003e were sampled from an artificial fish reef located at a depth of approximately 10 m in Nagasaki (Nagasaki Prefecture, Japan). Two experiments were performed in this study. Sixteen individuals were used in Experiment 1, including 12 individuals collected by scuba divers on September 27, 2019, and December 9, 2020, two collected by fishermen, and two that were provided by other researchers (both collected in Nagasaki Prefecture). In Experiment 2, six individuals collected from the same artificial fish reef on September 13, 2021, and November 17, 2021, were used. Each octopus was housed individually in a small rearing case (W 175 \u0026times; D 105 \u0026times; H 105 mm) and submerged in a 300-L trough filled with artificial seawater (salinity: 33\u0026permil;, water temperature: 20\u0026deg;C).\u003c/p\u003e \u003cp\u003e \u003cem\u003eExperiment 1.\u003c/em\u003e We examined whether exposure to predators in an aquarium over a 3-day period reduced TTX concentrations in the arms of \u003cem\u003eH.\u003c/em\u003e cf. \u003cem\u003efasciata.\u003c/em\u003e The experiment was conducted in a 90-cm-wide glass aquarium (W 900 mm \u0026times; D 300 mm \u0026times; H 360 mm) divided into two sections of 30 and 60 cm each by a clear acrylic plate with small holes. Before the experiment commenced, TTX was orally administered to 16 \u003cem\u003eH.\u003c/em\u003e cf. \u003cem\u003efasciata\u003c/em\u003e to sufficiently increase TTX levels in the body of all individuals and to compensate for any individuals with low TTX levels at the time of collection. If the TTX level was too low, then changes under the experimental conditions might be below the level of detection. TTX was administered by injecting crab surimi mixed with TTX into frozen crabs with a carapace length of 1\u0026ndash;2 cm, and all octopuses were fed the TTX-injected crabs until they stopped eating. The final amount of TTX administered ranged from 60 to 150 \u0026micro;g, depending on the number of crabs the octopus has eaten. In confirming the level of TTX in the muscle and skin before the experiment, all octopuses were anesthetized 24 h after TTX administration with 1% ethanol seawater (Andrews and Tansey \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Ikeda et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), and tissue samples from the arm tips (approximately 0.1 g) were collected to measure TTX levels. In previous research, TTX levels in the arm and mantle were not markedly different (Yamate et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, TTX levels in the arm may reflect TTX levels in the muscle and skin of the whole body. Once the octopuses had recovered from the anesthesia, they were allocated to the predator exposure group (n\u0026thinsp;=\u0026thinsp;8) or the control group (n\u0026thinsp;=\u0026thinsp;8). In the predator exposure group, a moray eel (\u003cem\u003eGymnothorax kidako\u003c/em\u003e) was introduced into the 60-cm compartment of the experimental tank, and the octopus was exposed to this predator for 3 days. In the control group, the octopus was kept for 3 days without a predator in the 60-cm compartment. At the end of the experiment, all octopuses were euthanized in 5% ethanol seawater (Andrews and Tansey \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Ikeda et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), and a portion of the arm tissue (approximately 0.1 g) was collected for TTX extraction.\u003c/p\u003e \u003cp\u003e \u003cem\u003eExperiment 2.\u003c/em\u003e This experiment aimed to investigate the presence of TTX in the mucus on the body surface after predator exposure. It was conducted in an experimental tank (W 450 mm \u0026times; D 300 mm \u0026times; H 300 mm) at a depth of 25 cm. The two experimental tanks of the system described in Experiment 1 were prepared per individual, one for predator exposure and the other for predator nonexposure (control). The same moray eels were used as predators in both experiments. Each octopus was housed in a small case (W 55 mm \u0026times; D 45 mm \u0026times; H 90 mm) made of clear acrylic plates with small holes, and the lid was closed with Parafilm to prevent them from escaping. Then, the case was hung by a string and suspended in the nonexposure tank for 10 min as the acclimation time and withdrawn from the tank after an additional 10 min. Next, the case was placed in a bath filled with seawater to a depth of approximately 2 cm. Then, the case was opened, and mucus was collected from the body surface of the octopus, with only the dorsal mantle of the octopus exposed out of the seawater. The mucus was collected using gauze (1 cm \u0026times; 1 cm) held by tweezers, which was slowly rubbed over 1 cm of the dorsal mantle of the octopus before turning the gauze over and rubbing the mantle again in the same manner. After collecting the mucus, the lid of the case was closed and sealed with Parafilm, and then the case was suspended in the predator exposure tank for 10 min for acclimation. Thereafter, a moray eel was introduced as a predator and exposed to the octopus for 10 min. Then, the case was pulled up again, and mucus was collected as previously described. In this experiment, the octopus may unintentionally secrete TTX because of contact stimulation by mucus collection rather than the presence of a predator. Therefore, after the first predator presentation, the series of steps from the nonexposure condition to the predator exposure condition was repeated. If no TTX was detected in the second nonexposure condition and TTX was detected in the predator presentation condition, then TTX was secreted by the octopus in response to the presence of the predator rather than in response to the contact stimuli associated with mucus collection.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTTX Analysis.\u003c/em\u003e TTX was extracted by adding 300 \u0026micro;L of 0.1% acetic acid per 0.1 g of arm tissue collected in Experiment 1 and 200 \u0026micro;L of 0.1% acetic acid to each mucus sample collected in Experiment 2. The mixture was homogenized by using an ultrasonic crusher and then heated for 10 min at 100\u0026deg;C in a heating block (CTU-Neo, Taitec, Aichi, Japan). After cooling the sample, it was centrifuged at 13,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 min. Then, the supernatant was filtered through a filter (FILTSTAR Syringe Filter Hydrophilic Nylon, Hawach Scientific, Xi\u0026rsquo;an City, China), transferred to a vial, and stored at \u0026minus;\u0026thinsp;30\u0026deg;C until assayed. The amount of TTX in each sample was quantified using liquid chromatography mass spectrometry (LC/MS, Online Resource 1) system. The limit of detection and the limit of quantification were 0.0009 nmol/mL (0.003 nmol/g tissue; signal-to-noise ratio [S/N]\u0026thinsp;=\u0026thinsp;3) and 0.003 nmol/mL (0.09 nmol/g tissue; S/N\u0026thinsp;=\u0026thinsp;10), respectively.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStatistical Analysis\u003c/em\u003e. In Experiment 1, multiple regression analysis was conducted using a generalized linear mixed model (GLMM) with TTX concentration in the arm tissue as the response variable, the presence/absence of predator exposure and pre- and post-experiment as explanatory variables, and individual ID as the random effect. \u003cem\u003eP\u003c/em\u003e values were calculated using likelihood ratio tests to determine whether the presence or absence of predator presentation was associated with pre- and post-experiment.\u003c/p\u003e \u003cp\u003eIn Experiment 2, the TTX concentration in the mucus was assumed to be zero if it was below the limit of detection. Multiple regression analysis was conducted using a GLMM with TTX concentration in mucus as the response variable, the presence or absence of predator exposure and the experimental cycle indicating the first (none, 1; presence, 1) and second (none, 2; presence, 2) presentation conditions as explanatory variables, and individual ID as the random effect. \u003cem\u003eP\u003c/em\u003e values were calculated by performing likelihood ratio tests for significant effects of predator presentation and experimental cycle.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cem\u003eExperiment 1.\u003c/em\u003e In the group that was not exposed to the predator, the TTX concentration in the arms increased because of the oral administration of TTX. On the contrary, the TTX concentration in the arms decreased in the group exposed to the predator (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A significant association was found between the presence or absence of predator presentation and the experimental phase (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.019), indicating that more TTX was consumed in the predator-exposed condition than was acquired by oral administration (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of multiple regression analysis of the effect of predator presentation and experimental stage to explain the TTX concentration in samples of arm tissue (ng/g)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEstimate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStandard error\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003et\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePr (Chi)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntercept\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e87.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e36.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.384\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePresence of predator\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e129.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e52.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.876\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePresence of predator: experimental stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;143.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e59.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;2.398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0190\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eExperiment 2.\u003c/em\u003e In 13 of the 20 cases, TTX was detected in the mucus after exposure to the predator. However, TTX was not detected in the mucus of any cases after exposure to predator-free tanks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The main effect of the presence of predator on TTX concentration in mucus was statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating that TTX in the muscle and skin was only secreted when a predator was presented (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of multiple regression analysis of the effect of predator presentation and experimental cycle to explain the TTX concentration in the mucus samples collected from the body surface (ng/g)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEstimate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStandard error\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003et value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePr (Chi)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntercept\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.852\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.693\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.637\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePresence of predator\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;7.205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.872\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;3.849\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.259E-04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperiment cycle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;1.294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.872\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;0.691\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.476\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAlthough many animals contain toxins in their body surface mucus (Noguchi and Arakawa \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Jared et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), with the exception of some species that spray toxins from their body surface (Brodie and Smatresk \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Jared et al. 2011; Mailho-Fontana et al. 2014), their secretion is generally not observable to the naked eye. In addition, studies examining whether their toxins are defense traits against predators are limited (Jared et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This study experimentally showed that TTX was present in the mucus on the body surface immediately after predator exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and TTX concentrations in the arms of \u003cem\u003eH.\u003c/em\u003e cf. \u003cem\u003efasciata\u003c/em\u003e decreased after exposure to the predator (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results indicate that TTX is secreted into the mucus from the arm muscles and epidermis in response to the presence of predators and is released (consumed) from the body, strongly suggesting that the TTX present in the arms of \u003cem\u003eH.\u003c/em\u003e cf. \u003cem\u003efasciata\u003c/em\u003e is used to induce defense against predators.\u003c/p\u003e \u003cp\u003eThe results of this study highlight several characteristics regarding the defensive use of TTX by \u003cem\u003eH.\u003c/em\u003e cf. \u003cem\u003efasciata\u003c/em\u003e. One of these is that TTX secretion into the body surface mucus of this species does not require physical contact stimulation with a predator. In general, the release of chemicals by poisonous organisms against predators is a passive defense, as it occurs simultaneously with or after an attack (Sugiura and Sato, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sugiura \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jared et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Toxin release in amphibians is passive because the poison glands on the body lack muscles. Considering that \u003cem\u003eH.\u003c/em\u003e cf. \u003cem\u003efasciata\u003c/em\u003e and moray eel were separated by a clear acrylic plate with small holes, this species actively secretes TTX from its body surface when it recognizes the presence of a predator by visual or olfactory cues. When the moray eel approached the octopus in this experiment, the characteristic blue ring and black stripes that develop during vigilance (M\u0026auml;thger et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) indicate that the octopus recognized the predator as a danger.\u003c/p\u003e \u003cp\u003eAlthough TTX secreted into the mucus by \u003cem\u003eH.\u003c/em\u003e cf. \u003cem\u003efasciata\u003c/em\u003e could be dissolved into seawater, the predator moray eels did not exhibit neither typical TTX intoxication reactions such as dyspnea or paralysis nor behaviors such as avoidance of the octopus observed in this study experiment, which was conducted in a closed aquarium. Therefore, TTX dissolved in the body surface mucus of this species may discourage attack against spatially distant predators prior to attack, as is known for fire salamander (\u003cem\u003eSalamandra salamandra\u003c/em\u003e; Brodie and Smatresk \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) and the smooth-sided toad \u003cem\u003eRhaebo guttatus\u003c/em\u003e (Mailho-Fontana et al. 2014). However, whether TTX dissolved in mucus or in seawater helps this species avoid predation remains unknown. The hatchling of the TTX-bearing grater blue-ringed octopus (\u003cem\u003eH. lunulata\u003c/em\u003e) has been observed to be spit out immediately after being taken into the predator\u0026rsquo;s mouth (Williams et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011b\u003c/span\u003e). Rainbow trout (\u003cem\u003eO. mykiss\u003c/em\u003e) and Arctic char (\u003cem\u003eS. alpinus\u003c/em\u003e) are known to reject food containing TTX because they have gustatory receptors that can detect even trace amounts of TTX (Yamamori et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Yamaha et al. 2006; Hara \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The TTX in the mucus of \u003cem\u003eH.\u003c/em\u003e cf. \u003cem\u003efasciata\u003c/em\u003e may also play a role in minimizing the cost of injury in case of a predator attack. In fact, many adult \u003cem\u003eH.\u003c/em\u003e cf. \u003cem\u003efasciata\u003c/em\u003e collected in the field have lost their arms (unpublished data), indicating that they may be able to avoid predation by only losing part of their arms when attacked by predators, although predator attack is not the only cause.\u003c/p\u003e \u003cp\u003eThe experimental results of this study also provide insights into the origin and internal transport of TTX possessed in this species. The TTX concentration in the arms of \u003cem\u003eH.\u003c/em\u003e cf. \u003cem\u003efasciata\u003c/em\u003e in Experiment 1 increased with the oral administration of TTX (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, control group). Therefore, \u003cem\u003eH.\u003c/em\u003e cf. \u003cem\u003efasciata\u003c/em\u003e absorbs TTX from its food and transports it to the muscle and skin via the digestive gland within 3 days. This result indicates that this species has a physiological mechanism for the accumulation of TTX in the body from outside the body, which would be one piece of evidence supporting the exogenous nature of TTX in this species. Similarly, orally administered TTX can accumulate in the body of the tiger puffer \u003cem\u003eT. pardalis\u003c/em\u003e within 24 h (Gao et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and the bivalve \u003cem\u003ePaphies australis\u003c/em\u003e within 7\u0026ndash;13 days (Biessy et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). By contrast, despite the administration of TTX, TTX levels in the arms of individuals exposed to predators decreased by about 30% in 3 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although the effect of the length of exposure period must be considered, the magnitude of this percentage reduction indicates that the magnitude of the effect of predation pressure on the amount of TTX in the muscle and skin is not negligible. Substantial differences in individual TTX levels have been observed in \u003cem\u003eH.\u003c/em\u003e cf. \u003cem\u003efasciata\u003c/em\u003e, with TTX concentrations in the muscle and skin ranging from 9.61 to 12,900 ng/g, and several individuals with undetectably low TTX levels have been found (Yamate et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These individual differences may be partially influenced by predation pressure in the habitat.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThis study was reviewed and approved by the Fish Experiment Committee of the Faculty of Fisheries, Nagasaki University, in accordance with the Fish Experiment Guidelines (established in February 2016, approval no.: NF-0036).\u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting Interests:\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research was financially supported by the Sasakawa Scientific Research Grant from The Japan Science Society (no. 2020\u0026ndash;4013). Yuta Yamate has received research support from The Japan Science Society.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eCollection of octopus: Yuta Yamate and Takeshi Takegaki. Conception and design of experiments: Yuta Yamate and Takeshi Takegaki. Guidance on chemical analysis: Tomohiro Takatani. Perform experiments and chemical analysis: Yuta Yamate. Writing manuscripts: Yuta Yamate. All authors read and approved the manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGMENTS\u003c/h2\u003e \u003cp\u003eWe thank the members of the Nomozaki Sanwa Fishery Cooperative Association and Mr. Endo of SNC Corporation for allowing us to survey the reefs for the collection of octopus specimens. This research was financially supported by the Sasakawa Scientific Research Grant from The Japan Science Society.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAndrews PLR, Tansey EM (1981) The effects of some anaesthetic agents in \u003cem\u003eOctopus vulgaris\u003c/em\u003e. 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Toxins 7:1497\u0026ndash;1531. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/toxins7051497\u003c/span\u003e\u003cspan address=\"10.3390/toxins7051497\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Toxic octopus, chemical defense, moray eel, antipredator defense, defensive secretion","lastPublishedDoi":"10.21203/rs.3.rs-3913047/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3913047/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMany taxa secrete chemicals to avoid predation. The Japanese blue-lined octopus \u003cem\u003eHapalochlaena\u003c/em\u003e cf. \u003cem\u003efasciata\u003c/em\u003e has high levels of potent lethal tetrodotoxin (TTX) in the muscles and skin; thus, it has been hypothesized that TTX is a defense mechanism. However, this hypothesis is based on the relationship between the location and level of TTX possession, and it has not been verified whether TTX is actually secreted in response to predators. In determining whether the external secretion of chemicals is a predator avoidance behavior, TTX must be verified as targeted to predators. In this study, TTX concentrations in the arms (muscle and skin) of octopus decreased after 3 days of predator (moray eel) presentation. In addition, TTX was only secreted in the mucus on the body surface of the octopus in the presence of a predator. Our findings showed that octopuses secrete TTX in the muscle and skin for defense, indicating that \u003cem\u003eH.\u003c/em\u003e cf. \u003cem\u003efasciata\u003c/em\u003e does not necessarily require a physical contact attack by the predator to stimulate TTX secretion and can recognize predators by visual or olfactory stimuli, secreting TTX in response.\u003c/p\u003e","manuscriptTitle":"Verification of Tetrodotoxin Utilization Against Predators in Japanese Blue-lined Octopus Hapalochlaena Cf. Fasciata","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-07 08:53:31","doi":"10.21203/rs.3.rs-3913047/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"090054dc-67bf-4b07-98fa-e116f9b619b5","owner":[],"postedDate":"February 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-04T14:39:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-07 08:53:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3913047","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3913047","identity":"rs-3913047","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}