Blue-ringed Octopus Research Articles

Bioinspired multi-dimensional anti-counterfeiting device by combining electrochromism and structural color

Multi-dimensional anti-counterfeiting technologies possess multiple color/pattern variations under different conditions, opening up new avenues in information security. Herein, inspired by the blue-ringed octopus, novel multi-dimensional anti-counterfeiting devices are reported by combining electrochromism and structural color with different coloration mechanisms, that is, by introducing colloidal photonic crystals (PCs) in electrolyte of the electrochromic devices (ECDs). Through actively manipulating voltage or passively changing environment background, the degree of light reflectance and transmittance of PCs can be finely modulated, thus realizing the display and hiding of structural colors of devices. Besides, they also feature distinct color evolution under different viewing angles, enabling more optical information to be carried. Laser-etched patterned devices have also been successfully achieved, showcasing superior triple anti-counterfeiting abilities in terms of optical information. Notably, such inherent patterns can further be innovatively switched to different preset background patterns through the synergy of voltage stimulation and background colors. Based on this, a high-level anti-counterfeiting system has been demonstrated, providing the ability to deliver multiple distinct optical information on-demand. Itis believed that such strategy of combining electrochromic and structural color will pave a new platform for designing advanced multi-dimensional anti-counterfeiting devices.

Blue-ringed Octopus inspired slippery coating with physico-chemical synergistic antifouling properties

Bionic superhydrophobic and slippery liquid-infused porous surfaces (SLIPS) have attracted great interest due to their antifouling properties, while the poor stability, complex preparation of porous structures and rapid loss of lubricants, as well as only possessing physical antifouling capability, have hindered their further application. Herein, inspired by the Blue-ringed Octopus’ defensive equipment of slippery skin and toxin secretion when faced with predators, an antifouling slippery coating containing repellent is easily prepared. The repellent capsaicin physically connected to the slippery networks can be activated to accelerate release in response to the changes in pH caused by bacterial secretions, and the adhesion resistance of the coating is improved by 99 % compared to aluminum plates, both conclusions confirmed by experimental and computational data. The anti-adhesive properties of the slippery surface, combined with the repellent properties of capsaicin provides the coating with excellent synergistic physico-chemical antifouling performance. This is demonstrated by the fact that the surface of the coating remains clear after contamination with proteins, bacteria and diatoms, as well as after 65 days of marine field testing. Additionally, the slippery surface has self-healing and outstanding anticorrosion ability. Therefore, the eco-friendly coating has promising application prospects in marine field for antifouling purposes.

Blue-ringed octopus-inspired microneedle patch for robust tissue surface adhesion and active injection drug delivery.

Intratissue topical medication is important for the treatment of cutaneous, mucosal or splanchnic diseases. However, penetrating surface barriers to providing adequate and controllable drug delivery while guaranteeing adhesion in bodily fluids remains challenging. Here, the predatory behavior of the blue-ringed octopus inspired us with a strategy to improve topical medication. For effective intratissue drug delivery, the active injection microneedles were prepared in a manner inspired by the teeth and venom secretion of blue-ringed octopus. With on demand release function guided by temperature-sensitive hydrophobic and shrinkage variations, these microneedles can supply adequate drug delivery at an early stage and then achieve the long-term release stage. Meanwhile, the bionic suction cups were developed to facilitate microneedles to stay firmly in place (>10 kilopascal) when wet. With wet bonding ability and multiple delivery mode, this microneedle patch achieved satisfactory efficacy, such as accelerating the ulcers' healing speed or halting early tumor progression.

SNP data reveals the complex and diverse evolutionary history of the blue-ringed octopus genus (Octopodidae: Hapalochlaena) in the Asia-Pacific

The blue-ringed octopus species complex (Hapalochlaena spp.), known to occur from Southern Australia to Japan, currently contains four formally described species (Hapalochlaena maculosa, Hapalochlaena fasciata, Hapalochlaena lunulata and Hapalochlaena nierstraszi). These species are distinguished based on morphological characters (iridescent blue rings and/or lines) along with reproductive strategies. However, the observation of greater morphological diversity than previously captured by the current taxonomic framework indicates that a revision is required. To examine species boundaries within the genus we used mitochondrial (12S rRNA, 16S rRNA, cytochrome c oxidase subunit 1 [COI], cytochrome c oxidase subunit 3 [COIII] and cytochrome b [Cytb]) and genome-wide SNP data (DaRT seq) from specimens collected across its geographic range including variations in depth from 3 m to >100 m. This investigation indicates substantially greater species diversity present within the genus Hapalochlaena than is currently described. We identified 10,346 SNPs across all locations, which when analysed support a minimum of 11 distinct clades. Bayesian phylogenetic analysis of the mitochondrial COI gene on a more limited sample set dates the diversification of the genus to ∼30 mya and corroborates eight of the lineages indicated by the SNP analyses. Furthermore, we demonstrate that the diagnostic lined patterning of H. fasciata found in North Pacific waters and NSW, Australia is polyphyletic and therefore likely the result of convergent evolution. Several “deep water” (>100 m) lineages were also identified in this study with genetic convergence likely to be driven by external selective pressures. Examination of morphological traits, currently being undertaken in a parallel morphological study, is required to describe additional species within the complex.

Structural Characterization and Spatial Mapping of Tetrodotoxins in Australian Polyclads.

Tetrodotoxin (TTX) is a potent marine neurotoxin that occurs in several Australian phyla, including pufferfish, toadfish, gobies, and the blue-ringed octopus. These animals are partially immune, and TTX is known to bioaccumulate and subject to trophic transfer. As such, it could be more ubiquitously distributed in animals than is currently known. Flatworms of the order Polycladida are commonly occurring invertebrates in intertidal ecosystems and are especially diverse in Australian waters. While TTX has been identified in polyclads from Japan and New Zealand, Australian species have yet to be tested. In this study, several eastern Australian polyclad flatworm species from the suborders Cotylea and Acotylea were tested for TTX and analogs by HILIC-HRMS to understand the distribution of this toxin within these suborders. Herein, we report the detection of TTX and some known analogs in polyclad species, one of which is a pest to shellfish aquaculture. We also report, for the first time, the application of MALDI mass spectrometry imaging utilized to map TTX spatially within the intestinal system of polyclads. The identification of TTX and its analogs in Australian flatworms illustrates a broader range of toxic flatworms and highlights that analogs are important to consider when studying the distributions of toxins in animals.

High-Density Genetic Linkage Map of the Southern Blue-ringed Octopus (Octopodidae: Hapalochlaena maculosa)

Genetic linkage maps provide a useful resource for non-model genomes and can aid in genome reassembly to form more contiguous pseudo-chromosomes. We present the first linkage map of any cephalopod, H. maculosa, composed of 47 linkage groups (LG). A total of 2166 single nucleotide polymorphisms and 2455 presence–absence variant loci were utilised by Lep-Map3 in linkage map construction. The map length spans 2016.62 cM with an average marker distance of 0.85 cM. Integration of the recent H. maculosa genome allowed 1151 scaffolds comprising 34% of the total genomic sequence to be orientated and/or placed using 1278 markers across all 47 LG. The linkage map generated provides a new perspective on HOX gene distribution in octopods. In the H. maculosa linkage map three (SCR, LOX4 and POST1) of six identified HOX genes (HOX1/LAB, SCR, LOX2, LOX4, LOX5, POST1) were located within the same LG (LG 9). The generation of a linkage map for H. maculosa has provided a valuable resource for understanding the evolution of cephalopod genomes and will provide a base for future work.

How the blue-ringed octopus flashes its rings

The animals flex and relax fast-acting muscles to expose light reflectors hidden in specialized skin pouches.

A cluster of tetrodotoxin poisoning in Oman

Introduction Tetrodotoxin (TTX) is a potent sodium channel blocker, with significant neurotoxicity, found in marine animals like pufferfish and blue-ringed octopus. The severity of toxicity depends on the amount of toxin ingested and the outcome depends on the time-lapse to appropriate medical care. Cases report We report five patients who presented with tetrodotoxin poisoning after consuming fried internal organs of local pufferfish from the coast of Oman. The patients’ clinical manifestations were consistent with the expected TTX toxidrome of perioral and generalized paresthesia, weakness of upper and lower extremities, gastrointestinal manifestations, dyspnea, dysarthria, ascending paralysis, hypotension, bradycardia and coma. The severity varied among the patients who recovered completely except one patient who developed a subarachnoid hemorrhage without underlying aneurysms on computed tomography-angiogram. This complication was potentially related to TTX poisoning and has not been previously reported. In addition to standard supportive management, patients with severe illness should potentially receive the intravenous acetylcholinesterase inhibitor neostigmine, and intermittent dialysis. Urine specimens were sent to CDC in Atlanta, where they were analyzed using online solid phase extraction (SPE) with LC-MS/MS and confirmed the diagnosis in all five cases. Discussion In general, the patients’ clinical manifestations were consistent with the expected TTX toxidrome except patient 3 who developed a subarachnoid hemorrhage early during his clinical course. Two patients received neostigmine and underwent dialysis with complete recovery.

A Pseudoalteromonas Clade with Remarkable Biosynthetic Potential

Pseudoalteromonas species produce a diverse range of biologically active compounds, including those biosynthesized by non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). Here we report the biochemical and genomic analysis of Pseudoalteromonas sp. HM-SA03, isolated from the blue-ringed octopus, Hapalochalaena sp. Genome mining for secondary metabolite pathways revealed seven putative NRPS/PKS biosynthesis gene clusters, including those for the biosynthesis of alterochromides, pseudoalterobactins, alteramides and four hitherto novel compounds. Among these was a novel siderophore biosynthesis gene cluster with unprecedented architecture (NRPS-PKS-NRPS-PKS-NRPS-PKS-NRPS). Alterochromide production in HM-SA03 was also confirmed by liquid chromatography-mass spectrometry. An investigation of the biosynthetic potential of 42 publicly available Pseudoalteromonas genomes indicated that some of these gene clusters are distributed throughout the genus. Through phylogenetic analysis, a particular subset of strains formed a clade with extraordinary biosynthetic potential, with an average density of ten biosynthesis gene clusters per genome. In contrast, the majority of Pseudoalteromonas strains outside this clade contained an average of three clusters encoding complex biosynthesis. These results highlight the under-explored potential of Pseudoalteromonas as a source of new natural products.Importance This study demonstrates that the Pseudoalteromonas strain, HM-SA03, isolated from the venomous blue-ringed octopus, Hapalochalaena sp., is a biosynthetically talented organism, capable of producing alterochromides and potentially six other specialized metabolites. We have identified a pseudoalterobactin biosynthesis gene cluster and proposed a pathway for the production of the associated siderophore. A novel siderophore biosynthesis gene cluster with unprecedented architecture was also identified in the HM-SA03 genome. Finally, we have demonstrated that HM-SA03 belongs to a phylogenetic clade of strains with extraordinary biosynthetic potential. While our results do not support a role of HM-SA03 in Hapalochalaena sp. venom (tetrodotoxin) production, they emphasize the untapped potential of Pseudoalteromonas as a source of novel natural products.

Adaptive venom evolution and toxicity in octopods is driven by extensive novel gene formation, expansion, and loss.

BackgroundCephalopods represent a rich system for investigating the genetic basis underlying organismal novelties. This diverse group of specialized predators has evolved many adaptations including proteinaceous venom. Of particular interest is the blue-ringed octopus genus (Hapalochlaena), which are the only octopods known to store large quantities of the potent neurotoxin, tetrodotoxin, within their tissues and venom gland.FindingsTo reveal genomic correlates of organismal novelties, we conducted a comparative study of 3 octopod genomes, including the Southern blue-ringed octopus (Hapalochlaena maculosa). We present the genome of this species and reveal highly dynamic evolutionary patterns at both non-coding and coding organizational levels. Gene family expansions previously reported in Octopus bimaculoides (e.g., zinc finger and cadherins, both associated with neural functions), as well as formation of novel gene families, dominate the genomic landscape in all octopods. Examination of tissue-specific genes in the posterior salivary gland revealed that expression was dominated by serine proteases in non–tetrodotoxin-bearing octopods, while this family was a minor component in H. maculosa. Moreover, voltage-gated sodium channels in H. maculosa contain a resistance mutation found in pufferfish and garter snakes, which is exclusive to the genus. Analysis of the posterior salivary gland microbiome revealed a diverse array of bacterial species, including genera that can produce tetrodotoxin, suggestive of a possible production source.ConclusionsWe present the first tetrodotoxin-bearing octopod genome H. maculosa, which displays lineage-specific adaptations to tetrodotoxin acquisition. This genome, along with other recently published cephalopod genomes, represents a valuable resource from which future work could advance our understanding of the evolution of genomic novelty in this family.

First occurrence of tetrodotoxins in bivalve mollusks from Northern Adriatic Sea (Italy)

Tetrodotoxins are a potent neurotoxic class of toxins with potentially fatal effects if ingested by humans, and thus, these toxins are a dangerous threat for food safety. Tetrodotoxins are widely distributed among various organisms of different taxa, i.e. pufferfish, blue-ringed octopuses and marine gastropods. Lately the presence of these toxins has been reported in mussels (Mytilus sp.). Countries like the United Kingdom, Greece, the Netherlands, and Italy have increasingly notified the detection of tetrodotoxins in these bivalves, which are widely diffused in European shellfish farming. In this context we report, for the first time, the detection of substantial amounts of tetrodotoxins in Mytilus galloprovincialis collected in Italy in a single area of the Northern Adriatic Sea (Marano Lagoon). Tetrodotoxin-positive mussels were identified in samples collected from that geographic area at the end of May in the context of official monitoring programs both in 2017 and 2018. After the positive results were obtained by means of mouse bioassay screening, tetrodotoxins were detected and quantified by HILIC-MS/MS, revealing the bioaccumulation in the mussels of 541 and 216 μg/kg in 2017 and 2018 respectively, the highest amount ever found in mollusks in Europe. The presence of tetrodotoxins at the concentration of 413 μg/kg was further confirmed with Liquid Chromatography coupled to High Resolution Mass Spectrometry in the mussels sampled in 2017.

Marine Tetrodotoxin as a Risk for Human Health.

Tetrodotoxin (TTX) is a toxin mainly occurring naturally in contaminated puffer fish, which are a culinary delicacy in Japan. It is also detected in various marine organisms like globefish, starfish, sunfish, stars, frogs, crabs, snails, Australian blue-ringed octopuses, and bivalve molluscs. TTX is produced by marine bacteria that are consumed mainly by fish of the Tetraodontidae family and other aquatic animals. TTX poisoning through consuming marine snails has recently begun to occur over a wider geographical extent through Taiwan, China, and Europe. This neurotoxin causes food intoxication and poses an acute risk to public health. The aim of this review is to present the most recent information about TTX and its analogues with particular regard to toxicity, methods of analysis, and risk to humans of exposure.

Convergent and parallel evolution in a voltage-gated sodium channel underlies TTX-resistance in the Greater Blue-ringed Octopus: Hapalochlaena lunulata

The natural history and pharmacology of tetrodotoxin (TTX) has long intrigued biologists. This toxin has a remarkable distribution that spans two domains of life (Bacteria and Eukarya). Within Eukaryotes, TTX has only been identified in animals but is known to be present in over five-dozen species of phylogenetically distant Metazoans. Despite decades of work, the origin and biosynthetic pathways of TTX remain unresolved. Investigations in puffer fishes and salamanders have provided insights into the acquisition of auto-resistance to TTX through the evolution of voltage-gated sodium ion channels (VGSCs) that have reduced binding affinity for TTX. To date there have been no studies of these proteins in tetrodotoxic Blue-Ringed Octopuses. Here we report data demonstrating that the Greater Blue-ringed Octopus (Hapalochlaena lunulata) expresses a VGSC (HlNaV1) gene with mutations that reduce the channel's TTX-binding affinity and likely render the organism TTX resistant. We identified three amino-acid substitutions in the TTX-binding site of HlNaV1 that likely confer TTX-resistance to both the channel and the organism. These substitutions are associated with organismal TTX-resistance in other TTX-bearing taxa and are convergent with substitutions that have evolved in fish, salamanders, and some TTX-resistant invertebrates.

Preliminary report of specific behaviours of juvenile greater blue-ringed octopus Hapalochlaena lunulata (Quoy and Gaimard, 1832)

ABSTRACTThe greater blue-ringed octopus (Hapalochlaena lunulata) is one of three venomous octopuses well-known for the iridescent blue rings on the skin and dangerous toxin. However, the understanding of this octopus at the juvenile phase is limited compared to the adult phase. The other two venomous octopuses (Hapalochlaena fasciata and Hapalochlaena maculosa) have also not been thoroughly examined at the juvenile stage. We collected a juvenile greater blue-ringed octopus from the coastal waters of the Ryukyu Archipelago, Japan, and maintained it in captivity for observation. Five days after beginning rearing, iridescent blue rings, similar to those in the adult, appeared on the entire skin of the octopus and continued to be expressed with varying intensity. When the octopus was behaving aggressively it flashed the blue rings. Episodic antagonistic behaviour against the experimenters was also observed.

Tetrodotoxin (TTX) Time–Resolved Absorption and Resonance FT–IR and Raman Biospectroscopy and Density Functional Theory (DFT) Investigation of Vibronic–Mode Coupling Structure in Vibrational Spectra Analysis

Abstract Tetrodotoxin (TTX) is a potent neurotoxin. Its name derives from Tetraodontiformes, an order that includes pufferfish, porcupinefish, ocean sunfish, and triggerfish; several of these species carry the toxin. Although tetrodotoxin was discovered in these fish and found in several other aquatic animals (e.g., in blue–ringed octopuses, rough–skinned newts, and moon snails), it is actually produced by certain infecting or symbiotic bacteria like Pseudoalteromonas, Pseudomonas, and Vibrio as well as other species found in animals. Parameters such as FT–IR and Raman vibrational wavelengths and intensities for single crystal Tetrodotoxin (TTX)are calculated using density functional theory and were compared with empirical results. The investigation about vibrational spectrum of cycle dimers in crystal with carboxyl groups from each molecule of acid was shown that it leads to create Hydrogen bounds for adjacent molecules. The current study aimed to investigate the possibility of simulating the empirical values. Analysis of vibrational spectrum of Tetrodotoxin (TTX) is performed based on theoretical simulation and FT–IR empirical spectrum and Raman empirical spectrum using density functional theory in levels of F/6–31G*, HF/6–31++G**, MP2/6–31G, MP2/6–31++G**, BLYP/6–31G, BLYP/6–31++G**, B3LYP/6–31G and B3LYP6–31–HEG**. Vibration modes of methylene, carboxyl acid and phenyl cycle are separately investigated. The obtained values confirm high accuracy and validity of results obtained from calculations. Molecular structure of Tetrodotoxin (TTX) 1–42.

Toxicity and Toxin Composition of the Greater Blue-Ringed Octopus Hapalochlaena lunulata from Ishigaki Island, Okinawa Prefecture, Japan.

The toxicity of the greater blue-ringed octopus Hapalochlaena lunulata, whose bite is fatal to humans, was examined to better understand and prevent deaths from accidental bites. Living specimens were collected from tide pools on Ishigaki Island, Okinawa Prefecture, Japan, in November and December of 2015, 2016, and 2017. The specimens were examined for the anatomical distribution of the toxicity, which was expressed in terms of mouse units (MU), by the standard bioassay method for tetrodotoxin (TTX) in Japan. Paralytic toxicity to mice was detected in all of the soft parts. The posterior salivary glands exhibited the highest toxicity score with a maximum level of 9276 MU/g, which was classified as “strongly toxic” (more than 1000 MU/g tissue) according to the classification of toxicity established by the Ministry of Health, Labor and Welfare of Japan, followed by the hepatopancreas (21.1 to 734.3 MU/g), gonads (not detectable to 167.6 MU/g), arms (5.3 to 130.2 MU/g), and other body areas (17.3 to 107.4 MU/g). Next, the toxin from the salivary glands was partially purified by a Sep-Pak C18 cartridge and an Amicon Ultra Centrifugal Filter with a 3000-Da cut-off, and analyzed by liquid chromatography-mass spectrometry (LC-MS) equipped with a φ2.0 × 150-mm (5 μm) TSKgel Amide-80 column (Tosoh, Tokyo, Japan) with a mixture of 16 mM ammonium formate buffer (pH 5.5) and acetonitrile (ratio 3:7, v/v) as a mobile phase. This study aimed to clarify the toxicity and the composition of TTX and its derivatives in this toxic octopus. The main toxin in this toxic octopus was identified as TTX, along with 4-epi TTX, 4, 9-anhydroTTX and 6-epi TTX. Further, the toxicity of this species is also significant from a food hygiene point of view.

Known unknowns: Conservation and research priorities for soft sediment fauna that supports a valuable SCUBA diving industry

Wildlife tourism can provide sustainable livelihoods, but can also significantly impact vulnerable species if improperly managed. To manage these impacts whilst continuing to support livelihoods, it is important to know the interests of tourists. Using the Best-Worst scaling method, we identified taxa that were most important to scuba dive tourism on shallow soft sediment habitats in Southeast Asia. We further identified differences in interest between demographic groups. We then investigated the current conservation status and research effort into the species driving this branch of tourism. The highest ranked taxa included fishes and invertebrates such as cephalopods and crustaceans. More than 200 respondents indicated that the species most important to muck dive tourism are mimic octopus/wunderpus, blue ringed octopus, rhinopias, flamboyant cuttlefish and frogfish. Diver interests were most influenced by sex, age and dive experience. The extinction risk of six of the top ten species has not yet been assessed by the International Union for Conservation of Nature. On average, the species driving this multi-million dollar tourism industry had less than one paper published every two years over the past two decades. The lack of research and conservation effort toward these species is at odds with their economic and social importance. Considering their high economic tourism value and unknown vulnerability, there is an urgent need for more research on fauna from shallow soft sediment and other habitats important to tourism.

Mating behaviour and postcopulatory fertilization patterns in the southern blue-ringed octopus, Hapalochlaena maculosa

Female octopuses are known to store sperm from multiple males they encounter throughout a breeding season, before laying a single clutch with mixed paternity. Although octopuses display a broad range of precopulatory behaviours, and both sperm competition and cryptic female choice have been hypothesized to occur, the current understanding of how these processes influence resulting paternity remains limited. This study aimed to identify behavioural factors associated with paternity patterns and the capacity of females to bias paternity postcopulation to specific males in the southern blue-ringed octopus, Hapalochlaena maculosa. Genetic markers and controlled, sequential, laboratory pairings of genotyped individuals were used to examine paternity patterns and compare them to relative signatures of male sperm remaining in female oviducal glands after egg laying. Multiple paternity was discovered in all 12 laboratory-reared clutches. There was no indication that the relative time spent in copulation affected the resulting paternity. Males that waited for females to terminate the copulation had greater paternity when they were the first candidate male, but this was not the case among second candidate males. The relative quantities of candidate male alleles detected in female oviducal glands after egg laying were consistent with relative paternity of the candidate males in all but three cases. In one of these, sibship analysis revealed that the male that obtained less paternity than expected was in fact the female's full-sibling brother. Although this study found no evidence for female postcopulatory selection of male sperm, anecdotal evidence suggests that female H. maculosa might benefit from polyandry if chemical processes can favour clutch fertilization by unrelated males. Future studies, investigating paternity bias among genotyped males of varying, but known relatedness to the female, might help to validate this pattern.

Marine Biotoxins: Occurrence, Toxicity, and Detection Methods

This review summarizes the role of marine organisms as vectors of marine biotoxins, and discusses the need for surveillance to protect public health and ensure the quality of seafood. I Paralytic shellfish poison (PSP) and PSP-bearing organisms-PSP is produced by toxic dinoflagellates species belonging to the genera Alexandrium, Gymnodinium, and Pyrodinium. Traditionally, PSP monitoring programs have only considered filter-feeding molluscs that concentrate these toxic algae, however, increasing attention is now being paid to higher-order predators that carry PSP, such as carnivorous gastropods and crustaceans. II. Tetrodotoxin (TTX) and TTX-bearing organisms - TTX is the most common natural marine toxin that causes food poisonings in Japan, and poses a serious public health risk. TTX was long believed to be present only in pufferfish. However, TTX was detected in the eggs of California newt Taricha torosa in 1964, and since then it has been detected in a wide variety of species belonging to several different phyla. In this study, the main toxic components in the highly toxic ribbon worm Cephalothrix simula and the greater blue-ringed octopus Hapalochlaena lunulata from Japan were purified and analysed.

Blue Ringed Octopus

Blue Ringed Octopus